Determination of ITS1 haplotypes of Fritillariae Cirrhosae Bulbus by amplicon sequencing.

BACKGROUND: Fritillariae Cirrhosae Bulbus is an antitussive and expectorant Chinese medicinal material derived from the dried bulbs of six Fritillaria species. In the 2015 edition of the Chinese Pharmacopoeia, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is the officially listed method for their authenfication. Specifically, the ~ 300-bp ITS1 amplicon of only Fritillariae Cirrhosae Bulbus but not other Fritillaria species can be cleaved into two smaller fragments with restriction enzyme SmaI. Considering repeated reported cases of incomplete digestion of ITS1 amplicon, this study aims to investigate the possibility of heterogeneous ITS1 sequences contained in the Fritillariae Cirrhosae Bulbus. METHODS: In this study, ITS1 amplicons of Fritillaria Cirrhosae Bulbus and four other Fritillaria species were sequenced on Illumina platform. We utilised high-throughout amplicon sequencing to determine ITS1 haplotypes and their frequencies in Fritillaria genomes. RESULTS: Our results showed that all six botanical sources of Fritillariae Cirrhosae Bulbus indeed possess ITS1 haplotypes with no SmaI restriction site, and the average percentages of ITS1 reads containing SmaI restriction site ranged from 63.60% to 91.81%. CONCLUSION: Our findings suggest that the incomplete digestion in PCR-RFLP analysis of Fritillariae Cirrhosae Bulbus is caused by the presence of ITS1 haplotypes without SmaI restriction site due to intragenomic heterogeneity.

[UPLC-QDA and machine learning for distinguishing different commodity specifications of Fritillariae Cirrhosae Bulbus and application of data augmentation technology].

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.

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.

Simulation of potential suitable distribution of original species of Fritillariae Cirrhosae Bulbus in China under climate change scenarios.

Fritillariae Cirrhosae Bulbus (FCB) is a famous traditional Chinese medicine, mainly used for relieving cough and resolving phlegm. According to Chinese Pharmacopoeia (2020), the medicine comes from dried bulbs of five species and one variety in Fritillaria. Due to climate change and human disturbance, the wild resources have become critically endangered in recent years. Following three climate change scenarios (SSP1-2.6, SSP2-4.5, and SSP5-8.5) under 2050s and 2070s, geographic information technology (GIS) and maximum entropy model (MaxEnt) were used to simulate the ecological suitability of FCB, a third-grade rare and endangered medicinal plant species. The results showed that the key environmental variables affecting the distribution of FCB were altitude, human activity intensity, and mean temperature of coldest quarter. Under current climate situation, the highly suitable areas were mainly located in the east of Qinghai Tibet Plateau, including Western Sichuan, southeastern Tibet, southern Gansu, Northwestern Yunnan, and Eastern Qinghai, with a total area of 31.47×104 km2, the area within the nature reserve was 7.13×104 km2, indicating that there was a large protection gap. Under the future climate change scenarios, the areas of the highly and poorly suitable areas of FCB showed a decreasing trend, while the areas of the moderately and total suitable areas showed an increasing trend. The geometric center of the total suitable area of the medicine will move to the northwest. The results could provide a strategic guidance for protection,development, and utilization of FCB though its prediction of potential distribution based on the key variables of climate change.

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.

[Nucleosides-based identification model for Fritillariae Cirrhosae Bulbus].

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.

Effects of different harvesting times and processing methods on the quality of cultivated Fritillaria cirrhosa D. Don.

Fritillaria cirrhosa D. Don is the major source plants of traditional Chinese medicine Fritillariae Cirrhosae Bulbus (FCB). Domestication, introduction, and cultivation is an important strategy to alleviate the shortage of endangered medicinal plants of F. cirrhosa. However, until now, the yield and quality changes of FCB in different harvest periods and drying treatments after harvest were not well understood. Therefore, in this paper, we investigated the yield and quality of cultivated F. cirrhosa at different harvest periods and postharvest processing methods. The results showed that dry weight per bulb ranged from 0.8913 to 1.4681 g and reached the highest at the wilting stage. The soluble sugar content ranged from 0.075% to 0.127% and reached the highest at the wilting stage. The content of total alkaloids ranged from 0.088% to 0.218% and reached the highest at the late-flowering stage. The contents of peimisine, sipeimine, peimine, and peiminine were 0.01178%-0.02615%, 0%-0.01713%, 0%-0.00745%, and 0%-0.00621% and reached the highest at the late-flowering period, wilting period, young fruit period, and initial flowering period, respectively. For the two different postharvest processing methods, the contents of total alkaloids and the 16 main characteristic peaks did not exhibit significant differences. Still, the alkaloid contents of the oven drying after washing were slightly higher than the sun drying. In conclusion, the best harvest period is the wilting period of F. cirrhosa, and oven drying after washing is more beneficial to ensure the quality of FCB and improve productivity.

Simultaneous identification of Fritillariae cirrhosae bulbus and its common adulterants in one reaction by multiplex ligation-dependent probe amplification and high-resolution melting curve assay.

Fritillariae cirrhosae bulbus, a well-known and precious medicinal and edible herb in China, causes remarkable effects on swelling and relieving cough, with fewer side effects than other congeneric medicine. It has been subject to various cheaper congeneric adulteration because of its high price and limited production. In this paper, a rapid, high throughput, sensitive and efficient technique was described for simultaneous identification of F. cirrhosae bulbus and its common adulterants by employing multiplex ligation-dependent probe amplification coupled with high-resolution melting (MLPA-HRM) curve assay in their internal transcribed spacer 1 (ITS1) regions. This assay was highly sensitive with a detection limit of 0.19 ng genomic DNA, and highly specific with no cross-reaction with common adulterants. Mixed sample analysis showed as low as 10% adulteration can be detected from F. cirrhosae bulbus in one MLPA-HRM reaction. Overall, the method described in this paper is well suited for detecting adulteration in F. cirrhosae bulbus.

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.

[A rapid identification of authenticity and specifications of Chinese medicine Fritillariae Cirrhosae Bulbus based on E-eye technology].

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.

Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway.

Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory properties. However, the anti-inflammatory effect and its mechanism have not been fully explored. In this study, the anti-inflammatory activitives and the potential mechanisms of five isosteroid alkaloids from F. Cirrhosae Bulbus were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. The pro-inflammatory mediators and cytokines were measured by Griess reagent, ELISA and qRT-PCR. The expression of MAPKs was investigated by western blotting. Treatment with the five isosteroid alkaloids in appropriate concentrations could reduce the production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in supernatant, and suppressed the mRNA expressions of TNF-α and IL-6. Meanwhile, the five isosteroid alkaloids significantly inhibited the phosphorylated activation of mitogen activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). These results demonstrated that isosteroid alkaloids from F. Cirrhosae Bulbus exert anti-inflammatory effects by down-regulating the level of inflammatory mediators via mediation of MAPK phosphorylation in LPS-induced RAW264.7 macrophages, thus could be candidates for the prevention and treatment of inflammatory diseases.

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.

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.

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.

High altitude species, high profits: Can the trade in wild harvested Fritillaria cirrhosa (Liliaceae) be sustained?

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillaria cirrhosa D. Don bulbs contain alkaloids and are one of the most intensively exploited alpine Himalayan medicinal species. In terms of proprietary medicines, our study shows that 210 F. cirrhosa products are offered by 46 suppliers, most of which (44) are situated in China and two in Nepal. A widespread commercial use is as one of the main ingredients in cough syrups. A well known example is "Nin Jiom Pei Pa Koa Herbal Cough & Throat Syrup", which typically contains more F. cirrhosa than any other herbal ingredient in the formulation. The biggest market for F. cirrhosa bulbs is China, where demand exceeds supply of this wild harvested species for use in traditional Chinese medicine (TCM). Cross-border trade from Nepal to China occurs in significant quantities. Bhutan also imports F. cirrhosa bulbs from Nepal. In addition, F. cirrhosa is registered as an active ingredient in traditional herbal medicinal preparations in Australia, Canada, Hong Kong SAR, Malaysia, Republic of Korea, Singapore and Taiwan. There is also an export trade in F. cirrhosa to Europe. Assessing how much F. cirrhosa is traded is complex, however, due to a "look-alike" challenge, as nine Chinese Fritillaria species are traded in Europe (Fritillaria cirrhosa, F. delavayi, F. hupehensis, F. pallidiflora, F. przewalskii, F. thunbergii, F. unibracteata, F. ussuriensis and F. walujewii). AIMS OF THE STUDY: The aims of this review were to assess the scale of the global trade in F. cirrhosa, and to synthesise studies of the impacts of wild harvest on F. cirrhosa populations and on the extent of emerging cultivation initiatives as an alternative to wild harvest. METHODS: Firstly, we reviewed published information on studies on impacts of wild F. cirrhosa harvest from across the geographic range of this species. Secondly, global trade data for F. cirrhosa were analysed. RESULTS: The principal demand for F. cirrhosa bulbs is in China, where hundreds of different companies produce Fritillaria preparations. Trade data also show that in 2013, China exported over 44 tonnes of F. cirrhosa bulbs to Taiwan and 26.7 tonnes to the Republic of Korea. Extensive commercial use and limited wild stocks result in a high price (2000 - 3800 CNY per kg (around US$ 303 -560 per kg in 2017)) for F. cirrhosa bulbs. Prices of cultivated Fritillaria bulbs are much lower (600-680 CNY per kg in 2017) than wild harvested bulbs. But due to very specific growth requirements of F. cirrhosa, cultivation is not yet able to meet total demand. The consequence is continued exploitation of wild stocks. At the same time, however, an increasing proportion of the demand is met by cultivation of alternative Fritillaria species that are easier to grow than F. cirrhosa. The air-dry mass of F. cirrhosa bulbs varies between 0.0917 and 0.1116 g per bulb. This represents 8960 - 10,900 bulbs/kg or 8.9 - 10.9 million bulbs per tonne. Current demand therefore represents billions of bulbs per year. CONCLUSIONS: Demand for F. cirrhosa bulbs, particularly from China, makes this species one of the most intensively harvested alpine Himalayan medicinal bulbs. Although F. cirrhosa is listed as a Class III protected species in China, billions of these tiny, wild harvested bulbs are sold per year. Due to demand exceeding supply, the price of F. cirrhosa bulbs has increased dramatically. Between 2002 and 2017, for example, the price of wild harvested F. cirrhosa bulbs increased over nine-fold, from the equivalent of US$60 in 2002 to US$560 per kg in 2017. To date, cultivation has been unable to meet the entire market demand for F. cirrhosa bulbs, although other Fritillaria species are successfully cultivated on a larger scale.

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.