CN-121994964-A - Construction method of finger-print of Isodon japonicus and quality detection method of anti-alcoholic liver injury effect of finger-print of Isodon japonicus

CN121994964ACN 121994964 ACN121994964 ACN 121994964ACN-121994964-A

Abstract

The invention relates to a construction method of a finger print of Isodon japonicus and a quality detection method of an anti-alcoholic liver injury effect of the finger print, belonging to the technical field of quality detection of traditional Chinese medicines. The invention provides a method for constructing a finger print of Isodon japonicus with narrow base line, which has the advantages of good specificity, good precision, good repeatability and higher accuracy. The construction method of the invention has wider universality. The fingerprint spectrum is obtained by using the construction method, the spectral efficiency relation is constructed by combining a gray correlation analysis method, a hierarchical analysis method and a PLSR analysis method with the efficacy of resisting alcoholic liver injury, the quantitative efficiency relation is further obtained, and the quality standard of the Rabdosia trichocarpa with narrow base line is established. The quality detection method constructed by the invention overcomes the defects that the quality markers are only measured by the content in the prior art and the component efficacy is verified one by utilizing a plurality of monomers, saves time and reagent cost, and effectively establishes the quality standard of the sample.

Inventors

YU XIAOYI
CHENG ZHILONG
LI JUNBO
HUANG ZIJIN
YU LIANG
CHEN CHANG
CHEN ZEYAN

Assignees

完美（广东）日用品有限公司
完美（中国）有限公司

Dates

Publication Date: 20260508
Application Date: 20260228

Claims (10)

1. The method for constructing the finger-print of the rabdosia rubescens is characterized by comprising the following steps of: S1, preparation of sample solution Dissolving a water extract sample of the Isodon japonicus in water, and filtering to obtain a sample solution; S2, preparation of reference substance solution Dissolving caffeic acid, rosmarinic acid, rutin, schaftoside and isosaftoside in methanol to obtain reference solution; S3, high performance liquid chromatography determination Respectively injecting the sample solution and the reference substance solution into a liquid chromatograph, and combining with an ultraviolet detector for measurement to respectively obtain a sample chromatogram and a reference substance chromatogram, and performing identification and calibration on the sample chromatograms to obtain a Isodon japonicus fingerprint spectrum with a narrow base line; The chromatographic conditions of the liquid chromatography are that a T3 chromatographic column is adopted, a mobile phase A is acetonitrile, a mobile phase B is formic acid aqueous solution, an elution process is that the mobile phase A is 0-6 min, the volume percentage of the mobile phase A is 5%, the volume percentage of the mobile phase A is changed from 5% to 20%, the volume percentage of the mobile phase A is changed from 20% to 25% in 20-35 min, the volume percentage of the mobile phase A is changed from 25% to 35-38 min, the volume percentage of the mobile phase A is changed from 25% to 5% in 38-48 min, and the volume percentage of the mobile phase A is changed from 25% to 5% in 48-58 min.
2. The construction method according to claim 1, wherein in the step S1, the preparation method of the Rabdosia angustifolia aqueous extract comprises the steps of extracting Rabdosia angustifolia with water under thermal reflux, collecting filtrate and freeze-drying to obtain the Rabdosia angustifolia aqueous extract; And/or, in the step S1, the concentration of the water extract of the Rabdosia trichocarpa in the sample solution is 10 mg/mL; and/or, in step S1, the particle size of the filtered filter membrane is 0.45 μm; and/or, in step S3, the T3 chromatographic column is a Hungpu T chromatographic column, the specification of which is 4.6 μm×250 μm×5 μm; and/or in the step S3, the column temperature in the chromatographic condition is 25-35 ℃, the flow rate is 0.6-1.0 mL/min, and the detection wavelength is 250-360 nm; and/or, in step S3, the mobile phase B is a 0.1% formic acid aqueous solution.
3. The use of a fingerprint constructed by the construction method of claim 1 or 2 in detecting the quality of rabdosia angustifolia.
4. The quality detection method for the alcohol-resistant liver injury efficacy of the rabdosia rubescens is characterized by comprising the following steps of: A. establishing a fingerprint of the Rabdosia angustifolia by using the construction method of claim 1 or 2; B. study on the efficacy of the rabdosia rubescens in resisting alcoholic liver injury; C. Establishing a spectral efficiency relation between a common peak in the finger print of the Isodon japonicus of the narrow-base line in the step A and the efficacy of resisting alcoholic liver injury in the step B by using a gray correlation analysis method, a hierarchical analysis method and a PLSR analysis method; D. And C, establishing a quality standard according to the spectrum efficiency relation in the step, and detecting the quality of the Rabdosia angustifolia.
5. The quality control method according to claim 4, wherein in step B, the study of the efficacy of the Rabdosia angustifolia in anti-alcoholic liver injury comprises the inhibition effect of Rabdosia angustifolia on alanine aminotransferase and aspartate aminotransferase.
6. The method according to claim 4, wherein in the step C, the gray correlation analysis method specifically comprises the steps of: C1, taking the anti-alcoholic liver injury efficacy index data as a reference sequence, and marking as x i (t), (i=1, 2,3,., n; t=1, 2,3,., k), and the peak areas of the common peaks in the rabdosia rubescens fingerprint with the narrow-base line as comparison sequences, and marking as y j (t), (j=1, 2,3, n; t=1, 2,3,., k); C2, carrying out dimensionless treatment on the reference sequence and the comparison sequence; And C3, calculating gray correlation coefficients xi ij (t) and a correlation degree beta ij of the reference sequence and the comparison sequence.
7. The quality control method according to claim 4, wherein in step C, the weight coefficient of each drug effect index is calculated by an analytic hierarchy process.
8. The method of claim 4, wherein in the step C, the PLSR analysis method comprises the steps of taking the peak area of each common peak in the finger print of the Rabdosia angustifolia as X, taking the total drug effect data of the anti-alcoholic liver injury efficacy of the Rabdosia angustifolia obtained by the hierarchical analysis as Y, and establishing a PLSR model by utilizing the partial least squares regression correlation analysis, wherein the regression equation of the PLSR model is that ：Y＝0.0164X 1 +0.0225X 2 +0.0427X 3 +0.0013X 4 -0.0021X 5 -0.0835X 6 +0.0268X 7 +0.0654X 8 -0.0005X 9 +0.0440X 10 .
9. The quality detection method according to any one of claims 4 to 8, wherein in step D, a common peak having a degree of association of more than 0.6 in the gray association degree analysis and showing a positive correlation with the efficacy of the rabdosia angustifolia in the PLSR analysis in resisting alcoholic liver injury is selected as a characteristic peak according to the gray association analysis and PLSR analysis results in the spectral efficiency relationship in step C, and the ratio of the sum of the areas of the characteristic peaks to the total peak area is calculated to formulate the quality detection method for the rabdosia angustifolia in the narrow base line.
10. The method according to claim 9, wherein in the step D, the characteristic peaks F1, F2, F4, F7, F8 and F10 in the common peaks of the fingerprints are not less than 50.27% of the total area of the chromatographic peaks, F1 represents a chromatographic peak with a retention time of 12.757 min, F2 represents a chromatographic peak with a retention time of 16.552 min, F4 represents a chromatographic peak with a retention time of 21.832 min, F7 represents a chromatographic peak with a retention time of 24.580 min, F8 represents a chromatographic peak with a retention time of 26.427 min, and F10 represents a chromatographic peak with a retention time of 35.181 min.

Description

Construction method of finger-print of Isodon japonicus and quality detection method of anti-alcoholic liver injury effect of finger-print of Isodon japonicus Technical Field The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a construction method of a finger print of Isodon japonicus selenka and a quality detection method of an anti-alcoholic liver injury effect of the finger print. Background The Rabdosia angustifolia (Isodon lophathoidesvar. Gerard iana) is one of the important basic plants of the herb of Rabdosia crenata, and is easily confused with the Rabdosia crenata, rabdosia crenata and Rabdosia crenata (Isodon serra) in morphology. The plant is a variety of rabdosia rubescens, has obvious difference with rabdosia lophanthide in chemical composition of volatile oil, and has unique material basis. The plant is higher, the leaves are larger, the plant is oval, the front end is tapered, the basal part is wedge-shaped, and the plant grows under the miscellaneous tree forest or in the shrub with the altitude of 430-2900 m. In China, the distribution range of the plant growth regulator covers a plurality of areas such as Tibet, yunnan, sichuan, gansu, guizhou, guangxi, guangdong, hunan and the like, and is also found in partial south Asia and southeast Asia areas. It is noted that the Rabdosia angustifolia has been officially approved by the national health committee in 2013 as a new food material (bulletin No. 10 of the Ministry of original health 2013), and is allowed to be used in food categories such as tea beverages and substituted tea. The identity highlights the edible safety and development value of the food, and provides a high-quality raw material source for the innovative development of related health foods. The pathogenesis of alcoholic liver injury is closely related to oxidative stress, inflammatory response, and hepatocyte apoptosis. At present, the quality evaluation of the rabdosia lophanthide mostly depends on component content or fingerprint similarity, and the spectrum effect relation of chemical components and liver injury resistance is not established, so that the rabdosia lophanthide which partially accords with the existing quality standard has poor actual clinical curative effect. Therefore, development of an integrated method for fusing the fingerprint characteristics and the anti-liver injury efficacy index is needed, and quality upgrading of the rabdosia lophanthide from 'qualified' to 'effective' is realized. In the prior art, the identification of the medicinal material of rabdosia lophanthide Huang Caolei is mainly to identify the basic sources, such as CN106053696A, which is a method for identifying the plant sources of rabdosia lophanthide, oridonin, sesbanin and caffeic acid ethyl ester, wherein rosmarinic acid, rabdosia lophanthide, or rabdosia lophanthide is used as standard substances, three basic sources of rabdosia lophanthide, namely rabdosia lophanthide, plant rabdosia lophanthide or rabdosia lophanthide are identified, only basic identification is focused, and the rabdosia lophanthide is not related to the basic efficacy, CN104614480A builds 12 common peak finger-prints of rabdosia lophanthide Huang Caoshui soluble total flavone, realizes quality control of an extract level, but does not aim at the basic line rabdosia lophanthide, and does not definitely form a corresponding relation with liver injury resistant activity, CN103776926A rabdosia lophanthide HPLC finger print is established, 4 basic herb finger-C acid, 6-C-arabinopyranosyl-8-glucopyranosyl apigenin, vitexin and rosmarinic acid are used as standard substances, and herba rabdosia lophatthide are used for carrying out basic identification, and the basic characteristics of rabdosia lophanthide is not found in basic herb, such as basic herb, namely, the basic herb is not examined by the basic herb is not examined, but is tested by the basic herb is not examined by the basic herb, and the basic herb is not examined by the basic herb, but is tested by the basic herb, and the basic herb is not tested by the basic herb, but is tested by the basic herb. However, the quality of rabdosia lophanthide with the same basic source is also uneven, and a related detection method aiming at the finger print of the rabdosia lophanthide with the specific basic source is lacked. The prior literature research shows that the Rabdosia angustifolia has liver protection effect, but has difference. At present, the Rabdosia angustifolia has no relevant quality evaluation standard in the aspect of liver protection. The rabdosia rubescens is used as a traditional liver-clearing and cholagogic herbal medicine, is widely applied to folk and Chinese patent medicine production, and the prior art can not realize the exclusive identification and quality control of rabdosia rubescens medicinal materials, so that the raw material sources are mixed. The composition content of the