UPLC-QDA and machine learning for distinguishing different commodity specifications of Fritillariae Cirrhosae Bulbus and application of data augmentation technology / 中国中药杂志

This study aimed to establish a method based on machine learning technology for accurately predicting the commodity specifications of Fritillariae Cirrhosae Bulbus and explore the application of data augmentation technology in the field of drug analysis. The correlation optimized warping(COW) algorithm was used to perform peak calibration on the UPLC-QDA multi-channel superimposed data of 30 batches of samples, and the data were normalized. Through unsupervised learning methods such as clustering analysis, principal component analysis(PCA), and correlation analysis, the general characteristics of the data were understood. Then, the logistic regression algorithm was used for supervised learning on the data, and the condition tabular generative adversarial networks(CTGAN) was used to generate a large amount of data. Logistic regression classification models were trained separately using the real data and the data generated by CTGAN, and these models were evaluated. The logistic regression model trained with real data achieved cross-validation and test set accuracies of 0.95 and 1.00, respectively, while the logistic regression model trained with both real and CTGAN-generated data achieved cross-validation and test set accuracies of 0.99 and 1.00, respectively. The results indicate that machine learning can accurately predict the classification of Songbei, Qingbei, and Lubeibased on UPLC-QDA detection data. CTGAN-generated data can partially compensate for the lack of data in drug analysis, improving the accuracy and predictive ability of machine learning models.

Rapid Detection of Adulteration of Fritillariae Cirrhosae Bulbus Based on Portable Near Infrared Spectroscopy / 中国实验方剂学杂志

ObjectiveIn order to establish a systematic quality evaluation system for Fritillariae Cirrhosae Bulbus adulteration， portable near-infrared （NIR） spectroscopy was used to identify Fritillariae Cirrhosae Bulbus and its adulterants and detect their adulteration quantity. MethodA total of 72 batches of Fritillariae Cirrhosae Bulbus samples were collected and 570 batches of adulterated products （dry bulbs of Fritillaria thunbergii， F. ussuriensis， F. pallidiflora and F. hupehensis， Bulbus Tulipae， flour） were prepared， NIR spectral data of samples were collected by the portable NIR spectrometer. Linear discriminant analysis （LDA） was used to establish the qualitative correction models of Fritillariae Cirrhosae Bulbus-adulterants and adulterants of different categories， partial least squares （PLS） was used to establish the quantitative correction models of adulteration quantity of different kinds of adulterants. ResultThe recognition rates of qualitative analysis model of Fritillariae Cirrhosae Bulbus and its adulterants were 99.49% （calibration set） and 100.00% （validation set）， respectively. In different adulterant models， the recognition rates of calibration set and validation set were 70.47% and 73.68%， respectively. Moreover， the correlation coefficients of validation set （R2P） of the six quantitative models of adulteration ratio were 0.840 2 （Fritillariae Cirrhosae Bulbus adulterated with F. thunbergii dry bulbs）， 0.960 2 （Fritillariae Cirrhosae Bulbus adulterated with F. ussuriensis dry bulbs）， 0.765 7 （Fritillariae Cirrhosae Bulbus adulterated with F. pallidiflora dry bulbs）， 0.902 5 （Fritillariae Cirrhosae Bulbus adulterated with F. hupehensis dry bulbs）， 0.957 4 （Fritillariae Cirrhosae Bulbus adulterated with Bulbus Tulipae）， 0.976 1 （Fritillariae Cirrhosae Bulbus adulterated with flour）， the root mean square error of prediction （RMSEP） were 10.948 5， 5.463 9， 13.256 4， 8.549 2， 5.655 3， 4.235 6， respectively. The two qualitative models and six quantitative models showed good prediction performance. ConclusionThe portable NIR spectroscopy can be used to identify Fritillariae Cirrhosae Bulbus and its adulterants in real time， the method is rapid and accurate， which can meet the requirements of nondestructive identification of Fritillariae Cirrhosae Bulbus on site.

Analysis on Feasibility of Electronic Nose Technology for Identification of Fritillariae Cirrhosae Bulbus from Authenticity and Specifications / 中国实验方剂学杂志

Objective:To establish a new fast and accurate method for identifying the authenticity and specifications of Fritillariae Cirrhosae Bulbus based on electronic nose technology， and to discuss the feasibility of this technology in the identification of decoction pieces. Method:Fritillariae Cirrhosae Bulbus was used as the research object， 80 batches of samples to be tested were collected， and the olfactory sensory data of the electronic nose were taken as independent variables （<italic>X</italic>）， the results of the method contained in the 2020 edition of <italic>Chinese Pharmacopoeia</italic> were taken as the focus， and the traditional empirical identification results were used as benchmarking information （<italic>Y</italic>）. Four chemometric methods， including discriminant analysis （DA）， least square support vector machine （LS-SVM）， principal component analysis-DA （PCA-DA） and partial least squares-DA （PLS-DA）， were used to establish the identification model ［<italic>Y</italic>=<italic>F</italic>（<italic>X</italic>）］ of authenticity and commodity specifications of Fritillariae Cirrhosae Bulbus， respectively. Wherein， the identification accuracy and time-consuming was taken as indicators to discuss the results. Result:After cross-verification by leave-one-out method， the correct rates of the above four models were 93.75%， 91.25%， 95.00% and 95.00%， respectively， and the PCA-DA and PLS-DA identification models were the best in terms of authenticity identification. In specification identification， the correct rates of these four models were 86.67%， 88.00%， 89.33% and 68.00%， respectively， and the PCA-DA identification model was the best. The electronic nose had a high accuracy in the identification of authenticity and specification model， and the time consuming was relatively short. Conclusion:Electronic nose technology can identify Fritillariae Cirrhosae Bulbus accurately and quickly， and has significant advantages in terms of timeliness and correct judgment rate.

Nucleosides-based identification model for Fritillariae Cirrhosae Bulbus / 中国中药杂志

A high performance liquid chromatography( HPLC) method was established for the fast,and precise determination of ten nucleosides in Fritillariae Cirrhosae Bulbus and its counterfeits. Then multivariate statistical analyses,such as clustering analysis,principal component analysis( PCA),and Fisher' s linear discriminant analysis( LDA),were conducted to establish a discriminant function model for an integrated analysis. The results indicated that data acquisition time of a single sample was shortened within 16 min by the HPLC method. In the range of 5-1 000 mg·kg~(-1),the mass concentrations of all nucleosides exhibited good linear relationships with the corresponding peak areas( R2> 0. 999). The spiked recoveries were in the range of 93. 83%-108. 9% with RSDs of0. 12%-1. 3%( n = 5). The limit of quantitation( LOQ) was 0. 98-4. 13 mg·kg~(-1). As revealed by the clustering analysis,Fritillariae Cirrhosae Bulbus and the counterfeits could be discriminated into two clusters based on the content of nucleosides. Fisher's LDA could achieve this discrimination,while PCA dimension reduction failed. The accuracy of the discriminant function model established on the screened characteristic indicators reached 97. 5%. The present study proposed a new identification method of Fritillariae Cirrhosae Bulbus with one-dimensional indicators,which is simple,accurate,and reliable. It can provide a scientific basis for further optimizing the identification techniques for Fritillariae Cirrhosae Bulbus and inspiration for quality control strategy development of Chinese medicinal materials.

Intelligent Identification of Fritillariae Cirrhosae Bulbus，Crataegi Fructus and Pinelliae Rhizoma Based on Deep Learning Algorithms / 中国实验方剂学杂志

Objective:To propose a new method for detecting and evaluating traditional Chinese medicine （TCM） by artificial intelligence and machine vision technology. Method:Taking Fritillariae Cirrhosae Bulbus， Crataegi Fructus and Pinelliae Rhizoma as the research objects， big data of pictures was collected by machine vision and the image database was established. Through the intelligent analysis of the external characteristics of TCM， the deep convolutional neural network model was established to realize the functions of location detection and variety identification by means of deep learning， so as to significantly improve the accuracy of rapid identification of TCM. Result:The classification accuracy of 11 kinds of Chinese herbal pieces （raw， fried， parched and charred products of Crataegi Fructus， Pinelliae Rhizoma， Pinelliae Rhizoma Praeparatum Cum Zingibere et Alumine， Pinelliae Rhizoma Praeparatum， Pinelliae Rhizoma Praeparatum Cum Alumine and three products of Fritillariae Cirrhosae Bulbus） could be more than 99%， and the average recognition accuracy of specific categories could reach more than 97%. Conclusion:The intelligent identification technology of TCM decoction pieces realized by deep learning algorithms has the advantages of simplicity， rapidity， high precision and quantifiable detection， which can provide technical support for the quality detection and evaluation of TCM， and enrich the research ideas of quality evaluation of TCM.

Quality Evaluation of Fritillariae Cirrhosae Bulbus Based on Traditional Character Analysis / 中国药学杂志

OBJECTIVE: To improve the quality evaluation method of Fritillariae Cirrhosae Bulbus by the means of objective quantization of appearance character. METHODS: The height and diameter were measured by a vernier caliper. The color of powder was determined by a colorimeter based on the human eye observation. The content of total alkaloids was measured by the ultraviolet spectroscopy. Some correlation analysis between the content of total alkaloids and height, diameter, and △E value were evaluated by a SPSS software, respectively. RESULTS: There were a very significant negative correlation (P0.01) between height and the total alkaloids, a negative correlation between diameter and the total alkaloids, and no significant correlation between △E value and the total alkaloids. CONCLUSION: Because the content of total alkaloids could reflect the quality of Fritillariae Cirrhosae Bulbus objectively, these RESULTS: of this study provide a scientific basis for the traditional ideas of “the smaller the best” and “the broken Fritillariae Cirrhosae Bulbus could not be used as medicine” and affirm the traditional processing technology that “should not be washed by water”. Songbei was the best Fritillariae Cirrhosae Bulbus of them all.

Research progress on wild source plant resources distribution and conservation of Fritillariae Cirrhosae Bulbus / 中草药

Genuine medicinal materials-Fritillariae Cirrhosae Bulbus and its active components are widely used in clinical medicine in China and even in many countries in the world because of their good medicinal effect. However, with the increase of the demand for the resources of Fritillariae Cirrhosae Bulbus, and the low yield of itself, the small number of seeds under natural conditions, the extremely low germination rate and survival rate and so on, the wild resources of Fritillariae Cirrhosae Bulbus are endangered. This paper summarized researches of several aspects like herbal research of Fritillariae Cirrhosae Bulbus, distribution of wild source plant resources and its influencing factors, conservation measures, and using endophyte to extract Fritillaria alkaloids, based on the articles published by China National Knowledge Internet and Web of Science. Hoping to provide some development ideas for the protection of wild source plant resources of Fritillariae Cirrhosae Bulbus from the perspectives of traditional conservation measures and active products extracted by microbial vigorously developed at this stage, so as to realize the balance of supply and demand of Fritillariae Cirrhosae Bulbus as soon as possible.

A rapid identification of authenticity and specifications of Chinese medicine Fritillariae Cirrhosae Bulbus based on E-eye technology / 中国中药杂志

The quality of traditional Chinese medicine tablets is correlated with clinical efficacy and drug safety, and plays a great role in promoting the development of traditional Chinese medicine. However, the existing traditional artificial identification and modern instrument detection in terms of accuracy and timeliness have both advantages and disadvantages. Therefore, how to quickly and accurately identify the quality of traditional Chinese medicine tablets has become a high-profile issue. The purpose of this paper is to explore the feasibility of the application of electronic eye technology in the study of rapid identification of traditional Chinese medicine quality. A total of 80 batches of samples were collected and tested by Fritillariae Cirrhosae Bulbus for traditional empirical identification(M_1) and modern pharmacopeia(M_2). The optical data was collected from electronic eyes, and the chemical metrology was used to establish suitable discrimination models(M_3). Four authenticity and commodity specification models, namely identification analysis(DA), minimum bidirectional support vector machine(LS-SVM), partial minimum two-multiplier analysis(PLS-DA), main component analysis identification analysis(PCA-DA), were established, respectively. The accuracies of the authenticity identification models were 82.5%, 90.0%, 96.2% and 93.8%, while the accuracies of the commodity specification identification models were 89.3%, 96.0%, 90.7% and 97.3%, respectively. The models were well judged, the authenticity identification was based on the final identification model of PLS-DA, and the commodity specification was based on the final identification model of PCA-DA. There was no significant difference between its accuracy and M_1, and the time of determination was much shorter than M_2(P<0.01). Therefore, electronic-eye technology could be used for the rapid identification of the quality of Fritillariae Cirrhosae Bulbus.

Phylogenetic relationship analysis of common medicinal submetals and multiple real-time fluorescence PCR identification of Fritillariae Cirrhosae Bulbus / 中草药

Objective To investigate the feasibility of multiple real-time PCR for the detection of Fritillariae Cirrhosae Bulbus and adulterants. Methods Based on the analysis of interspecies variation, genetic distance and phylogenetic relationship of ITS, psbA-trnH, rbcL and matK gene sequences, the genes with fast evolution rate, big interspecies variation and small intraspecies variation were selected as target genes. Fritillariae Cirrhosae Bulbus and adulterants specific primers and Taqman probes were designed to establish a multiplex real-time PCR assay. Methods were evaluated by comparison of specificity, sensitivity and mixed sample detection and sequencing. Results The ITS and psbA-trnH mutations were higher than rbcL and matK, and rbcL and matK were significantly lower than ITS and psbA-trnH genes by genetic distance analysis. And the sensitivity of the establish multiple real-time PCR using ITS as the target gene was 0.01 ng. Four samples of adulterants were detected in 18 samples, and the results were consistent with the results of NJ tree clustering analysis. Conclusion Based on the IIS region sequence as the target gene to establish multiple real-time fluorescence PCR detection method can successfully identify Fritillariae Cirrhosae Bulbus and its counterfeit goods, which provides a new basis for the authenticity of identification.

Application of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis in Identification of Fritillariae Cirrhosae Bulbus / 中国药学杂志

OBJECTIVE: To identify 70 samples of Fritillariae Cirrhosae Bulbus from 16 cities such as Beijing, Tianjin, Changchun,Jilin and so on by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis kit.METHODS: The genomic DNA of Fritillariae Cirrhosae Bulbus was extracted by kit method, and PCR reaction and RFLP identification were carried out. The method of the Chinese Pharmacopoeia of 2015 was used for comparison and re-examination.RESULTS: The purity of the genomic DNA of Fritillariae Cirrhosae Bulbus was very good, the OD260/OD280 value was between 1.90-2.1, and the concentration could reach the requirement of PCR. Seventy samples were tested, of which 37 were positive and 33 were counterfeit, and the false rate was 47.1%.CONCLUSION: The purity and concentration of the nucleic acid meet the requirements of PCR and RFLP. The test results of the samples from 16 cities show that the counterfeit rate of Fritillariae Cirrhosae Bulbus is high in the market. Relevant government departments should strengthen the quality management of the traditional Chinese medicine market.

Effects of Fritillariae Cirrhosae Bulbus on Airway Inflammation and ERK/MAPK Signal Pathway in Asthma Model Mice / 中国药房

OBJECTIVE: To investigate the effects of Fritillariae cirrhosae bulbus on airway inflammation and ERK/MAPK signal pathway of asthma model mice, and to explore its possible mechanism of the treatment of asthma. METHODS: The asthma model was induced by egg albumin. A total of 40 model mice were randomly divided into model group (0. 5％ carboxymethyl cellulose, intragastric administration), positive control group (0. 5 mg/kg dexamethasone, intraperitoneal injection), Fritillariae cirrhosae bulbus low-dose and high-dose groups (9. 0, 18. 0 mg/kg, intragastric administration), with 10 mice in each group. Other 10 normal mice were included in normal group (0. 5％ carboxymethyl cellulose, intragastric administration). They were given medicine once a day for consecutive 28 d. After medication, the number of total cells and differential cells (neutrophils, macrophages, lymphocytes and eosinophils) in bronchoalveolar lavage fluid (BALF) of mice were counted. The pathological morphology of bronchial smooth muscle in mice was observed under light microscope, and the inflammatory score was scored; the activities of ERK, phosphorylated ERK (p-ERK), p38 MAPK and phosphorylated p38 MAPK (p-p38 MAPK) were measured by ELISA. The protein expression of ERK, p-ERK, p38 MAPK and p-p38 MAPK in lung tissue were determined by Western blot assay. mRNA expression of ERK and p38 MAPK were determined by real time fluorescent quantitative PCR. RESULTS: Compared with normal group, the number of total cells and differential cells in BALF of mice, inflammation score, the activities of p-ERK and p-p38 MAPK in lung tissues were increased significantly of mice in model group (P<0. 01); the protein expression of p-ERK and p-p38 MAPK, mRNA expression of ERK and p38 MAPK were increased significantly in lung tissue of mice in model group (P<0. 01). Compared with model group, above indexes of treatment groups were all improved significantly (P<0. 05 or P<0. 01). CONCLUSIONS: Fritillariae Cirrhosae Bulbus can improve airway inflammation in asthma model mice, the mechanisms of which may be related to inhibiting the activation of ERK/MAPK signal pathway.

Effects of Fritillariae Cirrhosae Bulbus on MMP-2,MMP-9 and TIMP-1 in murine asthma model / 中国中药杂志

To investigate the effects of Fritillariae Cirrhosae Bulbus on airway remodeling and matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9), tissue inhibitor-1 of metalloproteinase(TIMP-1) of a murine asthma model, and explore its mechanism in treatment of asthma. BALB/C murines were randomly divided into the normal group, model group, high dose group, low dose group, and positive control group. Except for the normal group, all the other groups received ovalbumin(OVA) to establish murine asthma model. After successful modeling, the murines in high dose group and low dose group were orally administered with Fritillariae Cirrhosae Bulbus powder at the dose of 18.0 mg•kg⁻¹ and 9.0 mg•kg⁻¹, respectively; the murines in positive control group were injected intraperitoneally with dexamethasone at the dose of 0.5 mg•kg⁻¹; while the murines in normal group and the model group were orally administered with the same volume of normal saline. All the drugs were given to murines per day for 28 d. The variations of airway responsiveness, variations of the total cell count and leukocyte differential count in bronchoalveolar lavage fluid(BALF), and the variations of thicknesses of bronchial wall and airway smooth muscle of each group were observed. The levels of MMP-2, MMP-9 and TIMP-1 were measured by ELISA; and the expression levels of MMP-2, MMP-9 and TIMP-1 mRNA were detected by RT-PCR. The results showed that as compared with the normal group, the airway responsiveness, the count of total cells, neutrophils, macrophage, lymphocytes, eosinophils in BALF, and the thicknesses of bronchial wall and airway smooth muscle were increased significantly in the model group(P<0.01); as compared with the model group, the above indicators were decreased significantly in the high dose group, low dose group and positive control group (P<0.05 or P<0.01). As compared with the normal group, the levels and expressions of MMP-2, MMP-9 and TIMP-1 mRNA were increased significantly in the model group(P<0.01); while as compared with the model group, these levels were decreased significantly in the high dose group, low dose group and positive control group(P<0.01). In conclusion, Fritillariae Cirrhosae Bulbus can improve airway remodeling in a murine asthma model, and its mechanisms may be related to down-regulating MMP-2, MMP-9 and TIMP-1 levels.

Functional production regionalization for Fritillariae Cirrhosae Bulbus based on growth and quality suitability assessment / 中国中药杂志

The major contributing factors for growth of endangered medicinal plants of Fritillariae Cirrhosae Bulbus were screened on the GIS platform by using the MaxEnt model, and spatial distribution data of the medicine quality suitability were generated by geostatistics interpolation based on reported measured data of ecology and quality suitability assessment. On this basis, a functional production cultivation regionalization with high feasibility and operability were formatted for protection, wild monitoring, and cultivation of this plant by fuzzy superposition of spatial suitability data of ecology and quality, as well as integrated with land use and cover data. Therefore, a novel assessment and regionalization method were presented for ecology, growth and quality suitability of the Chinese traditional medicinal plants. This method is expected to overcome shortage of traditional regionalization methods difficult to distinguish the contribution of ecological factors and quality factors, which provide an innovative theory and methodology for regionalization, and is helpful to practical application of wild resource protection, monitoring, and commercialization cultivation for traditional Chinese medicinal plants.

Application of Real-time Fluorescent Quantitative PCR in the Identification of Fritillariae Cirrhosae Bulbus / 中国药师

Objective:To investigate the feasibility of real-time fluorescence quantitative PCR in the identification of Fritillariae Cirrhosae Bulbus.Methods:The DNA of samples was extracted by magnetic beads ,the primers were amplified by real-time fluores-cence quantitative PCR , and the Cq value and amplification curve were used to determine the samples .Results:The Cq values for five batches of Fritillariae Cirrhosae Bulbus were lower than 30, and the curve had obvious growth period .No Cq value was shown for Fritil-lariae Ussuriensis Bulbus , Fritillariae Thunbergii Bulbus and Bolbostemmatis Rhizoma with straight line curves .Conclusion:The meth-od is simple,feasible and effective in the identification of Bulbus Fritillariae Cirrhosae with high accuracy and good reproducibility .

Quality analysis of Fritillariae Cirrhosae Bulbus / 中国药学杂志

OBJECTIVE: To analyze the quality of crude drug materials and decoction pieces of Fritillariae Cirrhosae Bulbus by the national wide quality surveillance of 72 batches of samples in 2013, and study the suitability of polymerase chain reaction-restriction enzymes length polymorphism (PCR-RFLP) tests. METHODS: The samples from different origins were identified and detected according to the standard of Chinese Pharmacopoeia. RESULTS: Seventeen batches of samples did not meet the requirements, resulting in a disqualification rate of 25.4%. CONCLUSION: Further measures should be taken to improve the quality standard and strengthen the quality surveillance of Fritillariae Cirrhosae Bulbus and other species of Chinese material medica.

Curative effect comparison of Fritillariae Cirrhosae Bulbus in different species on recurrent asthma in mice / 中草药

Objective: To compare the curative effect of Fritillariae Cirrhosae Bulbus (FCB) in six species from five origins on recurrent asthma in mice. Methods: Eighty one female Kunming mice were randomly divided into nine groups with equal weights, such as model, control, Dexamethasone (DEX, positive control), and six FCB groups. The mice in the model, DEX, and FCB groups were sc injected with ovalbumin (OVA) on days 1, 7, and 14, then challenged with the aerosols of normal saline contained OVA on days 22-28. The mice in DEX and FCB groups were ig administered with DEX (0.6 mg/kg) and FCB (830 mg/kg) respectively for 28 d, and the mice in the control group were given 0.1% carboxymethyl cellulose. The surface tension of bronchoalveolar lavage was measured, the lung tissues were taken for hematoxylin-eosin (HE) staining, and the tracheal stenosis and volume difference of pulmonary alveolus were analyzed by the microphotograph. Results: Compared with the control group, the tracheal stenosis, surface tension, and volume difference of pulmonary alveolus were increased obviously in the model group (P < 0.01). The pathological section revealed vessel wall thickening in bronchiole, inflammatory cell infiltrating, gland hyperplasia, and mucus hypersecretion, but FCB could reverse these pathological changes (P < 0.01). Each FCB had its feature to cure asthma. F. unibracteata and F. delavayi had the most potency on increasing alveolar surfactant; F. unibracteata and F. taipaiensis had the most potency on attenuating tracheal stenosis; F. unibracteata and F. przewalskii had the most potency on relieving the inflammation; F. unibracteata and F. taipaiensis had the most potency on reducing glandular hyperplasia or intimal thickening. Each FCB had its feature to cure asthma, but F. unibracteata was the best. Conclusion: FCB has the best potency to prevent and cure the recurrent asthma in mice, but each FCB has its feature. The diversity of FCB species should be protected.