| Objective:Under the guidance of the the principle of "based on pharmacopoeia,based on market,simple and practical",establish a unified commodity grade evaluation standard for Flos Trollii,and through the analysis and evaluation of quality indicators,provide a scientific theoretical basis for the high quality and good price of Flos Trollii in the market circulation and the safety of clinical medication.Methods:1.Preliminary formulation of commodity specifications and grade standards of Flos Trollii based on appearance characteristics Consult Chinese Pharmacopoeia,local standards and related books;Investigate the main producing areas of Flos Trollii,such as Hubei,Shanxi and Inner Mongolia,as well as Medicinal materials market of Anguo,Bozhou and Hehuachi,and conduct origin and field investigation on the variety distribution,planting,processing and grade division of Flos Trollii;Collect different grades of Flos Trollii from different producing areas as experimental samples.The collected Flos Trollii samples were quantified according to traditional empirical indicators such as "flower diameter,impurity content,flower stalk length,color and etc",and the appearance character indicators for the classification of Flos Trollii were selected by using statistical methods such as principal component analysis,analysis of variance and correlation analysis.The commodity grade standard of Flos Trollii was preliminarily established by quartile method.2.Study on Extraction of quality indexes of Flos Trollii Flos Trollii was included in the 1977 edition of "Chinese Pharmacopoeia",and its quality standards only contains characters and identification items;Based on this,this subject first carried out character identification,microscopic identification and thin layer identification of the collected Flos Trollii;Reference to the relevant methods of the general principles of the Chinese Pharmacopoeia（Volume IV）in the 2020 edition,routine inspections were carried out on Flos Trollii,such as moisture,total ash,extract,etc;A method for the determination of total flavonoids in Flos Trollii was established by UV-vis spectrophotometry;A quantitative analysis of multi-components by single marker method was used to establish a suitable method for the determination of Flos Trollii by HPLC,and the relative correction factors and relative retention time values of the remaining components to be tested were established with orientin as the internal reference.HPLC method was used to establish the common pattern of the fingerprint of Flos Trollii,and the common peaks of the characteristic fingerprint of Flos Trollii were identified;The content determination and common peak data of fingerprint were analyzed by statistical analysis software.3.Formulation of commodity specification and grade standard of Flos Trollii based on the combination of character and quality Analysis software was used to count the range and mean of the commodity specification grades and internal quality of different Flos Trollii based on appearance characters,and correlation analysis and one-way analysis of variance were carried out.Results:1.（1）Through literature research,it is found that there is no record of the specifications and grades of Flos Trollii in the herbal monographs and related standards of the past dynasties,but only the quality evaluation of Flos Trollii and the descriptions are basically the same,that is,"the one with completeness,golden color and strong aroma is better".The local standard stipulates that Flos Trollii come from the Trollius chinensis Bge.,Trollius asiaticus L.,Trollius macropetalus Fr.Schmidt.,Trollius ledebouri Reichb dried flowers.（2）The results of the field investigation of Flos Trollii are as follows: there are few Chinese herbal medicine market firm selling Flos Trollii,and the specifications and grades of Flos Trollii in major markets and main producing areas are not clear,and they are all of the highest quality in golden color and large flowers.（3）The research group collected a total of 51 batches of Flos Trollii,which were identified as 27 batches of T.chinensis Bge.,14 batches of T.ledebouri Reichb,and 10 batches of T.maccropetalus Fr.Schmidt.（4）Adopting the principle of minimum grading,the specification and grading standard of Flos Trollii is formulated as follows:Trollius chinensis Bge.: The color of the flower is light yellow to golden yellow,the chromaticity value L*≥57,b*≥28,E*ab≥64 is the first grade,and the second grade flower color is brown to dark brown,and the chromaticity value is 52≤L *<57 or 22≤L*<28 or 57≤E*ab<64.Trollius ledebouri Reichb: The color of the flower is light yellow,the chromaticity value L*≥60,b*≥23,E*ab≥66 is the first grade,and the second grade flower color is dark brown,and the chromaticity value is 58≤L *<60 or19≤L*<23 or 61≤E*ab<66.Trollius macropetalus Fr.Schmidt.: The color of the flower is light yellow,the chromaticity value L*≥60,b*≥22,E*ab≥65 is the first grade,and the second grade flower color is dark brown,and the chromaticity value is 56≤L*<60 or16≤L*<22 or 58≤E*ab<65.Trollius asiaticus L.: Standard goods,regardless of color.2.（1）The characters of different varieties of Flos Trollii were obviously different,which can be distinguished mainly by the number of calyx and the length of petals.The main microscopic characteristics were pollen grains,epidermal mastoid cells on calyx,adventitious stomata,beaded epidermal cells of anther,inner wall cells of pollen sac and non glandular hairs.Polyamide thin-layer plate was used,orientin and vitexin were used as reference substances,chloroform-acetone-glacial acetic acid-absolute ethanol（4:1:3:2）was used as developing agent,and 1% aluminum trichloride ethanol solution was used as the developing agent,inspect under ultraviolet light（365nm）,the results showed that the test sample of Flos Trollii and the reference substances of orientin and vitexin displayed the same color spots in the corresponding positions.The characters,microscopic and thin layer identification items of Flos Trollii were established.（2）According to the relevant methods of the four general chapters of the 2020 edition of the "Chinese Pharmacopoeia",the moisture content of Flos Trollii ranges from 6.40% to 10.72%,the extract content ranges from 32.06 to 43.78%,and the total ash content ranges from7.79 to 12.35%;Statistical analysis results showed that the ranges of moisture,extract,and total ash content in different origins and producing areas all overlapped severely and had no significant difference.（3）The methodological study on the determination of total flavonoids in Flos Trollii showed that the total flavonoids in Flos Trollii had a good linear relationship in the concentration range of 0.01~0.1 mg/m L,and the regression equation was Y=10.211X+0.0089（r=0.9998,n=6）,the average sample recovery rate was 98.38%;Statistical analysis results showed that the ranges of total flavonoids content in different origins and producing areas all overlapped severely and had no significant difference.（4）The results of determination of five flavonoids components in Flos Trollii with quantitative analysis of multi-components by single marker show that: qualitatively,the relative retention time was stable on three columns of two high performance liquid chromatographs;Quantitatively,there is no difference between the external standard method and the relative correction factor method,quantitative analysis of multi-components by single marker can replace the external standard method to determine the content of multiple components in Flos Trollii;Statistical analysis results showed that the content ranges of the five flavonoids in Flos Trollii from different origins were seriously overlapping,There was no significant difference in the contents of Orientin-2’’-O-β-L-galactopyranosyl,Vitexin and Quercetin,Significant difference,while the orientin content of T.chinensis Bge.was significantly different from those of T.ledebouri Reichb and T.maccropetalus Fr.Schmidt.,while the orientin content of T.ledebouri Reichb and T.maccropetalus Fr.Schmidt.had no significant difference.There was no significant difference in the Diosmetin content of T.chinensis Bge.,T.ledebouri Reichb and T.maccropetalus Fr.Schmidt.The content ranges of five flavonoids in Flos Trollii from different producing areas overlapped severely,and there was no significant difference.（5）The HPLC fingerprint of Flos Trollii was established,by comparing the retention time of the chromatographic peaks in the chromatogram of the mixed reference solution,six common peaks were identified,which were Orientin-2’’-O-β-L-galactopyranosyl,Orientin,Vitexin,Naringenin,Quercetin,Diosmetin.The similarity of samples from different origins of T.chinensis Bge.,T.ledebouri Reichb and T.maccropetalus Fr.Schmidt.were all greater than 0.8.Cluster analysis basically gathered T.chinensis Bge.into one category,and T.ledebouri Reichb and T.maccropetalus Fr.Schmidt.into another category.The results of orthogonal partial least squares discriminant analysis are consistent with those of cluster analysis.3.Statistical analysis results show that the content ranges of internal quality indexes of different grades of T.chinensis Bge.have different degrees of overlap and no significant difference;Except for the content of total ash and total flavonoids,which have a significant range and change trend and have extremely significant differences,the other internal quality indicators of T.ledebouri Reichb have different degrees of overlap and no significant difference;In addition to extracts,the contents of total flavonoids in the T.maccropetalus Fr.Schmidt.have a significant range and change trend,and there are extremely significant differences.Among them,the content of orientin has a significant difference but the range is partially overlapped,and the other internal quality indicators have different degrees of overlap and no significant differences.Conclusion:On the basis of a large number of literatures and market origin research,referring to the standards and related literatures such as "225 Commonly Used Chinese Medicinal Materials Commodity Specification Grade Group Standards",the chromaticity indexes L*,b* and E*ab were screened by statistical analysis method as the main indexes for the classification of Flos Trollii,and the grading standard of Flos Trollii was preliminarily proposed.Moreover,combined with the correlation of the internal quality of Flos Trollii,a reasonable and feasible commodity grade standard for Flos Trollii is formulated,which provides a scientific basis for the high quality and good price of Flos Trollii in the market.In addition,51 batches of Flos trollii from different origins and different producing areas were studied on their characters,microscopic,thin layer identification,routine inspection items,content determination of five flavonoids,content determination of total flavonoids and fingerprint,which provided a scientific reference for improving the quality standard of Flos trollii. |
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