| Purpose:Mulberry leaf is a traditional Chinese herb in China,which is widely used in hypoglycemia and hypolipidemia,but there is a problem of species confusion in practical application,resulting in unclear.Based on this,this study was conducted to systematically evaluate the herbal quality of mulberry leaves of different species of the same genus in terms of traits,physicochemical identification and content,to provide a methodological basis for the identification of different species of mulberry leaves,and to provide an experimental basis for ensuring accurate clinical use of drugs.Material and method:1 Materials:The experimental materials were 13 batches of mulberry leaves of different species of the same genus from the National Germplasm Zhenjiang Mulberry Nursery,Zhejiang Academy of Agricultural Sciences Institute of Sericulture,Xinjiang Hotan Institute of Silkworm Science and China Academy of Food and Drug Administration,which were identified by Professor Kang Tingguo of Liaoning University of Traditional Chinese Medicine as different species of mulberry leaves under the genus Moraceae,and the medicinal parts were its dried leaves,and the certified specimens were stored in the Liaoning University of Traditional Chinese Medicine,protected from light.The specimens were stored in Laboratory 304 of the Engineering Technology Center for TCM Innovation.2Methods:(1)In this experiment,the traits of different species of mulberry leaves were identified by the direct observation method and vernier caliper measurement method in the first part of the Chinese Pharmacopoeia,2020 edition.The microstructural characteristics of different species of mulberry leaves were observed by chloral hydrate permeation method.The thin layer identification method method under mulberry leaves in the pharmacopoeia was used to compare the chromatograms of different species of mulberry leaves with the standard mulberry leaf herbs under UV light.The content of general substances and alcohol-soluble extracts in different species of mulberry leaf herbs was determined by the standard methods specified in the Chinese Pharmacopoeia(2020 edition)under general rules.(2)The total flavonoid content in different species of mulberry leaves was determined by the sodium nitrite-aluminum nitrate method,and the extraction process of total flavonoids in mulberry leaves was optimized by using L9(3)4orthogonal test with the total flavonoid extraction content as the evaluation index.The phenol-sulfuric acid method was used to determine the total polysaccharide content in mulberry leaves.The total alkaloid content in mulberry leaves was determined by the colorimetric method using Ray's ammonium salt.The total protein content in mulberry leaves was determined by using the Thomas Brilliant Blue method.(3)The fingerprinting study of 13 batches of mulberry leaf samples was performed by high performance liquid chromatography(HPLC),and the similarity was evaluated using the 2012 version of the Chinese medicine fingerprinting similarity evaluation software.Cluster analysis and principal component analysis were performed using SPSS 26.0 and SIMCA 14.1 software.Results:1.The results of the herbal examination show that there are many kinds of mulberry leaves for medicinal use in ancient times.In the Materia Medica,chicken mulberry is considered to be the most useful.2.The results of mulberry leaf trait identification showed that the shapes of different species of mulberry leaves included round,ovoid,heart-shaped,broad-hearted,obliquely ovate,and ovate-elliptic,etc.;leaf size was mostly in the range of 8?13 cm,with the larger leaves of Lu Sang about 8?16 cm and the smaller leaves of Bai Sang about 3?5 cm;mulberry leaf edge serrations were divided into acute serrations or blunt serrations,with the tip of the leaf edge of Meng Sang having a long spiny awn.Except for Xinjiang medicine mulberry and Xinjiang white mulberry,which have strong odor,other mulberry leaves have no obvious odor.3.Microscopic identification shows that mulberry leaf powder is yellowish green or yellowish brown.It has a stomatal structure,56?68?m in diameter,with stomata of indeterminate type and 5?9 paramecium cells.The non-glandular hair single cells are48?220 pm long,and the calcium oxalate clusters are 4?12?m in diameter,with threaded ducts,fenestrations and occasional square crystals.The thin-layer identification results showed that the mulberry leaf samples and the mulberry leaf standard herbal control had blue and red fluorescent spots at the same locations,and the spot locations were largely the same,with the main differences being in the intensity of the fluorescent spots.4.The results of the general substance examination showed that the moisture content in the samples of different species of mulberry leaves ranged from 5.35 to 7.98%,with an average moisture content of 6.30%.Ash content ranged from 2.12 to 3.99%,with an average content of 3.09%.The acid insoluble ash content ranged from 0.45 to1.06%,with an average content of 0.68%.The alcohol-soluble leachate content ranged from 9.97 to 14.72%,with an average content of 11.79%.5.The results of the determination of the content of the main components showed that among the different species of mulberry leaf samples,the highest content of total flavonoids(23.18 mg/g);the highest content of total polysaccharides(21.17 mg/g);the highest content of total alkaloids(4.79 mg/g);and the highest content of total protein(87.8 mg/g)were found in Chinese mulberry.Principal component analysis was performed using SPSS 26.0 software with total flavonoids,total polysaccharides and total alkaloids as evaluation indexes,and the top three were chicken mulberry,Chinese mulberry and Mongolian mulberry by weighting the principal components according to the principal component score coefficients.6.The fingerprint results showed that there were 15 peaks in total for different species of mulberry leaf samples,and the peaks 3,7 and 10 were identified as chlorogenic acid,isoquercitrin and zingiberin,respectively,by standard fingerprints.The similarity was found to be in the range of 0.697-0.988 using the fingerprinting software.7.The results of cluster analysis showed that the different species of mulberry leaf herbs could be classified into two major groups,with Guangdong mulberry,medicinal mulberry,Xinjiang white mulberry,white mulberry,and weeping mulberry as the first group,and long-fruited mulberry,Lu mulberry,Meng mulberry,Rui Sui mulberry,chicken mulberry,bilberry,and Chinese mulberry as the second group.The results of principal component analysis showed that chlorogenic acid,isoquercitrin,zingiberin and several other unknown components were involved in the quality expression of mulberry leaves as the main information.The results of orthogonal partial least squares discriminant analysis(OPLS-DA)showed that four signature components causing interspecies variation in mulberry leaf herbs were screened from the shared peaks of HPLC fingerprint profiles,and one of them was zingiberin.Conclusion:1.We established the methods for the characterization,microscopic and thin-layer identification,general substance examination and content determination of the main components of mulberry leaves of different species of the same genus,and developed the quality standard system.Established a method for the characterization,microscopic and thin-layer identification,general substance examination and determination of the content of the main components of different species of mulberry leaves of the same genus.2.The content data of total flavonoids,total polysaccharides,total alkaloids and total proteins in mulberry leaves of different species of the same genus were obtained to provide experimental basis for more accurate clinical use in the later stage.3.The characterization,microscopic and thin-layer identification,general substance examination,content determination and fingerprinting study of mulberry leaves reflect the differences of different species of mulberry leaves from different aspects and provide a reference basis for the identification of different species of mulberry leaves.4.Through multivariate statistical analysis,principal component analysis was performed with total flavonoids,total polysaccharides and total alkaloids as indicators to rank the comprehensive scores of different species of mulberry leaves and found that chicken mulberry had the highest score.Multivariate statistical analysis based on fingerprint data was conducted to find the signature substance that caused the difference between species,a known component as zingiberin.5.Modern scientific techniques were used to measure the content of the main medicinal components and to analyze them statistically.The results show that chicken mulberry has the first overall score,which verifies the conclusion that"chicken mulberry is the most useful for use"based on the practical application of the ancient people.The results showed that chicken mulberry was the first in the overall score,which verified the conclusion of"chicken mulberry is the most useful"based on the ancient practice. |
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