[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.

[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.

[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.

[Effects of Fritillariae Cirrhosae Bulbus on MMP-2,MMP-9 and TIMP-1 in murine asthma model].

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].

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.

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.

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.

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.

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.

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.

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.

A strategy to identify and quantify closely related adulterant herbal materials by mass spectrometry-based partial least squares regression.

In this study, a new strategy combining mass spectrometric (MS) techniques with partial least squares regression (PLSR) was proposed to identify and quantify closely related adulterant herbal materials. This strategy involved preparation of adulterated samples, data acquisition and establishment of PLSR model. The approach was accurate, sensitive, durable and universal, and validation of the model was done by detecting the presence of Fritillaria Ussuriensis Bulbus in the adulteration of the bulbs of Fritillaria unibracteata. Herein, three different MS techniques, namely wooden-tip electrospray ionization mass spectrometry (wooden-tip ESI/MS), ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and UPLC-triple quadrupole tandem mass spectrometry (UPLC-TQ/MS), were applied to obtain MS profiles for establishing PLSR models. All three models afforded good linearity and good accuracy of prediction, with correlation coefficient of prediction (rp2) of 0.9072, 0.9922 and 0.9904, respectively, and root mean square error of prediction (RMSEP) of 0.1004, 0.0290 and 0.0323, respectively. Thus, this strategy is very promising in tracking the supply chain of herb-based pharmaceutical industry, especially for identifying adulteration of medicinal materials from their closely related herbal species.

MOLECULAR IDENTIFICATION OF ORIGINAL PLANTS OF FRITILLARIAE CIRRHOSAE BULBUS, A TRADTIONAL CHINESE MEDICINE (TCM) USING PLANT DNA BARCODING.

BACKGROUND: DNA barcoding is a widely used tool that enables rapid and accurate identification of species based on standardized DNA regions. MATERIALS AND METHODS: In this study, potential DNA barcodes, namely three plastid regions (rbcL, trnH-psbA and matK) and one nuclear ribosomal internal transcribed spacer (ITS) were adopted for species identification of original plants of Fritillariae Cirrhosae Bulbus. RESULTS: The rbcL and trnH-psbA regions showed better success rate of PCR amplification and DNA sequencing, as well as superior discriminatory ability. On the contrary, ITS region did not possess effective genetic variation and matK was faced with low success rate of sequencing. Combination of multi-loci sequences could improve identification ability of DNA barcoding. The trnH-psbA + rbcL could discriminate 25% - 100% species based on the Blast, Tree-Building and Distance methods. CONCLUSION: The potential DNA barcodes could not completely solving species identification of botanic origins of Fritillariae Cirrhosae Bulbus. In future, we should pay more attention to super-barcoding or specific barcode that enhance ability to discriminate the closely related plants.

Rapid identification of plant materials by wooden-tip electrospray ionization mass spectrometry and a strategy to differentiate the bulbs of Fritillaria.

The counterfeit plant products, especially by using incorrect plant materials in pharmaceutical industry, have become a global problem. The plant materials belonging to closely related species but differing in medicinal properties are difficult to be identified. Here, a novel and generally applicable approach to identify the sources of plant materials was developed, which was based on the use of wooden-tip electrospray ionization mass spectrometry (wooden-tip ESI-MS) and multivariate statistical analysis of unidentified MS features (non-targeted). Using this approach, six officinal species of Fritillariae Cirrhosae Bulbus had been successfully differentiated. In addition, Fritillariae Pallidiflorae Bulbus, a common adulterant of Fritillariae Cirrhosae Bulbus, was also identified by using the strategy reported here. Compared with DNA phylogenetic trees, our approach provided finer resolution in distinguishing the closely related Fritillaria species. By combining wooden-tip ESI-MS and multivariate statistical analysis, a useful method was developed here for rapid identification of the sources of herbs, which showed promising perspectives in tracking the supply chain of pharmaceutical suppliers.