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CN-120703285-B - UHPLC fingerprint establishment method and application of traditional Chinese medicine composition

CN120703285BCN 120703285 BCN120703285 BCN 120703285BCN-120703285-B

Abstract

According to the UHPLC fingerprint establishment method and application of the traditional Chinese medicine composition, 15 parts of drynaria rhizome, 12 parts of rhizoma cibotii, 10 parts of glossy privet fruit, 10 parts of suberect spatholobus stem, 10 parts of pyrola herb and 10 parts of medicinal cyathula root are prepared into traditional Chinese medicine composition water decoction, the traditional Chinese medicine composition water decoction is subjected to reduced pressure concentration and vacuum freeze drying to prepare freeze-dried powder, quality control is carried out on the traditional Chinese medicine composition by establishing the UHPLC fingerprint, the content of multi-index components of the traditional Chinese medicine composition is measured under the same chromatographic condition, the multi-index component value transfer relation is further measured, and the full-chain quality monitoring from single object quality control to 'composition-medicinal material' is realized, and the comprehensiveness and effectiveness of quality control are remarkably improved.

Inventors

  • ZOU JUNBO
  • YANG FENG
  • SHI YAJUN
  • XIE WEI
  • LI WENXIONG
  • LIU JING

Assignees

  • 陕西中医药大学

Dates

Publication Date
20260512
Application Date
20250717

Claims (7)

  1. 1. The UHPLC fingerprint establishment method of the traditional Chinese medicine composition is characterized by comprising the following operation steps: (1) Diluting the Chinese medicinal composition with 60% methanol, performing ultrasonic treatment, cooling to room temperature, shaking uniformly, centrifuging, collecting supernatant, filtering, and collecting filtrate to obtain sample solution; The preparation method of the traditional Chinese medicine composition comprises 15 parts of rhizoma drynariae, 12 parts of rhizoma cibotii, 10 parts of glossy privet fruit, 10 parts of suberect spatholobus stem, 10 parts of pyrola herb and 10 parts of medicinal cyathula root, adding water for decocting twice, adding 11 times of water for decocting for 1.5 hours in the first time, adding 9 times of water for decocting for 1.5 hours in the second time, merging, and filtering to obtain the traditional Chinese medicine composition water decoction; concentrating the water decoction of the traditional Chinese medicine composition to 40mL under reduced pressure at 37 ̊ ℃, pre-freezing for 12 hours in a refrigerator at-80 ℃, and vacuum freeze-drying for 24: 24 h to obtain freeze-dried powder of the traditional Chinese medicine composition; in the step (1), the ultrasonic treatment condition is that the power is 400W, the frequency is 40 kHz, the ultrasonic treatment time is 30min, and the filtration condition is a 0.22 mu m microporous filter membrane; (2) Taking appropriate amounts of naringin, terprivet glucoside, hyperin, cyasterone and daidzein reference substances, placing into a measuring flask, and adding methanol for dilution to obtain mixed reference substance solution; (3) Taking the sample solution in the step (1) and the mixed reference substance solution in the step (2), respectively injecting the sample solution and the mixed reference substance solution into a liquid chromatograph, and measuring a fingerprint spectrum and calibrating characteristic peaks through the following chromatographic conditions; The chromatographic conditions are a chromatographic column ACQUITY UPLC BEH C, acetonitrile as a mobile phase A, 0.1% formic acid as a mobile phase B, a gradient elution program of ：0～1 min,14% A;1～5 min,14%～21.2% A;5～6 min,21.2%～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;13～25min,14%A;, 0.2 mL/min flow rate, 40 ℃ column temperature and 254 nm detection wavelength.
  2. 2. The method for establishing the UHPLC fingerprint of the traditional Chinese medicine composition according to claim 1, wherein 24 calibration characteristic peaks are measured in the step (3), wherein peaks 1, 2, 8 and 13 are exclusive peaks of rhizoma drynariae, peaks 8, 9, 10, 11, 12, 16, 18, 19 and 23 are exclusive peaks of glossy privet fruits, peaks 3 are exclusive peaks of glossy privet fruits and caulis spatholobi, peaks 4,5,6, 7, 17, 20, 21 and 22 are exclusive peaks of pyrola, and peaks 14 and 15 are exclusive peaks of radix cyathulae.
  3. 3. The method for establishing a UHPLC fingerprint of the traditional Chinese medicine composition according to claim 2, wherein the characteristic peaks marked in the step (3) are hyperin 6, terligustrin 12, naringin 13, amaranth 15 and daidzein 20.
  4. 4. The method for establishing a UHPLC fingerprint of a traditional Chinese medicine composition according to claim 1, wherein the mass concentration of hyperin, terprivet glycoside, naringin, cyamarone and daidzein in the mixed reference solution in the step (2) is 0.01047 mg/mL, 0.26413 mg/mL, 0.19300 mg/mL, 0.01614 mg/mL and 0.00108 mg/mL.
  5. 5. The method for measuring the content of the multi-index components of the traditional Chinese medicine composition is characterized by comprising the following implementation steps: (1) Diluting the Chinese medicinal composition with 60% methanol, performing ultrasonic treatment with power of 400W and frequency of 40 kHz at 30min, cooling to room temperature, shaking uniformly, centrifuging, collecting supernatant, filtering, and collecting filtrate to obtain sample solution; the traditional Chinese medicine composition consists of 15 parts of drynaria rhizome, 12 parts of rhizoma cibotii, 10 parts of glossy privet fruit, 10 parts of suberect spatholobus stem, 10 parts of pyrola herb and 10 parts of medicinal cyathula root; (2) Taking appropriate amounts of naringin, terprivet glucoside, hyperin, cyasterone and daidzein reference substances, placing into a measuring flask, and adding methanol for dilution to obtain mixed reference substance solution; (3) Taking the mixed reference substance solutions in the step (2) according to the unequal volumes of 1-5 mu L, respectively injecting the mixed reference substance solutions into a liquid chromatograph, and measuring the chromatograms of the reference substances under the following chromatographic conditions; The chromatographic conditions are that the chromatographic column is ACQUITY UPLC BEH C, the mobile phase A is acetonitrile, the mobile phase B is 0.1% formic acid water, the gradient elution program is ：0～1 min,14% A;1～5 min,14%～21.2% A;5～6 min,21.2%～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;13～25min,14%A;, the flow rate is 0.2 mL/min, the column temperature is 40 ℃, and the detection wavelength is 254 nm; (4) According to the color spectrum obtained in the step (3), determining the peak area of each reference substance, drawing a standard curve by taking the mass X of each reference substance as an abscissa and the corresponding peak area Y as an ordinate, and calculating a regression equation of each index component as follows: Y=aX-b Wherein a is the regression coefficient of the regression equation, b is the intercept; (5) Taking the sample solution obtained in the step (1) to enter a liquid chromatograph, obtaining a chromatogram according to the chromatographic conditions of the step (3), measuring the corresponding peak area, and substituting the chromatogram into the regression equation of the step (4) to respectively obtain the contents of naringin, terprivet glucoside, hyperin, cyamarone and daidzein serving as index components.
  6. 6. The method for measuring the content of multiple index components of a Chinese medicinal composition according to claim 5, wherein the regression equation corresponding to each index component in (4) is: the regression equation of hyperin is y= 7893056351.4804X-33886.3000, The regression equation of the terprivet glycoside is that Y= 3729169813.8589X-524309.3334, The regression equation of naringin is that y= 1700221807.6804X-171939.3334, The regression equation of the cyasterone is that Y= 3023463444.8576X-24914.0000, The regression equation of daidzein is Y= 17214691046.6582X-9964.0000.
  7. 7. A method for determining the transmission relation of multi-index component values of a traditional Chinese medicine composition is characterized by comprising the following steps: (1) Determining the traditional Chinese medicine composition by referring to a UHPLC fingerprint establishment method of the traditional Chinese medicine composition of claim 1; (2) Taking a traditional Chinese medicine composition and a single decoction piece, and measuring the content of index components in the traditional Chinese medicine composition and the single decoction piece according to the method for measuring the content of the multi-index components of the traditional Chinese medicine composition in claim 5; (3) Calculating the transfer rate of the content of the index component in the decoction piece-traditional Chinese medicine composition sample according to the following formula, Transfer rate (%) =w×m/w×m Wherein w and m respectively represent the content of index components in the traditional Chinese medicine composition and the quality of the traditional Chinese medicine composition, W, M respectively represent the content of index components in the medicinal materials and the quality of the prescription medicinal material decoction pieces; the index component is hyperin, terprivet glycoside, naringin, cyasterone and daidzein.

Description

UHPLC fingerprint establishment method and application of traditional Chinese medicine composition Technical Field The invention relates to a UHPLC fingerprint establishment method of a traditional Chinese medicine composition and application thereof, belonging to the field of traditional Chinese medicine pharmaceutical quality standard control. Background The technical guidelines (trial) of pharmaceutical research of traditional Chinese medicine compound preparation according to the ancient classical prescription directory define a plurality of basic principles of pharmaceutical research of 3.1 types of traditional Chinese medicine, wherein the preparation and quality control of substance references are the preconditions and the basis of the declaration of classical prescription preparation, and the construction of a comprehensive and accurate substance reference quality control system is significant for guaranteeing the quality of classical prescription compound preparation. In the field of traditional Chinese medicine quality control, the traditional Chinese medicine quality is an important guarantee of clinical curative effect and medication safety, but the establishment of an evaluation system faces double challenges of not only continuing the traditional cognition concept of 'overall view', but also overcoming the modern technical problems of complex component analysis. Because of the variety of Chinese medicine compositions and the treatment scheme following the principle of dialectical treatment, the detection of a single active ingredient cannot represent the overall efficacy. At present, researchers mostly adopt a method of combining HPLC characteristic spectrum analysis and high resolution mass spectrum analysis to reflect complex chemical information of traditional Chinese medicines, and the HPLC fingerprint spectrum method is also a common traditional Chinese medicine quality control means. However, in the prior art, when aiming at the specific traditional Chinese medicine composition comprising drynaria rhizome, rhizoma cibotii, glossy privet fruit, suberect spatholobus stem, pyrola herb, medicinal cyathula root and the like, the establishment and detection of a substance reference are insufficient, and the method is difficult to provide powerful support for the quality standard control of the traditional Chinese medicine composition comprehensively and accurately. In view of the above, the invention provides a method for establishing and detecting a substance standard of the traditional Chinese medicine composition, which can provide a solid theoretical basis for quality standard control. Disclosure of Invention The invention aims to provide a UHPLC fingerprint establishment method and application of a traditional Chinese medicine composition, which are convenient for further establishing a material standard, and the UHPLC fingerprint establishment method has good precision, stability and repeatability, can give consideration to analysis time and high detection efficiency, provides a basis for quality control of the traditional Chinese medicine composition, and can also simultaneously control the quality of medicinal materials of drynaria, rhizoma cibotii, glossy privet fruit, suberect spatholobus stem, pyrola and medicinal cyathula root, thereby achieving the purpose of more comprehensively and effectively controlling the quality. The invention adopts the technical means that: A UHPLC fingerprint establishment method of a traditional Chinese medicine composition comprises the following operation steps: (1) Diluting the Chinese medicinal composition with 60% methanol, performing ultrasonic treatment, cooling to room temperature, shaking uniformly, centrifuging, collecting supernatant, filtering, and collecting filtrate to obtain sample solution; (2) Taking appropriate amounts of naringin, terprivet glucoside, hyperin, cyasterone and daidzein reference substances, placing into a measuring flask, and adding methanol for dilution to obtain mixed reference substance solution; (3) Taking the sample solution in the step (1) and the mixed reference substance solution in the step (2), respectively injecting the sample solution and the mixed reference substance solution into a liquid chromatograph, and measuring a fingerprint spectrum and calibrating characteristic peaks through the following chromatographic conditions; The chromatographic conditions are a chromatographic column ACQUITY UPLC BEH C, acetonitrile as a mobile phase A, 0.1% formic acid as a mobile phase B, a gradient elution program of ：0～1 min,14% A;1～5 min,14%～21.2% A;5～6 min,21.2%～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;13～25min,14%A;, 0.2 mL/min flow rate, 40 ℃ column temperature and 254 nm detection wavelength. The method is further limited, 24 marked characteristic peaks in the map measured in the step (3) are total, wherein peaks 1, 2, 8 and 13 are exclusive peaks of rhizoma drynariae, peaks