UHPLC (Ultra High Performance Liquid Chromatography) fingerprint spectrum establishment method and application of traditional Chinese medicine composition

Abstract

According to the UHPLC fingerprint spectrum establishment method and application of the traditional Chinese medicine composition, 15 parts of rhizoma drynariae, 12 parts of rhizoma cibotii, 10 parts of glossy privet fruit, 10 parts of caulis spatholobi, 10 parts of herba pyrolae and 10 parts of radix cyathulae are prepared into traditional Chinese medicine composition water decoction, freeze-dried powder is prepared through vacuum concentration and vacuum freeze-drying, and the quality of the traditional Chinese medicine composition is controlled by establishing the UHPLC fingerprint spectrum. According to the method, the content of the multi-index components of the traditional Chinese medicine composition is measured under the same chromatographic condition, the multi-index component value transfer relation of the traditional Chinese medicine composition is further measured, the whole-chain quality monitoring from single-object quality control to composition-medicinal material quality control is achieved, and the comprehensiveness and effectiveness of quality control are remarkably improved.

Description

Technical Field

[0001] The present invention relates to a method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition and its application, and belongs to the field of quality standard control of traditional Chinese medicine pharmaceuticals. Background Art

[0002] The "Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Catalogue of Ancient Classic Prescriptions (Trial)" clarifies a number of basic principles for Class 3.1 pharmaceutical research of traditional Chinese medicine. Among them, the preparation and quality control of material standards are the premise and basis for the declaration of classic prescription preparations. Establishing a comprehensive and accurate material standard quality control system is of great significance to ensuring the quality of classic prescription compound preparations.

[0003] In the field of Traditional Chinese Medicine (TCM) quality control, TCM quality is an important guarantee for clinical efficacy and medication safety. However, the establishment of an evaluation system faces a dual challenge: it is necessary to maintain the traditional cognitive concept of "holistic view" while also overcoming the modern technical difficulties of complex component analysis. Due to the diverse composition of TCM and the principle of syndrome differentiation and treatment in treatment plans, the detection of a single active ingredient cannot represent its overall efficacy. Currently, researchers often use a method that combines HPLC characteristic spectrum analysis with high-resolution mass spectrometry to reflect the complex chemical information of TCM. HPLC fingerprinting has also become a commonly used method for TCM quality control.

[0004] However, when it comes to the establishment and testing of material benchmarks for this type of specific Chinese herbal composition composed of Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Millettia reticulata, Herba schizonepetae and Achyranthes bidentata, the existing technology has deficiencies, making it difficult to provide comprehensive and accurate support for the quality standard control of the Chinese herbal composition.

[0005] In view of this, the present invention provides a material benchmark establishment and detection method for the traditional Chinese medicine composition, which can provide a solid theoretical basis for its quality standard control. Summary of the Invention

[0006] The present invention aims to provide a method and application for establishing a UHPLC fingerprint of a traditional Chinese medicine composition, thereby facilitating further establishment of a material benchmark. The UHPLC fingerprint established by the present invention has good precision, stability, and repeatability, while taking into account analysis time and high detection efficiency, thereby providing a basis for quality control of the traditional Chinese medicine composition. Furthermore, the method can simultaneously perform quality control on the medicinal materials of Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Millettia reticulata, Herba schizonepetae, and Cyathulae chinensis, thereby achieving the purpose of more comprehensive and effective quality control.

[0007] The technical means adopted in the present invention are:

[0008] A method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition comprises the following steps:

[0009] (1) Take the Chinese medicine composition, add 60% methanol to dilute, ultrasonicate, cool to room temperature, shake evenly, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution;

[0010] (2) Take appropriate amounts of naringin, terunoside, hyperoside, cyasterone, and daidzein reference substances, place them in a volumetric flask, and dilute with methanol to prepare a mixed reference solution;

[0011] (3) The test solution of step (1) and the mixed reference solution of step (2) were injected into a liquid chromatograph respectively, and the fingerprints were measured under the following chromatographic conditions to calibrate the characteristic peaks;

[0012] The chromatographic conditions were as follows: chromatographic column: ACQUITY UPLC BEH C18; the mobile phase was acetonitrile (A)-0.1% formic acid water (B), and the gradient elution program was: 0-1 min, 14% A; 1-5 min, 14%-21.2 A; 5-6 min, 21%-21.8 A; 6-7 min, 21.8-23 A; 7-8 min, 23 A; 8-12 min, 23-30 A; 12-13 min, 30-14 A; 13-25 min, 14 A; the flow rate was 0.2 mL / min; the column temperature was 40°C; and the detection wavelength was 254 nm.

[0013] It is further defined that the step (3) measures a total of 24 calibrated characteristic peaks in the spectrum, of which peaks 1, 2, 8, and 13 are exclusive peaks of Drynaria fortunei; peaks 8, 9, 10, 11, 12, 16, 18, 19, and 23 are exclusive peaks of Ligustrum lucidum; peak 3 is a common peak of Ligustrum lucidum and Millettia reticulata; peaks 4, 5, 6, 7, 17, 20, 21, and 22 are exclusive peaks of Herba Lycopodii; and peaks 14 and 15 are exclusive peaks of Cyathulae chinensis.

[0014] It is further defined that the characteristic peaks measured in the spectrum in step (3) are: Peak 6 is hyperoside, Peak 12 is teruncleside, Peak 13 is naringin, Peak 15 is cyasterone, and Peak 20 is daidzein.

[0015] It is further defined that the preparation method of the Chinese medicine composition includes 15 parts of Drynaria fortunei, 12 parts of Cibotium bark, 10 parts of Ligustrum lucidum, 10 parts of Millettia reticulata, 10 parts of Herba Lycopodii and 10 parts of Cyathulae, decocting them twice with water, the first time adding 11 times the amount of water and decocting them for 1.5 hours, the second time adding 9 times the amount of water and decocting them for 1.5 hours, combining and filtering to obtain the water decoction of the Chinese medicine composition.

[0016] It is further defined that the water decoction of the traditional Chinese medicine composition is concentrated under reduced pressure at 37° C. to 40 mL, placed in a -80° C. refrigerator for pre-freezing for 12 hours, and then vacuum freeze-dried for 24 hours to obtain a freeze-dried powder of the traditional Chinese medicine composition.

[0017] It is further defined that in step (1), the ultrasonic treatment conditions are: power of 400 W, frequency of 40 kHz, and ultrasonic treatment time of 30 min; and the filtration conditions are: 0.22 μm microporous filter membrane.

[0018] It is further defined that the mass concentrations of hyperoside, teruncetoside, naringin, cyasterone, and daidzein in the mixed reference solution of step (2) are 0.01047 mg / mL, 0.26413 mg / mL, 0.19300 mg / mL, 0.01614 mg / mL, and 0.00108 mg / mL, respectively.

[0019] The present invention also provides a method for determining the content of multiple components of a traditional Chinese medicine composition, comprising the following steps:

[0020] (1) Take the Chinese medicine composition, add 60% methanol to dilute, ultrasonicate for 30 min, cool to room temperature, shake evenly, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution;

[0021] (2) Take appropriate amounts of naringin, terunoside, hyperoside, cyasterone, and daidzein reference substances, place them in a volumetric flask, and dilute with methanol to prepare a mixed reference solution;

[0022] (3) Taking 1 to 5 μL of the mixed reference solution prepared in step (2), respectively, the solution was injected into a liquid chromatograph, and the chromatogram of each reference solution was determined under the following chromatographic conditions;

[0023] Chromatographic conditions were as follows: chromatographic column: ACQUITY UPLC BEH C18; mobile phase: acetonitrile (A)-0.1% formic acid water (B); gradient elution program: 0-1 min, 14% A; 1-5 min, 14%-21.2 A; 5-6 min, 21%-21.8 A; 6-7 min, 21.8-23 A; 7-8 min, 23 A; 8-12 min, 23-30 A; 12-13 min, 30-14 A; 13-25 min, 14 A; flow rate: 0.2 mL / min; column temperature: 40°C; detection wavelength: 254 nm;

[0024] (4) Based on the color spectrum obtained in step (3), the peak area of ​​each reference substance is measured, and then a standard curve is drawn with the mass X of each reference substance as the abscissa and the corresponding peak area Y as the ordinate. The regression equation of each index component is calculated as follows:

[0025] Y=aX-b

[0026] Among them, a is the regression coefficient of the regression equation; b is the intercept;

[0027] (5) The test solution of step (1) is introduced into a liquid chromatograph, and a chromatogram is obtained according to the chromatographic conditions of step (3), and the corresponding peak area is measured. The peak area is substituted into the regression equation of step (4) to obtain the content of each index component, namely, naringin, teruncin, hyperoside, cyperasterone, and daidzein.

[0028] Further limiting, the regression equation corresponding to each indicator component in (4) is:

[0029] The regression equations of hyperoside are: Y = 7893056351.4804X - 33886.3000

[0030] The regression equation of terglucosidin is: Y = 3729169813.8589X - 524309.3334

[0031] The regression equation of naringin is: Y = 1700221807.6804X - 171939.3334

[0032] The regression equation of cyasterone is: Y = 3023463444.8576X - 24914.0000

[0033] The regression equation of daidzein is: Y=17214691046.6582X-9964.0000.

[0034] The present invention also provides a method for determining the transfer relationship of multiple index components of a traditional Chinese medicine composition, the method comprising the following steps:

[0035] (1) Determine the Chinese medicine composition by referring to the method for establishing the UHPLC fingerprint of the Chinese medicine composition according to claim 1;

[0036] (2) taking the Chinese medicine composition and the single decoction piece, and determining the content of the index components in the Chinese medicine composition and the single decoction piece according to the method for determining the content of multiple index components in the Chinese medicine composition according to claim 8;

[0037] (3) Calculate the transfer rate of the index component content in the slice-TCM composite sample according to the following formula: Transfer rate (%) = w×m / W×M

[0038] In the formula, w and m represent the content of the index component in the Chinese medicine composition and the quality of the Chinese medicine composition, respectively; W and M represent the content of the index component in the medicinal materials and the quality of the prescribed medicinal materials, respectively.

[0039] The index components are hyperoside, privetside, naringin, cyperasterone and daidzein.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The Chinese medicine composition provided by the present invention is composed of Drynaria fortunei, Cibotium barometz, Fructus Ligustri Lucidi, Millettia spatholobi, Herba Lucerae and Cyathulae chinensis. By systematically optimizing the conditions such as the instrument, chromatographic column, mobile phase, and detection wavelength for determining the fingerprint spectrum, a stable and reliable fingerprint spectrum detection condition is established. A comprehensive methodological investigation is carried out to establish the UHPLC fingerprint spectrum of the Chinese medicine composition. Not only can the Chinese medicine composition itself be accurately controlled, but it also breaks through the quality control of all the constituent medicinal materials such as Drynaria fortunei, Cibotium barometz, Fructus Ligustri Lucidi, Millettia spatholobi, Herba Lucerae and Cyathulae chinensis, realizing the transition from single-object quality control to "composition-medicinal material" full-chain quality monitoring, significantly improving the comprehensiveness and effectiveness of quality control, and reflecting the trend of Chinese medicine quality control technology evolving towards systematization and refinement.

[0042] (2) The fingerprint detection method of the present invention has good specificity and stability, strong durability, and can effectively ensure the stability and controllability of drug quality during industrial production. At the same time, it can improve the intrinsic quality control standard of the traditional Chinese medicine composition composed of Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Millettia reticulata, Herba Lycopodii and Cyathulae chinensis, further effectively ensuring the safety and effectiveness of the drugs.

[0043] (3) The UHPLC fingerprint spectrum established in the present invention has good precision, stability and repeatability, while taking into account the analysis time, with peaks appearing within 15 minutes and high detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The fingerprints of 15 batches of Chinese herbal decoctions (S1-S15) and the control fingerprint (R);

[0045] Figure 2 It is the characteristic peaks and their attribution in the fingerprint of the decoction of the Chinese medicine composition. DETAILED DESCRIPTION

[0046] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only 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 ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0047] The raw materials of the traditional Chinese medicine composition of the present invention are composed of drynaria rhizome, dog spine, glossy privet fruit, millettia vine, herba leucanthemum and cyathulae, and the preferred compatibility is: 15 parts of drynaria rhizome, 12 parts of dog spine, 10 parts of glossy privet fruit, 10 parts of millettia vine, 10 parts of herba leucanthemum and 10 parts of cyathulae.

[0048] Among them, Sichuan Achyranthes can also be replaced with Achyranthes of equal quality.

[0049] The Chinese medicine composition decoction of the present invention is prepared according to the following steps:

[0050] (1) Prepare the raw materials according to the recipe.

[0051] (2) Water extraction: Mix 15g of Drynaria fortunei, 12g of Cibotium barometz, 10g of Ligustrum lucidum, 10g of Millettia reticulata, 10g of Herba Lycopodii and 10g of Cyathulae chinensis, add water and boil twice. The first time, add 11 times the amount of water and boil for 1.5 hours. The second time, add 9 times the amount of water and boil for 1.5 hours.

[0052] (3) Filtration: Combine the two decoctions and filter them while hot using a double layer of nylon gauze to obtain a decoction of the Chinese medicine composition.

[0053] Furthermore, the water decoction of the traditional Chinese medicine composition can also be prepared into a freeze-dried powder by freeze-drying, that is, the water decoction of the traditional Chinese medicine composition is concentrated to 40 ml at 37°C under reduced pressure, placed in a -80°C refrigerator for pre-freezing for 12 hours, and then vacuum freeze-dried for 24 hours to obtain a reference freeze-dried sample of the traditional Chinese medicine composition for later use.

[0054] According to the same method, single-ingredient pieces of Drynaria fortunei, Cibotium barometz, Fructus Ligustri Lucidi, Caulis Spatholobi, Herba Lycopodii and Cyathulae were prepared, as well as negative freeze-dried powder samples of the missing single-ingredient herbs, and set aside.

[0055] The information of the decoction pieces used is shown in Table 1. All medicinal materials were purchased from Shaanxi Xingshengde Pharmaceutical Co., Ltd., and all six decoction pieces complied with the relevant provisions of the 2020 edition of the Chinese Pharmacopoeia.

[0056] Table 1 Medicinal material information

[0057]

[0058]

[0059] Example 1: Establishment of UHPLC fingerprint of Chinese medicine composition

[0060] The method for establishing the UHPLC fingerprint of the Chinese medicine composition in this embodiment is achieved by the following steps:

[0061] (1) Take the Chinese medicine composition, add 60% methanol to dilute, ultrasonically treat at 400W power and 40kHz frequency for 30min, cool to room temperature, shake evenly, centrifuge, take the supernatant, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution;

[0062] (2) Take appropriate amounts of naringin, terglucosidoside, hyperoside, cyasterone, and daidzein reference substances, place them in a volumetric flask, and add methanol to dilute them to prepare a mixed reference solution; the mass concentrations of hyperoside, terglucosidoside, naringin, cyasterone, and daidzein in the mixed reference solution are 0.01047 mg / mL, 0.26413 mg / mL, 0.19300 mg / mL, 0.01614 mg / mL, and 0.00108 mg / mL, respectively;

[0063] (3) The test solution of step (1) and the mixed reference solution of step (2) were injected into a liquid chromatograph respectively, and the fingerprints were measured under the following chromatographic conditions to calibrate the characteristic peaks;

[0064] The chromatographic conditions were as follows: chromatographic column: ACQUITY UPLC BEH C18; the mobile phase was acetonitrile (A)-0.1% formic acid water (B), and the gradient elution program was: 0-1 min, 14% A; 1-5 min, 14%-21.2 A; 5-6 min, 21%-21.8 A; 6-7 min, 21.8-23 A; 7-8 min, 23 A; 8-12 min, 23-30 A; 12-13 min, 30-14 A; 13-25 min, 14 A; the flow rate was 0.2 mL / min; the column temperature was 40°C; and the detection wavelength was 254 nm.

[0065] The fingerprint of the Chinese medicine composition was compared with that of the mixed reference solution, and a total of 24 characteristic peaks were identified by comparing the retention times of the chromatographic peaks, among which peaks 1, 2, 8, and 13 were exclusive to Rhizoma Drynariae; peaks 8, 9, 10, 11, 12, 16, 18, 19, and 23 were exclusive to Fructus Ligustri Lucidi; peak 3 was a common peak between Fructus Ligustri Lucidi and Spatholobi; peaks 4, 5, 6, 7, 17, 20, 21, and 22 were exclusive to Herba Lycopodii; and peaks 14 and 15 were exclusive to Cyathulae Bidentatae. Further calibration identified peak 6 as hyperoside, peak 12 as terunguisin, peak 13 as naringin, peak 15 as cyasterone, and peak 20 as daidzein.

[0066] Example 2: Method for determining the content of multiple components in a traditional Chinese medicine composition

[0067] The method for determining the content of multiple components of a traditional Chinese medicine composition of this embodiment is implemented by the following steps:

[0068] (1) Take the Chinese medicine composition, add 60% methanol to dilute, ultrasonically treat at 400W power and 40kHz frequency for 30min, cool to room temperature, shake evenly, centrifuge, take the supernatant, filter through a 0.22μm microporous membrane, and take the filtrate to obtain the test solution;

[0069] (2) Take appropriate amounts of naringin, terglucosidoside, hyperoside, cyasterone, and daidzein reference substances, place them in a volumetric flask, and add methanol to dilute them so that the mass concentrations of hyperoside, terglucosidoside, naringin, cyasterone, and daidzein are 0.01047 mg / mL, 0.26413 mg / mL, 0.19300 mg / mL, 0.01614 mg / mL, and 0.00108 mg / mL, respectively, to prepare a mixed reference solution;

[0070] (3) Taking different volumes of 1 to 5 μL of the mixed reference solution prepared in step (2), injecting them into a liquid chromatograph, and determining the fingerprint of each reference solution under the following chromatographic conditions;

[0071] Chromatographic conditions were as follows: chromatographic column: ACQUITY UPLC BEH C18; mobile phase: acetonitrile (A)-0.1% formic acid water (B); gradient elution program: 0-1 min, 14% A; 1-5 min, 14%-21.2 A; 5-6 min, 21%-21.8 A; 6-7 min, 21.8-23 A; 7-8 min, 23 A; 8-12 min, 23-30 A; 12-13 min, 30-14 A; 13-25 min, 14 A; flow rate: 0.2 mL / min; column temperature: 40°C; detection wavelength: 254 nm;

[0072] (4) Based on the fingerprint obtained in step (3), the peak area of ​​each reference substance is measured, and then the standard curve is drawn with the mass X of each reference substance as the horizontal coordinate and the corresponding peak area Y as the vertical coordinate. The regression equation of each index component is calculated as follows:

[0073] Y=aX-b

[0074] Among them, a is the regression coefficient of the regression equation; b is the intercept;

[0075] The regression equations of hyperoside are: Y=7893056351.4804X-33886.3000,

[0076] The regression equation of terglucosidin is: Y=3729169813.8589X-524309.3334,

[0077] The regression equation of naringin is: Y = 1700221807.6804X-171939.3334,

[0078] The regression equation of cyasterone is: Y = 3023463444.8576X - 24914.0000,

[0079] The regression equation of daidzein is: Y=17214691046.6582X-9964.0000.

[0080] (5) The test solution of step (1) is introduced into a liquid chromatograph, and a fingerprint spectrum is obtained according to the chromatographic conditions of step (3), and the corresponding peak area is measured. The peak area is substituted into the regression equation of step (4) to obtain the content of each index component, namely, naringin, teruncin, hyperoside, cyperasterone, and daidzein.

[0081] Example 3: Multi-index component quantity transfer relationship of traditional Chinese medicine composition

[0082] The method of determining the transfer relationship of the quantity values ​​of multiple index components of a traditional Chinese medicine composition in this embodiment is implemented by the following steps:

[0083] (1) Determine the Chinese medicine composition by referring to the method for establishing the UHPLC fingerprint of the Chinese medicine composition in Example 1;

[0084] (2) The Chinese medicine composition and the single decoction pieces were prepared into freeze-dried powders, and the content of the index components in the Chinese medicine composition and the single decoction pieces was determined according to the multi-index component content determination method of the Chinese medicine composition in Example 2;

[0085] (3) Calculate the transfer rate of the index component content in the slice-TCM composite sample according to the following formula: Transfer rate (%) = w×m / W×M

[0086] In the formula, w and m represent the content of the index component in the Chinese medicine composition and the quality of the Chinese medicine composition, respectively; W and M represent the content of the index component in the medicinal materials and the quality of the prescribed medicinal materials, respectively.

[0087] The index components are hyperoside, privetside, naringin, cyperasterone and daidzein.

[0088] The method provided by the present invention lays the foundation for further accurate determination of the quantitative transfer relationship of indicator components from decoction pieces to traditional Chinese medicine compositions and then to material benchmarks. Therefore, the method of the present invention can be further extended to the research of material benchmarks of traditional Chinese medicine compositions, thereby clarifying the quantitative transfer relationship of indicator components between decoction pieces, compositions, and material benchmarks, and is suitable for the systematic research of the whole process of material benchmarks of traditional Chinese medicine compositions.

[0089] To facilitate further research on material benchmarks, according to the "Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Catalog of Ancient Classic Prescriptions (Trial)", material benchmarks are usually dry products or concentrated extracts, and low-temperature concentration, freeze-drying and other methods can be considered for preparation. These are all common technical means used by technical personnel in this field.

[0090] The following is a further verification and investigation of the technical effects of Examples 1 to 3 of the present invention through experiments.

[0091] 1. Investigation of the fingerprint establishment method of traditional Chinese medicine compositions

[0092] In order to further verify the repeatability, stability and precision of the fingerprint establishment method of the present invention and analyze the transfer relationship of the fingerprint value of the traditional Chinese medicine decoction of the present invention, a comprehensive methodological investigation of the fingerprint detection of the present invention was conducted. The specific results are as follows:

[0093] The single-ingredient decoction pieces were randomly combined into 15 batches of Chinese medicine compositions using the Excel random number table method, numbered S1 to S15, as shown in Table 2.

[0094] Table 2S1-15 Chinese medicine composition

[0095]

[0096] Instruments and materials: ACQUITY I-scales ultra-high performance liquid chromatograph (Agilent, USA); Heraeus Fresco 17 high-speed desktop centrifuge (Thermo Fisher Scientific Inc.); BSA124S-CW 1 / 100 analytical balance (Sartorius); BT 25S 1 / 10000 analytical balance (Ultimate); FDL-1000 freeze dryer (Eyela-lab, Japan); Mingche D24 UV Merck water purifier (Millipore, USA); YM-080S ultrasonic cleaner (Shenzhen Fangao Microelectronics Co., Ltd.).

[0097] Reference substances (naringin (batch number: 110722-202417), terunguiside (batch number: 39011-92-2), hyperoside (482-36-0), cyasterone (batch number: 17086-76-9), and daidzein (batch number: 486-66-8), all with a purity ≥98%, were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Methanol, formic acid, and acetonitrile were all chromatographically graded (Thermo Fisher Scientific, USA). Ultrapure water was prepared using a Merck D24 UV water purifier.

[0098] (1) Precision

[0099] Take the Chinese medicine composition decoction sample S1, prepare the test solution according to the method of step (1) in Example 1, and continuously inject 6 times according to the operation of step (3). The obtained fingerprint spectrum is analyzed, and peak No. 12 (terglucosidoside) is used as the reference peak (its separation is good and the chromatographic peak is relatively stable). The relative standard deviation RSD of the relative retention time and relative peak area of ​​each common peak is calculated to be less than 0.045% and 1.29%, respectively, indicating that the instrument precision is good and the detection accuracy of the present invention is high.

[0100] (2) Repeatability

[0101] Take the Chinese medicine decoction sample S1, prepare 6 test solutions in parallel according to the method of step (1) in Example 1, and perform the determination according to the method of Example 1.

[0102] The results showed that the RSDs of the relative retention times and relative peak areas of the common peaks were less than 0.33% and 1.55%, respectively, using Peak 12 (teruncleside) as the reference peak, indicating that the method had good repeatability.

[0103] (3) Stability

[0104] A sample of the Chinese herbal decoction S1 was prepared in parallel with six test solutions according to the method of step (1) in Example 1. The samples were injected at 0, 2, 4, 8, 12, 24, and 48 hours after preparation, and the chromatographic conditions were referred to as those in Example 1. The results showed that the relative retention times and relative peak areas of the common peaks were less than 0.36% and 1.05%, respectively, using Peak 12 (terglucosidoside) as the reference peak, indicating that the method had good stability within 48 hours.

[0105] (4) Chromatographic peak identification

[0106] 15 batches of Chinese medicine compositions, single herbs, and negative control samples (single herb missing) were prepared. Test solutions were prepared according to the method of step (1) in Example 1, and the chromatographic conditions of Example 1 were used for determination. All sample data were imported into the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system. Using S1 as the reference spectrum, the similarity analysis was performed using the median method, as shown in Table 3. The UHPLC fingerprint of the Chinese medicine composition sample is shown in Figure 1 .

[0107] Table 3 Similarity evaluation of 15 batches of Chinese herbal decoctions (S1-S15)

[0108]

[0109] The similarity analysis in Table 3 above shows that the Chinese medicine composition of the present invention has stable quality and good consistency. Figure 1 The fingerprint map has a total of 24 common peaks. By comparing with the reference substances, 5 common peaks were identified, namely hyperoside (peak 6), teruncleside (peak 12), naringin (peak 13), amaranthone (peak 15), and daidzein (peak 20).

[0110] (5) Analysis of the relationship between quantity value transfer

[0111] The UHPLC chromatograms of single herbal medicine slices, single herbal medicine slice negative samples (missing single herbal medicine) and Chinese medicine composition samples were compared in the same manner as in Example 1. Figure 2 .

[0112] Depend on Figure 2Peaks 1, 2, 8, and 13 (naringin) in the fingerprint are specific to Drynaria fortunei. Peaks 8, 9, 10, 11, and 12 (terglucosidin), 16, 18, 19, and 23 are specific to Ligustrum lucidum fruit. Peak 3 is a common peak between Ligustrum lucidum fruit and Millettia reticulata. Peaks 4, 5, and 6 (hyperoside), 7, 17, and 20 (daidzein), and 21 and 22 are specific to Herba Lycopodii. Peaks 14 and 15 (cyasterone) are specific to Cyathulae chinensis.

[0113] This indicates that the characteristic peaks of the Chinese medicine composition of the present invention can be stably transferred between the decoction pieces and the combined samples as a whole, and the attribution relationship is clear.

[0114] 2. Investigation of the method for determining the content of multiple components in traditional Chinese medicine compositions

[0115] In order to investigate the precision, repeatability, stability, content determination and quantity transfer analysis of the multi-index component content determination method of the traditional Chinese medicine composition of the present invention, a comprehensive methodological investigation of the determination method of the present invention was conducted, as follows:

[0116] (1) Linear relationship

[0117] Accurately weigh appropriate amounts of reference substances for hyperoside, teruncleside, naringin, cyasterone, and daidzein and thoroughly dissolve them in methanol to prepare mixed reference substance stock solutions with mass concentrations of 0.01047 mg / mL, 0.26413 mg / mL, 0.19300 mg / mL, 0.01614 mg / mL, and 0.00108 mg / mL, respectively. Filter through a 0.22 μm filter membrane, collect the filtrate, and obtain the reference substance solution. Samples were injected for determination and a standard curve was plotted. The reference substance mass concentration (X) was plotted on the abscissa and the peak area (Y) on the ordinate. A standard curve was plotted and the regression equation was calculated. The results are shown in Table 4.

[0118] Table 4 Results of the methodological investigation on the content determination of multiple index components in traditional Chinese medicine compositions

[0119]

[0120] As can be seen from Figure 4, the determination method of the present invention presents a good linear relationship and a high correlation.

[0121] (2) Precision

[0122] The Chinese medicine composition solution with batch number S1 was taken, and the operation of Example 2 was referred to. The sample was injected continuously 6 times under the same chromatographic conditions. The peak area RSDs of hyperoside, teruncleside, naringin, cyasterone, and daidzein were calculated to be 0.73%, 0.24%, 0.46%, 0.30%, and 0.33%, respectively, indicating that the method of the present invention has good precision.

[0123] (3) Repeatability

[0124] The Chinese medicine composition solution with batch number S1 was taken, and 6 test solutions were prepared by referring to the operation of Example 2. The samples were continuously injected under the same chromatographic conditions, and the peak area RSDs of hyperoside, teruncleside, naringin, cyasterone, and daidzein were 0.22%, 0.23%, 0.40%, 0.22%, and 0.23%, respectively. The RSDs of the retention times were 0.15%, 0.19%, 0.15%, 0.22%, and 0.12%, respectively. The average content RSDs were 0.18%, 0.20%, 0.33%, 0.20%, and 0.19%, respectively, indicating that the method of the present invention has good repeatability.

[0125] (4) Stability

[0126] The Chinese medicine composition solution with batch number S1 was taken, and 6 test solutions were prepared by referring to the operation of Example 2. Under the same chromatographic conditions, the samples were injected at 0, 2, 4, 8, 12, 24, and 48 hours, respectively. The peak area RSDs of hyperoside, teruncleside, naringin, cyasterone, and daidzein were measured to be 0.40%, 0.19%, 0.34%, 0.14%, and 0.27%, respectively, indicating that the method of the present invention has good stability within 48 hours.

[0127] (5) Spike recovery

[0128] Take 6 portions of a traditional Chinese medicine composition solution with a known content, add a certain amount of a reference solution to each of them, and prepare a test solution according to the operation of Example 2 for sampling and determination.

[0129] The results showed that the average recoveries of hyperoside, teruncetoside, naringin, cyasterone and daidzein were 100%, 102%, 101%, 99.1% and 101.5, respectively, and the RSDs were 1.6%, 2.4%, 2.0%, 1.4% and 1.6%, respectively, which proved that the method of the present invention meets the requirements of methodological validation.

[0130] 3. Multi-index component quantity transfer analysis of traditional Chinese medicine compositions

[0131] (1) Transfer rate

[0132] Fifteen batches of Chinese medicine compositions and single decoction pieces were prepared into freeze-dried powders, and the test solution was prepared according to the operation of Example 2. The samples were injected under the same chromatographic conditions, and the content of each index component and the transfer rate of the decoction piece-TCM composition sample were determined. The results are shown in Table 5.

[0133] The transfer rate of the index component content in the slice-TCM composite sample is calculated according to formula (1):

[0134] Transfer rate (%) = w × m / W × M (1)

[0135] In formula (1), w and m represent the content of the index component in the traditional Chinese medicine composition and the mass of the traditional Chinese medicine composition, respectively; W and M represent the content of the index component in the medicinal material and the mass of the prescribed medicinal material slices, respectively.

[0136] Table 5 Content and transfer rate of index components in 15 batches of Chinese medicine compositions

[0137]

[0138] As shown in Table 5, the average transfer rate of hyperoside in 15 batches of traditional Chinese medicine composition samples was 74.76%, and the transfer rate ranged from 57.75% to 85.15%; the average transfer rate of teruncin was 105.19%, and the transfer rate ranged from 83.45% to 129.80%; the average transfer rate of naringin was 82.70%, and the transfer rate ranged from 70.01% to 99.01%; the average transfer rate of cyasterone was 35.40%, and the transfer rate ranged from 29.63% to 45.01%; and the average transfer rate of daidzein was 60.44%, and the transfer rate ranged from 43.61% to 78.90%.

[0139] According to the requirement that the content of the index components in the traditional Chinese medicine composition and their transfer rate should be maintained within the range of ±30% of the average value, the content and transfer rate of hyperoside, teruncleside, naringin, cyasterone, and daidzein in the present invention are almost within the required range, indicating that the traditional Chinese medicine composition can ensure the consistency and stability from the decoction pieces to the traditional Chinese medicine composition during the preparation process.

[0140] (2) Ointment yield

[0141] The yield of the herbal composition was further calculated by freeze-drying 15 batches of herbal composition decoctions and recording their mass to calculate the actual yield. Decoctions of single herbal pieces of Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Millettia reticulata, Herba Lycopodii, and Cyathulae chinensis were prepared from 15 prescribed batches, freeze-dried, and their mass recorded.

[0142] Actual paste yield = m / M

[0143] Wherein, m represents the mass of freeze-dried powder, and M represents the amount of slices weighed in each group.

[0144] The total weight of the Chinese medicine composition is 67 g.

[0145] The theoretical yield of the whole formula should be calculated based on the conversion of each single herb: theoretical yield = 15 / 67 × yield of the corresponding batch of Drynaria fortunei slices + 12 / 67 × yield of the corresponding batch of Cibotium barometz slices + 10 / 67 × yield of the corresponding batch of Ligustrum lucidum slices + 10 / 67 × yield of the corresponding batch of Millettia reticulata slices + 10 / 67 × yield of the corresponding batch of Herba Lycopodii slices + 10 / 67 × yield of the corresponding batch of Cyathulae chinensis slices. The calculation results are shown in Table 6.

[0146] Table 6: Paste yield and paste transfer rate of Chinese medicine composition samples and single medicinal materials

[0147]

[0148] As can be seen from Table 6, the paste yield rates of the 15 batches of Chinese medicine compositions ranged from 17.86% to 20.55%, with an average paste yield rate of 19.376%. The paste yield rates of each batch were within ±30% of the mean.

[0149] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition, characterized in that: The steps are as follows: (1) Take the Chinese medicine composition, add 60% methanol to dilute, ultrasonicate, cool to room temperature, shake evenly, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution; (2) Take appropriate amounts of naringin, terunoside, hyperoside, cyasterone, and daidzein reference substances, place them in a volumetric flask, and dilute with methanol to prepare a mixed reference solution; (3) The test solution of step (1) and the mixed reference solution of step (2) were injected into a liquid chromatograph respectively, and the fingerprints were measured under the following chromatographic conditions to calibrate the characteristic peaks; The chromatographic conditions were as follows: chromatographic column: ACQUITY UPLC BEH C18; the mobile phase was acetonitrile (A)-0.1% formic acid water (B), and the gradient elution program was: 0-1 min, 14% A; 1-5 min, 14%-21.2 A; 5-6 min, 21%-21.8 A; 6-7 min, 21.8-23 A; 7-8 min, 23 A; 8-12 min, 23-30 A; 12-13 min, 30-14 A; 13-25 min, 14 A; the flow rate was 0.2 mL / min; the column temperature was 40°C; and the detection wavelength was 254 nm.

2. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to claim 1, characterized in that: The step (3) measured a total of 24 calibrated characteristic peaks in the spectrum, of which peaks 1, 2, 8, and 13 were exclusive peaks of Drynaria fortunei; peaks 8, 9, 10, 11, 12, 16, 18, 19, and 23 were exclusive peaks of Ligustrum lucidum; peak 3 was a common peak of Ligustrum lucidum and Millettia reticulata; peaks 4, 5, 6, 7, 17, 20, 21, and 22 were exclusive peaks of Herba Lycopodii; and peaks 14 and 15 were exclusive peaks of Cyathulae chinensis.

3. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to claim 2, characterized in that: The characteristic peaks measured in the spectrum in step (3) are: peak 6 is hyperoside, peak 12 is teruncleside, peak 13 is naringin, peak 15 is cyasterone and peak 20 is daidzein.

4. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The preparation method of the traditional Chinese medicine composition comprises the following steps: adding 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Millettia reticulata, 10 parts of Herba Lycopodii and 10 parts of Cyathulae chinensis to water and decocting the mixture twice: adding 11 times the amount of water to decoct the mixture for 1.5 hours for the first time, and adding 9 times the amount of water to decoct the mixture for 1.5 hours for the second time; combining the mixture and filtering the mixture to obtain a water decoction of the traditional Chinese medicine composition.

5. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to claim 4, characterized in that: The water decoction of the traditional Chinese medicine composition was concentrated to 40 mL under reduced pressure at 37° C., placed in a −80° C. refrigerator for pre-freezing for 12 hours, and then vacuum-freeze-dried for 24 hours to obtain a freeze-dried powder of the traditional Chinese medicine composition.

6. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to claim 1, characterized in that: In the step (1), the ultrasonic treatment conditions are: power of 400 W, frequency of 40 kHz, and ultrasonic treatment time of 30 min; and the filtration conditions are: 0.22 μm microporous filter membrane.

7. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to claim 1, characterized in that: The mass concentrations of hyperoside, teruncetoside, naringin, cyasterone, and daidzein in the mixed reference solution of step (2) are 0.01047 mg / mL, 0.26413 mg / mL, 0.19300 mg / mL, 0.01614 mg / mL, and 0.00108 mg / mL, respectively.

8. A method for determining the content of multiple components of a traditional Chinese medicine composition, characterized in that: The implementation steps include: (1) Take the Chinese medicine composition, add 60% methanol to dilute, ultrasonicate for 30 min, cool to room temperature, shake evenly, centrifuge, take the supernatant, filter, and take the filtrate to obtain the test solution; (2) Take appropriate amounts of naringin, terunoside, hyperoside, cyasterone, and daidzein reference substances, place them in a volumetric flask, and dilute with methanol to prepare a mixed reference solution; (3) Taking 1 to 5 μL of the mixed reference solution prepared in step (2), respectively, the solution was injected into a liquid chromatograph, and the chromatogram of each reference solution was determined under the following chromatographic conditions; Chromatographic conditions were as follows: chromatographic column: ACQUITY UPLC BEH C18; mobile phase: acetonitrile (A)-0.1% formic acid water (B); gradient elution program: 0-1 min, 14% A; 1-5 min, 14%-21.2 A; 5-6 min, 21%-21.8 A; 6-7 min, 21.8-23 A; 7-8 min, 23 A; 8-12 min, 23-30 A; 12-13 min, 30-14 A; 13-25 min, 14 A; flow rate: 0.2 mL / min; column temperature: 40°C; detection wavelength: 254 nm; (4) Based on the color spectrum obtained in step (3), the peak area of ​​each reference substance is measured, and then a standard curve is drawn with the mass X of each reference substance as the abscissa and the corresponding peak area Y as the ordinate. The regression equation of each index component is calculated as follows: Y=aX-b Among them, a is the regression coefficient of the regression equation; b is the intercept; (5) The test solution of step (1) is introduced into a liquid chromatograph, and a chromatogram is obtained according to the chromatographic conditions of step (3), and the corresponding peak area is measured. The peak area is substituted into the regression equation of step (4) to obtain the content of each index component, namely, naringin, teruncin, hyperoside, cyperasterone, and daidzein.

9. The method for determining the content of multiple components in a traditional Chinese medicine composition according to claim 8, wherein: The regression equations corresponding to the index components in (4) are: The regression equations of hyperoside are: Y=7893056351.4804X-33886.3000, The regression equation of terglucosidin is: Y=3729169813.8589X-524309.3334, The regression equation of naringin is: Y = 1700221807.6804X-171939.3334, The regression equation of cyasterone is: Y = 3023463444.8576X - 24914.0000, The regression equation of daidzein is: Y=17214691046.6582X-9964.0000.

10. A method for determining the quantitative transfer relationship of multiple index components of a traditional Chinese medicine composition, characterized by: The method comprises the following steps: (1) Determine the Chinese medicine composition by referring to the method for establishing the UHPLC fingerprint of the Chinese medicine composition according to claim 1; (2) taking the Chinese medicine composition and the single decoction piece, and determining the content of the index components in the Chinese medicine composition and the single decoction piece according to the method for determining the content of multiple index components in the Chinese medicine composition according to claim 8; (3) Calculate the transfer rate of the index component content in the slice-TCM composite sample according to the following formula: Transfer rate (%) = w×m / W×M Wherein, w and m represent the content of the index component in the Chinese medicine composition and the quality of the Chinese medicine composition, respectively; W and M represent the content of the index component in the medicinal materials and the quality of the prescribed medicinal materials, respectively; The index components are hyperoside, privetside, naringin, cyperasterone and daidzein.

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