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CN-122017108-A - Construction method of traditional Chinese medicine compound preparation characteristic spectrum for Alzheimer disease

CN122017108ACN 122017108 ACN122017108 ACN 122017108ACN-122017108-A

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for constructing a characteristic map of a traditional Chinese medicine compound preparation for Alzheimer disease. Aiming at the complex chemical composition characteristics of the traditional Chinese medicine compound preparation, five characteristic components are scientifically selected as reference substances, and the characteristic patterns for simultaneously representing multiple components such as flavone, saponin, oligosaccharide ester and the like are established, so that the limitation of single index component control is overcome, and the overall quality of the product can be reflected more comprehensively and truly. The invention innovatively adopts an integrated pretreatment strategy of 'composite solvent extraction-liquid distribution low-temperature precipitation-mixed mode Solid Phase Extraction (SPE) purification'. The method realizes the high-efficiency synchronous extraction of various target components by optimizing the solvent composition.

Inventors

  • XING JIE
  • ZHAO WEIGUO
  • SHAO CHENGLEI
  • ZHANG JINXING

Assignees

  • 五源本草(山东)健康科技有限公司
  • 中山市人民医院

Dates

Publication Date
20260512
Application Date
20260410

Claims (8)

  1. 1. The method for constructing the characteristic spectrum of the traditional Chinese medicine compound preparation for treating Alzheimer's disease is characterized by comprising the following steps: Step one: Preparing reference solution by adding 70% methanol solution into icariin, epimedin C, tenuifolin, 3,6 '-sinapioyl sucrose and ginsenoside Rg1 reference substance, ultrasonic treating to dissolve thoroughly, cooling, diluting with 70% methanol, shaking uniformly, and filtering with microporous membrane to obtain mixed reference solution containing icariin, epimedin C10, tenuifolin, 3,6' -sinapioyl sucrose and ginsenoside Rg1 in each 1 mL; Step two: Preparing a sample solution, namely taking traditional Chinese medicine compound preparation powder for Alzheimer's disease, adding a methanol-acetonitrile-water mixed solvent, weighing, cooling after ultrasonic treatment, supplementing the reduced weight with the methanol-acetonitrile-water mixed solvent, shaking uniformly, filtering to obtain a subsequent filtrate, precisely taking the subsequent filtrate, placing the subsequent filtrate into a centrifuge tube, adding an ethyl acetate-n-butanol mixed solvent which has the same volume as the subsequent filtrate, swirling, standing for layering, taking an upper organic phase, standing overnight at low temperature, precipitating for removing impurities, centrifuging to obtain a supernatant, concentrating the supernatant in a water bath under reduced pressure to obtain residues, concentrating the supernatant in the water bath under reduced pressure to obtain residues, dissolving the residues obtained by drying in a 5% formic acid aqueous solution to obtain a solution, loading the solution to an activated Oasis mixed mode solid phase extraction column, sequentially washing with 5% formic acid aqueous solution and methanol, discarding the washing solution, eluting with 5mL of a methanol solution containing 2% ammonia, collecting an eluent, concentrating the eluent in a water bath under reduced pressure to obtain residues, concentrating the residues in a water bath under reduced pressure to obtain 70% residues, diluting the eluent with a microporous membrane, and diluting the filtrate to obtain a sample solution; Step three: The measurement method comprises precisely sucking 10 μl of reference solution and 10 μl of sample solution, respectively, injecting into a liquid chromatograph, and recording chromatogram; the chromatographic conditions comprise a chromatographic column GL SCIENCES InertSustain C, a mobile phase, a flow rate of 1.0mL/min, a column temperature of 30 ℃ and a detection wavelength of 320nm, wherein the mobile phase is prepared by taking 10mmol/L potassium dihydrogen phosphate buffer solution pH 3.5-methanol with a volume ratio of 95:5 as a mobile phase A, acetonitrile-isopropanol solution with a volume ratio of 80:20 as a mobile phase B, and performing gradient elution according to a specified condition; The gradient elution procedure was: 0-15 min, wherein the volume ratio of mobile phase A to mobile phase B is 80:20; 15-30 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 80:20 to 76:24; 30-50 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 76:24 to 75:25; 50-55 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 75:25 to 73:27; 55-75 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 73:27 to 72:28; 75-76 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 72:28 to 65:35; 76-85 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is 65:35; 85-110 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 65:35 to 64:36; 110-120 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 64:36 to 60:40; 120-121 min, wherein the volume ratio of the mobile phase A phase to the mobile phase B phase is changed from 60:40 to 80:20; and step four, generating a control characteristic map, namely selecting chromatographic peaks existing in chromatograms of the traditional Chinese medicine compound preparations in different batches as common peaks, and generating the control characteristic map of the traditional Chinese medicine compound preparation by using an average value calculation method.
  2. 2. The method for constructing a characteristic spectrum of a Chinese herbal compound preparation for Alzheimer's disease according to claim 1, wherein in the first step, ultrasonic conditions are 250W and 40 kHz ultrasonic treatment for 30 minutes.
  3. 3. The method for constructing a characteristic map of a Chinese herbal medicine compound preparation for Alzheimer's disease according to claim 1, wherein in the first step and the second step, the filtration pore diameter of the microporous membrane is 0.22 μm.
  4. 4. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating Alzheimer's disease according to claim 1, wherein in the second step, the volume ratio of the methanol-acetonitrile-water mixed solvent is 4:3:3.
  5. 5. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for treating Alzheimer's disease according to claim 1, wherein in the second step, the volume ratio of ethyl acetate to n-butanol is 1:1.
  6. 6. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, wherein in the second step, the specification of an Oasis MAX mixed mode solid phase extraction column is 60mg/3mL.
  7. 7. The method for constructing a characteristic spectrum of a traditional Chinese medicine compound preparation for Alzheimer's disease according to claim 1, wherein the characteristic spectrum of the contrast generated in the fourth step shows 5 chromatographic peaks, wherein peak 1, peak 2, peak 3, peak 4 and peak 5 respectively correspond to reference peaks of contrast 3,6' -sinapiyl sucrose, ginsenoside Rg1, epimedin C, tenuifolia saponin and icariin.
  8. 8. The method for constructing a characteristic spectrum of a Chinese herbal compound preparation for Alzheimer' S disease according to claim 7, wherein the contrast characteristic spectrum generated in the fourth step uses icariin reference peak as S peak, and calculates the relative retention time of each characteristic peak and S peak, wherein the relative retention time is within + -10% of a specified value, and the specified value is that peak 1 is 0.30, peak 2 is 0.84, peak 3 is 0.89, and peak 4 is 0.95.

Description

Construction method of traditional Chinese medicine compound preparation characteristic spectrum for Alzheimer disease Technical Field The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for constructing a characteristic map of a traditional Chinese medicine compound preparation for Alzheimer disease. Background Alzheimer's Disease (AD) is a common neurodegenerative Disease, and is clinically manifested mainly by progressive cognitive dysfunction and memory decline. Currently, the therapeutic drug options for this disease are limited, and most are symptomatic relief, making it difficult to radically delay or reverse progression. The Chinese herbal compound preparation is increasingly valued in the prevention and treatment of Alzheimer's disease due to the overall regulation characteristics of multiple components, multiple targets and multiple channels. The invention relates to a preparation for treating Alzheimer's disease, which is a compound granule prepared from epimedium, polygala tenuifolia, ginseng and natural borneol. In the preparation, the epimedium has the effects of tonifying kidney yang and strengthening tendons and bones, the flavonoid glycoside component contained in the epimedium is considered as an important material basis for improving cognitive functions, the polygala tenuifolia tranquillizes and benefits intelligence, the saponin and oligosaccharide ester components of the polygala tenuifolia have the effects of protecting a nervous system, the ginseng has the effects of reinforcing primordial qi and promoting intelligence and tranquillizing, the ginsenoside component has remarkable effects of improving memory and resisting aging, and the natural borneol has the effects of inducing resuscitation and refreshing mind, and is beneficial to guiding medicine upwards and promoting blood brain barrier permeation. The medicines are combined together to have the effects of improving intelligence, strengthening brain, tonifying kidney and soothing nerves, and is suitable for the auxiliary treatment of Alzheimer's disease and related cognitive dysfunction. However, the formulation belongs to a complex system of typical multi-medicinal, multi-class ingredients. The chemical components of the composition cover different categories with large polarity span, including weak polarity epimedium flavonoid glycoside, medium polarity ginsenoside, and strong polarity polygala tenuifolia saponin and oligosaccharide ester component. Under the existing analysis technical conditions, the single chromatographic method is difficult to realize the synchronous and effective separation and detection of multiple active ingredients with obvious property differences, so that the quality evaluation is not comprehensive enough, and the whole material basis and the internal quality of the preparation are difficult to truly reflect. In addition, the auxiliary materials introduced in the preparation process and complex matrixes such as polysaccharide, pigment, protein and the like contained in the medicinal materials can cause serious interference to the detection of target components. The traditional pretreatment method cannot effectively remove matrix interference while extracting various target components at high efficiency, and is easy to cause the problems of unstable baseline, masking of characteristic peaks, poor reproducibility of the method and the like in chromatographic analysis. Meanwhile, how to scientifically select a group of multi-component reference objects capable of representing the overall chemical characteristics of the preparation and construct a characteristic map with comprehensiveness, specificity and transferability is also a key technical bottleneck for realizing stable and controllable quality of the preparation. Therefore, aiming at the traditional Chinese medicine compound preparation for treating Alzheimer's disease, a characteristic spectrum construction method capable of systematically solving the problems of component complexity, matrix interference and method robustness is urgently needed to be established, so that scientific basis and technical guarantee are provided for stable and reliable production process control, quality consistency among batches and clinical curative effect. The characteristic spectrum detection of the preparation has the difficulty that the preparation contains various chemical components with large polarity span, and comprises weak-polarity epimedium flavonoid glycoside, medium-polarity ginsenoside, and strong-polarity polygala tenuifolia saponin and oligosaccharide esters. And meanwhile, a large amount of coexisting matrixes such as polysaccharide, pigment, auxiliary materials and the like in the compound have serious interference, and the traditional pretreatment method is difficult to balance between high extraction efficiency and high sample purity, so that the characteristic spectrum baselin