Comprehensive analysis using DNA metabarcoding, SCAR marker based PCR assay, and HPLC unveils the adulteration in Brahmi herbal products.

BACKGROUND: Brahmi is one of the important nootropic botanicals, widely sold in the market, with the name "Brahmi'' being used to describe both Bacopa monnieri and Centella asiatica species. The Brahmi herbal products market is expanding; hence, economically motivated adulteration is highly prevalent. METHODS AND RESULTS: This study aimed to develop DNA-based methods, including SCAR marker-based PCR and metabarcoding, to authenticate Brahmi herbal products and compare these methods with HPLC. These methods have been validated using mock controls (in-house blended formulations). All targeted plant species in mock controls were detected successfully with all three methods, whereas, in market samples, only 22.2%, 55.6%, and 50.0% were found positive for Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively. Metabarcoding can detect the presence of non-labeled plants together with targeted species, which is an advantage over PCR assay or HPLC. CONCLUSION: SCAR marker-based PCR is a rapid and cost-effective method for detecting the presence of B. monnieri and C. asiatica. However, in this study, the success rate of PCR amplification was relatively low because the primers targeted either RAPD or ITS-based SCAR markers. HPLC assay, although an alternative, was unable to detect the presence of other botanicals, just like the SCAR marker-based PCR assay. On the other hand, metabarcoding can be utilized to identify the target plants, even in very small quantities, while also providing simulated identification of other botanicals. This study successfully addressed the need for quality control of Brahmi herbal products and provided the first-time report of DNA metabarcoding for such products.

Linkage of Microbial Parameters with Sediment Physicochemical Properties in Subsurface Fluvial Sediment Deposits of the Mahi River Basin, Western India.

The linkage between sediment physicochemical and microbial parameters within river terrace sedimentary ecosystems of semiarid regions is still in infancy. Here, we investigated microbial enzyme activities involved in C, N, P, and S cycling, and quantitative PCR (qPCR) based gene abundance of two laterally deposited sediment cores (28 and 25 m deep) comprising the Late Quaternary sediments of the Mahi River (Gujarat, India). Gene abundance indicates the presence of a sustainable bacterial population throughout both cores. The stratified subsurface sediments had notable microbial enzyme activities indicating an important role of both cores in biogeochemical cycling. Correlation between microbial and geological parameters revealed that various trace elements, rare earth elements, K2O, P2O5, EC, TDS, and salinity link significantly with microbial parameters. However, the direction and magnitude of the correlation differ in both cores under study. These results emphasize that sediment physicochemical properties influence microbial parameters differently in the laterally deposited subsurface sediments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-021-00998-4.

Evaluation of bacterial strains isolated from Late Quaternary alluvial sediments spanning ~ 28 m in depth for heavy metal tolerance and Cr(VI) removal ability.

Microbial heavy metal tolerance in subsurface samples is indicative of long-term ecotoxicological impact of metals and could also reflect metal contamination of groundwater. However, the heavy metal tolerance characteristics of microbes isolated from subsurface river sediment profiles are still obscure. In the present study, determination of heavy metal tolerance of bacterial strains isolated from two Late Quaternary sediment profiles (~ 28 m and ~25 m deep) located at the Mahi river basin, Western India, was carried out. Identification of bacterial isolates by the 16S rRNA gene sequencing revealed that bacterial isolates affiliated with phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes were dominant in both sediment profiles. Heavy metal tolerance of bacterial strains as determined by plate diffusion assay revealed order of metal tolerance as follows: Hg(II)

Digital PCR: A Tool to Authenticate Herbal Products and Spices.

Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However, poor DNA quality and quantity are the major demerits of conventional PCR and real-time quantitative PCR (qPCR), as herbal products and spices are highly enriched in secondary metabolites such as polyphenolic compounds. The third-generation digital PCR (dPCR) technology is a highly sensitive, accurate, and reliable method to detect target DNA molecules as it is less affected by PCR inhibiting secondary metabolites due to nanopartitions. Therefore, it can be certainly used for the detection of adulteration in herbal formulations. In dPCR, extracted DNA is subjected to get amplification in nanopartitions using target gene primers, the EvaGreen master mix, or fluorescently labeled targeted gene-specific probes. Here, we describe the detection of Carica papaya (CP) adulteration in Piper nigrum (PN) products using species-specific primers. We observed an increase in fluorescence signal as the concentration of target DNA increased in PN-CP blended formulations (mock controls). Using species-specific primers, we successfully demonstrated the use of dPCR in the authentication of medicinal botanicals.

A combined approach of DNA metabarcoding collectively enhances the detection efficiency of medicinal plants in single and polyherbal formulations.

Introduction: Empirical research has refined traditional herbal medicinal systems. The traditional market is expanding globally, but inadequate regulatory guidelines, taxonomic knowledge, and resources are causing herbal product adulteration. With the widespread adoption of barcoding and next-generation sequencing, metabarcoding is emerging as a potential tool for detecting labeled and unlabeled plant species in herbal products. Methods: This study validated newly designed rbcL and ITS2 metabarcode primers for metabarcoding using in-house mock controls of medicinal plant gDNA pools and biomass pools. The applicability of the multi-barcode sequencing approach was evaluated on 17 single drugs and 15 polyherbal formulations procured from the Indian market. Results: The rbcL metabarcode demonstrated 86.7% and 71.7% detection efficiencies in gDNA plant pools and biomass mock controls, respectively, while the ITS2 metabarcode demonstrated 82.2% and 69.4%. In the gDNA plant pool and biomass pool mock controls, the cumulative detection efficiency increased by 100% and 90%, respectively. A 79% cumulative detection efficiency of both metabarcodes was observed in single drugs, while 76.3% was observed in polyherbal formulations. An average fidelity of 83.6% was observed for targeted plant species present within mock controls and in herbal formulations. Discussion: In the present study, we achieved increasing cumulative detection efficiency by combining the high universality of the rbcL locus with the high-resolution power of the ITS2 locus in medicinal plants, which shows applicability of multilocus strategies in metabarcoding as a potential tool for the Pharmacovigilance of labeled and unlabeled plant species in herbal formulations.