Fingerprint spectrum construction method and application of kadsura pepper stem medicinal material
By optimizing chromatographic conditions and building fingerprint maps, the problems of long-term detection of Dafeng Teng medicinal materials and fewer chromatographic peaks are solved, and simple and efficient quality control and basic research on pharmacokinetic substances are achieved.
Patent Information
- Application Number
- CN202311317950.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-11
- Publication Date
- 2025-04-11
AI Technical Summary
In the prior art, the detection methods of Dafeng Teng medicinal materials are reported fewer and time-consuming, and fewer chromatographic peaks are identified, making it difficult to achieve efficient quality control.
Using T3 chromatography column, mobile phase A is an acid aqueous solution, alkali aqueous solution and/or buffered saline solution, mobile phase B is acetonitrile, methanol and tetrahydrofuran, gradient elution procedure and high-performance liquid phase detection, combined with external standard method to calculate the component content, and build a fingerprint map of Dafeng Teng medicinal materials.
It realizes the simplicity, efficiency and rapid testing of Dafeng Teng medicinal materials, provides a theoretical basis for quality control, and ensures the quality stability and consistency of medicinal materials.
Smart Images
Figure CN119804746A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of medical technology, and in particular to a fingerprint construction method of a schefflera herb and an application thereof. Background Art
[0002] The Chinese medicinal material Cocculus Radix et Caulis is mostly produced in Guizhou Province. The Guizhou Provincial Chinese Medicinal Material Standard (2019 Edition) stipulates that it is the dried root and stem of Cocculus orbiculatus (L.) DC., a plant of the Menispermaceae family. Its aliases are recorded in the Chinese Materia Medica as Inula and Niu Mu Xiang. In my country, Cocculus Radix et Caulis is mainly distributed in Jiangsu, Zhejiang, Guizhou, Jiangxi, Chongqing, Sichuan and other places. Cocculus Radix et Caulis has a long history of medicinal use. It was first recorded in the Shennong's Herbal Classic under the name of Cocculus Radix et Caulis. It is bitter and cold in nature. It has the effects of dispelling wind and relieving pain, promoting diuresis and reducing swelling. It is mainly used to treat rheumatic arthralgia, edema, beriberi, dysuria, eczema and sores. In the Golden Chamber, it is indicated as the Cocculus Radix et Caulis Decoction, which has the effects of dispelling fluid and clearing heat, invigorating qi and warming yang. In recent years, basic experimental research on Mufangji Decoction has also become a hot topic, and it has been clinically used in cardiovascular diseases, bone and joint diseases, edema and effusion. At present, Radix Phellodendri is also used in many Chinese patent medicines, but it is currently mainly used as one of the seven raw medicinal materials of Guizhou Miao medicine Ningmitai Capsule, which has the effects of clearing away heat and detoxifying, promoting dampness and relieving stranguria, and is used to treat diseases such as urinary problems and chronic prostatitis.
[0003] In Miao language, Dafengteng is called "Mengnaoliu". The "Chinese Materia Medica Miao Medicinal Volume" points out that it has the effects of dispelling wind and relieving pain, promoting water circulation and reducing swelling, detoxifying, and lowering blood pressure. It is used for rheumatic pain, neuralgia, nephritic edema, urinary tract infection, traumatic injury, and snake bites. At present, the chemical components of Dafengteng known to us are all alkaloids, including isoquinolines, morphinanes, apophenes, erythrinanes, and protoberberines. Most of these compounds have antibacterial, cytotoxic, antihypertensive, anticancer, anti-inflammatory, and antimalarial pharmacological activities.
[0004] Cocculus plants belong to the family Menispermaceae of the order Ranunculales, and include 8 species. They are widely distributed in Asia, America, Africa, and other places. In China, they are mainly distributed in Shandong, Zhejiang, Guizhou, Shaanxi, Jiangsu, and other places. There are 2 species and 1 variant in China, namely C. orbiculatus (L.) DC., C. orbiculatus var. mollis, and C. laurifolius DC. Among them, C. pendulus (C. leaebe), C. hirsutus, C. carolinus, and C. genera have not been reported in China. At present, there are many reports on the research of Cocculus, and relatively few on C. laurifolius. Among them, Cocculus is a commonly used Chinese medicine in folk medicine. It was first recorded in the "Shennong Bencao Jing". It has a long history of medicinal use. It has the effects of dispelling wind and relieving pain, promoting diuresis and reducing swelling. It is often used to treat rheumatism, edema, athlete's foot, urinary incontinence, eczema, and sores.
[0005] There are few reports on the detection method of Radix Paeoniae Wind-Resistant in the prior art, and the detection takes a very long time and the identified chromatographic peaks are very few. Summary of the invention
[0006] Based on this, the present invention provides a method for detecting the medicinal material of Radix Paeoniae Wind-Firefly, and the detection method comprises the following steps:
[0007] Take the test solution and reference solution of Radix Paeoniae Daphne for testing, wherein the reference solution is magnolamine, sinomenine and tetrandrine.
[0008] The chromatographic conditions of the detection are as follows: a T3 chromatographic column is used, the mobile phase A is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, the mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the gradient elution program is as follows: 0-4 min, 7% B→11% B; 4-10 min, 11% B→11% B; 10-14 min, 11% B→15% B; 14-18 min, 15% B→17% B; 18-22 min, 17% B→23% B; 22-24 min, 2 3% B→24% B; 24-28 min, 24% B→25% B; 28-40 min, 25% B→33% B; 40-41 min, 33% B→36% B; 41-45 min, 36% B→36% B; 45-50 min, 36% B→50% B; 50-55 min, 50% B→98% B, flow rate 0.1-1.0 ml / min, column temperature 20-40°C, detection wavelength 200-400 nm, injection volume 1-10 μl;
[0009] According to the test results, the component information, or the component information and content information of the Radix Paeoniae Wind-Resistantis medicinal material is obtained.
[0010] Furthermore, the information is calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Radix Phellodendri var. Wind-Stemmingi medicinal material and the chromatogram of the reference solution.
[0011] Furthermore, the preparation method of the test solution of the Radix Eupatorii medicinal material includes the following steps: weighing an appropriate amount of Radix Eupatorii medicinal material powder, adding water or alcohol for extraction, cooling and fixing the volume, taking an appropriate volume of solution for centrifugation, taking the supernatant, and obtaining the test solution of the Radix Eupatorii medicinal material.
[0012] Furthermore, the preparation method of the reference solution includes: weighing appropriate amounts of magnolia alkaloids, sinomenine and tetrandrine, adding a methanol solution with a volume percentage concentration of 10% to 100%, such as a 100% methanol solution, to prepare the reference solution with magnolia alkaloids, sinomenine and tetrandrine concentrations of 0.01 to 100 mg / ml respectively.
[0013] Furthermore, the concentration of magnolidine in the reference solution is 0.01-0.1 mg / ml, for example, about 0.05 mg / ml.
[0014] Furthermore, the concentration of sinomenine in the reference solution is 0.01-0.1 mg / ml, for example, about 0.05 mg / ml.
[0015] Furthermore, the concentration of tetrandrine in the reference solution is 0.01-0.1 mg / ml, for example, about 0.05 mg / ml.
[0016] Furthermore, the flow rate of the HPLC detection is 0.1-0.5 ml / min, for example, about 0.3 ml / min.
[0017] Furthermore, the column temperature is 25-35°C, for example, about 28°C.
[0018] Furthermore, the detection wavelength is 210-280 nm, for example, 254 nm.
[0019] Furthermore, the injection volume is 1-5 μl, for example, about 2 μl.
[0020] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the reference substance is not less than 5000.
[0021] Furthermore, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 2.0.
[0022] Furthermore, the chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column.
[0023] Furthermore, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.
[0024] Furthermore, in the method for preparing the test solution of the Radix Eupatorii medicinal material, the mass of the Radix Eupatorii medicinal material powder is 0.1 to 10 g, for example, about 0.5 g.
[0025] Furthermore, the mass / volume (g / ml) ratio of the Radix Paeoniae Wind-Resistantis powder to the water or alcohol is (0.5-10):(1-100), for example, about 1:about 50.
[0026] Furthermore, the alcohol is ethanol.
[0027] Furthermore, the volume percentage concentration of the ethanol is 10% to 100%, for example, about 60%.
[0028] Furthermore, the volume of the ethanol is 10-50 ml, for example, about 25 ml.
[0029] Further, the extraction is ultrasonic extraction, cold soaking extraction or reflux extraction, such as ultrasonic extraction.
[0030] Furthermore, the power of the ultrasonic extraction is 20-120W, for example, about 100W.
[0031] Furthermore, the power of the ultrasonic extraction is 20-60 kHz, for example, about 40 kHz.
[0032] Furthermore, the ultrasonic extraction time is 5 to 60 minutes, for example, about 20 minutes.
[0033] Furthermore, the extraction is performed 1 to 3 times, for example, once.
[0034] Furthermore, the volume of the appropriate volume of solution is 1-10 ml, for example, about 2 ml.
[0035] Furthermore, the ratio of the appropriate volume of the solution to the volume (ml / ml) of the water or alcohol is (0.5-10):(1-20), for example, about 1:about 12.5.
[0036] Furthermore, the centrifugal rotation speed is 10000-14000 rpm, for example, about 12000 rpm.
[0037] Furthermore, the centrifugation time is 10 to 20 minutes, for example, about 15 minutes.
[0038] Furthermore, the mobile phase B is acetonitrile.
[0039] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.
[0040] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.
[0041] Furthermore, the acid aqueous solution is a 0.01% to 0.2% acid aqueous solution.
[0042] Furthermore, the acid aqueous solution is a 0.01% to 0.2% trifluoroacetic acid aqueous solution.
[0043] Furthermore, the acid aqueous solution is about 0.1% trifluoroacetic acid aqueous solution.
[0044] Furthermore, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.
[0045] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.
[0046] According to another aspect of the present invention, a method for constructing a fingerprint of a medicinal material of Radix Paeoniae Wind-Firefly is provided, and the method comprises the following steps:
[0047] Preparation of the test solution of Radix Glehniae: weigh an appropriate amount of Radix Glehniae powder, add water or alcohol to extract, cool, fix the volume, take an appropriate volume of the solution for centrifugation, take the supernatant, and obtain the test solution of the Radix Glehniae;
[0048] Preparation of reference solution: weigh appropriate amounts of magnolia alkaloids, sinomenine and tetrandrine, add a methanol solution with a volume percentage concentration of 10% to 100%, for example, 100% methanol solution to prepare a reference solution with a concentration of magnolia alkaloids, sinomenine and tetrandrine of 0.01 to 100 mg / ml respectively;
[0049] According to the results of high performance liquid chromatography detection of the test solution of the Radix Angelicae Pubescentis and the reference solution, a fingerprint of the Radix Angelicae Pubescentis is obtained;
[0050] The chromatographic conditions of the high performance liquid phase detection are as follows: a T3 chromatographic column is used, mobile phase A is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the gradient elution program is: 0-4 min, 7% B→11% B; 4-10 min, 11% B→11% B; 10-14 min, 11% B→15% B; 14-18 min, 15% B→17% B; 18-22 min, 17% B→23% B; 22-24 min , 23% B→24% B; 24~28min, 24% B→25% B; 28~40min, 25% B→33% B; 40~41min, 33% B→36% B; 41~45min, 36% B→36% B; 45~50min, 36% B→50% B; 50~55min, 50% B→98% B, the flow rate is 0.1~1.0ml / min, the column temperature is 20~40℃, the detection wavelength is 200~400nm, and the injection volume is 1~10μl.
[0051] Furthermore, the flow rate of the HPLC detection is 0.1-0.5 ml / min, for example, about 0.3 ml / min.
[0052] Furthermore, the column temperature is 25-35°C, for example, about 28°C.
[0053] Furthermore, the detection wavelength is 210-280 nm, for example, 254 nm.
[0054] Furthermore, the injection volume is 1-5 μl, for example, about 2 μl.
[0055] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the reference substance is not less than 5000.
[0056] Furthermore, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 2.0.
[0057] Further, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-8, wherein the retention times of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and peak 8 correspond to about 6.9, about 19.4, about 22.8, about 27.6, about 29.2, about 32.2, about 36.8 and about 37.6, respectively.
[0058] Furthermore, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-8, wherein peak 2 is magnolia alkaloids as a reference peak, and the relative retention time average values of peaks 1, 3, 4, 5, 6, 7 and 8 correspond to approximately 0.36, approximately 1.18, approximately 1.43, approximately 1.51, approximately 1.66, approximately 1.90 and approximately 1.95, respectively.
[0059] Furthermore, the peak No. 1 is sinomenine.
[0060] Furthermore, the peak No. 6 is tetrandrine.
[0061] Furthermore, the mobile phase B is acetonitrile.
[0062] Furthermore, the acid aqueous solution, alkali aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.
[0063] Further, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.
[0064] Furthermore, the acid aqueous solution is a 0.01% to 0.2% acid aqueous solution.
[0065] Furthermore, the acid aqueous solution is a 0.01% to 0.2% trifluoroacetic acid aqueous solution.
[0066] Furthermore, the acid aqueous solution is about 0.1% trifluoroacetic acid aqueous solution.
[0067] Furthermore, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.
[0068] Furthermore, the pH value of the buffered saline solution is not greater than 7.0.
[0069] Furthermore, the chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column.
[0070] Furthermore, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.
[0071] According to another aspect of the present invention, a quality control method for Radix Psoraleae Radix is provided, the quality control method comprising the following steps:
[0072] (1) Establishing a standard fingerprint of the reference sample of Radix Psoraleae Radix according to the above fingerprint construction method;
[0073] (2) taking a test solution of the Radix Phellodendri var. windii medicinal material, and performing detection according to the chromatographic conditions in the above-mentioned fingerprint spectrum construction method to obtain a fingerprint spectrum of the Radix Phellodendri var. windii medicinal material sample to be tested; and
[0074] (3) Compare the fingerprint of the tested sample of the Radix Angelicae Pubescentis obtained in step (2) with the standard fingerprint of the reference sample of the Radix Angelicae Pubescentis obtained in step (1). The product that meets the requirements is qualified, and the product that does not meet the requirements is unqualified.
[0075] Furthermore, the compliance requirement includes one or more of the following:
[0076] (1) The schefflera herb sample to be tested exhibits 8 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the schefflera herb reference sample;
[0077] (2) The peak corresponding to the magnolia alkaloid reference substance is taken as the S peak, and the relative retention time of each characteristic chromatographic peak in the fingerprint of the tested sample of Radix Angelicae Pubescentis and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Radix Angelicae Pubescentis reference sample; and
[0078] (3) According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the tested sample of Radix Psoraleae Herba and the standard fingerprint of the reference sample of Radix Psoraleae Herba shall not be less than 0.90.
[0079] According to another aspect of the present invention, there is provided a use of the above detection method, the above construction method or the above quality control method in quality detection and / or quality evaluation and / or quality control of Radix Paeoniae Dioscoreae medicinal materials.
[0080] Beneficial effects of the present invention:
[0081] The method of the invention is simple, efficient and rapid, and provides a theoretical basis for the quality control of the medicinal material of Radix Phellodendron chinense and the in-depth study of the material basis of the medicinal effect through the detection of the medicinal material of Radix Phellodendron chinense. BRIEF DESCRIPTION OF THE DRAWINGS
[0082] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.
[0083] Figure 1 This is the chromatogram after the chromatographic conditions of the Radix Paeoniae Dafeng were optimized.
[0084] Figure 2 This is the precision test chart of Radix Dioscoreae.
[0085] Figure 3 This is the stability test chart of Radix Paeoniae Dafeng.
[0086] Figure 4 This is the reproducibility test spectrum of the medicinal material Rhizoma Dioscoreae.
[0087] Figure 5 The fingerprints and reference maps of 15 batches of Radix Paeoniae Rhizoma. Among them, 1 represents sinomenine, 2 represents magnoliavine, and 6 represents tetrandrine). DETAILED DESCRIPTION
[0088] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.
[0089] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.
[0090] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.
[0091] To the extent that the terms "comprises," "including," and "having," "has," and "with," or variations thereof, are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."
[0092] As described in the background technology section, there are few reports on the detection methods of Radix Paeoniae Wind-Wooden Branch in the prior art, and the detection time is very long, and the chromatographic peaks identified are very few. In order to solve the above problems, the present invention provides a detection method of Radix Paeoniae Wind-Wooden Branch, which comprises the following steps:
[0093] Take the test solution and reference solution of Radix Paeoniae Daphne for testing, wherein the reference solution is magnolamine, sinomenine and tetrandrine.
[0094] The chromatographic conditions of the detection are as follows: a T3 chromatographic column is used, the mobile phase A is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, the mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the gradient elution program is as follows: 0-4 min, 7% B→11% B; 4-10 min, 11% B→11% B; 10-14 min, 11% B→15% B; 14-18 min, 15% B→17% B; 18-22 min, 17% B→23% B; 22-24 min, 2 3% B→24% B; 24-28 min, 24% B→25% B; 28-40 min, 25% B→33% B; 40-41 min, 33% B→36% B; 41-45 min, 36% B→36% B; 45-50 min, 36% B→50% B; 50-55 min, 50% B→98% B, flow rate 0.1-1.0 ml / min, column temperature 20-40°C, detection wavelength 200-400 nm, injection volume 1-10 μl;
[0095] According to the test results, the component information, or the component information and content information of the Radix Paeoniae Wind-Resistantis medicinal material is obtained.
[0096] In a preferred embodiment, the information is calculated according to the external standard method based on the corresponding peak areas in the recorded chromatogram of the test solution of the Radix Paeoniae Wind-Firefly and the chromatogram of the reference solution of the Radix Paeoniae Wind-Firefly.
[0097] In a preferred embodiment, the method for preparing the test solution of the Radix Eupatorii medicinal material comprises the following steps: weighing an appropriate amount of Radix Eupatorii medicinal material powder, adding water or alcohol for extraction, cooling and fixing the volume, taking an appropriate volume of the solution for centrifugation, taking the supernatant, and obtaining the Radix Eupatorii medicinal material test solution.
[0098] In a preferred embodiment, the preparation method of the reference solution includes: weighing appropriate amounts of magnolia alkaloids, sinomenine and tetrandrine, adding a methanol solution with a volume percentage concentration of 10% to 100%, such as a 100% methanol solution, to prepare the reference solution with a concentration of magnolia alkaloids, sinomenine and tetrandrine of 0.01 to 100 mg / ml respectively.
[0099] In a preferred embodiment, the concentration of magnolia alkaloids in the reference solution is 0.01-0.1 mg / ml, such as about 0.05 mg / ml.
[0100] In a preferred embodiment, the concentration of sinomenine in the reference solution is 0.01-0.1 mg / ml, such as about 0.05 mg / ml.
[0101] In a preferred embodiment, the concentration of tetrandrine in the reference solution is 0.01-0.1 mg / ml, such as about 0.05 mg / ml.
[0102] In a preferred embodiment, the flow rate of the HPLC detection is 0.1-0.5 ml / min, such as about 0.3 ml / min.
[0103] In a preferred embodiment, the column temperature is 25-35°C, such as about 28°C.
[0104] In a preferred embodiment, the detection wavelength is 210-280 nm, for example 254 nm.
[0105] In a preferred embodiment, the injection volume is 1-5 μl, such as about 2 μl.
[0106] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the reference substance is not less than 5000.
[0107] In a preferred embodiment, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 2.0.
[0108] In a preferred embodiment, the chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column.
[0109] In a preferred embodiment, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.
[0110] In a preferred embodiment, in the method for preparing the Radix Angelicae Pubescentis medicinal material test solution, the mass of the Radix Angelicae Pubescentis medicinal material powder is 0.1-10 g, for example, about 0.5 g.
[0111] In a preferred embodiment, the mass / volume (g / ml) ratio of the Radix Paeoniae Wind-Resistant Herbs powder to the water or alcohol is (0.5-10):(1-100), for example, about 1:about 50.
[0112] In a preferred embodiment, the alcohol is ethanol.
[0113] In a preferred embodiment, the volume percentage concentration of the ethanol is 10% to 100%, such as about 60%.
[0114] In a preferred embodiment, the volume of the ethanol is 10-50 ml, such as about 25 ml.
[0115] In a preferred embodiment, the extraction is ultrasonic extraction, cold soaking extraction or reflux extraction, such as ultrasonic extraction.
[0116] In a preferred embodiment, the power of the ultrasonic extraction is 20-120W, for example, about 100W.
[0117] In a preferred embodiment, the power of the ultrasonic extraction is 20-60 kHz, for example, about 40 kHz.
[0118] In a preferred embodiment, the ultrasonic extraction time is 5 to 60 minutes, such as about 20 minutes.
[0119] In a preferred embodiment, the extraction is performed 1 to 3 times, for example, once.
[0120] In a preferred embodiment, the volume of the appropriate volume of solution is 1-10 ml, for example, about 2 ml.
[0121] In a preferred embodiment, the ratio of the appropriate volume of the solution to the volume (ml / ml) of the water or alcohol is (0.5-10):(1-20), for example, about 1:about 12.5.
[0122] In a preferred embodiment, the centrifugal rotation speed is 10000-14000 rpm, such as about 12000 rpm.
[0123] In a preferred embodiment, the centrifugation time is 10 to 20 min, such as about 15 min.
[0124] In a preferred embodiment, the mobile phase B is acetonitrile.
[0125] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.
[0126] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.
[0127] In a preferred embodiment, the acid aqueous solution is a 0.01% to 0.2% acid aqueous solution.
[0128] In a preferred embodiment, the acid aqueous solution is a 0.01% to 0.2% trifluoroacetic acid aqueous solution.
[0129] In a preferred embodiment, the aqueous acid solution is about 0.1% trifluoroacetic acid in water.
[0130] In a preferred embodiment, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.
[0131] In a preferred embodiment, the pH value of the buffered saline solution is no greater than 7.0.
[0132] According to another aspect of the present invention, a method for constructing a fingerprint of a medicinal material of Radix Paeoniae Wind-Firefly is provided, and the method comprises the following steps:
[0133] Preparation of the test solution of Radix Glehniae: weigh an appropriate amount of Radix Glehniae powder, add water or alcohol to extract, cool, fix the volume, take an appropriate volume of the solution for centrifugation, take the supernatant, and obtain the test solution of the Radix Glehniae;
[0134] Preparation of reference solution: weigh appropriate amounts of magnolia alkaloids, sinomenine and tetrandrine, add a methanol solution with a volume percentage concentration of 10% to 100%, for example, 100% methanol solution to prepare a reference solution with a concentration of magnolia alkaloids, sinomenine and tetrandrine of 0.01 to 100 mg / ml respectively;
[0135] According to the results of high performance liquid chromatography detection of the test solution of the Radix Angelicae Pubescentis and the reference solution, a fingerprint of the Radix Angelicae Pubescentis is obtained;
[0136] The chromatographic conditions of the high performance liquid phase detection are as follows: a T3 chromatographic column is used, mobile phase A is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the gradient elution program is: 0-4 min, 7% B→11% B; 4-10 min, 11% B→11% B; 10-14 min, 11% B→15% B; 14-18 min, 15% B→17% B; 18-22 min, 17% B→23% B; 22-24 min , 23% B→24% B; 24~28min, 24% B→25% B; 28~40min, 25% B→33% B; 40~41min, 33% B→36% B; 41~45min, 36% B→36% B; 45~50min, 36% B→50% B; 50~55min, 50% B→98% B, the flow rate is 0.1~1.0ml / min, the column temperature is 20~40℃, the detection wavelength is 200~400nm, and the injection volume is 1~10μl.
[0137] In the present invention, when flow rate, temperature, wavelength, concentration, time, pressure, ratio, equivalent, concentration, or other values or parameters are expressed as ranges, preferred ranges, or a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "20-40" is disclosed, the described range should be interpreted as including the range "20-40", "20-35", "20-30", "20-25", "25-40", "30-40", "35-40", "30-40", "35-40", "30-40", "35-40", "30-35", etc. When a numerical range is described herein, unless otherwise specified, the range is intended to include its end values and all integers and fractions within the range, and the technical effects of the present invention can be achieved within the above range.
[0138] In a preferred embodiment, the flow rate of the HPLC detection is 0.1-0.5 ml / min, such as about 0.3 ml / min.
[0139] In the present 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.
[0140] In a preferred embodiment, the column temperature is 25-35°C, such as about 28°C.
[0141] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 28" includes ±5% of 28, or from 26.6 to 29.4.
[0142] In a preferred embodiment, the detection wavelength is 210-280 nm, for example 254 nm.
[0143] In a preferred embodiment, the injection volume is 1-5 μl, such as about 2 μl.
[0144] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1.
[0145] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the reference substance is not less than 5000.
[0146] In a preferred embodiment, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 2.0.
[0147] In a preferred embodiment, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-8, wherein the retention times of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and peak 8 are about 6.9, about 19.4, about 22.8, about 27.6, about 29.2, about 32.2, about 36.8 and about 37.6, respectively.
[0148] In the present invention, "about" refers to a value within a range of ±5% of a particular value. For example, “about 6.9” includes ±5% of 6.9, or from 6.555 to 7.245; “about 19.4” includes ±5% of 19.4, or from 18.43 to 20.37; “about 22.8” includes ±5% of 22.8, or from 21.66 to 23.94; “about 27.6” includes ±5% of 27.6, or from 26.22 to 28.98; “about 29.2” includes ±5% of 29.2, or from 27.74 to 30.66; “about 32.2” includes ±5% of 32.2, or from 30.59 to 33.81; “about 36.8” includes ±5% of 36.8, or from 34.96 to 38.64; “about 37.6” includes ±5% of 37.6, or from 35.72 to 39.48.
[0149] In a preferred embodiment, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1-8, wherein peak 2 is magnolia alkaloids as a reference peak, and the relative retention time average values of peaks 1, 3, 4, 5, 6, 7 and 8 are approximately 0.36, approximately 1.18, approximately 1.43, approximately 1.51, approximately 1.66, approximately 1.90 and approximately 1.95, respectively.
[0150] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.36" includes ±5% of 0.36, or from 0.342 to 0.378; "about 1.18" includes ±5% of 1.18, or from 1.121 to 1.239; "about 1.43" includes ±5% of 1.43, or from 1.3585 to 1.5015; "about 1.51" includes ±5% of 1.51, or from 1.4345 to 1.5855; "about 1.66" includes ±5% of 1.66, or from 1.577 to 1.743; "about 1.9" includes ±5% of 1.9, or from 1.805 to 1.995; "about 1.95" includes ±5% of 1.95, or from 1.8525 to 1.995.
[0151] In a preferred embodiment, the peak No. 1 is sinomenine.
[0152] In a preferred embodiment, the peak No. 6 is tetrandrine.
[0153] In a preferred embodiment, the mobile phase B is acetonitrile.
[0154] In a preferred embodiment, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations.
[0155] In a preferred embodiment, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations.
[0156] In a preferred embodiment, the acid aqueous solution is a 0.01% to 0.2% acid aqueous solution.
[0157] In a preferred embodiment, the acid aqueous solution is a 0.01% to 0.2% trifluoroacetic acid aqueous solution.
[0158] In a preferred embodiment, the aqueous acid solution is about 0.1% trifluoroacetic acid in water.
[0159] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 0.1" includes ±5% of 0.1, or from 0.095 to 0.105.
[0160] In a preferred embodiment, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution.
[0161] In a preferred embodiment, the pH value of the buffered saline solution is no greater than 7.0.
[0162] In a preferred embodiment, the chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column.
[0163] In a preferred embodiment, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.
[0164] According to another aspect of the present invention, a quality control method for Radix Psoraleae Radix is provided, the quality control method comprising the following steps:
[0165] (1) Establishing a standard fingerprint of the reference sample of Radix Psoraleae Radix according to the above fingerprint construction method;
[0166] (2) taking a test solution of the Radix Phellodendri var. windii medicinal material, and performing detection according to the chromatographic conditions in the above-mentioned fingerprint spectrum construction method to obtain a fingerprint spectrum of the Radix Phellodendri var. windii medicinal material sample to be tested; and
[0167] (3) Compare the fingerprint of the tested sample of the Radix Angelicae Pubescentis obtained in step (2) with the standard fingerprint of the reference sample of the Radix Angelicae Pubescentis obtained in step (1). The product that meets the requirements is qualified, and the product that does not meet the requirements is unqualified.
[0168] In a preferred embodiment, the compliance requirements include one or more of the following:
[0169] (1) The schefflera herb sample to be tested exhibits 8 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint of the schefflera herb reference sample;
[0170] (2) The peak corresponding to the magnolia alkaloid reference substance is taken as the S peak, and the relative retention time of each characteristic chromatographic peak in the fingerprint of the tested sample of Radix Angelicae Pubescentis and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Radix Angelicae Pubescentis reference sample; and
[0171] (3) According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the tested sample of Radix Psoraleae Herba and the standard fingerprint of the reference sample of Radix Psoraleae Herba shall not be less than 0.90.
[0172] According to another aspect of the present invention, there is provided a use of the above detection method, the above construction method or the above quality control method in quality detection and / or quality evaluation and / or quality control of Radix Paeoniae Dioscoreae medicinal materials.
[0173] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.
[0174] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.
[0175] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.
[0176] Example
[0177] The present invention investigates and optimizes the best chromatographic conditions by factors affecting chromatographic behavior, such as mobile phase composition, different chromatographic columns, different detection wavelengths, different acids, different acid concentrations, different flow rates, different column temperatures, and different injection volumes. The optimized chromatogram is shown in FIG. Figure 1 shown.
[0178] 1. Experimental instruments and materials
[0179] 1.1 Experimental instruments
[0180] Shimadzu Nexera-i LC 2040C 3D plus fast liquid chromatograph (equipped with SPD-40V detector; CTO-40C column oven; LC-40B XR infusion pump; SIL-40C autosampler; CBM-40Lite system controller; Shimadzu LabSolutions workstation); ME204E electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); XM-600UVF ultrasonic instrument (Xiaomei Ultrasonic Instrument (Kunshan) Co., Ltd.); H2050R centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.)
[0181] 1.2 Materials and Reagents
[0182] Methanol (chromatographic grade, Aladdin); acetonitrile (chromatographic grade, Thermo Fisher Scientific (China Co., Ltd.)); trifluoroacetic acid (analytical grade, Aladdin); Rhizoma Dioscoreae (batch numbers are shown in Table 1), Magnolia alkaloids (batch number: 808F021, Solebao Company); Sinomenine (batch number: BBP06007, Yunnan Xili Biotechnology Co., Ltd.), Tetrandrine (batch number: BBP03852, Yunnan Xili Biotechnology Co., Ltd.).
[0183] Table 1 Information on batch number, part and source of Radix Dioscoreae
[0184]
[0185]
[0186] 2. Optimization of chromatographic conditions
[0187] 2.1. Investigation of mobile phase composition
[0188] The present invention compares water-methanol and water-acetonitrile as mobile phases to analyze the truncatum fengdi sample under the same chromatographic conditions. The results show that compared with water-methanol, water-acetonitrile has better peak shape and more stable baseline.
[0189] 2.2. Investigation of different chromatographic columns
[0190] In the present invention, five chromatographic columns, including ACQUITY UPLC HSS T3 (2.1x100 mm, 1.8 μm), ACQUITY UPLC HSS T3 (2.1x150 mm, 1.8 μm), Gemini NX-C18 (4.6x150 mm, 1.8 μm), Agilent ZORBAX SB-C18 (4.6x250 mm, 5 μm) and ACQUITY UPLC BEH C18 (2.1x50 mm, 1.7 μm), are investigated under the same chromatographic conditions to analyze the same sample. The results show that compared with other chromatographic columns, the peak shape of the ACQUITY UPLC HSS T3 (2.1x100 mm, 1.8 μm) chromatographic column is better and more stable.
[0191] 2.3. Investigation of different detection wavelengths
[0192] The present invention investigates four detection wavelengths of 210nm, 254nm, 280nm and 320nm. The results show that, compared with other detection wavelengths, at 254nm, each chromatographic peak has better ultraviolet absorption, a stable baseline, better separation of each chromatographic peak, and a better chromatogram peak shape.
[0193] 2.4. Investigation of different acids
[0194] The present invention investigates using 0.1% and 0.5% trifluoroacetic acid (A)-acetonitrile (B), 0.1% and 0.5% formic acid (A)-acetonitrile (B), 0.1% and 0.5% acetic acid (A)-acetonitrile (B), and water (A)-acetonitrile (B) as mobile phases. Results show that using the peak areas of eight chromatographic peaks with larger responses in the chromatogram as evaluation indicators, the results show that, compared with other acids, under the condition of 0.1% trifluoroacetic acid (A)-acetonitrile (B), the number of peaks is moderate, the baseline is stable, the separation of each chromatographic peak is better, and the peak shape of the chromatogram is better.
[0195] 2.5. Investigation of different acid concentrations
[0196] The invention compares the extraction efficiency of trifluoroacetic acid at different concentrations (0.5%, 0.1%, 0.05% and 0.01%), and takes the peak areas of eight chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the effect of 0.1% trifluoroacetic acid is better than that of 0.5%, 0.05% and 0.01% trifluoroacetic acid.
[0197] 2.6. Investigation of different flow rates
[0198] The present invention takes the peak areas of the eight chromatographic peaks with larger responses in the chromatogram as evaluation indicators, and examines different flow rates (0.2mL / min, 0.3mL / min, and 0.4mL / min). The results show that compared with other flow rates, at 0.3mL / min, the baseline is stable, the separation of each chromatographic peak is better, and the chromatogram peak shape is better.
[0199] 2.7. Investigation of different column temperatures
[0200] The present invention compares the extraction efficiency at different column temperatures (25°C, 28°C, 30°C and 32°C), and takes the peak areas of eight chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the effect of 28°C is better than that of 25°C, 30°C and 32°C.
[0201] 2.8. Investigation of different injection volumes
[0202] This study compared the extraction efficiency of different injection volumes (1μL, 2μL, 4μL, 5μL and 8μL), and took the peak area of the eight chromatographic peaks with larger responses in the chromatogram as the evaluation index. The results showed that 2μL was better than 1μL, 4μL, 5μL and 8μL.
[0203] 2.9 Conclusion
[0204] The final chromatographic conditions were determined as follows: Waters ACQUITY HSS T3 (2.1×100mm, 1.8μm) chromatographic column, the mobile phase was 0.1% trifluoroacetic acid water (A)-acetonitrile (B), and the mobile phase elution gradient was: mobile phase elution gradient: 0-4min, 7-11% (B); 4-10min, 11-11% (B); 10-14min, 11-15% (B); 14-18min, 15-17% (B); 18-22min, 17-23% ( B); 22-24 min, 23-24% (B); 24-28 min, 24-25% (B); 28-40 min, 25-33% (B); 40-41 min, 33-36% (B); 41-45 min, 36-36% (B); 45-50 min, 36-50% (B); 50-55 min, 50-98% (B), column temperature 28 ° C, flow rate 0.3 mL min -1 , detection wavelength was 254 nm, and injection volume was 2 μL.
[0205] 3. Investigation of sample preparation methods
[0206] 3.1. Investigation of extraction methods
[0207] The present invention compares the extraction efficiencies of three extraction methods, namely, ultrasound, cold soaking and reflux, and uses the peak areas of eight chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the ultrasonic extraction effect is better than the reflux and cold soak extraction effects.
[0208] 3.2. Extraction time investigation
[0209] The present invention compares the extraction efficiency of different ultrasonic extraction times (10min, 20min, 40min and 60min), and takes the peak area of 8 chromatographic peaks with larger responses in the chromatogram as the evaluation index. The results show that the effect of 20min is better than that of 10min, 40min and 60min.
[0210] 3.3 Investigation of ultrasonic extraction power
[0211] The present invention compares the extraction efficiency of different ultrasonic extraction powers (40W, 60W, 80W and 100W), and takes the peak areas of 8 chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the effect of 100W is better than that of 60W and 80W.
[0212] 3.4. Investigation of extraction solvent
[0213] The present invention compares the extraction efficiencies of different extraction solvents (water, 50% methanol, 100% methanol, 50% ethanol and 100% ethanol), and takes the peak areas of 8 chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the effect of 50% ethanol is better than that of water, 50% methanol, 100% methanol and 100% ethanol, that is, the peak area of the chromatographic peak is larger.
[0214] 3.5. Investigation of extraction solvent concentration
[0215] The invention compares the extraction efficiency of different concentrations (20%, 40%, 50%, 60%, 80% and 100%) of the extraction solvent ethanol, and takes the peak areas of 8 chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the effect of 60% ethanol is better than that of 20%, 40%, 50%, 80% and 100% ethanol, that is, the peak area of the chromatographic peak is larger.
[0216] 3.6. Investigation of material-liquid ratio
[0217] The present invention compares the extraction efficiencies of different solid-liquid ratios of 1:25, 1:50, 1:75 and 1:100, and takes the peak areas of 8 chromatographic peaks with larger responses in the chromatogram as evaluation indicators. The results show that the effect of a solid-liquid ratio of 1:50 is better than that of 1:25, 1:75 and 1:100.
[0218] 3.7. Investigation of extraction times
[0219] This study compared the extraction efficiency of different extraction times (1 time, 2 times and 3 times), and took the peak area of the 8 chromatographic peaks with larger responses in the chromatogram as the evaluation index. The results showed that the effect of extraction once was better than that of extraction twice and three times, that is, the peak area of the chromatographic peak was larger.
[0220] 3.8 Conclusion
[0221] The peak areas of the eight chromatographic peaks with the largest response in the chromatogram were used as evaluation indicators to optimize the optimal extraction conditions by investigating the factors affecting the extraction of Radix Pleurotus eryngii, such as extraction method, extraction time, number of extractions, extraction solvent, solid-liquid ratio and extraction solvent concentration. The results determined that the extraction conditions were: ultrasonic extraction (100W, 40kHz), extraction time of 20min, once, extraction solvent of 60% ethanol, and solid-liquid ratio of (V / m) 1:50.
[0222] 3.9. Preparation of sample solution
[0223] Take 0.5g of 15 batches of Dafengteng sample powder (passed through No. 4 sieve), accurately weigh, place in a stoppered conical bottle, accurately measure 25mL of 60% ethanol solution and place in a stoppered conical bottle, weigh, seal, ultrasonically extract (100W, 40kHz) for 20min, cool, make up the lost weight with 60% ethanol solution, and mix. Take 2mL of the mixed solution in a centrifuge tube, centrifuge at 12000rpm for 15min, take the supernatant, and obtain the test solution for standby use.
[0224] 3.10. Preparation of reference solution:
[0225] Use a balance to accurately weigh 1 mg of magnolia alkaloids (batch number: 808F021), sinomenine (batch number: BBP06007), and tetrandrine (batch number: BBP03852) reference substances, place them in a 10 mL volumetric flask, dilute to 10 mL with methanol, sonicate for 20 min, prepare a 0.1 mg / mL magnolia alkaloid reference substance solution, and store in a 4°C refrigerator. (Dilution according to experimental requirements)
[0226] 50μg / ml magnolamine, sinomenine, and tetrandrine reference solution: Take 2mL of 0.1mg / mL stock solution in a 5mL centrifuge tube, add 2mL of methanol, and mix well.
[0227] 4. Methodological Validation
[0228] The fingerprint chromatogram uses the peak areas of the eight chromatographic peaks with larger responses in the chromatogram as a reference to evaluate the feasibility of the established method.
[0229] 4.1 Precision test
[0230] Preparation of the test solution: Take 0.5 g of QC (passed through a No. 4 sieve) of each batch of mixed powder of Radix Dioscoreae samples, accurately weigh it, and place it in a stoppered conical flask. Accurately measure 25 mL of 60% ethanol solution and place it in a stoppered conical flask, weigh it, seal it, and perform ultrasonic extraction (40W, 40kHz) for 20 min. Let it cool, make up the lost weight with 60% ethanol solution, and mix well. Take 2 mL of the mixed solution in a centrifuge tube, centrifuge it at 12000 rpm for 15 min, take the supernatant, and the test solution is obtained. Perform 6 consecutive injections to examine the consistency of the relative peak areas of the main chromatographic peaks in the spectra with different injection times. The results are as follows: Figure 2 shown.
[0231] Taking the eight chromatographic peaks with larger peak area response in the spectrum as reference, the retention time and peak area of the chromatographic peak with a retention time of 19.432 min were used as references to calculate the relative retention times and relative peak areas of the remaining chromatographic peaks, as shown in Tables 2 and 3.
[0232] Table 2 Relative retention time results of fingerprint precision test
[0233]
[0234] Table 3 Relative peak area results of fingerprint precision test
[0235]
[0236] 4.2 Stability test
[0237] Preparation of the test solution: Take 0.5 g of QC (passed through No. 4 sieve) of each batch of mixed powder of Radix Dioscoreae samples, accurately weigh it, and place it in a stoppered conical flask. Accurately measure 25 mL of 60% ethanol solution and place it in a stoppered conical flask, weigh it, seal it, and perform ultrasonic extraction (100 W, 40 kHz) for 20 min. Let it cool, make up the lost weight with 60% ethanol solution, and mix well. Take 2 mL of the mixed solution in a centrifuge tube, centrifuge it at 12000 rpm for 15 min, take the supernatant, and the test solution is obtained. The sample is injected at 0, 4, 8, 12, 16, 24, and 48 h of sample extraction, and the consistency of the relative peak areas of the main chromatographic peaks in the spectra of the samples in different time periods is examined. The resulting stability spectrum is as follows: Figure 3 shown.
[0238] Taking the eight chromatographic peaks with relatively high corresponding peak areas in the spectrum as reference, the retention time and peak area of the chromatographic peak with a retention time of 19.432 min were used as references to calculate the relative retention times and relative peak areas of the remaining chromatographic peaks, as shown in Tables 4 and 5.
[0239] Table 4 Relative retention time results of fingerprint stability test
[0240]
[0241] Table 5 Relative peak area results of fingerprint stability test
[0242]
[0243]
[0244] 4.3 Repeatability test
[0245] Preparation of the test solution: Take 6 portions of QC (passed through No. 4 sieve) of mixed powder from each batch of Radix Dioscoreae samples, 0.5 g each, accurately weigh, and place in a stoppered conical flask. Accurately measure 25 mL of 60% ethanol solution and place in a stoppered conical flask, weigh the weight, seal, and perform ultrasonic extraction (100 W, 40 kHz) for 20 min. Cool, make up the lost weight with 60% ethanol solution, and mix well. Take 2 mL of the mixed solution in a centrifuge tube, centrifuge at 12000 rpm for 15 min, take the supernatant, and the test solution is obtained. Samples were injected separately to examine the consistency of the relative peak areas of the main chromatographic peaks in the spectra of repeated samples. The resulting stability spectrum is as follows: Figure 4 shown.
[0246] Taking the eight chromatographic peaks with relatively high corresponding peak areas in the spectrum as reference, and taking the retention time and peak area of the chromatographic peak with a retention time of 19.432 min as reference, the relative retention times and relative peak areas of the remaining chromatographic peaks were calculated, as shown in Tables 6 and 7.
[0247] Table 6 Relative retention time results of fingerprint repeatability test
[0248]
[0249] Table 7 Relative peak area results of fingerprint stability test
[0250]
[0251]
[0252] As a result, the RSD values of relative retention time and relative peak area of each chromatographic peak were less than 3%, indicating that the method had good reproducibility.
[0253] The results of methodological validation showed that, using the 8 chromatographic peaks with larger responses in the chromatogram as reference, and the retention time and peak area of the chromatographic peak with a retention time of 19.432 min as reference for calculation, the relative retention time and relative area RSD values of precision, stability and reproducibility all met the relevant provisions of the 2020 edition of the "Chinese Pharmacopoeia" on methodological validation for the establishment of fingerprint methods.
[0254] 5. Establishment of fingerprints of different batches of Rhizoma Dioscoreae and calibration of common peaks
[0255] According to "3.9, Preparation of sample solution", the test solution was prepared, and the fingerprints of 15 batches of Radix Dioscoreae roots and stems were established and evaluated using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition)" of the National Pharmacopoeia Committee. The sample S2 spectrum was set as the reference spectrum, the time window was 0.50, and the similarity was calculated using the median method to generate the Radix Dioscoreae reference spectrum and fingerprint spectrum. By comparing with the reference substance, a total of 3 chromatographic peaks were identified. There were 8 common peaks in the 15 batches of Radix Dioscoreae medicinal materials, such as Figure 5 The similarity values of each batch of Radix Paeoniae Daphne are shown in Table 8.
[0256] Table 8 Similarity of 15 batches of Radix Psoraleae
[0257]
[0258]
[0259] The results of similarity analysis showed that the similarities of 15 different batches of Rhizoma Cibotii were all greater than 0.9, indicating that the quality of Rhizoma Cibotii products was stable.
[0260] 6. Chemical pattern recognition analysis
[0261] 6.1 Cluster Analysis
[0262] The eight common peak areas in the fingerprints of 15 batches of Radix Pleurotus eryngii were clustered and analyzed. The cluster analysis results showed that except for S11, S12 and S14, which were well classified with the other samples, the other samples were poorly separated, among which S11, S12 and S14 were all stems.
[0263] 6.2 Principal Component Analysis
[0264] The principal component analysis was performed on the 8 common peak areas of the fingerprints of 15 batches of Radix Paeoniae Dafeng. The results showed that the overlap between the roots and stems was large (i.e., the similarity between the roots and stems was still large), which was consistent with the cluster analysis results. The contribution rates of PC1 and PC2 were 68.3% and 14% respectively, and the cumulative variance contribution rate reached 83.4%, which could explain most of the variable information.
[0265] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.
Claims
1. A method for detecting the medicinal material of Rhizoma Dioscoreae, characterized in that: The detection method comprises the following steps: Take the test solution and reference solution of Radix Paeoniae Daphne for testing, wherein the reference solution is magnolamine, sinomenine and tetrandrine. The chromatographic conditions of the detection are as follows: a T3 chromatographic column is used, mobile phase A is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the gradient elution program is as follows: 0-4 min, 7% B→11% B; 4-10 min, 11% B→11% B; 10-14 min, 11% B→15% B; 14-18 min, 15% B→17% B; 18-22 min, 17% B→23% B; 22-24 min, 2 3% B→24% B; 24-28 min, 24% B→25% B; 28-40 min, 25% B→33% B; 40-41 min, 33% B→36% B; 41-45 min, 36% B→36% B; 45-50 min, 36% B→50% B; 50-55 min, 50% B→98% B, flow rate 0.1-1.0 ml / min, column temperature 20-40°C, detection wavelength 200-400 nm, injection volume 1-10 μl; According to the test results, the component information, or the component information and content information of the Radix Paeoniae Wind-Resistantis medicinal material is obtained.
2. The detection method according to claim 1, characterized in that: The information is calculated according to the external standard method based on the corresponding peak areas in the chromatogram of the test solution of the Radix Paeoniae Wind-Stemmed and the chromatogram of the reference solution of the Radix Paeoniae Wind-Stemmed; Preferably, the method for preparing the test solution of the Radix Paeoniae Wind-Wearing Herb comprises the following steps: weighing an appropriate amount of Radix Paeoniae Wind-Wearing Herb powder, adding water or alcohol for extraction, cooling, fixing the volume, taking an appropriate volume of the solution for centrifugation, taking the supernatant, and obtaining the test solution of the Radix Paeoniae Wind-Wearing Herb; More preferably, the preparation method of the reference solution comprises: weighing appropriate amounts of magnolia alkaloids, sinomenine and tetrandrine, adding a methanol solution with a volume percentage concentration of 10% to 100%, for example, a 100% methanol solution, to prepare the reference solution with a concentration of magnolia alkaloids, sinomenine and tetrandrine of 0.01 to 100 mg / ml respectively; Also preferably, the concentration of magnolidine in the reference solution is 0.01 to 0.1 mg / ml, for example, about 0.05 mg / ml; Also preferably, the concentration of sinomenine in the reference solution is 0.01-0.1 mg / ml, for example, about 0.05 mg / ml; Also preferably, the concentration of tetrandrine in the reference solution is 0.01-0.1 mg / ml, for example, about 0.05 mg / ml; Still more preferably, the flow rate of the HPLC detection is 0.1 to 0.5 ml / min, for example, about 0.3 ml / min; Still more preferably, the column temperature is 25-35°C, such as about 28°C; Still more preferably, the detection wavelength is 210-280 nm, for example 254 nm; Still more preferably, the injection volume is 1 to 5 μl, for example, about 2 μl; Particularly preferably, the theoretical plate number of the chromatographic peak corresponding to the reference substance is not less than 5000; Particularly preferably, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 2.0; Particularly preferably, the chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column; Particularly preferably, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.
3. The detection method according to claim 2, characterized in that: In the method for preparing the test solution of the Radix Eupatorii medicinal material, the mass of the Radix Eupatorii medicinal material powder is 0.1 to 10 g, for example, about 0.5 g; Preferably, the mass / volume (g / ml) ratio of the Radix Paeoniae Wind-Resistantis powder to the water or alcohol is (0.5-10):(1-100), for example, about 1:about 50; More preferably, the alcohol is ethanol; More preferably, the volume percentage concentration of the ethanol is 10% to 100%, for example, about 60%; More preferably, the volume of the ethanol is 10 to 50 ml, for example about 25 ml; Also preferably, the extraction is ultrasonic extraction, cold soaking extraction or reflux extraction, such as ultrasonic extraction; Particularly preferably, the power of the ultrasonic extraction is 20 to 120 W, for example, about 100 W; Particularly preferably, the power of the ultrasonic extraction is 20 to 60 kHz, for example, about 40 kHz; Particularly preferably, the ultrasonic extraction time is 5 to 60 min, for example, about 20 min; Particularly preferably, the extraction is performed 1 to 3 times, for example, once; Particularly preferably, the volume of the appropriate volume of solution is 1 to 10 ml, for example, about 2 ml; Particularly preferably, the ratio of the appropriate volume of the solution to the volume (ml / ml) of the water or alcohol is (0.5-10):(1-20), for example, about 1:about 12.5; Particularly preferably, the centrifugal speed is 10000-14000 rpm, for example about 12000 rpm; Particularly preferably, the centrifugation time is 10 to 20 min, such as about 15 min.
4. The detection method according to any one of claims 1 to 3, characterized in that: The mobile phase B is acetonitrile; Preferably, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations; More preferably, the acid aqueous solution is a 0.01% to 0.2% acid aqueous solution; More preferably, the acid aqueous solution is a 0.01% to 0.2% trifluoroacetic acid aqueous solution; More preferably, the aqueous acid solution is about 0.1% trifluoroacetic acid aqueous solution; Also preferably, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution; Also preferably, the pH value of the buffered saline solution is not greater than 7.
0.
5. A method for constructing a fingerprint of a medicinal material of Radix Paeoniae Daphne, characterized in that: The construction method comprises the following steps: Preparation of the test solution of Radix Angelicae Pubescentis: weigh an appropriate amount of Radix Angelicae Pubescentis powder, add water or alcohol to extract, cool, fix the volume, take an appropriate volume of the solution for centrifugation, take the supernatant, and obtain the test solution of the Radix Angelicae Pubescentis; Preparation of reference solution: weigh appropriate amounts of magnolia alkaloids, sinomenine and tetrandrine, add a methanol solution with a volume percentage concentration of 10% to 100%, for example, 100% methanol solution to prepare the reference solution with a concentration of magnolia alkaloids, sinomenine and tetrandrine of 0.01 to 100 mg / ml respectively; Obtaining a fingerprint of the Radix Angelicae Pubescentis medicinal material according to the results of high performance liquid chromatography-mass spectrometry (HPLC) detection of the Radix Angelicae Pubescentis medicinal material test solution and the reference solution; The chromatographic conditions of the high performance liquid phase detection are as follows: a T3 chromatographic column is used, mobile phase A is an acid aqueous solution, an alkaline aqueous solution and / or a buffered saline solution, mobile phase B is selected from one or more of acetonitrile, methanol and tetrahydrofuran, and the gradient elution program is as follows: 0-4 min, 7% B→11% B; 4-10 min, 11% B→11% B; 10-14 min, 11% B→15% B; 14-18 min, 15% B→17% B; 18-22 min, 17% B→23% B; 22-24 min , 23% B→24% B; 24~28min, 24% B→25% B; 28~40min, 25% B→33% B; 40~41min, 33% B→36% B; 41~45min, 36% B→36% B; 45~50min, 36% B→50% B; 50~55min, 50% B→98% B, the flow rate is 0.1~1.0ml / min, the column temperature is 20~40℃, the detection wavelength is 200~400nm, and the injection volume is 1~10μl.
6. The construction method according to claim 5, characterized in that: The flow rate of the HPLC detection is 0.1 to 0.5 ml / min, for example, about 0.3 ml / min; Preferably, the column temperature is 25-35°C, for example about 28°C; More preferably, the detection wavelength is 210 to 280 nm, for example 254 nm; More preferably, the injection volume is 1 to 5 μl, for example, about 2 μl; Preferably, the theoretical plate number of the chromatographic peak corresponding to the reference substance is not less than 5000; Still more preferably, the separation degree of the chromatographic peaks corresponding to the reference substance is greater than 2.0; Particularly preferably, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1 to 8, wherein the retention times of peaks 1, 2, 3, 4, 5, 6, 7 and 8 correspond to about 6.9, about 19.4, about 22.8, about 27.6, about 29.2, about 32.2, about 36.8 and about 37.6, respectively; Particularly preferably, when the detection wavelength is 254 nm, the fingerprint spectrum includes peaks 1 to 8, wherein peak 2 is magnolia alkaloids as a reference peak, and the relative retention time averages of peaks 1, 3, 4, 5, 6, 7 and 8 correspond to about 0.36, about 1.18, about 1.43, about 1.51, about 1.66, about 1.90 and about 1.95, respectively; Particularly preferably, the peak No. 1 is sinomenine; Particularly preferably, the No. 6 peak is tetrandrine.
7. The construction method according to claim 5, characterized in that: The mobile phase B is acetonitrile; Preferably, the acid aqueous solution, base aqueous solution and / or buffered saline solution is selected from one or more of weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acid aqueous solution, alkali aqueous solution and / or buffered saline 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 in different concentrations; More preferably, the acid aqueous solution is a 0.01% to 0.2% acid aqueous solution; More preferably, the acid aqueous solution is a 0.01% to 0.2% trifluoroacetic acid aqueous solution; More preferably, the aqueous acid solution is about 0.1% trifluoroacetic acid aqueous solution; Also preferably, the buffered saline solution is a phosphate aqueous solution and / or an acetate aqueous solution; Also preferably, the pH value of the buffered saline solution is not greater than 7.0; Particularly preferably, the chromatographic column is an ACQUITY UPLC HSS T3 chromatographic column; Particularly preferably, the specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.8 μm.
8. A quality control method for Radix Paeoniae Dafengii medicinal material, characterized in that: The quality control method comprises the following steps: (1) Establishing a standard fingerprint of a reference sample of Radix Dioscoreae as the fingerprint construction method according to any one of claims 5 to 7; (2) taking a test solution of Radix Phellodendri var. windii and detecting it according to the chromatographic conditions in the fingerprint construction method according to any one of claims 5 to 7 to obtain a fingerprint of the Radix Phellodendri var. windii sample; as well as (3) Compare the fingerprint of the tested sample of the Radix Paeoniae Wind-Firefly obtained in step (2) with the standard fingerprint of the reference sample of the Radix Paeoniae Wind-Firefly obtained in step (1). The product that meets the requirements is qualified, and the product that does not meet the requirements is unqualified.
9. The quality control method according to claim 8, characterized in that: The compliance requirements include one or more of the following: (1) The schefflera herba medicinal material sample to be tested exhibits 8 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference substance chromatographic peak in the standard fingerprint of the schefflera herba medicinal material reference sample; (2) The peak corresponding to the magnolia alkaloid reference substance is taken as the S peak, and the relative retention time of each characteristic chromatographic peak in the fingerprint of the tested sample of the Chinese truncatum medicinal material and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint of the Chinese truncatum medicinal material reference sample; and (3) According to the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint, the similarity between the fingerprint of the tested sample of the Radix Psoraleae Herba and the standard fingerprint of the reference sample of the Radix Psoraleae Herba shall not be less than 0.
90.
10. Use of the detection method according to any one of claims 1 to 4, the construction method according to any one of claims 5 to 7, or the quality control method according to claim 8 or 9 in quality detection and / or quality evaluation and / or quality control of Radix Paeoniae Dioscoreae.