Construction of characteristic spectrum and quality detection method for standard decoction of Taxus chinensis var. mairei

Abstract

The present invention discloses a construction and quality detection method for a characteristic spectrum of a standard decoction of Taxus chinensis. In this technical solution, an HPLC characteristic spectrum is established based on a high performance liquid chromatograph, and the HPLC characteristic spectrum of more than 15 batches of standard decoctions of Taxus chinensis are synthesized using a Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) to establish a control characteristic spectrum of the standard decoction of Taxus chinensis. The characteristic spectrum is then detected on an unknown decoction to identify the authenticity of the unknown decoction. The method can control the characteristic components in the standard decoction of Taxus chinensis as a whole, and the characteristic peaks in the obtained characteristic spectrum are rich in information, high in precision, good in reproducibility, high in separation, and high in accuracy. The stability of the overall quality of the standard decoction of Taxus chinensis can be effectively guaranteed, and an effective basis is provided for the quality identification of Chinese medicinal material decoctions.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection, and in particular to a method for constructing a characteristic spectrum of a standard decoction of Taxus chinensis var. mairei and a method for quality detection. Background Art

[0002] Taxus chinensis (Taurea chinensis) is the dried branches and leaves of cultivated varieties of the Taxaceae family. It has the functions of promoting menstruation, dispersing stagnation, promoting diuresis, and reducing swelling. It is primarily used to treat accumulation of masses, rheumatic pain, edema, and dysuria. Standard decoctions of Taxus chinensis (Taurea chinensis) are freeze-dried powders derived from Taurus chinensis slices through water extraction, concentration, and drying. Compared to the raw materials or slices, these decoctions have lost their original properties, and the corresponding active ingredient content has also changed accordingly. Therefore, quantitative analysis using only the content-indicating components specified in the quality standards for the raw materials or slices is insufficient to fully reflect their intrinsic quality. Currently, there are few qualitative testing technologies for standard decoctions of Taxus chinensis (Taurus chinensis).

[0003] Characteristic profiling is a quality assessment method that reflects the overall characteristics of the chemical components of traditional Chinese medicines. It can effectively detect and control the authenticity, quality consistency, and stability of traditional Chinese medicines and their preparations. Therefore, it is necessary to establish a method for rapidly identifying the authenticity of standard decoctions of Taxus chinensis.

[0004] In the related art, "Preliminary Establishment of HPLC Characteristic Chromatographic Method for Taxus chinensis Water Decoction" in the Journal of Traditional Chinese Medicine Oncology discloses a method for establishing the HPLC characteristic chromatogram of Taxus chinensis water decoction, which uses a ZORBAX SB-Aq chromatographic column; the mobile phase is methanol-water (gradient elution); the detection wavelength is 310 nm; the column temperature is 25 ° C; the flow rate is 1.0 ml min -1 The HPLC characteristic profiles of four batches of Taxus chinensis decoctions were determined. This method, based on the gradient elution procedure, detection wavelength, and chromatographic column used, primarily examined the characteristic profiles of non-taxane and non-flavonoid compounds. The main content indicator component in Taxus chinensis is 10-deacetylbaccatin III, a taxane compound. Other pharmacodynamically valuable taxanes and flavonoids were not examined. Therefore, this characteristic profile cannot accurately and stably evaluate the quality of standard decoctions of Taxus chinensis. Summary of the Invention

[0005] In view of this, the present application provides a method for constructing a characteristic spectrum and quality detection of a standard decoction of Taxus chinensis var. mairei, which has rich characteristic peak information, high precision, good reproducibility, high separation and high accuracy, and can effectively ensure the stability of the overall quality of the standard decoction of Taxus chinensis var. mairei.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a method for constructing an HPLC characteristic spectrum of a standard decoction of Taxus chinensis var. mairei, comprising the following steps:

[0008] M1. Inject 10-deacetylbaccatin III reference solution, 7-xylose 10-deacetylpaclitaxel reference solution, reference medicinal material solution I, and test solution I into a high-performance liquid chromatography for determination to establish an HPLC characteristic spectrum I. Use the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition) to synthesize the HPLC characteristic spectrum I of more than 15 batches of test solution I to establish a reference spectrum I of the characteristic spectrum I of the standard decoction of Taxus chinensis. The preparation process of the reference medicinal material solution I is as follows: obtain Taxus chinensis reference medicinal material powder, add methanol, sonicate, filter, evaporate the filtrate to dryness under reduced pressure, then add a mixed solution of water and dichloromethane for extraction, evaporate the extract to dryness, and dissolve it in methanol to obtain the reference medicinal material solution I.

[0009] The preparation process of the test solution I is as follows: taking the test sample, adding a mixed solution of water and dichloromethane to extract, evaporating the extract to dryness and then dissolving it in methanol to obtain the test solution I, and the test sample is a standard decoction of Taxus chinensis var. mairei.

[0010] The conditions for the high performance liquid chromatography described in step M1 are as follows: an octadecylsilane bonded silica gel column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution is performed. The gradient elution procedure is as follows:

[0011] 0-12 min, mobile phase A: 80%, mobile phase B: 20%;

[0012] 12-22 min, mobile phase A: from 80% to 70%, mobile phase B: from 20% to 30%;

[0013] 22-30 min, mobile phase A: 70%, mobile phase B: 30%;

[0014] 30-36 min, mobile phase A: from 70% to 68%, mobile phase B: from 30% to 32%;

[0015] 36-39 min, mobile phase A: from 68% to 60%, mobile phase B: from 32% to 40%;

[0016] 39-65 min, mobile phase A: 60%, mobile phase B: 40%;

[0017] M2. Inject rutin reference solution, cyperus rotundus reference solution, reference medicinal material solution II, and test solution II into a high-performance liquid chromatograph for determination and establishment of HPLC characteristic spectrum II. Using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition), synthesize HPLC characteristic spectrum II of more than 15 batches of test solution II to establish a reference characteristic spectrum II for the standard decoction of Taxus chinensis.

[0018] The preparation process of the control medicinal material solution II is as follows: taking Taxus chinensis var. mairei control medicinal material powder, adding methanol for ultrasonic treatment, replenishing the methanol lost by ultrasonic treatment, and then filtering to obtain the control medicinal material solution II;

[0019] The preparation process of the test solution II is as follows: taking the test sample, adding methanol and ultrasonically treating it, replenishing the methanol lost by ultrasonication and filtering it to obtain the test solution II, wherein the test sample is a standard decoction of Taxus chinensis var. mairei;

[0020] The conditions for the high performance liquid chromatography described in step M2 are as follows: an octadecylsilane bonded silica gel column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; mobile phase A is pure water, mobile phase B is methanol, and gradient elution is performed. The gradient elution procedure is as follows:

[0021] 0-5 min, mobile phase A: from 65% to 45%, mobile phase B: from 35% to 55%;

[0022] 5-12 min, mobile phase A: 45%, mobile phase B: 55%;

[0023] 12-16 min, mobile phase A: from 45% to 0%, mobile phase B: from 55% to 100%;

[0024] 16-20 min, mobile phase A: 0%, mobile phase B: 100%;

[0025] 20-25 min, mobile phase A: from 0% to 65%, mobile phase B: from 100% to 45%;

[0026] M3. Merge the HPLC characteristic spectrum I and the HPLC characteristic spectrum II to obtain the characteristic spectrum of the southern yew standard decoction; merge the control characteristic spectrum I and the control characteristic spectrum II to obtain the control characteristic spectrum of the southern yew standard decoction.

[0027] Preferably, the HPLC characteristic spectrum I includes eight characteristic peaks, each characteristic peak corresponding to the retention time of the characteristic peak of the reference medicinal material solution I reference substance, the peak corresponding to the 10-deacetylbaccatin III reference substance is peak 2, i.e., S peak, and the peak corresponding to the 7-xylose 10-deacetylpaclitaxel reference substance is peak 6. The relative retention times of peaks 1-8 and S peak are calculated, and their relative retention times are within ±10% of the specified values. The specified value of peak 1 is 0.352, the specified value of peak 3 is 1.182, and the specified value of peak 4 is 1.238; the specified value of peak 5 is 1.468, the specified value of peak 6 is 1.946, the specified value of peak 7 is 2.024, and the specified value of peak 8 is 2.449.

[0028] Preferably, the HPLC characteristic spectrum II includes six characteristic peaks, each characteristic peak corresponding to the retention time of the characteristic peak of the reference substance of the control medicinal material solution II. The peak corresponding to the rutin reference substance is peak 3, namely the S1 peak. The relative retention time of peaks 1-4 and the S1 peak is calculated, and the relative retention time is within ±10% of the specified value. The specified value of peak 1 is 0.844, the specified value of peak 2 is 0.948, and the specified value of peak 4 is 1.157; the peak corresponding to the golden pine biflavonoids reference substance is peak 6, namely the S2 peak. The relative retention time of peak 5 and the S2 peak is calculated, and the relative retention time is within ±10% of the specified value. The specified value of peak 5 is 0.965.

[0029] Preferably, the chromatographic column temperature of the high performance liquid chromatography in step M1 is 25-35°C, the flow rate of the mobile phase is 0.8-1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 228-232 nm; further preferably, the chromatographic column temperature of the high performance liquid chromatography in step M1 is 30-35°C, the flow rate of the mobile phase is 0.9-1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 230-232 nm; more preferably, the chromatographic column temperature of the high performance liquid chromatography in step M1 is 30°C, the flow rate of the mobile phase is 0.9 mL / min, the injection volume is 20 μL, and the detection wavelength is 230 nm.

[0030] Preferably, the chromatographic column temperature of the high performance liquid chromatography in step M2 is 25-35°C, the flow rate of the mobile phase is 0.6-1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 240-310 nm; further preferably, the chromatographic column temperature of the high performance liquid chromatography in step M1 is 30-35°C, the flow rate of the mobile phase is 0.6-0.9 mL / min, the injection volume is 20 μL, and the detection wavelength is 260-270 nm; more preferably, the chromatographic column temperature of the high performance liquid chromatography in step M2 is 30°C, the flow rate of the mobile phase is 0.75 mL / min, the injection volume is 20 μL, and the detection wavelength is 260 nm.

[0031] Preferably, the ultrasonic power of step M2 is 600W, the frequency is 40kHz, and the concentration of the methanol solution in step M2 is 100%; the volume ratio of water to dichloromethane in step M1 is 1:1-2, and the sum of the volumes of water and dichloromethane is 40-60ml; more preferably, the volume ratio of water to dichloromethane in step M1 is 1:1.5, and the sum of the volumes of water and dichloromethane is 50ml; in step M1, when the volume ratio of water to dichloromethane is 1:1.5 and the sum of the volumes of water and dichloromethane is 50ml, the control medicinal material solution I and the test sample solution I can be extracted with the least solvent, and a higher corresponding peak area can be obtained. In step M2, when the concentration of the methanol solution is 100%, the chromatographic peak information amount is the largest and the extraction efficiency is the highest.

[0032] Preferably, the extraction number of dichloromethane and water in step M1 is 1-3 times, and the extraction time of methanol solution in step M2 is 20-40 minutes. More preferably, the extraction number in step M1 is twice, and the extraction time in step M2 is 30 minutes. Under this extraction number and extraction time, the extraction number and extraction time can be minimized to obtain a better extraction effect.

[0033] Preferably, the concentration of the 10-deacetylbaccatin III reference solution is 500 μg / ml, the concentration of the 7-xylose 10-deacetylpaclitaxel reference solution is 50 μg / ml, the concentration of the rutin reference solution is 200 μg / ml, and the concentration of the scutellaria baicalensis reference solution is 200 μg / ml.

[0034] Preferably, in step M1 and step M2, the mass ratio of the Taxus chinensis standard decoction to the Taxus chinensis control medicinal material powder is 1:1-4.

[0035] Preferably, the preparation method of the standard decoction of Taxus chinensis is as follows: taking a control medicinal material of Taxus chinensis, decocting it twice with water, filtering, combining the two filtrates, concentrating under reduced pressure to form a fluid extract, and then freeze-drying and grinding to obtain the standard decoction of Taxus chinensis.

[0036] In a second aspect, the present application provides a method for quality testing of a standard decoction of Taxus chinensis var. mairei, comprising the following steps:

[0037] K1. Pre-treat two identical sample samples separately: extract one sample with a mixture of water and dichloromethane, evaporate the extract to dryness, and dissolve it in methanol to obtain sample solution I. Ultrasonicate the other sample in methanol, replace any methanol lost during sonication, and filter to obtain sample solution II.

[0038] K2. Take the test solution I and measure it according to the HPLC conditions of step M1 to obtain the HPLC Figure I, take the test solution II and measure it according to the HPLC conditions of step M2 to obtain the HPLC Figure II , the determination of high performance liquid chromatography Figure I Compared with the control characteristic spectrum I, the determination of high performance liquid chromatography Figure II Compared with the control characteristic spectrum II, if the relative retention time of each characteristic peak corresponding to the control characteristic spectrum is within ±10% of the specified value, the test sample meets the quality standard of the standard decoction of Taxus chinensis.

[0039] The beneficial effects of this application are as follows:

[0040] The present invention provides a method for constructing and quality testing characteristic spectra (I and II) of a standard decoction of Taxus chinensis var. mairei, which identifies eight characteristic peaks, including 10-deacetylbaccatin III and 7-xylosyl 10-deacetylpaclitaxel, and six characteristic peaks, including rutin and cypermethrin. The constructed HPLC characteristic spectra (I and II) achieve good separation of chromatographic peaks, are rich in characteristic spectra information, and have good chromatographic peak shapes. The method can comprehensively reflect the main chemical component information of the standard decoction of Taxus chinensis var. mairei. The detection method has good stability and repeatability, and is convenient for large-scale promotion and application.

[0041] The HPLC characteristic spectra (I and II) of the standard decoction of Taxus chinensis constructed in the present invention fill the gap in the quality control of the standard decoction of Taxus chinensis in the prior art and provide an effective research basis for subsequent research on the formula granules of Taxus chinensis.

[0042] The present invention establishes a characteristic spectrum based on a high performance liquid chromatography instrument and performs characteristic spectrum detection, can comprehensively control the characteristic components in the southern yew standard decoction, can effectively ensure the stability of the overall quality of the southern yew standard decoction, and makes the quality control technology of the southern yew standard decoction more complete and scientific; and the method is simple to operate, has the advantages of high precision, good stability, good repeatability and high accuracy, and provides an effective basis for the quality identification of traditional Chinese medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 :Chromatogram of Taxus chinensis standard decoction under different extraction solvents Figure I ;

[0044] Figure 2 :Chromatogram of Taxus chinensis standard decoction under different extraction solvents Figure II ;

[0045] Figure 3 :Chromatograms of Taxus chinensis standard decoction under different extraction methods Figure II ;

[0046] Figure 4 :Chromatograms of Taxus chinensis standard decoction under different extraction times Figure I ;

[0047] Figure 5 :Chromatograms of Taxus chinensis standard decoction under different extraction times Figure II ;

[0048] Figure 6 :Chromatogram of Taxus chinensis standard decoction under different solvent addition amounts Figure I ;

[0049] Figure 7 :Chromatogram of Taxus chinensis standard decoction under different solvent addition amounts Figure II ;

[0050] Figure 8 : UV absorption spectra of 10-deacetylbaccatin III and 7-xylosyl 10-deacetylpaclitaxel;

[0051] Figure 9 :Comparison of chromatograms at different wavelengths for standard decoction of Taxus chinensis var. mairei Figure I ;

[0052] Figure 10 : It is the ultraviolet absorption spectrum of rutin and cyperus rotundus;

[0053] Figure 11 :Comparison of chromatograms at different wavelengths for standard decoction of Taxus chinensis Figure II ;

[0054] Figure 12 :Chromatogram of Taxus chinensis standard decoction under different column temperatures Figure I ;

[0055] Figure 13 :Chromatogram of Taxus chinensis standard decoction under different column temperatures Figure II ;

[0056] Figure 14 :Chromatogram of Taxus chinensis standard decoction under different flow rates Figure I ;

[0057] Figure 15 :Chromatogram of Taxus chinensis standard decoction under different flow rates Figure II ;

[0058] Figure 16 :Chromatography of Taxus chinensis standard decoction under delayed investigation Figure I ;

[0059] Figure 17 :Chromatography of Taxus chinensis standard decoction under delayed investigation Figure II ;

[0060] Figure 18 :Characteristic spectrum of the chromatographic peak identification of the standard decoction of Taxus chinensis Ⅰ;

[0061] Figure 19 :Characteristic spectrum of the chromatographic peak identification of the standard decoction of Taxus chinensis Ⅱ;

[0062] Figure 20 :Chromatograms of Taxus chinensis standard decoction under different instruments Figure I ;

[0063] Figure 21 :Chromatograms of Taxus chinensis standard decoction under different instruments Figure II ;

[0064] Figure 22 :Chromatographic column durability investigation of Taxus chinensis standard decoction Figure I ;

[0065] Figure 23 :Chromatographic column durability investigation of Taxus chinensis standard decoction Figure II ;

[0066] Figure 24 : Characteristic spectrum of standard decoction of Taxus chinensis Ⅰ (wherein, S1-S16 correspond to NHFDSBT220501, NHFDSBT220502, NHFDSBT220503, NHFDSBT220504, NHFDSBT220505, NHFDSBT220506, NHFDSBT220507, NHFDSBT220508, NHFDSBT220508, NHFDSBT220510, NHFDSBT220511, NHFDSBT220512, NHFDSBT220601, NHFDSBT220602, NHFDSBT220603, and NHFDSBT220604, respectively);

[0067] Figure 25 :Comparative characteristic spectrum of standard decoction of Taxus chinensis Ⅰ (wherein, Peak 2 (S): 10-deacetylbaccatin III, Peak 6: 7-xylose 10-deacetylpaclitaxel)

[0068] Figure 26 :Chromatography of Taxus chinensis var. mairei Figure I ;

[0069] Figure 27:Characteristic spectrum of standard decoction of Taxus chinensis Ⅱ (wherein, S1-S16 correspond to NHFDSBT220501, NHFDSBT220502, NHFDSBT220503, NHFDSBT220504, NHFDSBT220505, NHFDSBT220506, NHFDSBT220507, NHFDSBT220508, NHFDSBT220508, NHFDSBT220510, NHFDSBT220511, NHFDSBT220512, NHFDSBT220601, NHFDSBT220602, NHFDSBT220603, NHFDSBT220604, respectively);

[0070] Figure 28 : Comparative characteristic spectrum of standard decoction of Taxus chinensis Ⅱ (wherein, peak 3 (S1): rutin, peak 5: isoginkgo biloba flavonoids, peak 6 (S2): cyperus chinensis flavonoids);

[0071] Figure 29 :Chromatography of Taxus chinensis var. mairei Figure II ;

[0072] Figure 30 :Identification chromatogram of the test sample Figure I ;

[0073] Figure 31 :Identification chromatogram of the test sample Figure II . DETAILED DESCRIPTION

[0074] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0075] Source of raw materials

[0076] Taxus wallichiana var. mairei (Lemée et H. Léveillé) LKFu et NanLi (Taxus wallichiana var. mairei) reference medicinal material: The Taxus wallichiana used in this protocol is divided into 16 batches based on origin and harvest season. Lots 1-6 were collected from Yangzi Town, Zhongxiang City, Hubei Province; lots 7-11 were collected from Changshou Town, Zhongxiang City, Hubei Province; and lots 12-16 were collected from Fengxin County, Yichun City, Jiangxi Province. All 16 batches were tested and found to meet the requirements for "Taxus wallichiana var. mairei (Lemée et H. Léveillé) LKFu et NanLi" under the "Quality Standards for Traditional Chinese Medicines in Hubei Province."

[0077] The preparation method of the standard decoction of southern yew is as follows: take 100g of the southern yew control medicinal material of this application, add water and decoct twice, add 10 times the amount of water for the first decoction, stir for 1 minute to make the leaves completely immersed in water, soak for 40 minutes, boil and then decoct for 30 minutes, filter with 200 mesh filter cloth; add 8 times the amount of water to the residue of the second decoction and decoct for 20 minutes, filter, combine the two filtrates, concentrate under reduced pressure at 60°C to a fluid extract with a material-liquid ratio of about 1:1, freeze-dry in a stainless steel plate, grind the obtained dry paste and mix evenly to obtain the standard decoction of southern yew; the obtained standard decoction of southern yew is a brown to brown powder with a unique odor and bitter taste. The preparation process meets the preparation requirements for standard decoctions of Chinese medicinal materials in the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules" issued by the State Pharmacopoeia Commission. According to the different batch numbers of the reference medicinal materials of Taxus chinensis collected, the reference medicinal materials No. 1-16 corresponded to the standard decoctions of Taxus chinensis numbered S1-S16 (the implementation standard is the "Quality Standard of Traditional Chinese Medicines in Hubei Province"). In this plan, S1-S16 were named NHFDSBT220501, NHFDSBT220502, NHFDSBT220503, NHFDSBT220504, NHFDSBT220505NFHDSBT220506, NHFDSBT220507, NHFDSBT220508, NHFDSBT220508NFHDSBT220510, NHFDSBT220511, NHFDSBT220512, NHFDSBT220601, NHFDSBT220602, NHFDSBT220603, and NHFDSBT220604 respectively.

[0078] 10-Deacetylbaccatin III (Nanjing Yuanzhi Biotechnology Co., Ltd., cas number: 32981-86-5 batch number: YZ201102).

[0079] 7-Xylose 10-deacetylpaclitaxel (Shanghai Yihe Biotechnology Co., Ltd., CAS No.: 90332-63-1, batch No.: 032020).

[0080] Rutin (Nanjing Yuanzhi Biotechnology Co., Ltd., cas number: 153-18-4 batch number: YZ202820);

[0081] Jinsong biflavonoids (Nanjing Yuanzhi Biotechnology Co., Ltd., cas number: 521-34-6 batch number: YZ200211).

[0082] The 2012 edition of the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” in this application is the “Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software” well known to those skilled in the art.

[0083] The high performance liquid chromatography detection instrument of the present application includes but is not limited to one or more of Shimadzu 20-AD, Agilent 1260, and Waters 2965. In some preferred embodiments, the high performance liquid chromatography detection instrument is Waters 2965.

[0084] The chromatographic column of the high performance liquid chromatography of the present application includes one or more of Agilent Eclipse Plus C18 (5μm, 4.6×250mm), CNW Athena C18-WP (5μm, 4.6×250mm), and Diamonsil Plus C18 (5μm, 4.6×250mm). In some preferred embodiments, the chromatographic column of the high performance liquid chromatography is Agilent ZORBAX Eclipse Plus C18 (5μm, 4.6×250mm).

[0085] The present invention is further described below through specific examples.

[0086] Example 1

[0087] A method for constructing a characteristic spectrum of a standard decoction of Taxus chinensis var. mairei comprises the following steps:

[0088] M1. Establishment of HPLC characteristic spectrum I and reference characteristic spectrum I: 10-deacetylbaccatin III reference solution, 7-xylose 10-deacetylpaclitaxel reference solution, reference medicinal material solution I, and test solution I were injected into high performance liquid chromatography for determination to establish HPLC characteristic spectrum I. The HPLC characteristic spectrum I (such as Figure 24 As shown in the figure), the control characteristic spectrum I of the standard decoction of Taxus chinensis var. mairei was established (as shown in the figure) Figure 25 shown);

[0089] The preparation process of each solution is as follows:

[0090] Preparation of reference solution I: 10-deacetylbaccatin III (10-deacetylbaccatin III) and 7-xylose 10-deacetylpaclitaxel reference substances were accurately weighed and methanol was added to prepare reference solution I-(1) containing 500 μg of 10-deacetylbaccatin III per 1 mL, i.e., 10-deacetylbaccatin III reference solution and reference solution I-(2) containing 50 μg of 7-xylose 10-deacetylpaclitaxel per 1 mL, i.e., 7-xylose 10-deacetylpaclitaxel reference solution;

[0091] Preparation of control medicinal material solution I: Take 1.0 g of southern yew control medicinal material powder (No. 1 powder, passed through a 200-mesh sieve), accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 min, filter it, wash the container and the residue with a small amount of methanol, filter it, combine the filtrate, evaporate to dryness under reduced pressure, and then add 50 mL of a water: dichloromethane mixed solution with a volume ratio of 1:1.5 for extraction, dissolve it in batches and transfer it to a separatory funnel, take the dichloromethane liquid, and extract the water layer with dichloromethane oscillation once. Combine the dichloromethane liquids, evaporate to dryness under reduced pressure below 40°C, dissolve the residue in 10 mL of methanol, cool it, shake it well, filter it through a 0.22 μm filter membrane, and take the filtrate to obtain it.

[0092] Preparation of test solution I: Take 0.5 g of standard decoction of Taxus chinensis var. mairei, accurately weigh it, place it in a stoppered conical flask, and then add 50 ml of a water:dichloromethane mixed solution with a volume ratio of 1:1.5 for extraction. Dissolve it in batches and transfer it to a separatory funnel. Take the dichloromethane liquid separately, and extract the aqueous layer with dichloromethane again by shaking once. Combine the dichloromethane liquids, evaporate to dryness under reduced pressure below 40°C, dissolve the residue in 10 mL of methanol, cool, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the product.

[0093] High performance liquid chromatography chromatographic conditions I meet:

[0094] Chromatographic column: octadecylsilane bonded silica gel as filler, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; column temperature: 30°C; mobile phase: pure water as mobile phase A, acetonitrile as mobile phase B; mobile phase flow rate: 0.9 mL / min; injection volume: 20 μL; detection wavelength: 230 nm; the theoretical plate number calculated based on the 10-deacetylbaccatin III peak should be no less than 5000; mobile phase gradient elution program I according to Table 1 is:

[0095] Table 1: Gradient elution program I

[0096]

[0097] Final regulations: Eight characteristic peaks should be presented in the characteristic spectrum I of the standard decoction of Taxus chinensis var. mairei, and should correspond to the retention times of the characteristic peaks of the reference substance in the control medicinal material solution I. The peak corresponding to the 10-deacetylbaccatin III reference substance is peak 2 (S peak), and the peak corresponding to the 7-xylosyl 10-deacetylpaclitaxel reference substance is peak 6. The relative retention times of peaks 1-8 and the S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified value of peak 1 is 0.352, the specified value of peak 3 is 1.182, the specified value of peak 4 is 1.238, the specified value of peak 5 is 1.468, the specified value of peak 6 is 1.946, the specified value of peak 7 is 2.024, and the specified value of peak 8 is 2.449.

[0098] That is, the HPLC characteristic spectrum I of the standard decoction of Taxus chinensis var. mairei includes eight characteristic peaks, wherein each characteristic peak corresponds to the retention time of the characteristic peak of the reference substance of the control medicinal material solution I, the peak corresponding to the 10-deacetylbaccatin III reference substance is peak 2 (S peak), and the relative retention times of peaks 1-8 and the S peak are calculated. The relative retention times should be within ±10% of the specified value. The specified value of peak 1 is 0.352, the specified value of peak 3 is 1.182, the specified value of peak 4 is 1.238, the specified value of peak 5 is 1.468, the specified value of peak 6 is 1.946, the specified value of peak 7 is 2.024, and the specified value of peak 8 is 2.449.

[0099] M2. Establishment of HPLC characteristic spectrum II and reference characteristic spectrum II: Take rutin reference solution, cyperus rotundus flavonoids reference solution, reference medicinal material solution II, and test solution II and inject them into high performance liquid chromatography for determination to establish HPLC characteristic spectrum II. Use the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition) to evaluate the HPLC characteristic spectrum II (such as Figure 27 As shown in the figure), the control characteristic spectrum II of the standard decoction of Taxus chinensis was established (as shown in the figure) Figure 28 shown).

[0100] The preparation process of each solution is as follows:

[0101] Preparation of reference solution II: Take rutin and cyperus rotundus reference standards, weigh accurately, and add methanol to prepare reference solution II-(1) containing 200 μg of rutin per 1 mL, i.e., rutin reference solution, and reference solution II-(2) containing 200 μg of cyperus rotundus reference solution per 1 mL, i.e., cyperus rotundus reference solution.

[0102] Preparation of reference medicinal material solution II: Take 1.0 g of Taxus chinensis reference medicinal material powder (No. 1 powder, passed through a 200-mesh sieve), accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes; weigh it, make up for the loss of methanol, absorb the supernatant, filter, and take the filtrate to obtain.

[0103] Preparation of test solution II: Take 0.5 g of standard decoction powder of Taxus chinensis var. mairei, accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 min; weigh it, make up for the loss of methanol, aspirate the supernatant, filter it through a 0.22 μm filter membrane, and take the filtrate.

[0104] High performance liquid chromatography chromatographic conditions II meet:

[0105] Chromatographic column: Octadecylsilane bonded silica gel as filler, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; column temperature: 30°C; mobile phase: pure water as mobile phase A, methanol as mobile phase B; mobile phase flow rate: 0.75 mL / min; injection volume: 20 μL; detection wavelength: 260 nm; the theoretical plate number calculated based on the rutin peak should be no less than 5000; mobile phase gradient elution program II according to Table 2 is:

[0106] Table 2: Gradient elution program II

[0107]

[0108] It is finally stipulated that the HPLC characteristic spectrum II of the standard decoction of southern yew should show six characteristic peaks, and should correspond to the retention time of the characteristic peaks of the reference substance in the control medicinal material solution II. The peak corresponding to the rutin reference substance is peak 3 (S1). The relative retention times of peaks 1-4 and S1 are calculated, and their relative retention times should be within ±10% of the specified value. The specified value of peak 1 is 0.844, the specified value of peak 2 is 0.948, and the specified value of peak 4 is 1.157; the peak corresponding to the golden pine flavonoids reference substance is peak 6 (S2). The relative retention time of peak 5 and S2 is calculated, and its relative retention time should be within ±10% of the specified value. The specified value of peak 5 is 0.965.

[0109] That is: the HPLC characteristic spectrum II of the standard decoction of southern yew includes six characteristic peaks, among which each characteristic peak corresponds to the retention time of the characteristic peak of the reference substance of the control medicinal material solution II. The peak corresponding to the rutin reference substance is peak 3 (S1). The relative retention time of peaks 1-4 and S1 peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified value of peak 1 is 0.844, the specified value of peak 2 is 0.948, and the specified value of peak 4 is 1.157; the peak corresponding to the golden pine biflavonoid reference substance is peak 6 (S2). The relative retention time of peak 5 and S2 peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified value of peak 5 is 0.965.

[0110] M3. Combine HPLC characteristic spectrum I and HPLC characteristic spectrum II to obtain the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei.

[0111] The control characteristic spectrum I and the control characteristic spectrum II are combined to obtain the control characteristic spectrum of the standard decoction of Taxus chinensis var. mairei.

[0112] Example 2

[0113] A quality testing method for a standard decoction of Taxus chinensis var. mairei comprises the following steps:

[0114] K1. Two identical test samples were pretreated separately. One of the test samples was pretreated in the same manner as the test sample solution I in Example 1, and the other was pretreated in the same manner as the test sample solution II in Example 1: 0.5 g of a lyophilized sample of the standard decoction of Taxus chinensis var. mairei was accurately weighed and placed in a stoppered conical flask. 50 ml of a water:dichloromethane mixture with a volume ratio of 1:1.5 was added for extraction. The solution was dissolved in portions and transferred to a separatory funnel. The dichloromethane solution was separated and the aqueous layer was then extracted with dichloromethane. Extract once with ane oscillation, combine the dichloromethane solutions, evaporate to dryness under reduced pressure below 40°C, dissolve the residue in 10 mL of methanol, cool, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution I. Take another 0.5 g of freeze-dried powder of the standard decoction of southern yew to be tested, accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, and sonicate (power 600 W, frequency 40 kHz) for 30 min; weigh it, make up for the loss of methanol, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain the test solution II.

[0115] K2 detection: The test solution obtained in step K1 is measured according to the HPLC conditions of step M1 to obtain a high performance liquid chromatography (HPLC) Figure I , take the test solution II and measure it according to the HPLC conditions of step M2 to obtain the HPLC Figure II , the determination of high performance liquid chromatography Figure I Compared with the control characteristic spectrum I, the determination of high performance liquid chromatography Figure II Compared with the reference characteristic spectrum II, if the relative retention time of each characteristic peak corresponding to the reference spectrum is within ±10% of the specified value, the test sample meets the quality standard of the standard decoction of Taxus chinensis.

[0116] Example 3

[0117] Based on Example 1, this example examines the extraction solvent, extraction time, number of extractions, extraction method and solvent addition amount in the preparation of the test solution, and further illustrates this example.

[0118] 1. Investigation of extraction solvent

[0119] The extraction solvents of the test solution I in step M1 of Example 1 were set to 40 mL of water: dichloromethane (1:1), 50 mL of water: dichloromethane (1:1.5), and 60 mL of water: dichloromethane (1:2). The other conditions were the same as those in Example 1. The results were as follows: Figure 1The results show that when the extraction solvent is 50 mL of water: dichloromethane (1:1.5), relatively less solvent is used and the chromatographic peak information is large. Therefore, the extraction solvent for test solution I is preferably 50 mL of water: dichloromethane (1:1.5).

[0120] The extraction solvent of the test solution II in step M2 of Example 1 was set to 25 ml of water, 25 ml of 50% methanol, and 25 ml of methanol. The other conditions were the same as in Example 1. The results were as follows: Figure 2 The results show that when methanol is used as the extraction solvent, the chromatographic peak information content is the largest and the extraction efficiency is the highest. Therefore, methanol is determined to be the extraction solvent for test solution II.

[0121] 2. Investigation of extraction methods

[0122] The extraction method of the test solution II in step M2 of Example 1 was set as follows: take two portions of the same number of the southern yew standard decoction, one portion was subjected to ultrasonic extraction (power 600W, frequency 40kHz) for 30min, and the other portion was subjected to reflux extraction for 30min. The other conditions were the same as those in Example 1. The results are shown in FIG. Figure 3 The results showed that the effects of ultrasonic extraction and reflux extraction were the same; when ultrasonic extraction was used, the operation was simpler and more convenient, so ultrasonic extraction was the preferred extraction method for the test solution II.

[0123] 3. Investigation of extraction times

[0124] The number of dichloromethane extractions in the test solution I in step M1 of Example 1 was set to 1, 2, and 3 times respectively, and the other conditions were the same as in Example 1. The results were as follows Figure 4 The results show that when the number of extractions is 2, relatively less solvent is used and the chromatographic peak information is large. Therefore, the number of extractions for the test solution I is preferably 2.

[0125] 4. Investigation of extraction time

[0126] The extraction time of the test solution II in step M2 of Example 1 was set to 20 min, 30 min, and 40 min respectively, and the other conditions were the same as those of Example 1. The results were as follows: Figure 5 The results show that when the extraction time is 30 minutes, the time used is relatively short and the chromatographic peak information content is large. Therefore, the extraction time of test solution II is preferably 30 minutes.

[0127] 5. Investigation of solvent addition amount

[0128] The amount of methanol added to the test solution I in step M1 of Example 1 was set to 5 mL, 10 mL, and 20 mL respectively, and the other conditions were the same as in Example 1. The results were as follows: Figure 6The results show that when the amount of methanol added is 10 mL, the peak shape is better and the amount of solvent used is relatively small. Therefore, the amount of solvent added to the test solution I is preferably 10 mL.

[0129] The amount of methanol added to the test solution II in step M2 of Example 1 was set to 15 mL, 25 mL, and 35 mL respectively, and the other conditions were the same as in Example 1. The results were as follows: Figure 7 The results show that when the amount of methanol added is 25 mL, the peak shape is better and the amount of solvent used is relatively small. Therefore, the amount of solvent added to the test solution II is preferably 25 mL.

[0130] In summary, the preparation method of the test solution I of the characteristic spectrum of the standard decoction of Taxus chinensis Ⅰ is preferably as follows: 0.5 g of the product is accurately weighed, placed in a stoppered conical flask, 50 mL of a mixed solution of water: dichloromethane (1:1.5) is added to dissolve the solution in portions and transferred to a separatory funnel, the dichloromethane liquid is separated, the aqueous layer is extracted once with dichloromethane by oscillation, the dichloromethane liquids are combined, and the solution is evaporated to dryness under reduced pressure below 40°C, 10 mL of methanol is added to the residue, the solution is cooled, shaken, filtered through a 0.22 μm filter membrane, and the filtrate is obtained; the preparation method of the test solution II of the characteristic spectrum of the standard decoction of Taxus chinensis Ⅱ is preferably as follows: 0.5 g of the powder of the product is accurately weighed, placed in a stoppered conical flask, 25 mL of methanol is added, and the solution is ultrasonically treated (power 600 W, frequency 40 kHz) for 30 min; the solution is weighed, the lost methanol is supplemented, the solution is filtered through a 0.22 μm filter membrane, and the filtrate is obtained.

[0131] Example 4

[0132] Based on Example 1, this example examines the chromatographic conditions (wavelength, column temperature, flow rate and delay) in the construction of the characteristic spectrum, and further illustrates them in this example.

[0133] 1. Wavelength Investigation

[0134] The 10-deacetyl baccatin III, 7-xylose 10-deacetyl paclitaxel and the test solution I in Example 1 were respectively subjected to UV full-band scanning using a diode array detector, and the chromatograms of the test solution I at wavelengths of 228 nm, 230 nm and 232 nm were extracted. The other conditions were the same as those in Example 1, and the results were as follows: Figure 8 、 9 The results show that when the detection wavelength is 230 nm, the peak heights and ratios of the characteristic peaks are relatively moderate, so the detection wavelength is preferably 230 nm.

[0135] The rutin, golden pine biflavonoids and isoginkgo biflavonoids in Example 1 and the test solution II were scanned in the full band using a diode array detector, and the chromatograms of the test solution II at wavelengths of 240 nm, 250 nm, 260 nm, 270 nm, 290 nm and 310 nm were extracted. The other conditions were the same as in Example 1. The results were as follows: Figure 10 、 11 The results show that when the detection wavelength is 260nm, the peak height and ratio of each characteristic peak are more moderate, and it is more conducive to distinguishing and identifying each characteristic peak, so the detection wavelength of 260nm is preferred.

[0136] 2. Column temperature inspection

[0137] The column temperature of the HPLC in step M1 of Example 1 was set to 25°C, 30°C, and 35°C, and the other conditions were the same as in Example 1. The results were as follows: Figure 12 The results show that when the column temperature is 25-35℃, the chromatogram peak shape is relatively symmetrical and the separation of each characteristic peak is also very good. Since the column temperature of 30℃ is more conducive to operation and control, the column temperature is preferably 30℃.

[0138] The column temperature of the HPLC in step M2 of Example 1 was set to 25°C, 30°C, and 35°C, and the other conditions were the same as in Example 1. The results were as follows: Figure 13 The results showed that when the column temperature was 30℃, the chromatogram peak shape was more symmetrical and the separation was better, so the column temperature was preferably set at 30℃.

[0139] 3. Investigation of mobile phase flow rate

[0140] The flow rates of the HPLC chromatography in step M1 in Example 1 were set to 0.8 mL / min, 0.9 mL / min, and 1.0 mL / min, and the other conditions were the same as in Example 1. The results were as follows: Figure 14 The results show that when the flow rate is 0.9-1.0 mL / min, the chromatographic peak information content is large, the peak shape of each characteristic peak is good, and the separation is moderate. When the flow rate is 0.9 mL / min, the peak shape is better and the solvent used is relatively less. Therefore, the flow rate is preferably 0.9 mL / min.

[0141] The flow rates of the HPLC chromatography in step M2 of Example 1 were set to 0.6 mL / min, 0.75 mL / min1, 0.9 mL / min, and 1.0 mL / min, and the other conditions were the same as in Example 1. The results were as follows: Figure 15The results show that at a flow rate of 0.6-0.9 mL / min, the peak shapes of the characteristic peaks are good and the resolution is moderate. However, at a flow rate of 0.75 mL / min, the peak shapes are even better and relatively less solvent is used. Therefore, the flow rate is preferably set at 0.75 mL / min.

[0142] 4. Delayed Investigation

[0143] According to the method for constructing the characteristic spectrum of the standard decoction of southern yew in Example 1, the chromatographic peaks of the test solution were examined to see whether they were all eluted within the specified time. The acquisition time of the HPLC conditions in step M1 was extended to 130 min, and the other conditions were the same as in Example 1. The results were as follows: Figure 16 The results showed that after 65 minutes, the elution was continued with 40% acetonitrile isocratically until 130 minutes, and it was found that the chromatographic information was completely collected within 65 minutes. Therefore, the collection time is preferably 65 minutes.

[0144] The acquisition time of the HPLC conditions in step M2 was extended to 50 min, and the other conditions were the same as in Example 1. The results were as follows: Figure 17 The results showed that after 25 minutes, the elution was continued with 45% methanol isocratically for 50 minutes, and it was found that the chromatographic information was completely collected within 25 minutes. Therefore, the collection time is preferably 25 minutes.

[0145] In summary, the HPLC characteristic spectrum chromatographic conditions and system suitability test of the standard decoction of Taxus chinensis were determined as follows: Chromatographic condition I was: octadecylsilane bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), pure water as mobile phase A, acetonitrile as mobile phase B, gradient elution, and the gradient elution program was as follows:

[0146] 0-12 min, mobile phase A: 80%, mobile phase B: 20%;

[0147] 12-22 min, mobile phase A: from 80% to 70%, mobile phase B: from 20% to 30%;

[0148] 22-30 min, mobile phase A: 70%, mobile phase B: 30%;

[0149] 30-36 min, mobile phase A: from 70% to 68%, mobile phase B: from 30% to 32%;

[0150] 36-39 min, mobile phase A: from 68% to 60%, mobile phase B: from 32% to 40%;

[0151] 39-65 min, mobile phase A: 60%, mobile phase B: 40%.

[0152] Chromatographic conditions II are: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), pure water as mobile phase A, methanol as mobile phase B, gradient elution, the gradient elution procedure is as follows:

[0153] 0-5 min, mobile phase A: from 65% to 45%, mobile phase B: from 35% to 55%;

[0154] 5-12 min, mobile phase A: 45%, mobile phase B: 55%;

[0155] 12-16 min, mobile phase A: from 45% to 0%, mobile phase B: from 55% to 100%;

[0156] 16-20 min, mobile phase A: 0%, mobile phase B: 100%;

[0157] 20-25 min, mobile phase A: from 0% to 65%, mobile phase B: from 100% to 45%.

[0158] Example 5

[0159] Based on the method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis in Example 1, the following methodological investigation was conducted:

[0160] 1. Chromatographic peak identification

[0161] Methanol was used as blank control solution, and reference solution and test solution were taken to identify peak I of HPLC characteristic spectrum of standard decoction of Taxus chinensis var. mairei. The results were as follows: Figure 18 As shown, peak 2 (S) is 10-deacetylbaccatin III and peak 6 is 7-xylose 10-deacetylbaccatin III. In the subsequent methodological investigation, the eight characteristic peaks in the test sample spectrum were investigated. The peak II of the HPLC characteristic spectrum of the standard decoction of southern yew was identified, and the results were as follows Figure 19 Among them, peak 3 (S1) is rutin and peak 6 (S2) is scutellaria baicalensis flavonoids. In the subsequent methodological investigation, the six characteristic peaks in the test sample spectrum were investigated.

[0162] 2. Precision test

[0163] Based on the method for constructing the characteristic spectrum I of the standard decoction of Taxus chinensis in Example 1, the test solution I of the standard decoction of Taxus chinensis (batch number: NFHDSBT220501) was taken and injected six times continuously. The retention time and peak area of ​​each characteristic peak were calculated. The results are shown in Tables 3-4 below.

[0164] Table 3 Precision investigation-retention time

[0165]

[0166] Table 4 Precision Investigation-Peak Area

[0167]

[0168] As can be seen from Table 3 and Table 4, the results show that the instrument has good precision.

[0169] Take the test solution II of the standard decoction of Taxus chinensis (batch number: NFHDSBT220501) and inject it six times continuously. Calculate the retention time and peak area of ​​each characteristic peak. The results are shown in Table 5-6 below.

[0170] Table 5 Precision investigation-retention time

[0171]

[0172] Table 6 Precision Investigation-Peak Area

[0173]

[0174] As can be seen from Tables 5 and 6, the results show that the instrument has good precision.

[0175] 3. Repeatability Investigation

[0176] Six portions of the standard decoction of Taxus chinensis (batch number: NFHDSBT220501) were accurately weighed, and the test solution I was prepared and measured according to the method for constructing the HPLC characteristic spectrum I of the standard decoction of Taxus chinensis in Example 1. The results are shown in Tables 7-8 below.

[0177] Table 7 Repeatability study - relative retention time

[0178]

[0179]

[0180] Table 8 Repeatability study - relative peak area

[0181]

[0182] The results showed that the RSD values ​​of the relative retention times of the six samples were 0.020% to 0.232%, and the RSD values ​​of the relative peak areas were 0.580% to 1.228%, indicating that the method had good repeatability.

[0183] Six portions of the standard decoction of Taxus chinensis var. mairei (batch number: NFHDSBT220501) were accurately weighed, and the test solution II was prepared and measured according to the method for constructing the HPLC characteristic spectrum II of the standard decoction of Taxus chinensis var. mairei in Example 1. The results are shown in Tables 9-10 below.

[0184] Table 9 Repeatability study - relative retention time

[0185]

[0186]

[0187] Table 10 Repeatability study - relative peak area

[0188]

[0189] The results showed that the RSD values ​​of the relative retention times of the six samples were 0.054% to 0.071%, and the RSD values ​​of the relative peak areas were 0.174% to 2.022%, indicating that the method had good repeatability.

[0190] 4. Stability Investigation

[0191] Based on the construction method of the characteristic spectrum I of the standard decoction of southern yew in Example 1, the same southern yew standard decoction test solution I (batch number: NFHDSBT220501) was taken and measured at 0h, 4h, 8h, 12h, 18h, and 24h, respectively. The results are shown in Tables 11-12 below.

[0192] Table 11 Stability Study-Retention Time

[0193]

[0194] Table 12 Stability Study-Peak Area

[0195]

[0196] The results showed that the RSD values ​​of the corresponding characteristic peak retention times were 0.010% to 0.058%, and the RSD values ​​of the characteristic peak areas were 0.503% to 1.593%. The sample solution was relatively stable within 24 hours.

[0197] Based on the construction method of the characteristic spectrum II of the standard decoction of Taxus chinensis in Example 1, the test solution II of the standard decoction of Taxus chinensis from the same batch was taken and measured at 0h, 4h, 8h, 12h, 18h, and 24h, respectively. The results are shown in Tables 13-14 below.

[0198] Table 13 Stability Study-Retention Time

[0199]

[0200] Table 14 Stability Study-Peak Area

[0201]

[0202]

[0203] The results showed that the RSD values ​​of the corresponding characteristic peak retention times were 0.041-0.123%, and the RSD values ​​of the characteristic peak areas were 0.335-1.860%. The sample solution was relatively stable within 24 hours.

[0204] 5. Intermediate precision inspection

[0205] 1. Different instruments

[0206] Based on the method for constructing the characteristic spectrum I of the standard decoction of Taxus chinensis in Example 1, the same Taxus chinensis standard decoction test solution I (batch number: NFHDSBT220501) was taken and measured using an Agilent Eclipse Plus C18 (5 μm, 4.6×250 mm) chromatographic column on three high performance liquid chromatographs: Shimadzu 20-AD, Agilent 1260, and Waters 2965. The results are shown in Tables 15-16, and Attached. Figure 20 The results showed that the RSD values ​​of the relative retention times of the eight characteristic peaks obtained from the three brands of liquid phases ranged from 0.079% to 4.545%; and the RSD values ​​of the relative peak areas of the eight characteristic peaks obtained from the three brands of liquid phases ranged from 1.905% to 21.388%.

[0207] Table 15 Instrument durability investigation - relative retention time

[0208]

[0209]

[0210] Table 16 Instrument durability investigation - relative peak area

[0211]

[0212] Based on the method for constructing the characteristic spectrum II of the standard decoction of Taxus chinensis in Example 1, the same Taxus chinensis standard decoction test solution II (batch number: NFHDSBT220501) was taken and measured on three high performance liquid chromatographs, Shimadzu 20-AD, Agilent 1260, and Waters 2965, using an Agilent Eclipse Plus C18 (5 μm, 4.6×250 mm) column. The results are shown in Tables 17-18 and Attached. Figure 21 The results showed that the RSD values ​​of the relative retention times of the six characteristic peaks obtained from the three brands of liquid phases were between 0.180% and 1.765%; the RSD values ​​of the relative peak areas of the eight characteristic peaks obtained from the three brands of liquid phases were between 6.487% and 8.937%.

[0213] Table 17 Instrument durability investigation - relative retention time

[0214]

[0215] Table 18 Instrument durability investigation - relative peak area

[0216]

[0217] 2. Inspection by different personnel and time

[0218] Based on the method for constructing characteristic spectrum I of the standard decoction of Taxus chinensis in Example 1, different personnel (A and B) accurately weighed the standard decoction of Taxus chinensis (Batch No.: NFHDSBT220501) at different times (T1 and T2) to prepare test solution I, and the results were shown in Tables 19-20. The results showed that when different personnel measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks ranged from 0.026% to 0.164%; the RSDs of the relative peak areas of the characteristic peaks ranged from 0.461% to 2.118%, indicating good method stability.

[0219] Table 19 Personnel and time inspection - relative retention time

[0220]

[0221] Table 20 Personnel and time investigation-relative peak area

[0222]

[0223] Based on the method for constructing characteristic spectrum II of the standard decoction of Taxus chinensis in Example 1, different personnel (A and B) accurately weighed the standard decoction of Taxus chinensis (Batch No.: NFHDSBT220501) at different times (T1 and T2) to prepare test solution II, and the results were shown in Tables 21-22. The results showed that when different personnel measured the same sample at different times, the RSDs of the relative retention times of the characteristic peaks were between 0.043% and 0.099%; the RSDs of the relative peak areas of the characteristic peaks were between 0.155% and 2.922%, indicating good method stability.

[0224] Table 21 Personnel and time inspection - relative retention time

[0225]

[0226] Table 22 Personnel and time investigation-relative peak area

[0227]

[0228] 6. Durability inspection:

[0229] Based on the method for constructing the characteristic spectrum I of the standard decoction of Taxus chinensis in Example 1, three chromatographic columns, Agilent Eclipse Plus C18 (5 μm, 4.6×250 mm), CNW Athena C18-WP (5 μm, 4.6×250 mm), and Diamonsil Plus C18 (5 μm, 4.6×250 mm), were investigated. The results are shown in Tables 23-24 and Attached. Figure 22 shown.

[0230] Table 23 Column durability investigation - relative retention time

[0231]

[0232] Table 24 Column durability investigation - relative peak area

[0233]

[0234]

[0235] The results showed that the RSDs for the relative retention times of characteristic peaks in solution I of the standard decoction of Taxus chinensis var. mairei using the three columns ranged from 0.786% to 9.528%, and the RSDs for the relative peak areas ranged from 1.099% to 24.730%. The relative retention times of characteristic peaks obtained by different columns varied significantly, particularly for peak 1. Peak 1, obtained using the CNWAthena C18-WP column, exceeded the specified value by 10%, while peak 8 did not elute. Using a Plus column, the RSDs for the relative retention times of characteristic peaks ranged from 0.434% to 3.707%, and the RSDs for the relative peak areas ranged from 0.773% to 7.443%. Therefore, this method has certain requirements for the HPLC column, and the use of a Plus column, which offers better analytical performance, is recommended.

[0236] Based on the construction method of characteristic spectrum II of the standard decoction of Taxus chinensis in Example 1, three chromatographic columns, Agilent Eclipse Plus C18 (5 μm, 4.6×250 mm), CNW Athena C18-WP (5 μm, 4.6×250 mm), and Diamonsil Plus C18 (5 μm, 4.6×250 mm), were investigated. The results are shown in Tables 25-26 and Attachment 2. Figure 23 shown.

[0237] Table 25 Column durability investigation - relative retention time

[0238]

[0239] Table 26 Column durability investigation - relative peak area

[0240]

[0241] The results showed that when the three chromatographic columns were used to detect the test solution II of the standard decoction of Taxus chinensis var. mairei, the RSD values ​​of the relative retention times of the characteristic peaks were 1.231% to 3.559%, and the RSD values ​​of the relative peak areas of the characteristic peaks were 5.701% to 13.402%. If the Plus column was used, the RSD values ​​of the relative retention times of the characteristic peaks were 0.753% to 1.152%, and the RSD values ​​of the relative peak areas of the characteristic peaks were 1.105% to 7.579%. It is recommended to use the Plus column for better analytical performance.

[0242] In summary, under the condition of selecting the Plus chromatographic column, the RSD values ​​of the relative retention times of each characteristic peak met the requirements in the above investigations. This method is good, and the above-mentioned characteristic peaks will be included in subsequent investigations.

[0243] Determination of characteristic peaks, establishment of characteristic spectra and reference characteristic spectra

[0244] Based on the method for constructing the characteristic spectrum of the standard decoction of Taxus chinensis in Example 1, 16 batches of standard decoction of Taxus chinensis in this scheme were taken to prepare test solution I, and the solution was measured according to chromatographic condition I to obtain HPLC characteristic spectrum I. The HPLC characteristic spectrum I of 16 batches of standard decoction of Taxus chinensis was synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) to establish the control characteristic spectrum I of the standard decoction of Taxus chinensis. The results are shown in the attached figure. Figure 24 and 25 ; Prepare control medicinal material solution I by taking control medicinal material, and then measure it according to chromatographic condition I, record the HPLC chart, and the results are shown in the attached Figure 26 , Figure 24 , S1-S16 correspond to NFHDSBT220501, NFHDSBT220502, NFHDSBT220503, NFHDSBT220504, NFHDSBT220505, NFHDSBT220506, NFHDSBT220507, NFHDSBT220508, NFHDSBT220508, NFHDSBT220510, NFHDSBT220511, NFHDSBT220512, NFHDSBT220601, NFHDSBT220602, NFHDSBT220603, and NFHDSBT220604, respectively; Figure 25 In the middle, peak 2 (S): 10-deacetylbaccatin III, peak 6: 7-xylose 10-deacetylpaclitaxel.

[0245] Based on the method for constructing characteristic spectrum I of the standard decoction of Taxus chinensis in Example 1, characteristic spectrum I was analyzed for sixteen batches of Taxus chinensis test samples, and the relative retention times and relative peak areas were calculated. The relative retention times and relative peak areas of each chromatographic peak are shown in Tables 27-28 below.

[0246] Table 27 Characteristic peaks-relative retention times of sixteen batches of Taxus chinensis standard decoctions

[0247]

[0248] Table 28 Characteristic peaks - relative peak areas of sixteen batches of Taxus chinensis standard decoctions

[0249]

[0250]

[0251] Based on the principles of stable relative retention times, consistent detection across all batches of test samples, and relatively high peak heights, eight peaks with good robustness were selected as characteristic peaks. The results showed that when Peak 2 was used as the characteristic peak, the relative peak area RSDs for the characteristic peaks in 16 batches of Taxus chinensis standard decoctions ranged from 26.34% to 50.62%, and the relative retention time RSDs for all eight characteristic peaks in 16 batches of Taxus chinensis standard decoctions were all less than 0.19%. However, when tested on different instruments and different chromatographic columns, the relative retention time RSDs ranged from 0.079% to 4.545%. Therefore, the relative retention time of the test sample should tentatively be within ±10% of the specified value.

[0252] Final regulations: Eight characteristic peaks should be presented in the characteristic spectrum I of the standard decoction of Taxus chinensis var. mairei, and should correspond to the retention times of the characteristic peaks of the reference substance in the control medicinal material solution I. The peak corresponding to the 10-deacetylbaccatin III reference substance is peak 2 (S peak), and the peak corresponding to the 7-xylosyl 10-deacetylpaclitaxel reference substance is peak 6. The relative retention times of peaks 1-8 and S peak are calculated, and their relative retention times should be within ±10% of the specified values. The specified value for peak 1 is 0.352, the specified value for peak 3 is 1.182, and the specified value for peak 4 is 1.238; the specified value for peak 5 is 1.468, the specified value for peak 6 is 1.946, the specified value for peak 7 is 2.024, and the specified value for peak 8 is 2.449.

[0253] Based on the method for constructing the characteristic spectrum of the standard decoction of southern yew in Example 1, 16 batches of southern yew standard decoction in this scheme were taken to prepare test solution II, and the solution was measured according to chromatographic conditions II to obtain HPLC characteristic spectrum II. The HPLC characteristic spectrum II of 16 batches of southern yew standard decoction was synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) to establish the control characteristic spectrum II of southern yew standard decoction. The results are shown in the attached Figure 16 and 19 ; Figure 23 , S1-S16 correspond to NFHDSBT220501, NFHDSBT220502, NFHDSBT220503, NFHDSBT220504, NFHDSBT220505, NFHDSBT220506, NFHDSBT220507, NFHDSBT220508, NFHDSBT220508, NFHDSBT220510, NFHDSBT220511, NFHDSBT220512, NFHDSBT220601, NFHDSBT220602, NFHDSBT220603, and NFHDSBT220604, respectively; Figure 19 In the middle, peak 3 (S1): rutin, peak 6 (S2): scutellaria baicalensis.

[0254] Based on the method for constructing characteristic spectrum II of the standard decoction of Taxus chinensis in Example 1, characteristic spectrum II was analyzed for sixteen batches of Taxus chinensis test samples, and the relative retention times and relative peak areas were calculated. The relative retention times and relative peak areas of each chromatographic peak are shown in Tables 29-30 below, respectively.

[0255] Table 29 Characteristic peaks-relative retention times of sixteen batches of Taxus chinensis standard decoctions

[0256]

[0257]

[0258] Table 30 Characteristic peaks - relative peak areas of sixteen batches of Taxus chinensis standard decoctions

[0259]

[0260]

[0261] Based on the principles of stable relative retention times, consistent detection across all batches of test samples, and relatively high peak heights, six peaks with good robustness were selected as characteristic peaks. The results showed that when Peaks 3 and 6 were used as characteristic peaks, the relative peak area RSDs for the characteristic peaks for all 16 batches of Taxus chinensis standard decoctions ranged from 9.97% to 19.72%, and the relative retention time RSDs for all six characteristic peaks for all 16 batches of Taxus chinensis standard decoctions were less than 0.33%. However, when tested on different instruments and columns, the relative retention time RSDs ranged from 0.180% to 1.765%. Therefore, the relative retention time of the test sample should tentatively be within ±10% of the specified value.

[0262] It is finally stipulated that the HPLC characteristic spectrum II of the standard decoction of southern yew should show six characteristic peaks, and should correspond to the retention time of the characteristic peaks of the reference substance in the control medicinal material solution II. The peak corresponding to the rutin reference substance is peak 3 (S1). The relative retention times of peaks 1-4 and S1 are calculated, and their relative retention times should be within ±10% of the specified value. The specified value of peak 1 is 0.844, the specified value of peak 2 is 0.948, and the specified value of peak 4 is 1.157. The peak corresponding to the golden pine flavonoids reference substance is peak 6 (S2). The relative retention time of peak 5 and S2 is calculated, and its relative retention time should be within ±10% of the specified value. The specified value of peak 5 is 0.965.

[0263] Example 6

[0264] Take 3 batches of test samples, prepare test sample solution I and test sample solution II according to step K1 of Example 2, and measure according to step K2. The results are as follows: Figure 30 、 31 The results showed that the relative retention times of the characteristic peaks of the three batches of test samples were essentially consistent with those of the reference characteristic spectrum of the standard decoction of Taxus chinensis, all within 10% of the specified values. This means that the three batches of test samples met the quality standards of the standard decoction of Taxus chinensis.

[0265] In summary, in the present technical solution, a characteristic spectrum was established based on a high performance liquid chromatography instrument, and sixteen batches of standard decoctions of Taxus chinensis var. mairei were synthesized using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition). A reference spectrum of the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei was established, which is the reference characteristic spectrum of the standard decoction of Taxus chinensis var. mairei. The characteristic components in the standard decoction of Taxus chinensis var. mairei are controlled as a whole, and the deficiencies of the existing quality control technology are compensated. The characteristic peaks in the obtained characteristic spectrum are rich in information, which can effectively ensure the stability of the overall quality of the standard decoction of Taxus chinensis var. mairei, making the quality control technology of the standard decoction of Taxus chinensis var. mairei more complete and scientific. Moreover, the present method is simple to operate, and has the advantages of high precision, good stability, good repeatability and high accuracy, providing an effective basis for the quality identification of standard decoctions of Chinese medicinal materials.

[0266] The above are only preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC characteristic spectrum of a standard decoction of Taxus chinensis var. mairei, characterized in that: The following steps are involved: M1. Inject 10-deacetylbaccatin III reference solution, 7-xylose 10-deacetylpaclitaxel reference solution, reference medicinal material solution I, and test sample solution I into a high-performance liquid chromatography (HPLC) column for determination and establishment of HPLC characteristic spectrum I. Use a traditional Chinese medicine chromatographic fingerprint similarity evaluation system to synthesize HPLC characteristic spectrum I of more than 15 batches of test sample solution I to establish a reference characteristic spectrum I for a standard decoction of Taxus chinensis var. mairei. The preparation process of the control medicinal material solution I is as follows: taking Taxus chinensis var. mairei control medicinal material powder, adding methanol, ultrasonically filtering, evaporating the filtrate to dryness under reduced pressure, then adding a mixed solution of water and dichloromethane for extraction, evaporating the extract to dryness, and dissolving it in methanol to obtain the control medicinal material solution I; The preparation process of the test solution I is as follows: taking the test sample, adding a mixed solution of water and dichloromethane to extract, evaporating the extract to dryness and then dissolving it in methanol to obtain the test solution I, wherein the test sample is a standard decoction of Taxus chinensis var. mairei; The conditions for the high performance liquid chromatography described in step M1 are as follows: an octadecylsilane bonded silica gel column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; mobile phase A is pure water, mobile phase B is acetonitrile, and gradient elution is performed. The gradient elution procedure is as follows: 0-12 min, mobile phase A: 80%, mobile phase B: 20%; 12-22 min, mobile phase A: from 80% to 70%, mobile phase B: from 20% to 30%; 22-30 min, mobile phase A: 70%, mobile phase B: 30%; 30-36 min, mobile phase A: from 70% to 68%, mobile phase B: from 30% to 32%; 36-39 min, mobile phase A: from 68% to 60%, mobile phase B: from 32% to 40%; 39-65 min, mobile phase A: 60%, mobile phase B: 40%; M2. Inject rutin reference solution, cyperus chinensis flavonoids reference solution, reference medicinal material solution II, and test solution II into a high-performance liquid chromatography (HPLC) column, determine the concentration, and establish HPLC characteristic spectrum II. Using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, synthesize HPLC characteristic spectrum II from more than 15 batches of test solution II to establish a reference characteristic spectrum II for the standard decoction of Taxus chinensis. The preparation process of the control medicinal material solution II is as follows: taking Taxus chinensis var. mairei control medicinal material powder, adding methanol for ultrasonic treatment, replenishing the methanol lost by ultrasonic treatment, and then filtering to obtain the control medicinal material solution II; The preparation process of the test solution II is as follows: taking the test sample, adding methanol and ultrasonically treating it, replenishing the methanol lost by ultrasonication and filtering it to obtain the test solution II, wherein the test sample is a standard decoction of Taxus chinensis var. mairei; The conditions for the high performance liquid chromatography described in step M2 are as follows: an octadecylsilane bonded silica gel column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; mobile phase A is pure water, mobile phase B is methanol, and gradient elution is performed. The gradient elution procedure is as follows: 0-5 min, mobile phase A: from 65% to 45%, mobile phase B: from 35% to 55%; 5-12 min, mobile phase A: 45%, mobile phase B: 55%; 12-16 min, mobile phase A: from 45% to 0%, mobile phase B: from 55% to 100%; 16-20 min, mobile phase A: 0%, mobile phase B: 100%; 20-25 min, mobile phase A: from 0% to 65%, mobile phase B: from 100% to 45%; M3. The control characteristic spectrum I and the control characteristic spectrum II are combined to obtain the control characteristic spectrum of the southern yew standard decoction; the control characteristic spectrum I includes eight characteristic peaks, each characteristic peak corresponds to the retention time of the characteristic peak of the reference substance of the control medicinal material solution I, the peak corresponding to the 10-deacetylbaccatin III reference substance is peak 2, i.e., S peak, the peak corresponding to the 7-xylose 10-deacetylpaclitaxel reference substance is peak 6, the relative retention time of peaks 1-8 and S peak is calculated, and the relative retention time is within ±10% of the specified value, the specified value of peak 1 is 0.352, the specified value of peak 3 is 1.182, the specified value of peak 4 is 1.238; the specified value of peak 5 is 1.468, the specified value of peak 6 is 1 .946, the specified value of peak 7 is 2.024, and the specified value of peak 8 is 2.449; the control characteristic spectrum II includes six characteristic peaks, each characteristic peak corresponds to the retention time of the characteristic peak of the reference substance of the control medicinal material solution II, the peak corresponding to the rutin reference substance is peak 3, namely S1 peak, the relative retention time of peaks 1-4 and S1 peak is calculated, and the relative retention time is within ±10% of the specified value, the specified value of peak 1 is 0.844, the specified value of peak 2 is 0.948, and the specified value of peak 4 is 1.157; the peak corresponding to the golden pine biflavonoid reference substance is peak 6, namely S2 peak, the relative retention time of peak 5 and S2 peak is calculated, and the relative retention time is within ±10% of the specified value, and the specified value of peak 5 is 0.

965.

2. The method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei according to claim 1, wherein The column temperature of the high performance liquid chromatography in step M1 is 25-35° C., the flow rate of the mobile phase is 0.8-1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 228-232 nm.

3. The method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei according to claim 1, wherein The column temperature of the high performance liquid chromatography in step M2 is 25-35° C., the flow rate of the mobile phase is 0.6-1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 240-310 nm.

4. The method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei according to claim 1, wherein: The ultrasonic power of step M2 is 600W, the frequency is 40kHz, the concentration of the methanol solution in step M2 is 100%; the volume ratio of water to dichloromethane in step M1 is 1:1-2.

5. The method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei according to claim 1, wherein The concentration of the 10-deacetylbaccatin III reference solution was 500 μg / ml, the concentration of the 7-xylose 10-deacetylpaclitaxel reference solution was 50 μg / ml, the concentration of the rutin reference solution was 200 μg / ml, and the concentration of the scutellaria baicalensis reference solution was 200 μg / ml.

6. The method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei according to claim 1, wherein: In step M1 and step M2, the mass ratio of the standard decoction of Taxus chinensis to the control medicinal material powder of Taxus chinensis is 1:1-4.

7. The method for constructing the HPLC characteristic spectrum of the standard decoction of Taxus chinensis var. mairei according to claim 1, wherein: The preparation method of the standard decoction of Taxus chinensis is as follows: taking a reference medicinal material of Taxus chinensis, adding water and boiling it twice, filtering, combining the two filtrates, concentrating under reduced pressure to form a fluid extract, and then freeze-drying and grinding to obtain the standard decoction of Taxus chinensis.

8. A quality testing method for a standard decoction of Taxus chinensis var. mairei, characterized in that: The following steps are involved: K1. Pre-treat two identical sample samples separately: extract one sample with a mixture of water and dichloromethane, evaporate the extract to dryness, and dissolve it in methanol to obtain sample solution I. Ultrasonicate the other sample in methanol, replace any methanol lost during sonication, and filter to obtain sample solution II. K2. Take the test sample solution I to be tested and measure it according to the HPLC conditions of step M1 as described in claim 1 to obtain a measurement HPLC chromatogram I. Take the test sample solution II to be tested and measure it according to the HPLC conditions of step M2 as described in claim 1 to obtain a measurement HPLC chromatogram II. Compare the measurement HPLC chromatogram I with the reference characteristic spectrum I as claimed in claim 1, and compare the measurement HPLC chromatogram II with the reference characteristic spectrum II as claimed in claim 1. If the relative retention time of each characteristic peak corresponding to the reference characteristic spectrum is within ±10% of the specified value, then the test sample meets the quality standard of the standard decoction of Taxus chinensis var. mairei.

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