Wenjuan Zhang

Background: Fritillaria hupehensis (HBBM) is frequently found as an adulterant in Fritillaria thunbergii Miq. (ZBM) owing to its similar appearance and lower price, resulting in risk of unstable medicinal effects of ZBM. Methods: A highly specific TaqMan real-time quantitative polymerase chain reaction (qPCR) method was established to identify HBBM from ZBM based on the 47 bp amplicon located on the internal transcribed spacer region of the nuclear ribosomal DNA. Results: The qPCR assay developed herein could specifically distinguish ZBM from HBBM. The sensitivity study showed that the detectable DNA template concentration of HBBM for this assay was 0.001 ng/μL. The efficiency of the optimized qPCR assay was evaluated to be 90.2% (R ² = 0.9990, slope = −3.581). A standard-curve-based quantification method was established to determine the proportion of HBBM adulteration in a binary mixture with ZBM, and the equation of the standard curve was determined as y = −3.192x + 18.316 with R ² = 0.9949. This quantification approach was evaluated using the ΔCt parameter (difference in Ct values between sample and reference standard); furthermore, the test accuracy was calculated using the recovery rate that ranged from 81.60% to 113.85% for mixed powder samples containing different proportions of HBBM (1%, 5%, 25%, 50%, and 100%). The short amplicon of length 47 bp ensured efficient detection and accurate quantification of dried decoction pieces. Conclusion: The TaqMan real-time qPCR assay developed in this study has high efficiency, sensitivity, and specificity; this method is applicable for not only authentication of ZBM and HBBM but also quantitative detection of a binary mixture or adulterated botanical drugs. Furthermore, it provides a reference for qualitative control of other traditional Chinese medicines.

Natural compounds have experienced increasing clinical application, but their association with rapid-onset anaphylactoid reactions (ARs) present a significant challenge to their safe use. These ARs, clinically resembling Type I hypersensitivity, are non-IgE-mediated and involve direct mast cell activation, primarily through the human Mas-related G protein-coupled receptor X2 (MRGPRX2). We computationally screened a natural compound library for MRGPRX2 activation. A human MRGPRX2-expressing cell model was established. Cell viability assays (0–80 μM) were performed to determine appropriate drug concentrations. Compared to the controls, Baohuoside I (10 μM), along with Kaempferol-3-O-rutinoside, Epigallocatechin gallate (EGCG), Isochlorogenic Acid B, Baicalin, Andrographolide, Isorhamnetin, and Dehydroandrographolide (all at 20 μM), significantly increased intracellular calcium flux (p < 0.05) and boosted tryptase and β-hexosaminidase secretion (ELISA) (p < 0.05) in mast cells. Furthermore, the degranulation induced by these compounds was inhibited by the MRGPRX2 inhibitor Z3578 at 20 μM. Neutral red staining was employed to observe cellular morphological changes. Specific compounds capable of mediating ARs through MRGPRX2 activation on mast cells were identified. This contributes to safer and more effective drug use by elucidating the potential triggers of ARs.

Due to morphological similarities, adulterants are frequently substituted for Qingfengteng (QFT, Sinomenium acutum) in regional markets. This study developed a TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay targeting a 57-base pair (bp) fragment of the internal transcribed spacer 2 (ITS2) region for the specific detection of QFT. The method was validated using a diverse set of samples, including: (1) 19 batches of QFT and 8 batches of Beidougen (BDG, Menispermum dauricum), comprising both medicinal materials and decoction pieces; (2) 5 batches each of decoction pieces from Qingsheteng (QST, Periploca calophylla), Jishiteng (JST, Paederia foetida), Kuanjinteng (KJT, Tinospora sinensis), and Huibeiqingfengteng (HBQFT, Sabia discolor); (3) 6 batches of commercial QFT-containing tablets (with different batch numbers) and 6 batches of laboratory-prepared QFT aqueous decoctions (with different decocting time). Distinct cycle threshold (Ct) values and amplification curves unambiguously differentiated QFT from all adulterants. An external standard-based quantification approach was established to detect adulteration with BDG, the morphologically and genetically most similar adulterant. Recovery rates ranged from 81.79 to 102.38% in herbal mixed powders spiked with 1%, 5%, 50%, and 100% BDG. The method reliably detected QFT in processed tablets and freeze-dried decoctions, demonstrating high tolerance to deoxyribonucleic acid (DNA) degradation. This qPCR assay enables specific and quantitative detection of QFT in dried and processed samples using short amplicons (57 bp), thereby supporting quality control throughout the herbal production chain.

Fritillariae Cirrhosae Bulbus (FCB) is a well-known and precious traditional Chinese medicine with a medicinal history spanning thousands of years. In recent years, it has been reported that fungal and mycotoxin contamination influenced the safety and quality of FCB. It is essential to systematically study the fungal community for the early warning of fungal and mycotoxin contamination in this herb. A total of 15 FCB samples were collected from five provinces in China, and the fungal communities in the FCB samples were analyzed via amplifying the internal transcribed spacer 2 region through the Illumina Miseq PE300 platform. Furthermore, we compared the differences in fungal community in five groups based on collection areas. Results showed that Ascomycota (41.58–99.66%) and Mucoromycota (0–57.42%) were dominant at the phylum level. Eurotiomycetes (8.49–63.93%), Eurotiales (8.49–63.53%), and Aspergillaceae (8.49–63.51%) were the most abundant at the class, order, and family levels. Aspergillus (8.49–63.41%), Rhizopus (0–57.42%), Fusarium (0–22.81%), Cladosporium (0.16–9.14%), and Alternaria (0.06–17.95%) were the main genera in FCB samples. A total of 34 fungal taxa were identified at the species level, including five potentially toxigenic fungi namely Penicillium brevicompactum, P. citrinum, P. oxalicum, Trichothecium roseum, and Aspergillus restrictus. The differences in fungal community between the five groups were observed. Our findings provide references for the safe utilization and quality improvement of FCB.