Construction method of characteristic spectrum or fingerprint spectrum of preparation or intermediate for strengthening body resistance and dispersing blood stasis and application of construction method

Abstract

The invention discloses a characteristic chromatogram construction method of a preparation or an intermediate for strengthening body resistance and dispersing blood stasis. The construction method comprises the following steps: preparing a test solution; preparing a reference substance solution; according to the results of the test solution and the reference solution detected by the ultra-high performance liquid chromatography, obtaining the characteristic spectrum of the body resistance strengthening and blood stasis dispersing preparation or the intermediate; the preparation for strengthening the body resistance and dispersing blood stasis or the intermediate comprises the following raw material medicines: salvia miltiorrhiza, fermented cordyceps sinensis powder, pine pollen, schisandra chinensis, peach kernel and gynostemma pentaphylla. The chemical components of the preparation or the intermediate for strengthening the body resistance and removing blood stasis are comprehensively and systematically analyzed, and a theoretical basis is provided for deep research on quality control and pharmacodynamic material basis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for constructing characteristic spectra or fingerprint spectra of a tonifying and blood-activating preparation or intermediate, and its application. Background Technology

[0002] Chronic liver disease is a long and complex condition, but from a Traditional Chinese Medicine (TCM) perspective, it is mainly caused by the body's infection with pathogenic factors, leading to a struggle between the body's resistance and the pathogenic factors. If the body's resistance is weak, it cannot expel the pathogenic factors, resulting in a prolonged and intractable disease. The basic pathogenesis is a coexistence of deficiency and excess. The deficiency mainly involves both Qi and Yin deficiency, while the excess involves heat accumulation and dampness, blood stasis and obstruction of the collaterals, resulting in Qi and Yin deficiency and the retention of damp-heat and pathogenic factors internally. From a Western medical perspective, liver fibrosis refers to the pathological process of abnormal proliferation of fibrous connective tissue in the liver when hepatocytes undergo necrosis and inflammation. The vast majority of chronic liver diseases involve liver fibrosis, which is the most important pathological feature of chronic liver disease. Liver fibrosis and cirrhosis are related but distinct. Pathologically, diffuse fibrosis alone is called liver fibrosis, while diffuse liver fibrosis accompanied by destruction of the liver lobule structure is cirrhosis. From a pathogenesis perspective, liver fibrosis is a pre-cirrhotic lesion and is reversible, while cirrhosis is the result of further development of liver fibrosis. Clinical observations show that liver fibrosis and cirrhosis are a continuous process. Liver fibrosis generally does not lead to liver dysfunction, but it can cause portal hypertension. Liver fibrosis is a repair process following liver injury, causing changes in the amount and composition of the liver's extracellular matrix (ECM).

[0003] Liver fibrosis is an inevitable pathological process in the progression of various chronic liver diseases to cirrhosis. Finding drugs to prevent, delay, or even reverse liver fibrosis is an urgent problem to be solved in liver disease research. For a long time, traditional Chinese medicine has played an important role in combating liver fibrosis. Traditional Chinese medicine believes that liver fibrosis involves both blood stasis and deficiency of vital energy; therefore, strengthening the body's resistance and removing blood stasis is an important treatment principle. Fuzheng Huayu Capsules (tablets) are specifically formulated to address the "internal stagnation of blood and deficiency of vital energy" in chronic hepatitis, liver fibrosis, and cirrhosis. Based on repeated pharmacological studies, the formula is composed of drugs that invigorate blood circulation, remove blood stasis, and nourish essence and replenish deficiency. Among them, Danshen (Salvia miltiorrhiza) invigorates blood circulation and removes blood stasis, serving as the principal drug; Cordyceps mycelium nourishes deficiency and replenishes essence; Peach kernel removes blood stasis and breaks up stagnation, serving as the assistant drug; Pine pollen nourishes and disperses liver stagnation; Gynostemma pentaphyllum clears heat and detoxifies, serving as the adjuvant drug; and Schisandra chinensis, with its sour and warm properties, nourishes the liver, serving as the guiding drug. Pharmacological experiments have shown that this compound preparation has the effects of anti-liver fibrosis, reducing portal pressure, protecting hepatocytes, reducing lipid peroxidation damage, regulating immune function, and reducing liver inflammation. Clinical trials have verified that this tonifying and stasis-removing preparation can achieve a liver fibrosis stage transformation rate of 52%–58.3%, therefore, it has a significant therapeutic effect on decompensated liver cirrhosis.

[0004] Fuzheng Huayu preparations are composed of six herbs: Danshen, fermented Cordyceps militaris powder, peach kernel, pine pollen, Gynostemma pentaphyllum, and Schisandra chinensis. They have the effect of treating liver, lung, and kidney fibrosis. Currently, there are two dosage forms on the market: (1) Fuzheng Huayu capsules: obtained the National New Drug Certificate of China in 2002 (National Drug Certificate No. Z20020053) and approved the National Drug Standard WS3-459(Z-79)-2005(Z); (2) Fuzheng Huayu tablets: obtained the National New Drug Certificate of China in 2005 (National Drug Certificate No. Z20050564) and approved the National Drug Standard YBZ19332005-2009Z. The preparations for strengthening the body and removing blood stasis are mainly used for hepatitis B liver fibrosis belonging to the syndrome of blood stasis obstructing the collaterals and liver and kidney deficiency. Symptoms include lumps under the ribs, pain in the ribs, sallow complexion, or red streaks of erythema, soreness and weakness of the waist and knees, fatigue, dizziness and blurred vision, dark red tongue or ecchymosis, thin or slightly yellow coating, and wiry and thready pulse.

[0005] Currently, there are no literature reports on a comprehensive analysis of the chemical composition of Fuzheng Huayu dry paste powder, or on the study of its characteristic spectral or fingerprint spectral characteristics. Summary of the Invention

[0006] Based on this, the present invention provides a method for constructing the characteristic spectrum of a tonifying and blood-activating preparation or intermediate, the method comprising the following steps:

[0007] Preparation of the test solution: Weigh an appropriate amount of the tonifying and blood-activating preparation or intermediate, place it in a container, add the first solvent and seal it tightly, weigh it, sonicate it for a period of time, cool it, weigh it again, add the second solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant to obtain the test solution.

[0008] Preparation of reference solution: Weigh appropriate amounts of adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masonone, schisandrol A, schisandrol B, schisandrin A, gomicin N, and schisandrin B reference standards. Add solvent to prepare a mixed solution with concentrations of 1–400 μg / ml for adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masonone, schisandrol A, schisandrol B, schisandrin A, gomicin N, and schisandrin B. Shake well, filter, and collect the filtrate to obtain the reference solution.

[0009] Based on the results of ultra-high performance liquid chromatography detection of the test solution and the reference solution, characteristic chromatograms of the tonifying and stasis-removing preparation or intermediate were obtained.

[0010] The chromatographic conditions for this ultra-high performance liquid chromatography (UHPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was an acid-water, alkaline-water, and / or buffer salt aqueous solution; mobile phase B was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and the gradient elution program was as follows: 0–2 min, 100% A; 2–6 min, 100% → 91% A; 6–11 min, 91% A; 11–13 min, 91% → 82% A; 13–18 min, 82% → 78% A; 18–25 min, ... 78%→66%A; 25–29 min, 66%→46%A; 29–36 min, 46%A; 36–38 min, 46%→18%A; 38–42 min, 18%→5%A; 42–44 min, 5%A; 44–44.1 min, 5%→100%A; 44.1–50 min, 100%A; flow rate 0.1–1 ml / min, column temperature 20–40℃, detection wavelength 180–300 nm, injection volume 0.1–5 μl;

[0011] The raw materials for this tonifying and blood-activating preparation or intermediate include Salvia miltiorrhiza, fermented Cordyceps militaris powder, pine pollen, Schisandra chinensis, peach kernel, and Gynostemma pentaphyllum.

[0012] According to another aspect of the present invention, a method for constructing a fingerprint spectrum of a tonifying and blood-activating preparation or intermediate is provided, the method comprising the following steps:

[0013] Preparation of the test solution: Weigh an appropriate amount of the tonifying and blood-activating preparation or intermediate, place it in a container, add the first solvent and seal it tightly, weigh it, sonicate it for a period of time, cool it, weigh it again, add the second solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant to obtain the test solution.

[0014] Based on the results of ultra-high performance liquid chromatography (UHPLC) analysis of the test solution, a fingerprint spectrum of the tonifying and stasis-removing preparation or intermediate was obtained.

[0015] The chromatographic conditions for this ultra-high performance liquid chromatography (UHPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was an acid-water, alkaline-water, and / or buffer salt aqueous solution; mobile phase B was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and the gradient elution program was as follows: 0–2 min, 100% A; 2–6 min, 100% → 91% A; 6–11 min, 91% A; 11–13 min, 91% → 82% A; 13–18 min, 82% → 78% A; 18–25 min, ... 78%→66%A; 25–29 min, 66%→46%A; 29–36 min, 46%A; 36–38 min, 46%→18%A; 38–42 min, 18%→5%A; 42–44 min, 5%A; 44–44.1 min, 5%→100%A; 44.1–50 min, 100%A; flow rate 0.1–1 ml / min, column temperature 20–40℃, detection wavelength 180–300 nm, injection volume 0.1–5 μl;

[0016] The raw materials for this tonifying and blood-activating preparation or intermediate include Salvia miltiorrhiza, fermented Cordyceps militaris powder, pine pollen, Schisandra chinensis, peach kernel, and Gynostemma pentaphyllum.

[0017] Furthermore, by weight, the raw materials of this tonifying and blood-activating preparation or intermediate include: 100-200 parts of peach kernel, 500-600 parts of salvia miltiorrhiza, 300-500 parts of gynostemma pentaphyllum, 200-300 parts of fermented cordyceps mycelium powder, 100-200 parts of pine pollen, 100-200 parts of schisandra chinensis, and an appropriate amount of starch.

[0018] Furthermore, by weight, the raw materials of the tonifying and stasis-removing preparation or intermediate include: approximately 130 parts of peach kernel, approximately 530 parts of salvia miltiorrhiza, approximately 400 parts of gynostemma pentaphyllum, approximately 260 parts of fermented cordyceps mycelium powder, approximately 130 parts of pine pollen, approximately 130 parts of schisandra chinensis, and an appropriate amount of starch.

[0019] Furthermore, the preparation for strengthening the body and removing blood stasis is either a solid preparation or a liquid preparation for strengthening the body and removing blood stasis.

[0020] Furthermore, the solid preparation for strengthening the body and removing blood stasis is in the form of powder, granules, tablets, capsules, films, or dry ointment.

[0021] Furthermore, this tonifying and blood-activating liquid preparation is an oral liquid preparation or an injectable liquid preparation.

[0022] Furthermore, the oral liquid preparation is a mixture, emulsion, suspension, drops, or syrup.

[0023] Furthermore, the preparation method of this tonifying and blood-activating dry paste powder includes the following steps:

[0024] (1) Weigh out the salvia miltiorrhiza, peach kernel and gynostemma pentaphyllum according to the weight ratio, add water and decoct, combine the decoctions, take the supernatant, concentrate into an extract, cool, add ethanol, precipitate, and filter.

[0025] (2) Take fermented Cordyceps militaris powder, add Schisandra chinensis to ethanol, heat and reflux, cool, combine the ethanol liquids, filter, concentrate and dry the filtrate;

[0026] (3) Alternatively, pine pollen is added to ethanol and warm-soaked. The extracts are combined, concentrated, and dried into a dry paste; and

[0027] (4) Take the above three dry pastes, mix and crush them, add an appropriate amount of starch, mix well, and obtain the dry paste powder for supporting the body and removing blood stasis.

[0028] Furthermore, the container is a conical flask, such as a stoppered conical flask.

[0029] Furthermore, the solvent is an alcohol, such as methanol.

[0030] Furthermore, the concentration of methanol is 10% to 90%, for example, about 50%.

[0031] Furthermore, the mass / volume (g / ml) ratio between the tonifying and stasis-removing preparation or intermediate and the first solvent is 0.01 to 0.5, for example, about 0.02.

[0032] Furthermore, the power of the ultrasound is 150–400W, for example, about 300W.

[0033] Furthermore, the frequency of the ultrasound is 20–60 kHz, for example, about 40 kHz.

[0034] Furthermore, the duration of the ultrasonic treatment is 5 to 30 minutes, for example, about 15 minutes.

[0035] Furthermore, the centrifuge speed is 10,000 to 15,000 rpm, for example, about 12,000 rpm.

[0036] Furthermore, the centrifugation time is 1 to 10 minutes, for example, about 5 minutes.

[0037] Furthermore, the concentration of adenosine in the reference solution was approximately 80 μg / ml.

[0038] Furthermore, the concentration of guanosine in the reference solution was approximately 80 μg / ml.

[0039] Furthermore, the concentration of sodium tanshinone in the reference solution was approximately 240 μg / ml.

[0040] Furthermore, the concentration of protocatechuic aldehyde in the reference solution was approximately 20 μg / ml.

[0041] Furthermore, the concentration of amygdalin in the reference solution was approximately 60 μg / ml.

[0042] Furthermore, the concentration of rutin in the reference solution was approximately 6 μg / ml.

[0043] Furthermore, the concentration of salvianolic acid D in the reference solution was approximately 30 μg / ml.

[0044] Furthermore, the concentration of rosmarinic acid in the reference solution was approximately 40 μg / ml.

[0045] Furthermore, the concentration of shikonin in the reference solution was approximately 30 μg / ml.

[0046] Furthermore, the concentration of salvianolic acid B in the reference solution was approximately 160 μg / ml.

[0047] Furthermore, the concentration of phytolaccaside in the reference solution was approximately 6 μg / ml.

[0048] Furthermore, the concentration of salvianolic acid A in the reference solution was approximately 100 μg / ml.

[0049] Furthermore, the concentration of naringenin in the reference solution was approximately 6 μg / ml.

[0050] Furthermore, the concentration of genistein in the reference solution was approximately 6 μg / ml.

[0051] Furthermore, the concentration of masoylide in the reference solution was approximately 30 μg / ml.

[0052] Furthermore, the concentration of schisandrol A in the reference solution was approximately 30 μg / ml.

[0053] Furthermore, the concentration of schisandrin ethyl in the reference solution was approximately 24 μg / ml.

[0054] Furthermore, the concentration of schisandrin A in the reference solution was approximately 16 μg / ml.

[0055] Furthermore, the concentration of gomisin N in the reference solution was approximately 10 μg / ml.

[0056] Furthermore, the concentration of schisandrin B in the reference solution was approximately 20 μg / ml.

[0057] Furthermore, the flow rate is 0.2–0.4 ml / min, for example, about 0.3 ml / min.

[0058] Furthermore, the column temperature is 25–35°C, for example, about 30°C.

[0059] Furthermore, the detection wavelength is 190–230 nm, for example, 210 nm.

[0060] Furthermore, the injection volume is 0.5–1.5 μl, for example, about 1.0 μl.

[0061] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the salvianolic acid B is not less than 200,000.

[0062] Furthermore, the resolution between the chromatographic peak corresponding to naringenin and the chromatographic peak corresponding to genistein is greater than 1.0.

[0063] Furthermore, the chromatographic column is WatersACQUITY. HSS T3 column.

[0064] Furthermore, the specifications of the chromatographic column are as follows: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.

[0065] 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.

[0066] 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.

[0067] Furthermore, the acidic aqueous solution is a 0.01% to 0.1% acidic aqueous solution.

[0068] Furthermore, the acidic aqueous solution is a 0.01% to 0.1% phosphoric acid aqueous solution.

[0069] Furthermore, the acidic aqueous solution is a 0.04% to 0.06% phosphoric acid aqueous solution.

[0070] Furthermore, the acidic aqueous solution is an aqueous solution of approximately 0.05% phosphoric acid.

[0071] Furthermore, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0072] Furthermore, the pH value of the buffer salt solution is no greater than 7.0.

[0073] Furthermore, the mobile phase B is acetonitrile.

[0074] Furthermore, at the detection wavelength of 210 nm, the fingerprint spectrum includes 18 characteristic peaks: peak 1 is the chromatographic peak of adenosine, peak 2 is the chromatographic peak of guanosine, peak 3 is the chromatographic peak of tanshinone, peak 4 is the chromatographic peak of protocatechuic aldehyde, peak 5 is the chromatographic peak of D-amygynol, peak 6 is the chromatographic peak of rutin, peak 7 is the chromatographic peak of salvianolic acid D, peak 8 is the chromatographic peak of rosmarinic acid, and peak 9 is the chromatographic peak of shikonin. The chromatographic peaks are as follows: peak 10 is the chromatographic peak of salvianolic acid B; peak 11 is the chromatographic peak of salvianolic acid A and phytolaccaside; peak 12 is the chromatographic peak of naringenin; peak 13 is the chromatographic peak of genistein; peak 14 is the chromatographic peak of masonin; peak 15 is the chromatographic peak of schisandrol A; peak 16 is the chromatographic peak of schisandrol B; peak 17 is the chromatographic peak of schisandrin A; and peak 18 is the chromatographic peak of schisandrin B and gomisin N.

[0075] Furthermore, peak 1 originates from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 2 originates from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 3 originates from salvia miltiorrhiza; peak 4 originates from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 5 originates from peach kernel; peak 6 originates from gynostemma pentaphyllum and schisandra chinensis; and peak 7 originates from salvia miltiorrhiza. The medicinal materials are as follows: Peak 8 comes from Salvia miltiorrhiza, Peak 9 comes from Salvia miltiorrhiza, Peak 10 comes from Salvia miltiorrhiza, Peak 11 comes from Salvia miltiorrhiza and Gynostemma pentaphyllum, Peak 12 comes from pine pollen, Peak 13 comes from fermented Cordyceps militaris powder, Peak 14 comes from fermented Cordyceps militaris powder, Peak 15 comes from Schisandra chinensis, Peak 16 comes from Schisandra chinensis, Peak 17 comes from Schisandra chinensis, and Peak 18 comes from Schisandra chinensis.

[0076] Furthermore, at the detection wavelength of 210 nm, the fingerprint spectrum includes 18 characteristic peaks. Taking the chromatographic peak of salvianolic acid B (peak number 10) as the reference peak, the relative retention times of the other 17 characteristic peaks are as follows: peak 1 0.235±10%, peak 2 0.267±10%, peak 3 0.341±10%, peak 4 0.431±10%, peak 5 0.587±10%, peak 6 0.757±10%, peak 7... Peak 0.855±10%, Peak 8 0.924±10%, Peak 9 0.948±10%, Peak 11 1.054±10%, Peak 12 1.162±10%, Peak 13 1.172±10%, Peak 14 1.325±10%, Peak 15 1.357±10%, Peak 16 1.405±10%, Peak 17 1.764±10%, Peak 18 1.792±10%.

[0077] Furthermore, at the detection wavelength of 210 nm, the fingerprint spectrum includes 18 characteristic peaks. Taking the chromatographic peak of salvianolic acid B (peak number 10) as the reference peak, the relative peak areas of the other 17 characteristic peaks are as follows: peak 1 0.282±10%, peak 2 0.122±10%, peak 3 0.714±10%, peak 4 0.205±10%, peak 5 0.041±10%, peak 6 0.028±10%, peak 7... Peak 0.179±10%, Peak 8 0.138±10%, Peak 9 0.128±10%, Peak 11 0.424±10%, Peak 12 0.017±10%, Peak 13 0.049±10%, Peak 14 0.128±10%, Peak 15 0.429±10%, Peak 16 0.127±10%, Peak 17 0.099±10%, Peak 18 0.182±10%.

[0078] According to another aspect of the present invention, a quality control method for a tonifying and blood-activating preparation or intermediate is provided, the quality control method comprising the following steps:

[0079] (1) Establish standard characteristic chromatograms or fingerprint chromatograms of reference samples for tonifying and stasis-removing preparations or intermediates based on the above construction method;

[0080] (2) Take the test solution of the tonifying and stasis-removing preparation or intermediate, and perform detection according to the chromatographic conditions in the above construction method to obtain the characteristic chromatogram or fingerprint chromatogram of the tonifying and stasis-removing preparation or intermediate to be tested; and

[0081] (3) Compare the characteristic spectrum or fingerprint spectrum of the test sample of the tonifying and blood-activating preparation or intermediate obtained in step (2) with the standard characteristic spectrum or fingerprint spectrum of the reference sample of the tonifying and blood-activating preparation or intermediate obtained in step (1). If the sample meets the requirements, it is a qualified product; otherwise, it is an unqualified product.

[0082] Furthermore, the compliance requirement includes one or more of the following:

[0083] (1) The characteristic chromatogram or fingerprint chromatogram of the test sample of the tonifying and stasis-removing preparation or intermediate shows 18 characteristic chromatographic peaks. At a detection wavelength of 210 nm, with the chromatographic peak of salvianolic acid B as the S peak, the relative retention time of each characteristic chromatographic peak in the characteristic chromatogram or fingerprint chromatogram of the test sample of the tonifying and stasis-removing preparation or intermediate and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard characteristic chromatogram or fingerprint chromatogram of the reference sample of the tonifying and stasis-removing preparation or intermediate; and

[0084] (2) When the detection wavelength is 210 nm, with the chromatographic peak of salvianolic acid B as the S peak, the relative peak area of ​​each characteristic chromatographic peak in the characteristic chromatographic spectrum or fingerprint spectrum of the test sample of the tonifying and blood-activating preparation or intermediate is within ±10% of the relative peak area of ​​each characteristic chromatographic peak in the standard characteristic chromatographic spectrum or fingerprint spectrum of the tonifying and blood-activating preparation or intermediate reference sample; and

[0085] (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the characteristic chromatogram or fingerprint of the test sample of the tonifying and blood-activating preparation or intermediate and the standard characteristic chromatogram or fingerprint of the reference sample of the tonifying and blood-activating preparation or intermediate shall not be less than 0.90.

[0086] 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, quality evaluation or quality control of the tonifying and stasis-removing preparation or intermediate.

[0087] The beneficial effects of this invention are:

[0088] This invention provides a comprehensive and systematic analysis of the chemical composition of tonifying and blood-activating preparations or intermediates, offering a theoretical basis for in-depth research on quality control and the material basis of pharmacodynamics. Attached Figure Description

[0089] 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.

[0090] Figure 1 These are chromatograms of multiple batches of tonifying and stasis-removing preparations or intermediates at a wavelength of 210 nm.

[0091] Figure 2This is a reference fingerprint chromatogram of a tonifying and stasis-removing preparation or intermediate at a wavelength of 210 nm. The compound in peak 1 is adenosine, peak 2 is guanosine, peak 3 is tanshinone, peak 4 is protocatechuic aldehyde, peak 5 is D-amygdalin, peak 6 is rutin, peak 7 is salvianolic acid D, peak 8 is rosmarinic acid, peak 9 is shikonin, peak 10 is salvianolic acid B, peak 11 is salvianolic acid A + phytolaccaside, peak 12 is naringenin, peak 13 is genistein, peak 14 is masonone, peak 15 is schisandrol A, peak 16 is schisandrol B, peak 17 is schisandrin A, and peak 18 is schisandrin B + gomisin N. Detailed Implementation

[0092] 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.

[0093] 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.

[0094] 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.

[0095] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0096] 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.

[0097] As described in the background section, there is no literature report on the comprehensive analysis of the chemical composition and fingerprinting of tonifying and stasis-removing preparations or intermediates. To address this issue, the present invention provides a method for constructing fingerprints of tonifying and stasis-removing preparations or intermediates, comprising the following steps:

[0098] Preparation of the test solution: Weigh an appropriate amount of the tonifying and blood-activating preparation or intermediate, place it in a container, add the first solvent and seal it tightly, weigh it, sonicate it for a period of time, cool it, weigh it again, add the second solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant to obtain the test solution.

[0099] Preparation of reference solution: Weigh appropriate amounts of adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masonone, schisandrol A, schisandrol B, schisandrin A, gomicin N, and schisandrin B reference standards. Add solvent to prepare a mixed solution with concentrations of 1–400 μg / ml for adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masonone, schisandrol A, schisandrol B, schisandrin A, gomicin N, and schisandrin B. Shake well, filter, and collect the filtrate to obtain the reference solution.

[0100] Based on the results of ultra-high performance liquid chromatography (UHPLC) analysis of the test solution and the reference solution, fingerprint chromatograms of the tonifying and stasis-removing preparation or intermediate were obtained.

[0101] The chromatographic conditions for this ultra-high performance liquid chromatography (UHPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was an acid-water, alkaline-water, and / or buffer salt aqueous solution; mobile phase B was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and the gradient elution program was as follows: 0–2 min, 100% A; 2–6 min, 100% → 91% A; 6–11 min, 91% A; 11–13 min, 91% → 82% A; 13–18 min, 82% → 78% A; 18–25 min, ... 78%→66%A; 25–29 min, 66%→46%A; 29–36 min, 46%A; 36–38 min, 46%→18%A; 38–42 min, 18%→5%A; 42–44 min, 5%A; 44–44.1 min, 5%→100%A; 44.1–50 min, 100%A; flow rate 0.1–1 ml / min, column temperature 20–40℃, detection wavelength 180–300 nm, injection volume 0.1–5 μl;

[0102] The raw materials for this tonifying and blood-activating preparation or intermediate include Salvia miltiorrhiza, fermented Cordyceps militaris powder, pine pollen, Schisandra chinensis, peach kernel, and Gynostemma pentaphyllum.

[0103] According to another aspect of the present invention, a method for constructing a fingerprint spectrum of a tonifying and blood-activating preparation or intermediate is provided, the method comprising the following steps:

[0104] Preparation of the test solution: Weigh an appropriate amount of the tonifying and blood-activating preparation or intermediate, place it in a container, add the first solvent and seal it tightly, weigh it, sonicate it for a period of time, cool it, weigh it again, add the second solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant to obtain the test solution.

[0105] Based on the results of ultra-high performance liquid chromatography (UHPLC) analysis of the test solution, a fingerprint spectrum of the tonifying and stasis-removing preparation or intermediate was obtained.

[0106] The chromatographic conditions for this ultra-high performance liquid chromatography (UHPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was an acid-water, alkaline-water, and / or buffer salt aqueous solution; mobile phase B was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and the gradient elution program was as follows: 0–2 min, 100% A; 2–6 min, 100% → 91% A; 6–11 min, 91% A; 11–13 min, 91% → 82% A; 13–18 min, 82% → 78% A; 18–25 min, ... 78%→66%A; 25–29 min, 66%→46%A; 29–36 min, 46%A; 36–38 min, 46%→18%A; 38–42 min, 18%→5%A; 42–44 min, 5%A; 44–44.1 min, 5%→100%A; 44.1–50 min, 100%A; flow rate 0.1–1 ml / min, column temperature 20–40℃, detection wavelength 180–300 nm, injection volume 0.1–5 μl;

[0107] The raw materials for this tonifying and blood-activating preparation or intermediate include Salvia miltiorrhiza, fermented Cordyceps militaris powder, pine pollen, Schisandra chinensis, peach kernel, and Gynostemma pentaphyllum.

[0108] In a preferred embodiment, the raw materials of the tonifying and blood-activating preparation or intermediate, by weight, include: 100-200 parts of peach kernel, 500-600 parts of salvia miltiorrhiza, 300-500 parts of gynostemma pentaphyllum, 200-300 parts of fermented cordyceps mycelium powder, 100-200 parts of pine pollen, 100-200 parts of schisandra chinensis, and an appropriate amount of starch.

[0109] In this invention, when quantities, concentrations, times, flow rates, column temperatures, injection volumes, ratios, power, frequencies, rotational speeds, 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 "100–200" is disclosed, the described range should be interpreted as including ranges such as "100–200", "100–180", "100–160", "100–140", "100–120", "120–200", "120–180", "120–160", "120–140", "140–200", "140–180", "140–160", "160–200", "160–180", "180–200", etc. When a range of values ​​is described in this document, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0110] In a preferred embodiment, the raw materials of the tonifying and blood-activating preparation or intermediate, by weight, include: about 130 parts of peach kernel, about 530 parts of salvia miltiorrhiza, about 400 parts of gynostemma pentaphyllum, about 260 parts of fermented cordyceps mycelium powder, about 130 parts of pine pollen, about 130 parts of schisandra chinensis, and an appropriate amount of starch.

[0111] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 130" includes ±5% of 130, or from 123.5 to 136.5; "about 530" includes ±5% of 530, or from 503.5 to 556.5; "about 400" includes ±5% of 400, or from 380 to 420; "about 260" includes ±5% of 260, or from 247 to 273.

[0112] In a preferred embodiment, the tonifying and stasis-removing preparation is a solid preparation or a liquid preparation for tonifying and stasis removal.

[0113] In a preferred embodiment, the solid preparation for strengthening the body and removing blood stasis is a powder, granules, tablets, capsules, film, or dry ointment.

[0114] In a preferred embodiment, the tonifying and blood-activating liquid preparation is an oral liquid preparation or an injectable liquid preparation.

[0115] In a preferred embodiment, the oral liquid preparation is a mixture, emulsion, suspension, drops, or syrup.

[0116] In a preferred embodiment, the preparation method of the tonic and stasis-removing dry ointment powder includes the following steps:

[0117] (1) Weigh out the salvia miltiorrhiza, peach kernel and gynostemma pentaphyllum according to the weight ratio, add water and decoct, combine the decoctions, take the supernatant, concentrate into an extract, cool, add ethanol, precipitate, and filter.

[0118] (2) Take fermented Cordyceps militaris powder, add Schisandra chinensis to ethanol, heat and reflux, cool, combine the ethanol liquids, filter, concentrate and dry the filtrate;

[0119] (3) Alternatively, pine pollen is added to ethanol and warm-soaked. The extracts are combined, concentrated, and dried into a dry paste; and

[0120] (4) Take the above three dry pastes, mix and crush them, add an appropriate amount of starch, mix well, and obtain the dry paste powder for supporting the body and removing blood stasis.

[0121] In a preferred embodiment, the container is a conical flask, such as a stoppered conical flask.

[0122] In a preferred embodiment, the solvent is an alcohol, such as methanol.

[0123] In a preferred embodiment, the concentration of methanol is 10% to 90%, for example, about 50%.

[0124] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50%" includes 50% ± 5%, or from 47.5% to 52.5%.

[0125] In a preferred embodiment, the mass / volume (g / ml) ratio between the tonifying and stasis-removing preparation or intermediate and the first solvent is 0.01 to 0.5, for example, about 0.02.

[0126] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.02" includes ±5% of 0.02, or from 0.019 to 0.021.

[0127] In a preferred embodiment, the power of the ultrasound is 150 to 400 W, for example, about 300 W.

[0128] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 300" includes ±5% of 300, or from 285 to 315.

[0129] In a preferred embodiment, the frequency of the ultrasound is 20 to 60 kHz, for example, about 40 kHz.

[0130] 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.

[0131] In a preferred embodiment, the ultrasonic treatment time is 5 to 30 minutes, for example, about 15 minutes.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0133] In a preferred embodiment, the centrifugation speed is 10,000 to 15,000 rpm, for example, about 12,000 rpm.

[0134] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 12000" includes ±5% of 12000, or from 11400 to 12600.

[0135] In a preferred embodiment, the centrifugation time is 1 to 10 minutes, for example, about 5 minutes.

[0136] 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.

[0137] In a preferred embodiment, the concentration of adenosine in the reference solution is about 80 μg / ml.

[0138] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0139] In a preferred embodiment, the concentration of guanosine in the reference solution is about 80 μg / ml.

[0140] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 80" includes ±5% of 80, or from 76 to 84.

[0141] In a preferred embodiment, the concentration of sodium tanshinone in the reference solution is about 240 μg / ml.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 240" includes ±5% of 240, or from 228 to 252.

[0143] In a preferred embodiment, the concentration of protocatechuic aldehyde in the reference solution is about 20 μg / ml.

[0144] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0145] In a preferred embodiment, the concentration of amygdalin in the reference solution is about 60 μg / ml.

[0146] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 60" includes ±5% of 60, or from 57 to 63.

[0147] In a preferred embodiment, the concentration of rutin in the reference solution is about 6 μg / ml.

[0148] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0149] In a preferred embodiment, the concentration of salvianolic acid D in the reference solution is about 30 μg / ml.

[0150] 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.

[0151] In a preferred embodiment, the concentration of rosmarinic acid in the reference solution is about 40 μg / ml.

[0152] 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.

[0153] In a preferred embodiment, the concentration of shikonin in the reference solution is about 30 μg / ml.

[0154] 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.

[0155] In a preferred embodiment, the concentration of salvianolic acid B in the reference solution is about 160 μg / ml.

[0156] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 160" includes ±5% of 160, or from 152 to 168.

[0157] In a preferred embodiment, the concentration of phytolaccaside in the reference solution is about 6 μg / ml.

[0158] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0159] In a preferred embodiment, the concentration of salvianolic acid A in the reference solution is about 100 μg / ml.

[0160] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 100" includes ±5% of 100, or from 95 to 105.

[0161] In a preferred embodiment, the concentration of naringenin in the reference solution is about 6 μg / ml.

[0162] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0163] In a preferred embodiment, the concentration of genistein in the reference solution is about 6 μg / ml.

[0164] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3.

[0165] In a preferred embodiment, the concentration of masoylide in the reference solution is about 30 μg / ml.

[0166] 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.

[0167] In a preferred embodiment, the concentration of schisandrol A in the reference solution is about 30 μg / ml.

[0168] 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.

[0169] In a preferred embodiment, the concentration of schisandrin ethyl in the reference solution is about 24 μg / ml.

[0170] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 24" includes ±5% of 24, or from 22.8 to 25.2.

[0171] In a preferred embodiment, the concentration of schisandrin A in the reference solution is about 16 μg / ml.

[0172] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 16" includes ±5% of 16, or from 15.2 to 16.8.

[0173] In a preferred embodiment, the concentration of gomisin N in the reference solution is about 10 μg / ml.

[0174] 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.

[0175] In a preferred embodiment, the concentration of schisandrin B in the reference solution is about 20 μg / ml.

[0176] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0177] In a preferred embodiment, the flow rate is 0.2 to 0.4 ml / min, for example, about 0.3 ml / min.

[0178] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0179] In a preferred embodiment, the column temperature is 25–35°C, for example, about 30°C.

[0180] 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.

[0181] In a preferred embodiment, the detection wavelength is 190–230 nm, for example, 210 nm.

[0182] In a preferred embodiment, the injection volume is 0.5 to 1.5 μl, for example, about 1.0 μl.

[0183] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0184] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the salvianolic acid B is not less than 200,000.

[0185] In a preferred embodiment, the resolution between the chromatographic peak corresponding to naringenin and the chromatographic peak corresponding to genistein is greater than 1.0.

[0186] In a preferred embodiment, the chromatographic column is Waters ACQUITY. HSS T3 column.

[0187] In a preferred embodiment, the chromatographic column has the following specifications: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.

[0188] 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.

[0189] 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.

[0190] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 0.1% acidic aqueous solution.

[0191] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 0.1% aqueous solution of phosphoric acid.

[0192] In a preferred embodiment, the acidic aqueous solution is a 0.04% to 0.06% aqueous solution of phosphoric acid.

[0193] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.05% phosphoric acid.

[0194] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.05%" includes 0.05% ± 5%, or from 0.0475% to 0.0525%.

[0195] In a preferred embodiment, the buffer salt solution is an aqueous solution of phosphate and / or an aqueous solution of acetate.

[0196] In a preferred embodiment, the pH value of the buffer salt solution is not greater than 7.0.

[0197] In a preferred embodiment, the mobile phase B is acetonitrile.

[0198] In a preferred embodiment, when the detection wavelength is 210 nm, the fingerprint spectrum includes 18 characteristic peaks: peak 1 is the chromatographic peak of adenosine, peak 2 is the chromatographic peak of guanosine, peak 3 is the chromatographic peak of tanshinone, peak 4 is the chromatographic peak of protocatechuic aldehyde, peak 5 is the chromatographic peak of D-amygynol, peak 6 is the chromatographic peak of rutin, peak 7 is the chromatographic peak of salvianolic acid D, peak 8 is the chromatographic peak of rosmarinic acid, and peak 9 is the chromatographic peak of lithospermum erythrorhizon. The chromatographic peaks of the acids are as follows: peak 10 is the chromatographic peak of salvianolic acid B; peak 11 is the chromatographic peak of salvianolic acid A and phytolaccaside; peak 12 is the chromatographic peak of naringenin; peak 13 is the chromatographic peak of genistein; peak 14 is the chromatographic peak of masonin; peak 15 is the chromatographic peak of schisandrol A; peak 16 is the chromatographic peak of schisandrol B; peak 17 is the chromatographic peak of schisandrin A; and peak 18 is the chromatographic peak of schisandrin B and gomisin N.

[0199] In a preferred embodiment, peak 1 is derived from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 2 is derived from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 3 is derived from salvia miltiorrhiza; peak 4 is derived from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 5 is derived from peach kernel; peak 6 is derived from gynostemma pentaphyllum and schisandra chinensis; and peak 7... Peak 8 comes from Salvia miltiorrhiza, Peak 9 comes from Salvia miltiorrhiza, Peak 10 comes from Salvia miltiorrhiza, Peak 11 comes from Salvia miltiorrhiza and Gynostemma pentaphyllum, Peak 12 comes from pine pollen, Peak 13 comes from fermented Cordyceps militaris powder, Peak 14 comes from fermented Cordyceps militaris powder, Peak 15 comes from Schisandra chinensis, Peak 16 comes from Schisandra chinensis, Peak 17 comes from Schisandra chinensis, Peak 18 comes from Schisandra chinensis.

[0200] In a preferred embodiment, when the detection wavelength is 210 nm, the fingerprint spectrum includes 18 characteristic peaks. Using the chromatographic peak of salvianolic acid B (peak number 10) as the reference peak, the relative retention times of the other 17 characteristic peaks are as follows: peak 1 0.235 ± 10%, peak 2 0.267 ± 10%, peak 3 0.341 ± 10%, peak 4 0.431 ± 10%, peak 5 0.587 ± 10%, and peak 6 0.757 ± 10%. Peak 7: 0.855±10%; Peak 8: 0.924±10%; Peak 9: 0.948±10%; Peak 11: 1.054±10%; Peak 12: 1.162±10%; Peak 13: 1.172±10%; Peak 14: 1.325±10%; Peak 15: 1.357±10%; Peak 16: 1.405±10%; Peak 17: 1.764±10%; Peak 18: 1.792±10%.

[0201] In a preferred embodiment, when the detection wavelength is 210 nm, the fingerprint spectrum includes 18 characteristic peaks. Using the chromatographic peak of salvianolic acid B (peak number 10) as the reference peak, the relative peak areas of the other 17 characteristic peaks are as follows: peak 1 0.282±10%, peak 2 0.122±10%, peak 3 0.714±10%, peak 4 0.205±10%, peak 5 0.041±10%, and peak 6 0.028±10%. Peak 7: 0.179±10%; Peak 8: 0.138±10%; Peak 9: 0.128±10%; Peak 11: 0.424±10%; Peak 12: 0.017±10%; Peak 13: 0.049±10%; Peak 14: 0.128±10%; Peak 15: 0.429±10%; Peak 16: 0.127±10%; Peak 17: 0.099±10%; Peak 18: 0.182±10%.

[0202] According to another aspect of the present invention, a quality control method for a tonifying and blood-activating preparation or intermediate is provided, the quality control method comprising the following steps:

[0203] (1) Establish standard characteristic chromatograms or fingerprint chromatograms of reference samples for tonifying and stasis-removing preparations or intermediates based on the above construction method;

[0204] (2) Take the test solution of the tonifying and stasis-removing preparation or intermediate, and perform detection according to the chromatographic conditions in the above construction method to obtain the characteristic chromatogram or fingerprint chromatogram of the tonifying and stasis-removing preparation or intermediate to be tested; and

[0205] (3) Compare the characteristic spectrum or fingerprint spectrum of the test sample of the tonifying and blood-activating preparation or intermediate obtained in step (2) with the standard characteristic spectrum or fingerprint spectrum of the reference sample of the tonifying and blood-activating preparation or intermediate obtained in step (1). If the sample meets the requirements, it is a qualified product; otherwise, it is an unqualified product.

[0206] In a preferred embodiment, the compliance requirement includes one or more of the following:

[0207] (1) The characteristic chromatogram or fingerprint chromatogram of the test sample of the tonifying and stasis-removing preparation or intermediate shows 18 characteristic chromatographic peaks. At a detection wavelength of 210 nm, with the chromatographic peak of salvianolic acid B as the S peak, the relative retention time of each characteristic chromatographic peak in the characteristic chromatogram or fingerprint chromatogram of the test sample of the tonifying and stasis-removing preparation or intermediate and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard characteristic chromatogram or fingerprint chromatogram of the reference sample of the tonifying and stasis-removing preparation or intermediate; and

[0208] (2) When the detection wavelength is 210 nm, with the chromatographic peak of salvianolic acid B as the S peak, the relative peak area of ​​each characteristic chromatographic peak in the characteristic chromatographic spectrum or fingerprint spectrum of the test sample of the tonifying and blood-activating preparation or intermediate is within ±10% of the relative peak area of ​​each characteristic chromatographic peak in the standard characteristic chromatographic spectrum or fingerprint spectrum of the tonifying and blood-activating preparation or intermediate reference sample; and

[0209] (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the characteristic chromatogram or fingerprint of the test sample of the tonifying and blood-activating preparation or intermediate and the standard characteristic chromatogram or fingerprint of the reference sample of the tonifying and blood-activating preparation or intermediate shall not be less than 0.90.

[0210] 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, quality evaluation or quality control of the tonifying and stasis-removing preparation or intermediate.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] Example

[0215] 1. Instruments and reagents

[0216] 1.1 Instruments and Equipment

[0217] Ultra-high performance liquid chromatograph: Waters H-Class (online degasser, quaternary gradient pump, autosampler, PDA detector, column oven) Waters Technology (Shanghai) Co., Ltd.; 0.01% balance: Mettler Toledo ME104; 0.01% balance: Mettler Toledo MS105DU; 0.01% balance: Mettler Toledo XPR2; High-precision centrifuge: SIGMA 3K15; CNC ultrasonic instrument: KQ-300DB Kunshan Ultrasonic Instrument Co., Ltd.

[0218] 1.2 Samples and Reagents

[0219] Fuzheng Huayu Dry Ointment Powder, batch number: 190520, serial number: S1, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 190601, serial number: S2, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 190608, serial number: S3, provided by Shanghai Huanghai Pharmaceutical Co., Ltd. (method development and validation batch); Fuzheng Huayu Dry Ointment Powder, batch number: 190611, serial number: S4, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 190913, serial number: S5, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 191001, serial number: S6, provided by Shanghai Huanghai Pharmaceutical Co., Ltd. Provided by: Fuzheng Huayu Dry Ointment Powder, batch number: 191113, serial number: S7, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 191118, serial number: S8, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 191205, serial number: S9, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 191211, serial number: S10, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 191219, serial number: S11, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: 200202, serial number: S12, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder; The following are listed as examples of pharmaceutical products: **Blood Stasis Removal Powder (Batch No.: 200207, Item No.: S13):** * **Blood Stasis Removal Powder (Batch No.: 200302, Item No.: S14):** * **Blood Stasis Removal Powder (Batch No.: 200306, Item No.: S15):** * **Blood Stasis Removal Powder (Batch No.: 200312, Item No.: S16):** * **Blood Stasis Removal Powder (Batch No.: S190501, Item No.: S17):** * **Blood Stasis Removal Powder (Batch No.: S191101, Item No.: S18):** * **Blood Stasis Removal Powder (Batch No.: S191101, Item No.: S18):** * **Blood Stasis Removal Powder (Batch No.: S191101, Item No.: S18):** * **Blood Stasis Removal Powder (Batch No.: S190207, Item No.: S13 ... The following are listed as product names: **Blood Stasis Removal Powder (Batch No.: S191102, Item No.: S19):** Provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; **Blood Stasis Removal and Tonifying Powder (Batch No.: S200301, Item No.: S20):** Provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; **Blood Stasis Removal and Tonifying Powder (Batch No.: S200401, Item No.: S21):** Provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; **Blood Stasis Removal and Tonifying Powder (Batch No.: S200402, Item No.: S22):** Provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; **Blood Stasis Removal and Tonifying Powder (Batch No.: S200404, Item No.: S23):** Provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; **Blood Stasis Removal and Tonifying Powder (Batch No.: S200405, Item No.: S24):** Provided by Shanghai Huanghai Pharmaceutical Co., Ltd.Fuzheng Huayu Dry Ointment Powder, batch number: S200406, serial number: S25, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: S200407, serial number: S26, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Dry Ointment Powder, batch number: S200501, serial number: S27, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Fuzheng Huayu Danshen Yin-Yang Dry Ointment Powder, serial number: BDS-DSYX, provided by Shanghai Huanghai Pharmaceutical Co., Ltd. Provided by: Fermented Cordyceps sinensis (negative) dry extract powder for strengthening the body and removing blood stasis (product code: BDS-CCYX), Shanghai Huanghai Pharmaceutical Co., Ltd.; Peach kernel (negative) dry extract powder for strengthening the body and removing blood stasis (product code: BDS-TRYX), Shanghai Huanghai Pharmaceutical Co., Ltd.; Pine pollen (negative) dry extract powder for strengthening the body and removing blood stasis (product code: BDS-SHFYX), Shanghai Huanghai Pharmaceutical Co., Ltd.; Gynostemma pentaphyllum (negative) dry extract powder for strengthening the body and removing blood stasis (product code: BDS-JGLYX), Shanghai Huanghai Pharmaceutical Co., Ltd. The company provides: Schisandra chinensis negative-labeled dry extract powder for strengthening the body and removing blood stasis, product number: BDS-WWZYX, provided by Shanghai Huanghai Pharmaceutical Co., Ltd.; Water: purified water, 20190801H, Guangzhou Watsons Food & Beverage Co., Ltd.; Methanol: LC-MS grade, batch number: I1099035904, Merck Chemical Technology (Shanghai) Co., Ltd.; Acetonitrile: LC-MS grade, batch number: I0965929833, Merck Chemical Technology (Shanghai) Co., Ltd.; Phosphoric acid: HPL C, Batch No.: 190900, Thermo Fisher Scientific (China) Co., Ltd.; Formic acid: Grade: Mass spectrometry pure, Batch No.: A1000090, CNW Company; Acetic acid: Grade: Mass spectrometry pure, Batch No.: 178042, Thermo Fisher Scientific (China) Co., Ltd.; Ammonium formate: Grade: Mass spectrometry pure, Batch No.: 143079, Thermo Fisher Scientific (China) Co., Ltd.; Adenosine: Purity: 99.90%, Batch No.: IOM365, USPreference Standard; Guanosine: Content: 93.60%, Batch No.: 111977-201501, China National Institutes for Food and Drug Control; Sodium Tanshinone: Content: 98.10%, Batch No.: 110855-201614, China National Institutes for Food and Drug Control; Protocatechuic Aldehyde: Content: 99.30%, Batch No.: 110810-201608, China National Institutes for Food and Drug Control; Amygdalin: Content: 88.2%, Batch No.: 110820-201808, China National Institutes for Food and Drug Control; Rutin: Content: 91.3%, Batch No.: R054J0, USP reference standard; Tanshinone D: Content: ≥96.0%, Batch No.: wkq18041602, Sichuan Weikeqi Biotechnology Co., Ltd.; Rosmarinic Acid: Content: 99.4%, Batch No.: F0M076, USP reference standard;Shisocaric acid: Content: ≥95%, Batch No.: 7831, Shanghai Shidander Standard Technical Service Co., Ltd.; Tanshinone B: Content: 95.00%, Batch No.: F0M013, USP reference standard; Phytolaccaside: Content: ≥98%, Batch No.: 6766, Shanghai Shidander Standard Technical Service Co., Ltd.; Tanshinone A: Content: ≥98%, Batch No.: 4881, Shanghai Shidander Standard Technical Service Co., Ltd.; Naringin: Content: ≥95.0%, Batch No.: 3597, Shanghai Shidander Standard Technical Service Co., Ltd.; Genistein: Content: ≥98%, Batch No.: 6310, Shanghai Shidander Standard Technical Service Co., Ltd.; Masoyanol: Content: ≥90%, Batch No.: 8541, Shanghai Shidander Standard Technical Service Co., Ltd.; Schisandrol A: Content: 99.40%, Batch No.: F00810, USP reference Standard; Schisandrin E: Content: ≥98%, Batch No.: 3249, Shanghai Shidander Standard Technical Service Co., Ltd.; Schisandrin A: Content: 99.50%, Batch No.: 110764-201915, China National Institutes for Food and Drug Control; Gomicin N: Content: ≥98%, Batch No.: 8550, Shanghai Shidander Standard Technical Service Co., Ltd.; Schisandrin B: Content: 99.10%, Batch No.: 110765-201813, China National Institutes for Food and Drug Control.

[0220] 2. Methodology Development

[0221] Preparation of the test solution: Weigh approximately 0.5 g of the powder and place it in a 50 ml stoppered conical flask. Add 25 ml of methanol, seal tightly, weigh, and extract using ultrasound (300 W, 40 kHz) for 30 min. Cool, weigh again, and replenish the lost weight with methanol. Shake well, centrifuge at 12000 rpm for 5 min, and collect the supernatant.

[0222] Chromatographic conditions: Column: Waters ACQUITY HSS T3 (2.1×100mm, 1.8μm); Flow rate: 0.3ml / min; Column temperature: 25℃; Detection wavelength: 210nm; Injection volume: 1μl; Mobile phase A: 0.1% phosphoric acid-water solution; Mobile phase B: acetonitrile; Mobile phase gradient is shown in Table 1:

[0223] Table 1. Mobile phase gradient

[0224]

[0225]

[0226] 2.1 Selection of Indicator Peaks

[0227] Based on the chemical composition analysis results of the Fuzheng Huayu dry paste powder using liquid chromatography-mass spectrometry (LC-MS), and considering the peak area and specificity of each component, compounds with good absorption under ultraviolet light were prioritized while also taking into account the attribution of all chromatographic peaks. Twelve index peaks for method development were selected, as shown in Table 2.

[0228] Table 2 List of Indicator Peaks

[0229] Serial Number compound Belonging 1 Danshensu Salvia miltiorrhiza 2 Protocatechuic aldehyde Salvia miltiorrhiza 3 D-Amygdalin peach kernel 4 Rutin Gynostemma pentaphyllum / Schisandra chinensis 5 Tanshinone B Salvia miltiorrhiza 6 Tanshinone Y+ Phytolaccaside Salvia miltiorrhiza / Gynostemma pentaphyllum 7 Naringin Pine pollen 8 Genistein Cordyceps 9 Masoyalactone Cordyceps 10 Schisandra chinensis alcohol A Schisandra chinensis 11 Schisandra chinensis alcohol ethyl Schisandra chinensis 12 Schisandrin B Schisandra chinensis

[0230] 2.2 Preparation method of test solution - Selection of extraction solvent

[0231] Different extraction solvents (water, methanol, 50% methanol, 80% methanol, 50% ethanol, 70% ethanol, anhydrous ethanol) were investigated. Two samples were prepared in parallel for each extraction solvent. The number of chromatographic peaks at 210 nm and the peak area / sample weight value of the index peak were calculated. The samples were prepared according to the following method:

[0232] Take approximately 0.5g of the powder, accurately weigh it, and place it in a 50ml stoppered conical flask. Accurately add 25ml of different extraction solvents, seal tightly, weigh, and sonicate (300W power, 40kHz frequency) for 30 minutes. Cool, weigh again, and replenish the lost weight with the corresponding solvent. Shake well, centrifuge at 12000rpm for 5 minutes, and take the supernatant.

[0233] Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph for determination.

[0234] The experimental results and analysis show that the number of peaks at 210 nm from anhydrous ethanol extraction is significantly lower than that from other solvent extractions, while there is no significant difference in the number of peaks at 210 nm among the other six solvents. The peak area of ​​the index peaks from anhydrous ethanol extraction is significantly lower than that from other solvent extractions, so it is not considered. The peak areas of index peaks 11 and 12 from water extraction are significantly lower than those from the other five extraction solvents, so they are not considered. The peak area of ​​index peak 5 from methanol extraction is significantly lower than that from the other four extraction solvents, so it is not considered. The total peak areas of the index peaks from 70% ethanol, 50% ethanol, 80% methanol, and 50% methanol extractions are roughly the same. Considering the overall profile and peak shape of some chromatographic peaks from 50% methanol extraction (e.g., RT = 2–6 min), 50% methanol is selected as the extraction solvent for preparing the test solution of the Fuzheng Huayu dry paste powder.

[0235] 2.3 Preparation method of test solution - Selection of extraction method

[0236] The extraction methods (heating reflux, ultrasonic extraction, and warm maceration) were investigated. Two samples were prepared in parallel for each extraction method. The number of chromatographic peaks at 210 nm and the peak area / sample weight value of the index peak were calculated. The samples were prepared as follows:

[0237] Heating under reflux: Weigh approximately 0.5g of the powder accurately and place it in a 100ml round-bottom flask. Add 25ml of 50% methanol accurately, seal tightly, weigh, and heat under reflux for 1 hour. Cool, replenish the lost weight with 50% methanol solution, shake well, centrifuge at 12000rpm for 5 minutes, and collect the supernatant.

[0238] Ultrasonic extraction: Weigh approximately 0.5g of the powder accurately and place it in a 50ml stoppered conical flask. Add 25ml of 50% methanol accurately, seal tightly, weigh, and ultrasonically extract for 1 hour. Cool, replenish the lost weight with 50% methanol solution, shake well, centrifuge at 12000rpm for 5 minutes, and collect the supernatant.

[0239] Warm soaking: Weigh approximately 0.5g of the powder accurately and place it in a 50ml stoppered conical flask. Add 25ml of 50% methanol accurately, seal tightly, weigh, and soak in a 50℃ water bath for 4 hours. Cool, replenish the lost weight with 50% methanol solution, shake well, centrifuge at 12000rpm for 5 minutes, and collect the supernatant.

[0240] Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph for determination.

[0241] Experimental results showed no significant difference in the number of peaks and peak area of ​​the index peaks at 210 nm when using different extraction methods. Considering all factors, ultrasonic extraction was chosen as the extraction method for the test sample.

[0242] 2.4 Preparation method of test solution - Selection of extraction time

[0243] The extraction times were examined (15 min, 30 min, 45 min, 60 min). Two samples were prepared in parallel for each extraction time. The number of chromatographic peaks at 210 nm and the peak area / sample weight value of the index peak were calculated. The samples were prepared according to the following method:

[0244] Accurately weigh approximately 0.5g of the powder and place it in a 50ml stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, and weigh. Extract using ultrasound (300W power, 40kHz frequency) for different times (15min, 30min, 45min, 60min). Cool, replenish the lost weight with 50% methanol, shake well, centrifuge at 12000rpm for 5min, and collect the supernatant.

[0245] The results showed no significant difference in the number of peaks and peak area at 210 nm when different extraction times were used. Considering all factors, 15 min was selected as the extraction time for the test sample.

[0246] 2.5. Preparation method of test solution - Investigation of solid-liquid ratio

[0247] The material-liquid ratios (1:100, 1:50, 1:20) were investigated. Two samples were prepared in parallel for each material-liquid ratio. The peak area of ​​the index at 210 nm was calculated as a percentage of the sample weight. The samples were prepared according to the following method:

[0248] Material-to-liquid ratio 1:100: Weigh approximately 0.25g of the powder accurately and place it in a 50ml stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, weigh, and extract ultrasonically (300W power, 40kHz frequency) for 15min. Cool, replenish the lost weight with 50% methanol, shake well, centrifuge at 12000rpm for 5min, and collect the supernatant.

[0249] Material-liquid ratio 1:50: Take about 0.5g of the powder, accurately weigh it, and put it into a 50ml stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, weigh, and extract ultrasonically (power 300W, frequency 40kHz) for 15min. Cool, replenish the lost weight with 50% methanol, shake well, centrifuge at 12000rpm for 5min, and take the supernatant.

[0250] Material-to-liquid ratio 1:20: Weigh approximately 1.25g of the powder accurately and place it in a 50ml stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, weigh, and extract ultrasonically (300W power, 40kHz frequency) for 15min. Cool, replenish the lost weight with 50% methanol, shake well, centrifuge at 12000rpm for 5min, and collect the supernatant.

[0251] Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph for determination.

[0252] Experimental results showed no significant difference in extraction efficiency between different solid-liquid ratios, indicating that the extraction solvent was not saturated with the main compounds in the sample. Therefore, a solid-liquid ratio of 1:50 was ultimately selected as the optimal extraction ratio for the test sample.

[0253] 2.6 Test Solution Preparation Method—Filtration Method Investigation

[0254] Different filtration methods were investigated (high-speed centrifugation, filtration with microporous membranes of different pore sizes: 0.22 μm and 0.45 μm). Two samples were prepared in parallel for each filtration method. The number of chromatographic peaks at 210 nm, the peak area / sample weight ratio, resolution, tailing factor, and theoretical plate number were calculated. The samples were prepared according to the following method:

[0255] Accurately weigh approximately 0.5g of the powder and place it in a 50ml stoppered conical flask. Accurately add 25ml of 50% methanol, seal tightly, weigh, and extract using ultrasonic extraction (300W power, 40kHz frequency) for 15 minutes. Cool, replenish the lost weight with 50% methanol, shake well, and use three different filtration methods (high-speed centrifugation, 0.22μm and 0.45μm filter membranes). Collect the supernatant / filtrate to obtain the product.

[0256] Accurately pipette 1 μl of the test solution and inject it into the liquid chromatograph for determination.

[0257] Experimental results showed that under the same conditions, there was no significant difference in the number of peaks obtained by the three filtration methods. There was also no significant difference in the resolution, tailing factor, and theoretical plate number. However, considering that the filter membrane has a certain adsorption effect on the index peaks 5, 6, 7 and 8, centrifugation was finally chosen as the filtration method for the test solution of the Fuzheng Huayu dry paste powder.

[0258] 2.7 Determination of the preparation method for the test solution

[0259] Through optimization of the above-mentioned test sample solution preparation method, the final determined test sample solution preparation method is as follows:

[0260] Take approximately 0.5 g of the test sample, accurately weigh it, and place it in a 50 ml stoppered conical flask. Accurately add 25 ml of 50% methanol, seal the flask tightly, weigh it, and extract it by sonication (300 W power, 40 kHz frequency) for 15 min. Cool the flask, replenish the lost weight with 50% methanol, shake well, centrifuge at 12000 rpm for 5 min, and collect the supernatant.

[0261] 2.8 Optimization of Chromatographic Conditions - Wavelength Selection

[0262] Prepare the test solution according to the method for preparing the test solution, and examine the wavelengths (254nm, 210nm, 280nm, 330nm), and calculate the number of chromatographic peaks and the peak area / sample weight value of the index peak.

[0263] The results showed that the number of chromatographic peaks was highest and the overall peak response was highest at 210 nm. The number of chromatographic peaks of the test sample was significantly less at 330 nm than at the other three wavelengths; the number of chromatographic peaks was roughly the same at 254 nm and 280 nm, but the overall response was lower than that at 210 nm. Considering all factors, 210 nm was selected as the wavelength for optimizing the chromatographic conditions of the fingerprint spectrum of the Fuzheng Huayu dry paste powder.

[0264] 2.9 Optimization of Chromatographic Conditions—Column Investigation

[0265] Different chromatographic columns were examined, with column numbers and models shown in Table 4. Two samples were prepared for each column in parallel, and the number of peaks, peak area / sample weight of the index peak, resolution, tailing factor, and theoretical plate number were calculated. The results are shown in Tables 5 to 9.

[0266] Because some chromatographic columns cannot tolerate pure aqueous phase, the mobile phase gradient was appropriately modified, as shown in Table 3.

[0267] Table 3. Mobile phase gradient after appropriate modifications

[0268] Time (min) A(％) B(％) 0 95 5 2 95 5 6 91 9 11 91 9 13 80 20 16 77 23 23 66 34 25 45 55 30 45 55 32 18 82 38 5 95 41 5 95 41.1 95 5 43 95 5

[0269] Table 4. Column Numbers and Corresponding Models and Specifications

[0270]

[0271]

[0272] Table 5. Statistics on the average number of peaks for different chromatographic columns (n ​​= 2 × 2)

[0273]

[0274] Table 6. Comparison of peak area / sample volume for different chromatographic columns (n ​​= 2 × 2)

[0275]

[0276]

[0277] Table 7 Comparison of Peak Resolution of Different Chromatographic Columns

[0278]

[0279] Table 8 Comparison of Peak Tailing Factors for Different Chromatographic Columns

[0280]

[0281]

[0282] Table 9 Comparison of theoretical plate numbers of different chromatographic column peaks

[0283]

[0284] Experimental results showed that under the same conditions, column 4 had poor peak shape at RT = 30–32 min and was not considered. Because some of the index peaks had low responses, to prevent low reproducibility, peak selection should focus on these components. Index peak 7 had low responses in columns 2 and 6 and was not considered. Columns 1, 3, and 5 showed little difference under these analytical conditions, but considering the poor peak shape and resolution of highly polar components with earlier retention times, columns with good resistance to aqueous phases should be selected; therefore, column 5 was not considered. Taking all factors into account, Waters ACQUITY was chosen. HSS T3 (column 1) and Waters Further investigation was conducted using column T3 (column 3). The results are shown in Tables 10 to 14.

[0285] Table 10. Statistics on the number of peaks in different chromatographic columns

[0286] chromatographic column Column 1 Column 3 Number of peaks (210nm) 228 236

[0287] Table 11 Comparison of Peak Area / Sample Volume for Different Chromatographic Columns

[0288]

[0289] Table 12 Comparison of Peak Resolution of Different Chromatographic Columns

[0290]

[0291]

[0292] Table 13 Comparison of Peak Tailing Factors for Different Chromatographic Columns

[0293]

[0294] Table 14 Comparison of theoretical plate numbers of index peaks on different chromatographic columns

[0295]

[0296]

[0297] The results show that, under the same conditions, Waters ACQUITY HSS T3 (column 1) and Waters There was no significant difference in the number of peaks at T3 (column 3); further considering the low responses of peaks 3, 4, 7, and 8, and taking into account the reproducibility of the method, a column with a higher response was selected; taking into account resolution, tailing factor, theoretical plate number, and overall peak shape, Waters ACQUITY was ultimately chosen. HSS T3 (column 1) is a column with optimized chromatographic conditions for fingerprinting.

[0298] 2.10 Optimization of Chromatographic Conditions—Investigation of Mobile Phase System

[0299] Different mobile phase systems (methanol-water, acetonitrile-water system) were investigated. Two samples were prepared in parallel for each mobile phase system, and the overall peak shape, retention time and other factors of the chromatograms were compared.

[0300] Experimental results show that the elution capacity is stronger in the acetonitrile system, and the cutoff wavelength of acetonitrile is lower than that of methanol. Since 210 nm was used as the fingerprint spectrum acquisition wavelength in this study, the acetonitrile-water system was selected as the mobile phase system for fingerprint chromatographic conditions.

[0301] 2.11 Optimization of Chromatographic Conditions—Investigation of Mobile Phase Additives

[0302] Different mobile phase additives (formic acid, acetic acid, phosphoric acid, ammonium formate) were investigated. The types of mobile phase additives are shown in Table 15. Two samples were prepared for each mobile phase additive, and the number of peaks at 210 nm, peak area / sample weight, resolution, tailing factor, and theoretical plate number were calculated. The results are shown in Tables 16 to 20.

[0303] Table 15 Types of Mobile Phase Additives

[0304] Mobile phase additives Phase A Phase B 1 0.1% acetic acid water 0.1% Acetonitrile 2 0.1% formic acid solution 0.1% Formic Acid Acetonitrile 3 0.1% phosphoric acid solution Acetonitrile 4 2 mmol / L ammonium formate solution Acetonitrile

[0305] Table 16. Statistics on the number of peaks for additives in different mobile phases

[0306] Mobile phase additives 0.1% acetic acid water 0.1% formic acid solution 0.1% phosphoric acid solution 2 mmol / L ammonium formate solution Number of peaks (210nm) 221 231 238 241

[0307] Table 17 Comparison of Peak Area / Sample Amount of Additives in Different Mobile Phases

[0308]

[0309] Table 18 Comparison of Peak Resolution of Additives in Different Mobile Phases

[0310]

[0311]

[0312] Table 19 Comparison of Peak Tailing Factors of Additives in Different Mobile Phases

[0313]

[0314] Table 20 Comparison of theoretical plate numbers for additive index peaks in different mobile phases.

[0315]

[0316]

[0317] Experimental results show that, under the same conditions, there is no significant difference in the number of peaks among different mobile phase additives. When ammonium formate is used as an additive, the peak areas of indexes 5, 6, and 7 are lower than those of the other three additives, so they are not considered. When acetic acid and phosphoric acid are used as mobile phase additives, indexes 1 and 5 are significantly higher than those of the other two additives. Considering that formic acid and acetic acid are prone to causing uneven baselines when used as additives, phosphoric acid is selected as the mobile phase additive after comprehensive consideration.

[0318] 2.12 Optimization of Chromatographic Conditions—Investigation of Mobile Phase Additive Concentration

[0319] Different mobile phase additive concentrations (0.02% phosphoric acid-water, 0.05% phosphoric acid-water, 0.1% phosphoric acid-water) were investigated. Two samples were prepared in parallel for each mobile phase additive concentration. The number of peaks at 210 nm and the peak area / sample weight value, resolution, tailing factor, and theoretical plate number were calculated.

[0320] Experimental results showed that, under the same conditions, there was no significant difference in the number and area of ​​peaks with different concentrations of additives. Considering the pH tolerance of the chromatographic column, 0.05% phosphoric acid was selected as the mobile phase additive.

[0321] 2.13 Optimization of Chromatographic Conditions—Column Temperature Study

[0322] The study investigated different column temperatures (25℃, 30℃, 35℃), with two samples prepared in parallel at each column temperature. The number of peaks at 210 nm and the peak area / sample weight, resolution, tailing factor, and theoretical plate number were calculated.

[0323] Experimental results showed that under the same conditions, there was no significant difference in the number of peaks at the three column temperatures. By comparing factors such as resolution, tailing factor, theoretical plate number, and peak shape, 30℃ was selected as the optimal column temperature for fingerprint chromatographic conditions.

[0324] 2.14 Optimization of Chromatographic Conditions—Flow Rate Investigation

[0325] Different flow rates (0.30 ml / min, 0.35 ml / min, 0.40 ml / min) were investigated. Two samples were prepared in parallel for each flow rate. The number of peaks at 210 nm and the peak area / sample weight, resolution, tailing factor, and theoretical plate number were calculated.

[0326] Experimental results showed that there was no significant difference in the number of peaks at different flow rates; the sum of peak areas was largest at 0.30 ml / min. Considering factors such as resolution, tailing factor, and theoretical plate number, 0.30 ml / min was selected as the flow rate for fingerprint chromatographic conditions.

[0327] 2.15. Optimization of Chromatographic Conditions—Injection Volume Investigation

[0328] Different injection volumes (0.5 μl, 1 μl, 2 μl, 5 μl) were investigated. Two samples were prepared in parallel for each injection volume. The number of peaks at 210 nm and the peak area / sample volume, resolution, tailing factor, and theoretical plate number were calculated.

[0329] Experimental results show that, under the same conditions, some index peaks have low responses at an injection volume of 0.5 μl, so they are not considered; the baseline is uneven at RT = 13-16 min when the injection volume is 2 μl and 5 μl; and the first index peak is saturated at an injection volume of 5 μl. By comparing the resolution, tailing factor, theoretical plate number, and overall peak shape, 1 μl was finally selected as the injection volume for the fingerprint chromatographic conditions.

[0330] 2.16. Optimization of Chromatographic Conditions—Experimentation of Elution Gradient

[0331] The test sample was analyzed by ultra-high performance liquid chromatography, and the mobile phase gradient is shown in Tables 21 to 23.

[0332] Table 21 Mobile phase gradient 1

[0333] Time (min) A(％) B(％) 0 100 0 2 100 0 6 91 9 11 91 9 13 81 19 17 77 23 19 75 25 21 75 25 25 66 34 27 45 55 34 45 55 36 18 82 42 5 95 45 5 95 45.1 100 0 47 100 0

[0334] Table 22 Mobile Phase Gradient 2

[0335] Time (min) A(％) B(％) 0 100 0 2 100 0 6 91 9 11 91 9 13 80 20 19 77 23 26 66 34 28 45 55 33 45 55 35 18 82 39 8 92 40 5 95 42 5 95 42.1 100 0 48 100 0

[0336] Table 23 Mobile Phase Gradient 3

[0337]

[0338]

[0339] Experimental results show that, under the same conditions, when using mobile phase gradient 1, the chromatographic peaks are not completely eluted, which may affect the next injection, and the chromatographic peak resolution is poor. When using mobile phase gradient 2, the baseline is uneven from RT to 14 to 16 min, and the chromatographic peak resolution is poor. When using mobile phase gradient 3, the baseline is more stable from RT to 13 to 16 min, and the chromatographic peak resolution is good. Furthermore, by comparing the overall peak shape, the peak shape of mobile phase gradient 3 is significantly better than that of mobile phase gradients 1 and 2. Finally, mobile phase gradient 3 was selected as the elution gradient for the fingerprint chromatographic conditions.

[0340] 2.17 Determination of chromatographic conditions

[0341] Column: Waters ACQUITY HSS T3 (2.1 mm × 100 mm, 1.8 μm); Column temperature: 30 °C; Mobile phase A: 0.05% phosphoric acid-water solution; Mobile phase B: acetonitrile;

[0342] Flow rate: 0.30 ml / min; detection wavelength: 210 nm; injection volume: 1 μl; mobile phase gradient: gradient 3, as shown in Table 23 above.

[0343] 3. Generation of fingerprint patterns

[0344] 3.1 Fingerprint Acquisition

[0345] Twenty-seven batches of Fuzheng Huayu dry extract powder samples were prepared according to the test solution preparation method. The samples were then analyzed under fingerprint chromatographic conditions, and data from all 27 batches were collected. The chromatograms are shown below. Figure 1 As shown, the integration parameters are set as follows: ① Integration algorithm: Traditional; minimum peak area: 0; minimum peak height: 0; minimum peak width is 4.5, and baseline noise is subtracted to be 300. ② System suitability parameters: dead volume: 0.1 min; USP separation tangent percentage: 50; USP theoretical plate number tangent percentage: 61.

[0346] 3.2 Fingerprint Similarity Evaluation

[0347] The 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software was used to import data from 27 batches of Fuzheng Huayu dry paste powder test samples into the software. The data processing time range was set to 1-42 minutes, and the parameters were set as follows: the reference chromatogram number was S14, the reference chromatogram generation method was the mean method, and the time window width was 0.1 minutes.

[0348] A common peak refers to a chromatographic peak that has the same relative retention time within a certain comparison time window. After peak position correction to match the chromatographic peaks, 18 common peaks were selected based on the chromatographic data of the Fuzheng Huayu dry paste powder test sample. The results of the common peak identification are shown in Table 24.

[0349] Finally, the fingerprint spectrum (R) of the Fuzheng Huayu dry ointment powder was obtained (e.g.) Figure 2 As shown in the figure, 18 common peaks were marked in the control fingerprint spectrum. The similarity between each batch of Fuzheng Huayu dry powder and the control fingerprint spectrum was calculated using software. The results showed that the similarity was greater than 0.90, which is good and meets the technical requirements of fingerprint spectrum.

[0350] Table 24. Identification results of common peaks in the fingerprint spectrum of Fuzheng Huayu dry ointment powder.

[0351] Compound Name Plant source 1 adenosine Cordyceps / Pine Pollen / Salvia miltiorrhiza / Schisandra chinensis / Peach Kernel / Gynostemma pentaphyllum 2 Guanosine Cordyceps / Pine Pollen / Salvia miltiorrhiza / Schisandra chinensis / Peach Kernel / Gynostemma pentaphyllum 3 Danshensu Salvia miltiorrhiza 4 Protocatechuic aldehyde Cordyceps / Pine Pollen / Salvia miltiorrhiza / Schisandra chinensis / Peach Kernel / Gynostemma pentaphyllum 5 D-Amygdalin peach kernel 6 Rutin Gynostemma pentaphyllum / Schisandra chinensis 7 Danshensu D Salvia miltiorrhiza 8 Rosemary acid Salvia miltiorrhiza 9 Shikonin Salvia miltiorrhiza 10 Tanshinone B Salvia miltiorrhiza 11 Tanshinone A + Phytolaccaside Salvia miltiorrhiza / Gynostemma pentaphyllum 12 Naringin Pine pollen 13 Genistein Cordyceps 14 Masoyalactone Cordyceps 15 Schisandra chinensis alcohol A Schisandra chinensis 16 Schisandra chinensis alcohol ethyl Schisandra chinensis 17 Schisandrin A Schisandra chinensis 18 Schisandrin B + Gomicin N Schisandra chinensis

[0352] 4. Validation of fingerprinting methodology

[0353] Method validation parameters include specificity, precision (instrument precision, repeatability and intermediate precision), stability and robustness of the test solution.

[0354] 4.1 Specificity

[0355] Preparation of blank solution: Take an appropriate amount of 50% methanol solution, centrifuge at 12000 rpm for 5 min, and take the supernatant.

[0356] Preparation of the test solution: Weigh approximately 0.5 g of this product accurately and place it in a 50 ml stoppered conical flask. Accurately add 25 ml of 50% methanol, seal tightly, weigh, and extract ultrasonically (300 W, 40 kHz) for 15 min. Cool, weigh again, and replenish the lost weight with 50% methanol. Shake well, centrifuge at 12000 rpm for 5 min, and collect the supernatant.

[0357] Preparation of negative urea-sensitive dry extract powder solution: Take about 0.5g of negative urea-sensitive dry extract powder, accurately weigh it, put it into a 50ml stoppered conical flask, accurately add 25ml of 50% methanol, stopper tightly, weigh it, extract by sonication (power 300W, frequency 40kHz) for 15min, cool it, weigh it again, replenish the lost weight with 50% methanol, shake well, centrifuge at 12000rpm for 5min, and take the supernatant to obtain the solution.

[0358] Preparation of reference solution: Accurately weigh appropriate amounts of adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masoylide, schisandrol A, schisandrol B, schisandrin A, gomisin N, and schisandrin B reference standards, and add 50% methanol to prepare a solution containing 80 μg adenosine, 80 μg guanosine, 240 μg sodium tanshinone, 20 μg protocatechuic acid, 60 μg amygdalin, 6 μg rutin, 30 μg salvianolic acid D, 40 μg rosmarinic acid, lithospermic acid, 160 μg salvianolic acid B, 6 μg phytolaccaside, and 10 μg salvianolic acid A per ml. A mixed solution of 100 μg of naringenin, 6 μg of genistein, 30 μg of masoylide, 30 μg of schisandrol A, 24 μg of schisandrol B, 16 μg of schisandrin A, 10 μg of gomisin N, and 20 μg of schisandrin B was prepared, shaken well, filtered, and the filtrate was collected.

[0359] Accurately pipette 1 μl each of the blank solution, test solution, and reference solution, and inject them into the liquid chromatograph for determination.

[0360] Comparing the chromatograms of the test solution and the reference solution, the blank solution chromatogram showed no obvious interference peaks at RT = 4–43 min; compared with the reference solution chromatogram, the test solution eluted at the same time, and each negative control powder solution showed no obvious interference peaks at the corresponding positions, indicating that the method has good specificity and meets the requirements.

[0361] 4.2 Precision

[0362] 4.2.1 Instrument Precision

[0363] For the same test solution, inject it 6 times consecutively under chromatographic conditions. Using peak 10 as the reference peak, calculate the relative retention time and relative peak area of ​​the common peaks. The RSD of the relative retention time of each common peak in the 6 injections should not be greater than 5%, and the RSD of the relative peak area of ​​each common peak should not be greater than 10%.

[0364] The experimental results show that the relative retention time RSD of the common peak of each of the 6 test samples is (0.01%-2.39%) and the relative peak area RSD is (0.06%-4.06%). The method has good repeatability and meets the requirements.

[0365] 4.2.2 Repeatability

[0366] Prepare 6 parallel samples of this product according to the preparation method of the test sample, and determine them according to the chromatographic conditions. Take peak 10 as the reference peak (S), calculate the relative retention time and relative peak area of ​​the common peaks. The RSD of the relative retention time of each common peak of the 6 test samples should not be greater than 5%; the RSD of the relative peak area of ​​each common peak should not be greater than 10%.

[0367] The experimental results show that the relative retention time (RSD) of the common peaks of the six test samples is (0.01%-0.18%), and the relative peak area (RSD) is (0.05%-1.68%). The method has good repeatability and meets the requirements.

[0368] 4.2.3 Intermediate Precision

[0369] In the same laboratory, at different times, by different analysts, and with different equipment, six parallel samples were prepared according to the method for preparing the test solution. The samples were then measured under chromatographic conditions. The relative retention time RSD of each common peak should not exceed 5%, and the relative peak area RSD of each common peak should not exceed 10%. The results of 12 measurements, including 6 intermediate precision experiments and 6 repeatability experiments, should also show that the relative retention time RSD of each common peak should not exceed 5%, and the relative peak area RSD should not exceed 10%.

[0370] The experimental results show that the RSD of the relative retention time of the common peak in the six intermediate precision experiments ranged from 0.01% to 0.16%, and the RSD of the relative peak area of ​​each common peak ranged from 0.08% to 5.88%. For the intermediate precision and repeatability experiments, the RSD of the relative retention time of the common peak ranged from 0.07% to 2.20%, and the RSD of the relative peak area of ​​each common peak ranged from 0.08% to 7.33%. These results indicate that the method has good intermediate precision and meets the requirements.

[0371] 4.3 Stability

[0372] The test solution is placed at room temperature for 0, 1, 2, 4, 6, 8, 12, 24, and 48 hours before measurement. The relative retention time RSD of each common peak in the test solution should not be greater than 5%, and the relative peak area RSD of each common peak should not be greater than 10%.

[0373] The experimental results show that within 48 hours, the relative retention time (RSD) of the common peaks in the test solution was (0.02%-0.36%), and the relative peak area (RSD) was (0.04%-3.05%), which meets the requirements. Therefore, the test solution is considered to have good stability within 48 hours.

[0374] 4.4 Durability

[0375] The robustness of the fingerprinting method was investigated by subtly varying various parameters during the fingerprinting process. The investigation included different column temperatures, injection volumes, flow rates, detection wavelengths, mobile phase pH, mobile phase gradients, and column types. The analysis was conducted using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," and the similarity was calculated with a control fingerprint (R). A similarity result greater than 0.90 indicated good robustness to the parameters.

[0376] For each project, 1-3 injections of blank solution, 5 injections of system suitability solution, and 2 parallel preparations of the test sample solution were performed, with 2 injections per sample. Analysis and verification were conducted using the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System," and similarity calculations were performed with the control fingerprint (R). Chromatographic condition variations are shown in Table 25.

[0377] Table 25 Parameters for Chromatographic Condition Variation

[0378]

[0379] 4.4.1 Different column temperatures

[0380] The robustness of this method to column temperature was investigated. Measurements were performed at different column temperatures (25℃, 30℃, and 35℃). The experimental results showed that the similarity between the measured Fuzheng Huayu dry extract powder at different column temperatures and the control fingerprint (R) was calculated. The results indicated that the similarity was greater than 0.90, demonstrating that the analytical method is robust to small changes in column temperature (25–35℃).

[0381] 4.4.2 Different injection volumes

[0382] The robustness of this method to different injection volumes was investigated. Measurements were performed using different injection volumes (0.5 μl, 1 μl, and 2 μl). The results showed that the similarity between the measured Fuzheng Huayu dry extract powder and the control fingerprint (R) was greater than 0.90 at different injection volumes, indicating that the analytical method is robust to small variations in injection volume (0.5-2 μl).

[0383] 4.4.3 Different Flow Rates

[0384] The robustness of this method to different flow rates was investigated. Measurements were performed at flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min. The experimental results show that the similarity between the measured fingerprints of the Fuzheng Huayu dry extract powder at different flow rates and the control fingerprint (R) is greater than 0.90, indicating that the analytical method is robust to small changes in flow rate (0.25-0.35 ml / min).

[0385] 4.4.4 Different detection wavelengths

[0386] The robustness of this method to different detection wavelengths was investigated. Measurements were performed at detection wavelengths of 208 nm, 210 nm, and 212 nm. The experimental results show that the similarity between the measured Fuzheng Huayu dry powder at different detection wavelengths and the control fingerprint spectrum (R) was calculated. The results showed that the similarity was greater than 0.90, indicating that the analytical method has good robustness to small changes in detection wavelength (208-212 nm).

[0387] 4.4.5. pH of different mobile phases

[0388] The robustness of this method to mobile phase pH was investigated. Measurements were performed at aqueous phases of 0.02% phosphoric acid-water, 0.05% phosphoric acid-water, and 0.1% phosphoric acid-water. The results showed that the similarity between the measured fingerprints of the Fuzheng Huayu dry extract powder at different mobile phase pH values ​​and the control fingerprint (R) was greater than 0.90, indicating that the analytical method is robust to small changes in mobile phase pH (0.02% phosphoric acid to 0.1% phosphoric acid).

[0389] 4.4.6 Different mobile phase gradients

[0390] The robustness of this method to mobile phase gradients was investigated. Measurements were performed under three conditions: a 1% reduction in the aqueous phase ratio, a normal mobile phase gradient, and a 1% increase in the aqueous phase ratio. The experimental results show that the similarity between the measured fingerprints of the Fuzheng Huayu dry extract powder under different mobile phase gradients and the control fingerprint (R) is greater than 0.90, indicating that the analytical method is robust to small changes in the mobile phase gradient (aqueous phase ratio ±1%).

[0391] 4.4.7 Different chromatographic columns

[0392] The robustness of this method to different column types was investigated. Four columns were used for the determination, and the column numbers and corresponding models are shown in Table 26. The relevant data are shown in Table 27.

[0393] Table 26 Chromatographic Column Numbers and Corresponding Models and Specifications

[0394]

[0395] Table 27 Robustness Test - Results for Different Columns

[0396] chromatographic column R Column 4 Column 7 Column 1 Column 3 R 1.000 0.855 0.987 0.985 0.985 Column 4 0.855 1.000 0.850 0.846 0.846 Column 7 0.987 0.850 1.000 1.000 0.999 Column 1 0.985 0.846 1.000 1.000 0.999 Column 3 0.985 0.846 0.999 0.999 1.000

[0397] The experimental results show that the Agilent ZORBAX RRHD SB-Aq column (column 4) has a significant impact on this fingerprinting method. Similarity calculations with the control fingerprint (R) show a similarity of less than 0.90. Waters ACQUITY HSS T3, Waters The T3 column has minimal impact on this fingerprinting method. Similarity calculations with the reference fingerprint (R) showed similarities greater than 0.90. Therefore, the analytical method is not significantly affected by the fingerprinting pattern in WatersACQUITY. HSS T3, Waters The T3 column is durable.

[0398] 4.5 Summary of Method Validation

[0399] In summary, this fingerprinting method exhibits good specificity, precision, and solution stability. It demonstrates good robustness to variations in column temperature, injection volume, flow rate, detection wavelength, mobile phase pH, and mobile phase gradient. It also meets the requirements of WatersACQUITY. HSS T3, Waters The T3 column has good durability.

[0400] 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 a tonifying and blood-activating preparation or intermediate, characterized in that, The construction method includes the following steps: Preparation of the test solution: Weigh an appropriate amount of the tonifying and blood-activating preparation or intermediate, place it in a container, add the first solvent and seal it tightly, weigh it, sonicate it for a period of time, cool it, weigh it again, add the second solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant to obtain the test solution. Preparation of reference solution: Weigh appropriate amounts of adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masonone, schisandrol A, schisandrol B, schisandrin A, gomicin N, and schisandrin B reference standards. Add solvent to prepare a mixed solution with concentrations of 1–400 μg / ml for adenosine, guanosine, sodium tanshinone, protocatechuic acid, amygdalin, rutin, salvianolic acid D, rosmarinic acid, lithospermic acid, salvianolic acid B, phytolaccaside, salvianolic acid A, naringenin, genistein, masonone, schisandrol A, schisandrol B, schisandrin A, gomicin N, and schisandrin B. Shake well, filter, and collect the filtrate to obtain the reference solution. Based on the results of ultra-high performance liquid chromatography detection of the test solution and the reference solution, characteristic chromatograms of the tonifying and blood-activating preparation or intermediate were obtained. The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is an acid-water, alkaline-water, and / or buffer salt aqueous solution; mobile phase B is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and the gradient elution program is as follows: 0–2 min, 100% A; 2–6 min, 100% → 91% A; 6–11 min, 91% A; 11–13 min, 91% → 82% A; 13–18 min, 82% → 78% A; 18–25 min… 78%→66%A; 25–29 min, 66%→46%A; 29–36 min, 46%A; 36–38 min, 46%→18%A; 38–42 min, 18%→5%A; 42–44 min, 5%A; 44–44.1 min, 5%→100%A; 44.1–50 min, 100%A; flow rate 0.1–1 ml / min, column temperature 20–40℃, detection wavelength 180–300 nm, injection volume 0.1–5 μl; The raw materials for the tonifying and blood-activating preparations or intermediates include Salvia miltiorrhiza, fermented Cordyceps militaris powder, pine pollen, Schisandra chinensis, peach kernel, and Gynostemma pentaphyllum.

2. A method for constructing the fingerprint spectrum of a tonifying and blood-activating preparation or intermediate, characterized in that, The construction method includes the following steps: Preparation of the test solution: Weigh an appropriate amount of the tonifying and blood-activating preparation or intermediate, place it in a container, add the first solvent and seal it tightly, weigh it, sonicate it for a period of time, cool it, weigh it again, add the second solvent to make up for the lost weight, shake it well, centrifuge it, take the supernatant to obtain the test solution. Based on the results of ultra-high performance liquid chromatography (UHPLC) analysis of the test solution, a fingerprint spectrum of the tonifying and stasis-removing preparation or intermediate was obtained. The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is an acid-water, alkaline-water, and / or buffer salt aqueous solution; mobile phase B is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; and the gradient elution program is as follows: 0–2 min, 100% A; 2–6 min, 100% → 91% A; 6–11 min, 91% A; 11–13 min, 91% → 82% A; 13–18 min, 82% → 78% A; 18–25 min… 78%→66%A; 25–29 min, 66%→46%A; 29–36 min, 46%A; 36–38 min, 46%→18%A; 38–42 min, 18%→5%A; 42–44 min, 5%A; 44–44.1 min, 5%→100%A; 44.1–50 min, 100%A; flow rate 0.1–1 ml / min, column temperature 20–40℃, detection wavelength 180–300 nm, injection volume 0.1–5 μl; The raw materials for the tonifying and blood-activating preparations or intermediates include Salvia miltiorrhiza, fermented Cordyceps militaris powder, pine pollen, Schisandra chinensis, peach kernel, and Gynostemma pentaphyllum.

3. The construction method according to claim 1 or 2, characterized in that, The raw materials of the tonifying and stasis-removing preparation or intermediate, by weight, include: 100-200 parts of peach kernel, 500-600 parts of salvia miltiorrhiza, 300-500 parts of gynostemma pentaphyllum, 200-300 parts of fermented cordyceps mycelium powder, 100-200 parts of pine pollen, 100-200 parts of schisandra chinensis, and an appropriate amount of starch. Preferably, by weight, the raw materials of the tonifying and stasis-removing preparation or intermediate include: about 130 parts of peach kernel, about 530 parts of salvia miltiorrhiza, about 400 parts of gynostemma pentaphyllum, about 260 parts of fermented cordyceps mycelium powder, about 130 parts of pine pollen, about 130 parts of schisandra chinensis, and an appropriate amount of starch. Preferably, the tonifying and stasis-removing preparation is a solid preparation or a liquid preparation for tonifying and stasis-removing; Preferably, the solid preparation for strengthening the body and removing blood stasis is a powder, granules, tablets, capsules, film, or dry ointment. Preferably, the tonifying and blood-activating liquid preparation is an oral liquid preparation or an injectable liquid preparation; Preferably, the oral liquid preparation is a mixture, emulsion, suspension, drops, or syrup; Preferably, the preparation method of the tonic and stasis-removing dry paste powder includes the following steps: (1) Weigh out the salvia miltiorrhiza, peach kernel and gynostemma pentaphyllum according to the weight ratio, add water and decoct, combine the decoctions, take the supernatant, concentrate into an extract, cool, add ethanol, precipitate, and filter. (2) Take fermented Cordyceps militaris powder, add Schisandra chinensis to ethanol, heat and reflux, cool, combine the ethanol liquids, filter, concentrate and dry the filtrate; (3) Alternatively, pine pollen is added to ethanol and warm-soaked. The extracts are combined, concentrated, and dried into a dry paste; and (4) Take the above three dry pastes, mix and crush them, add an appropriate amount of starch, mix well, and obtain the dry paste powder for supporting the body's resistance and removing blood stasis; Preferably, the container is a conical flask, such as a stoppered conical flask; Preferably, the solvent is an alcohol, such as methanol; Preferably, the concentration of methanol is 10% to 90%, for example, about 50%; More preferably, the mass / volume (g / ml) ratio between the tonifying and stasis-removing preparation or intermediate and the first solvent is 0.01 to 0.5, for example, about 0.02; Preferably, the power of the ultrasound is 150-400W, for example, about 300W; Preferably, the frequency of the ultrasound is 20–60 kHz, for example, about 40 kHz; Preferably, the ultrasonic treatment time is 5 to 30 minutes, for example, about 15 minutes; Preferably, the centrifugation speed is 10,000 to 15,000 rpm, for example, about 12,000 rpm; Preferably, the centrifugation time is 1 to 10 minutes, for example, about 5 minutes; More preferably, the concentration of adenosine in the reference solution is about 80 μg / ml; More preferably, the concentration of guanosine in the reference solution is about 80 μg / ml; More preferably, the concentration of sodium tanshinone in the reference solution is about 240 μg / ml; More preferably, the concentration of protocatechuic aldehyde in the reference solution is about 20 μg / ml; More preferably, the concentration of amygdalin in the reference solution is about 60 μg / ml; More preferably, the concentration of rutin in the reference solution is about 6 μg / ml; More preferably, the concentration of salvianolic acid D in the reference solution is about 30 μg / ml; More preferably, the concentration of rosmarinic acid in the reference solution is about 40 μg / ml; More preferably, the concentration of shikonin in the reference solution is about 30 μg / ml; More preferably, the concentration of salvianolic acid B in the reference solution is about 160 μg / ml; More preferably, the concentration of phytolaccaside in the reference solution is about 6 μg / ml; More preferably, the concentration of salvianolic acid A in the reference solution is about 100 μg / ml; More preferably, the concentration of naringenin in the reference solution is about 6 μg / ml; More preferably, the concentration of genistein in the reference solution is about 6 μg / ml; More preferably, the concentration of masoylide in the reference solution is about 30 μg / ml; More preferably, the concentration of schisandrol A in the reference solution is about 30 μg / ml; More preferably, the concentration of schisandrin ethyl in the reference solution is about 24 μg / ml; More preferably, the concentration of schisandrin A in the reference solution is about 16 μg / ml; More preferably, the concentration of gomizine N in the reference solution is about 10 μg / ml; More preferably, the concentration of schisandrin B in the reference solution is about 20 μg / ml; Particularly preferably, the flow rate is 0.2 to 0.4 ml / min, for example, about 0.3 ml / min; Particularly preferred, the column temperature is 25–35°C, for example, about 30°C; Particularly preferably, the detection wavelength is 190–230 nm, for example, 210 nm; Particularly preferably, the injection volume is 0.5–1.5 μl, for example, about 1.0 μl; Particularly preferred, the theoretical plate number of the chromatographic peak corresponding to the salvianolic acid B is not less than 200,000; Particularly preferably, the resolution between the chromatographic peak corresponding to naringenin and the chromatographic peak corresponding to genistein is greater than 1.0; Particularly preferred is that the chromatographic column is a Waters ACQUITY column. HSS T3 column; Particularly preferred is that the chromatographic column has the following specifications: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.

4. The construction method according to claim 1 or 2, characterized in that, The acid aqueous solution, alkaline aqueous solution and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are 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; More preferably, the acid aqueous solution is a 0.01% to 0.1% acid aqueous solution; More preferably, the acidic aqueous solution is a 0.01% to 0.1% phosphoric acid aqueous solution; More preferably, the acidic aqueous solution is a 0.04% to 0.06% phosphoric acid aqueous solution; More preferably, the acidic aqueous solution is an aqueous solution of about 0.05% phosphoric acid; More preferably, the buffer salt aqueous solution is a phosphate aqueous solution and / or an acetate aqueous solution; More preferably, the pH value of the buffer salt solution is not greater than 7.0; Preferably, the mobile phase B is acetonitrile.

5. The construction method according to claim 2, characterized in that, When the detection wavelength is 210 nm, the fingerprint spectrum includes 18 characteristic peaks: peak 1 is the chromatographic peak of adenosine, peak 2 is the chromatographic peak of guanosine, peak 3 is the chromatographic peak of tanshinone, peak 4 is the chromatographic peak of protocatechuic aldehyde, peak 5 is the chromatographic peak of D-amygdalin, peak 6 is the chromatographic peak of rutin, peak 7 is the chromatographic peak of salvianolic acid D, peak 8 is the chromatographic peak of rosmarinic acid, and peak 9 is the chromatographic peak of shikonin. Peak 10 is the chromatographic peak of salvianolic acid B, peak 11 is the chromatographic peak of salvianolic acid A and phytolaccatoin, peak 12 is the chromatographic peak of naringenin, peak 13 is the chromatographic peak of genistein, peak 14 is the chromatographic peak of masonin, peak 15 is the chromatographic peak of schisandrol A, peak 16 is the chromatographic peak of schisandrol B, peak 17 is the chromatographic peak of schisandrin A, and peak 18 is the chromatographic peak of schisandrin B and gomisin N. Preferably, peak 1 is derived from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 2 is derived from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 3 is derived from salvia miltiorrhiza; peak 4 is derived from fermented cordyceps mycelium powder, pine pollen, salvia miltiorrhiza, schisandra chinensis, peach kernel, and gynostemma pentaphyllum; peak 5 is derived from peach kernel; peak 6 is derived from gynostemma pentaphyllum and schisandra chinensis; and peak 7 is derived from salvia miltiorrhiza. Peak 8 is from Salvia miltiorrhiza, peak 9 is from Salvia miltiorrhiza, peak 10 is from Salvia miltiorrhiza, peak 11 is from Salvia miltiorrhiza and Gynostemma pentaphyllum, peak 12 is from pine pollen, peak 13 is from fermented Cordyceps militaris powder, peak 14 is from fermented Cordyceps militaris powder, peak 15 is from Schisandra chinensis, peak 16 is from Schisandra chinensis, peak 17 is from Schisandra chinensis, and peak 18 is from Schisandra chinensis.

6. The construction method according to claim 2, characterized in that, At the detection wavelength of 210 nm, the fingerprint spectrum includes 18 characteristic peaks. Using the chromatographic peak of salvianolic acid B (peak number 10) as the reference peak, the relative retention times of the other 17 characteristic peaks are as follows: Peak 1 0.235±10%, Peak 2 0.267±10%, Peak 3 0.341±10%, Peak 4 0.431±10%, Peak 5 0.587±10%, Peak 6 0.757±10%, Peak 7 0. Peak 8: 0.855±10%; Peak 9: 0.948±10%; Peak 11: 1.054±10%; Peak 12: 1.162±10%; Peak 13: 1.172±10%; Peak 14: 1.325±10%; Peak 15: 1.357±10%; Peak 16: 1.405±10%; Peak 17: 1.764±10%; Peak 18: 1.792±10%.

7. The construction method according to claim 2, characterized in that, When the detection wavelength is 210 nm, the fingerprint spectrum includes 18 characteristic peaks. Using the chromatographic peak of salvianolic acid B (peak number 10) as the reference peak, the relative peak areas of the other 17 characteristic peaks are as follows: Peak 1 0.282±10%, Peak 2 0.122±10%, Peak 3 0.714±10%, Peak 4 0.205±10%, Peak 5 0.041±10%, Peak 6 0.028±10%, Peak 7 0. Peak 0.179±10%, Peak 8 0.138±10%, Peak 9 0.128±10%, Peak 11 0.424±10%, Peak 12 0.017±10%, Peak 13 0.049±10%, Peak 14 0.128±10%, Peak 15 0.429±10%, Peak 16 0.127±10%, Peak 17 0.099±10%, Peak 18 0.182±10%.

8. A quality control method for a tonifying and blood-activating preparation or intermediate, characterized in that, The quality control method includes the following steps: (1) Establish a standard characteristic spectrum or fingerprint spectrum of a reference sample of a tonifying and blood-activating preparation or intermediate according to any one of claims 1 to 7; (2) Take the test solution of the tonifying and blood-activating preparation or intermediate and detect it under the chromatographic conditions in the construction method according to any one of claims 1 to 7 to obtain the characteristic spectrum or fingerprint spectrum of the tonifying and blood-activating preparation or intermediate to be tested; as well as (3) Compare the characteristic spectrum or fingerprint spectrum of the test sample of the tonifying and blood-activating preparation or intermediate obtained in step (2) with the standard characteristic spectrum or fingerprint spectrum of the reference sample of the tonifying and blood-activating preparation or intermediate obtained in step (1). If the sample meets the requirements, it is a qualified product; otherwise, it is an unqualified product.

9. The quality control method according to claim 8, characterized in that, The compliance requirements include one or more of the following: (1) The characteristic chromatogram or fingerprint chromatogram of the test sample of the tonifying and stasis-removing preparation or intermediate shows 18 characteristic chromatographic peaks. At the detection wavelength of 210 nm, with the chromatographic peak of salvianolic acid B as the S peak, the relative retention time of each characteristic chromatographic peak in the characteristic chromatogram or fingerprint chromatogram of the test sample of the tonifying and stasis-removing preparation or intermediate and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard characteristic chromatogram or fingerprint chromatogram of the reference sample of the tonifying and stasis-removing preparation or intermediate; and (2) When the detection wavelength is 210 nm, with the chromatographic peak of salvianolic acid B as the S peak, the relative peak area of ​​each characteristic chromatographic peak in the characteristic chromatographic spectrum or fingerprint spectrum of the tonifying and stasis-removing preparation or intermediate to be tested and the S peak is within ±10% of the relative peak area of ​​each characteristic chromatographic peak in the characteristic chromatographic spectrum or standard fingerprint spectrum of the tonifying and stasis-removing preparation or intermediate reference sample; and (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the characteristic chromatogram or fingerprint of the test sample of the tonifying and blood-activating preparation or intermediate and the standard characteristic chromatogram or fingerprint of the reference sample of the tonifying and blood-activating preparation or intermediate shall not be less than 0.

90.

10. The use of the construction method according to any one of claims 1 to 7 or the quality control method according to claim 8 or 9 in the quality detection, quality evaluation or quality control of the tonifying and stasis-removing preparation or intermediate.

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