Application of DNA barcoding to the entire traditional Chinese medicine industrial chain: A case study of Rhei Radix et Rhizoma.

BACKGROUND: Safety concerns, caused by complex and unpredictable adulterants, run through the entire industrial chain of traditional Chinese medicines (TCMs). However, the conventional circulation traceability system only focuses on a certain end or link at the back end of the TCM industrial chain, ignoring the integrity of the links cross the entire industrial chain and lacking traceability. In consequence, a strict and rational supervision system is urgently required for the entire industrial chain. HYPOTHESIS/PURPOSE: We hypothesize that DNA barcoding would be a suitable measure for the traceability of adulterants in the entire TCM industrial chain. METHODS: In this study, Rhei Radix et Rhizoma was selected as a model to establish a traceability system for the entire TCM industrial chain. A total of 110 samples, including leaves, seeds, roots, decoction pieces, and traditional Chinese patent medicines (TCPMs), were collected upstream, midstream, and downstream of the entire industrial chain of Rhei Radix et Rhizoma. The ndhF-rpl32 fragment rather than the universal DNA barcodes, which could not distinguish the three original species of Rhei Radix et Rhizoma, was selected as a specific DNA barcode to evaluate the practical application of DNA barcoding in the chain. RESULTS: The results showed that the ndhF-rpl32 fragment in all samples could be amplified and bi-directionally sequenced. Based on the standard operating procedures of DNA barcoding, the ndhF-rpl32 fragment clearly distinguished the seven Rheum species collected upstream of the entire industrial chain. For the samples collected midstream and downstream of the entire industrial chain, 25% of the 36 commercial decoction pieces samples were identified as adulterants, whereas the eight TCPM samples were all derived from genuine Rhei Radix et Rhizoma. CONCLUSIONS: This study shows that DNA barcoding is a powerful and suitable technology that can be applied to trace TCMs in the entire industrial chain, thereby assuring clinical medication safety.

Droplet digital PCR for the identification of plant-derived adulterants in highly processed products.

BACKGROUND: The high sensitivity of droplet digital PCR (ddPCR) contributes to its excellent performance in animal and microorganism identification, but the utilization of ddPCR is limited in plant adulterant identification of highly processed products for which effective methods are lacking. PURPOSE: This study investigated the feasibility of ddPCR in the identification of plant adulterants in Chinese patent medicine (CPM) as groundwork to develop ddPCR assays for other highly processed goods. METHODS: The original plant, processed and highly processed products of Mutong (Akebiae Caulis) and its two adulterants were used to analyze the specificity, sensitivity, and practical performance of the developed singleplex and triplex ddPCR assays. RESULTS: The results revealed that the limit of detection (LOD) and limit of quantification (LOQ) for the selective ddPCR assays developed to identify Mutong and its adulterants were 0.00002 ng/µl and 0.00016 ng/µl, respectively, and that the regression equations representing the relationships between DNA concentration and target copy number all exhibited good linearity. Furthermore, the common adulterant of Mutong in three samples of Longdan Xiegan pills was successfully identified through ddPCR assays and confirmed by Sanger sequencing. CONCLUSION: This work comprehensively revealed the great ability of ddPCR technology in detecting plant adulterants in traditional Chinese medicine (TCM), providing a method for the quality control of highly processed plant products with complex components for commonly used goods.

TaqMan Probe-Based Quantitative Real-Time PCR to Detect Panax notoginseng in Traditional Chinese Patent Medicines.

Background: There has been global concern about the safety and accuracy of traditional Chinese patent medicines (TCPMs). Panax notoginseng, also known as sanqi, is an important constituent of TCPMs. However, identifying the species contained in TCPMs is challenging due to the presence of multiple ingredients and the use of various preparation processes. Objective: To detect P. notoginseng in TCPMs. Methods: A TaqMan probe-based qPCR assay was constructed and validated with DNA extracted from P. notoginseng and adulterants. In total, 75 samples derived from 25 batches of TCPMs were tested using the constructed qPCR method. Results: A TaqMan probe-based qPCR assay targeting P. notoginseng was established. The constructed qPCR assay could specifically discriminate P. notoginseng from Panax ginseng, Panax quinquefolium and Curcuma aromatica Salisb. cv. Wenyujin. The sensitivity study showed that the detectable DNA template concentration of P. notoginseng for this qPCR assay was 0.001 ng/µl. All 75 samples from TCPMs were confirmed to contain P. notoginseng by the qPCR assay. Conclusions: The qPCR method can accurately identify P. notoginseng in TCPMs and is promising as a powerful tool for quality control and market regulation.

Degradation of sulfonamides in aquaculture wastewater by laccase-syringaldehyde mediator system: Response surface optimization, degradation kinetics, and degradation pathway.

As a new type of environmental pollutant, environmental antibiotic residues have attracted widespread attention, and the degradation and removal of antibiotics has become an engaging topic for scholars. In this paper, Novozym 51003 industrialized laccase and syringaldehyde were combined to degrade sulfonamides in aquaculture wastewater. Design Expert10 software was used for multiple regression analysis, and a response surface regression model was established to obtain the optimal degradation parameters. In the actual application, the degradation system could maintain a stable performance within 9 h, and timely supplement of the mediator could achieve a better continuous degradation effect. Low concentrations of heavy metals and organic matter would not significantly affect the degradation performance of the laccase-mediator system, making the degradation system suitable for a wide range of water quality. Enzymatic reaction kinetics demonstrated a strong affinity of sulfadiazine to the substrate. Ten degradation products were speculated using high-resolution mass spectrum based on the mass/charge ratios and the publication results. Four types of possible degradation pathways of sulfadiazine were deduced. This work provides a practical method for the degradation and removal of sulfonamide antibiotics in actual sewage.

Low-dose biochar added to sediment improves water quality and promotes the growth of submerged macrophytes.

Biochar is a good adsorbent for water pollutants. However, the effects of biochar on aquatic organisms are not well understood. In this study, different amounts of biochar (CK, 0 mg/g; T1, 10 mg/g; T2, 30 mg/g) were added to sediment to study changes in water quality and its impact on three submerged macrophytes (Hydrilla verticillata, Vallisneria natans, and Ceratophyllum demersum) and the sediment microbial community. The results indicated that biochar treatments significantly increased the water pH and conductivity. Compared with the initial values, the total phosphorus (P) contents in the water of the CK, T1, and T2 treatments decreased by 78.5%, 95.0%, and 58.3%, respectively, while the total nitrogen contents increased by 26.26%, -5.81%, and 19.70%, respectively. Compared with those in CK, the relative growth rates of H. verticillata, V. natans, and C. demersum in T1 increased by 28.4%, 163.1%, and 61.3%, respectively, while those in T2 showed no significant difference except that the growth rates of H. verticillata decreased by 17.7%. The P contents of the three submerged macrophytes increased with the increase of biochar addition, except that there was no significant difference between T2 and CK for H. verticillata. Biochar treatments reduced the biomass of total microbial, bacterial, and fungal phospholipid fatty acids in the sediment for H. verticillata and V. natans, and they increased fungal: bacterial ratios in the low-dose biochar treatments for V. natans and C. demersum. This study demonstrates that the addition of biochar to sediment significantly increased the pH and conductivity, and decreased total P contents in the water. Low-dose biochar treatments were more beneficial for water quality improvements and the growth of submerged macrophytes than high-dose biochar.

Anthocyanin Profiles in Flowers of Grape Hyacinth.

Grape hyacinth (Muscari spp.) is a popular ornamental bulbous perennial famous for its blue flowers. To understand the chemical basis of the rich blue colors in this plant, anthocyanin profiles of six blue flowering grape hyacinths as well as one pink and one white cultivar were determined using high-performance liquid chromatography and mass spectrometry. Along with two known compounds, eight putative anthocyanins were identified in the tepals of grape hyacinth for the first time. The accumulation and distribution of anthocyanins in the plant showed significant cultivar and flower development specificity. Violet-blue flowers mainly contained simple delphinidin-type anthocyanins bearing one or two methyl-groups but no acyl groups, whereas white and pink flowers synthesised more complex pelargonidin/cyanidin-derivatives with acyl-moieties but no methyl-groups. The results partially reveal why solid blue, orange or red flowers are rare in this plant in nature. In addition, pelargonidin-type anthocyanins were found for the first time in the genus, bringing more opportunities in terms of breeding of flower color in grape hyacinth.