A transcriptome-based association study of growth, wood quality, and oleoresin traits in a slash pine breeding population.

Slash pine (Pinus elliottii Engelm.) is an important timber and resin species in the United States, China, Brazil and other countries. Understanding the genetic basis of these traits will accelerate its breeding progress. We carried out a genome-wide association study (GWAS), transcriptome-wide association study (TWAS) and weighted gene co-expression network analysis (WGCNA) for growth, wood quality, and oleoresin traits using 240 unrelated individuals from a Chinese slash pine breeding population. We developed high quality 53,229 single nucleotide polymorphisms (SNPs). Our analysis reveals three main results: (1) the Chinese breeding population can be divided into three genetic groups with a mean inbreeding coefficient of 0.137; (2) 32 SNPs significantly were associated with growth and oleoresin traits, accounting for the phenotypic variance ranging from 12.3% to 21.8% and from 10.6% to 16.7%, respectively; and (3) six genes encoding PeTLP, PeAP2/ERF, PePUP9, PeSLP, PeHSP, and PeOCT1 proteins were identified and validated by quantitative real time polymerase chain reaction for their association with growth and oleoresin traits. These results could be useful for tree breeding and functional studies in advanced slash pine breeding program.

Halobaculum halophilum sp. nov. and Halobaculum salinum sp. nov., isolated from salt lake and saline soil.

Two halophilic archaeal strains, Gai3-2T and NJ-3-1T, were isolated from salt lake and saline soil samples, respectively, collected in PR China. The 16S rRNA gene sequences of the two strains were 97.5% similar to each other. Strains Gai3-2T and NJ-3-1T had the highest sequence similarities to 'Halobonum tyrrellense' G22 (96.7 and 97.8%, respectively), and displayed similarities of 91.5-93.5% and 92.3-94.7%, respectively, to Halobaculum members. Phylogenetic analysis revealed that the two strains formed different branches and clustered tightly with 'H. tyrrellense' G22 and Halobaculum members. The average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH) and amino acid identity (AAI) values between the two strains were 83.1, 26.9 and 77.9%, respectively, much lower than the threshold values proposed as a species boundary. These values between the two strains and 'H. tyrrellense' G22 (ANI 77.9-78.2%, isDDH 22.5-22.6% and AAI 68.8-69.3%) and Halobaculum members (ANI 77.53-77.63%, isDDH 21.8-22.3% and AAI 68.4-69.4%) were almost identical, and much lower than the recommended threshold values for species delimitation. These results suggested that strains Gai3-2T and NJ-3-1T represent two novel species of Halobaculum. Based on phenotypic, chemotaxonomic and phylogenetic properties, strains Gai3-2T (=CGMCC 1.16080T=JCM 33550T) and NJ-3-1T (=CGMCC 1.16040T=JCM 33552T) represent two novel species of the genus Halobaculum, for which the name Halobaculum halophilum sp. nov. and Halobaculum salinum sp. nov. are proposed.

Coupled in vitro transcription/translation based on wheat germ extract for efficient expression from PCR-generated templates in short-time batch reactions.

We successfully constructed a coupled in vitro transcription/translation (cIVTT) system based on wheat germ extract (WGE) for efficient expression from PCR-generated DNA templates in short-time (∼3-h) batch reactions. The productivity of this system under optimized conditions was 85 µg (2.8 nmol) per 1 mL of reaction solution (corresponding to 425 µg per 1 mL of WGE), which was about 9-fold higher than that by the conventional batch method using mRNA as a template. The DNA template concentration required for efficient cIVTT was as low as 2.5 nM, which is much lower than those required for other eukaryotic cIVTT systems to maximize their productivity (30-50 nM). The productivity of the present system with a 2.5 nM template was 80-fold and 4-fold higher than that of a commercially available WGE-based cIVTT system with a 2.5 nM and a 40 nM template, respectively. In addition, the present system functioned well in a liposome (i.e., in an artificial cell) without a loss of productivity. Given that WGE-based systems have the advantage of being suitable for the expression of a broad range of proteins, the present cIVTT system is expected to be widely used in future cell-free synthetic biology.

Association of Genetic Structure and Diversity in Iranian Wild Germplasms of Mentha longifolia L. Based on Phenotypical, Biochemical, and Molecular Markers.

Mentha longifolia L. is well-known to be one of the most pervasive wild-growing species of the Lamiaceae family, which has extensive beneficial properties in the fields of pharmacology and biological products. In the present study, the correlation between Inter-simple sequence repeat (ISSR) markers and morpho-chemical parameters of twenty different M. longifolia accessions (MLACs) were assessed. The geographic information system (GIS) has been employed to interpret the original habitat of the accessions in Iran. ISSR analysis indicated a remarkable difference in the studied accessions, segregated them into three main groups, constructed by an unweighted pair-group method with arithmetic (UPGMA) and principal coordinate analysis (PCoA). A total of 89 bands were generated by 12 ISSR primers, among which 82 (91.97 %) of them were polymorphic. The cluster analysis based on agro-morphological data scattered MLACs into two main groups. The essential oils (EOs) were analyzed through GC/FID/MS, and four chemotypes were characterized according to the major constituents. Pulegone ranged from 0.17 to 69.50 % was the main oil constituent with the highest content. Also, HPLC-PDA was employed to identify and to quantify the phenolic compounds in the MeOH extracts of MLACs. Heatmap cluster based on phenolic compounds produced three main categories of accessions. The components identified in the extracts were rosmarinic acid, rutin, vanillic acid, ferulic acid, chlorogenic acid, caffeic acid, 3,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and p-coumaric acid, which among them rosmarinic acid (RA) varied from 39.16 to 261.55 mg/100 g (DW) as a predominant constituent. Subsequently, multiple regression analyses between ISSR fragments and morpho-chemical data illustrated considerable relationships in the plant materials. The high variation and correlation observed in metabolic and phenotypic traits of MLACs establish an adequate source to conduct reserves conservation programs.

Chemical and Genetic Diversity of Ligularia kanaitzensis in the Hengduan Mountains Area. Chemical Relationship with L. subspicata.

Root chemicals and the sequences of the internal transcribed spacers (ITSs) were analyzed for 9 Ligularia kanaitzensis and 3 L. subspicata samples collected in northwestern Yunnan and southwestern Sichuan, China. Subspicatins A and C were isolated from two L. kanaitzensis samples. Introgression of genes responsible for these compounds from L. subspicata was suggested by their strong connection with L. subspicata/L. lamarum and the geographical proximity of the samples to L. subspicata. DNA analysis of a set of 27 L. kanaitzensis samples including those analyzed previously showed that they belong to two clades, designated A and B. Together with the presence/absence of furanoeremophilane, the 27 samples were sorted into three groups: clade A/furan, clade B/furan, and clade B/non-furan. The ancestral plant presumably belonged to clade B/non-furan, because furanoeremophilanes are biosynthesized from eremophilan-8-ones. 1ß-Angeloyloxyfukinone, a likely intermediate between fukinone and subspicatin C, was isolated for the first time. This finding allowed us to propose plausible biosynthetic pathways of subspicatins A and C.

A transcriptomic variation map provides insights into the genetic basis of Pinus massoniana Lamb. evolution and the association with oleoresin yield.

BACKGROUND: Masson pine (Pinus massoniana Lamb.), the dominant native coniferous species in southern China, is commercially important for supplying timber and oleoresin. However, knowledge of the genetic variability of masson pine germplasm is still limited. In this study, the genetic diversity and population structure of masson pine germplasm were assessed using 204 wild accessions from 10 main distribution regions using 94,194 core single-nucleotide polymorphisms (SNPs) obtained from transcriptome sequencing data. RESULTS: The average expected heterozygosity was 0.2724, implying abundant genetic diversity within masson pine germplasm. Analysis of molecular variance (AMOVA) revealed that 3.29% of the variation was sourced from genetic differentiation. Structure analysis identified two geographically distinct groups. Discriminant analysis of principal components (DAPC) showed that one of those groups was further divided into two clusters. Sichuan and Chongqing provenance is the geographical origin, which diffused outward along two different lines. Oleoresin yield is reflected in the evolution of the two groups, and exhibits two different trends along the two lines of diffusion. The oleoresin yield may be associated with the genes of chitinase, CYP720B, cytochrome P450, ABC transporter, and AP2/ethylene-responsive transcription factor (ERF) based on SNPs and expression. CONCLUSIONS: SNP markers from transcriptome sequencing are highly capable of evaluating genetic diversity within different species, as well as the genetic control of objective traits. The functions of these genes will be verified in future studies, and those genes strongly associated with oleoresin yield will be used to improve yields by means of early genotype selection and genetic engineering.

Transcriptomic and metabolomic analyses reveal several critical metabolic pathways and candidate genes involved in resin biosynthesis in Pinus massoniana.

Pine resin, which typically consists of terpenoids, is a natural product used in various industrial applications. Oleoresin can be obtained from the xylem tissue by wounding the stem bark. Pinus massoniana (masson pine) is an important resin-tapping tree species that originated in southern China. Masson pines with different genetic backgrounds typically have different resin-yielding capacities (RYCs). However, the mechanisms underlying high resin yield in masson pines are unclear. The aim of this study was to identify the possible genetic regulation pathways and functional genes that influence the resin yield. In this study, we conducted transcriptomic and metabolomic studies of masson pine secondary xylem with high, medium, and low RYCs. A total of 230,068 unigenes and 3894 metabolites were identified from the tissue of the secondary xylem. Several differentially expressed regulation factors, including WRKY, bHLH, and ERF, and functional genes such as PKc and LRR-RLKs, were identified among these masson pines. The Kyoto Encyclopedia of Genes and Genomes pathways were mainly focused on diterpenoid biosynthesis, plant hormone signal transduction, and ABC transporters. Furthermore, integration of the transcriptomic and metabolomic data indicated that the PKc- and LRR-RLK-related regulatory and metabolic pathways may play critical roles in the biosynthesis of terpenoids. These above results improve our understanding of the biosynthesis mechanism of oleoresin in P. massoniana and facilitate further research work into the functional analysis of these candidate genes.

To Elucidate the Inhibition of Excessive Autophagy of Rhodiola crenulata on Exhaustive Exercise-Induced Skeletal Muscle Injury by Combined Network Pharmacology and Molecular Docking.

Autophagy can remodel skeletal muscle in response to exercise. However, excessive autophagy can have adverse effects on skeletal muscle. Although Rhodiola crenulata (R. crenulata) is thought to regulate autophagy, its active ingredients and mechanisms of action remain unclear. In this study, molecular docking and network pharmacology were used to screen for autophagy-related targets of R. crenulata. Subsequently, protein-protein interaction (PPI) analysis was used to find the relationships between the inverse docking targets and autophagy-related targets and therefore highlight the key targets. And then the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database was recruited to explain the functions and enrichment pathways of the target proteins. Finally, the potential targets were validated by immunohistochemistry of a mouse model of exhaustive exercise-induced skeletal muscle injury. We found a network of 15 major constituents of R. crenulata with 30 autophagy-related and 105 inverse-docking targets by molecular docking and network pharmacology. The results of PPI analysis indicated that 16 inverse-docking targets interacted 8 autophagy-related proteins. Further pathway analysis showed that R. crenulata could regulate exercise-induced skeletal muscle autophagy through mammalian target of rapamycin (mTOR), AMP activated protein kinase (AMPK) and Forkhead box protein O (FoxO). The results of our animal experiments indicated that R. crenulata could suppress the expression of Ubiquitin-like protein ATG12 (ATG12), Beclin-1 (BECN1), and Serine/threonine-protein kinase ULK1 (ULK1), while increasing the expression of MTOR, NAD-dependent protein deacetylase sirtuin-1 (SIRT1), and Microtubule-associated protein tau (MAPT). In conclusion, this study demonstrated that R. crenulata may protect skeletal muscle injury induced by exhaustive exercise via regulating the mTOR, AMPK, and FoxO singling pathway.

Mutation of the start codon to enhance Cripavirus internal ribosome entry site-mediated translation in a wheat germ extract.

Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5' terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3-4-fold higher than that of its wild type, and also 3-4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.

Structure-guided identification of function: role of Capsicum annuum vicilin during oxidative stress.

Proteins belonging to cupin superfamily are known to have critical and diverse physiological functions. However, 7S globulins family, which is also a part of cupin superfamily, were undermined as only seed storage proteins. Structure determination of native protein - Vic_CAPAN from Capsicum annuum - was carried out, and its physiological functions were explored after purifying the protein by ammonium sulfate precipitation followed by size exclusion chromatography. The crystal structure of vicilin determined at 2.16 Šresolution revealed two monomers per asymmetric unit which are juxtaposed orthogonal with each other. Vic_CAPAN consists predominately of ß-sheets that folds to form a ß-barrel structure commonly called cupin fold. Each monomer of Vic_CAPAN consists of two cupin fold domains, N-terminal and C-terminal, which accommodate two different ligands. A bound ligand was identified at the C-terminal cupin fold in the site presumably conserved for metabolites in the crystal structure. The ligand was confirmed to be salicylic acid through mass spectrometric analysis. A copper-binding site was further observed near the conserved ligand-binding pocket, suggesting possible superoxide dismutase activity of Vic_CAPAN which was subsequently confirmed biochemically. Vicilins from other sources did not exhibit this activity indicating functional specificity of Vic_CAPAN. Discovery of bound salicylic acid, which is a known regulator of antioxidant pathway, and revelation of superoxide dismutase activity suggest that Vic_CAPAN has an important role during oxidative stress. As salicylic acid changes the redox state of cell, it may act as a downstream signal for various pathways involved in plant biotic and abiotic stress rescue.

Evolution of allosteric regulation in chorismate mutases from early plants.

Plants, fungi, and bacteria synthesize the aromatic amino acids: l-phenylalanine, l-tyrosine, and l-tryptophan. Chorismate mutase catalyzes the branch point reaction of phenylalanine and tyrosine biosynthesis to generate prephenate. In Arabidopsis thaliana, there are two plastid-localized chorismate mutases that are allosterically regulated (AtCM1 and AtCM3) and one cytosolic isoform (AtCM2) that is unregulated. Previous analysis of plant chorismate mutases suggested that the enzymes from early plants (i.e. bryophytes/moss, lycophytes, and basal angiosperms) formed a clade distinct from the isoforms found in flowering plants; however, no biochemical information on these enzymes is available. To understand the evolution of allosteric regulation in plant chorismate mutases, we analyzed a basal lineage of plant enzymes homologous to AtCM1 based on sequence similarity. The chorismate mutases from the moss/bryophyte Physcomitrella patens (PpCM1 and PpCM2), the lycophyte Selaginella moellendorffii (SmCM), and the basal angiosperm Amborella trichopoda (AmtCM1 and AmtCM2) were characterized biochemically. Tryptophan was a positive effector for each of the five enzymes examined. Histidine was a weak positive effector for PpCM1 and AmtCM1. Neither tyrosine nor phenylalanine altered the activity of SmCM; however, tyrosine was a negative regulator of the other four enzymes. Phenylalanine down-regulates both moss enzymes and AmtCM2. The 2.0 ŠX-ray crystal structure of PpCM1 in complex with the tryptophan identified the allosteric effector site and reveals structural differences between the R- (more active) and T-state (less active) forms of plant chorismate mutases. Molecular insight into the basal plant chorismate mutases guides our understanding of the evolution of allosteric regulation in these enzymes.

Species identification approach for both raw materials and end products of herbal supplements from Tinospora species.

BACKGROUND: Nowadays herbal products used in traditional medicine are sold in processed forms and thus morphological authentication is almost impossible. With herbal industry rapidly growing size, consumer safety becomes an important issue that requires special attention. Identification of herbal species in the products is therefore needed. METHODS: Sequences from the selected regions (matK, rbcL, trnL and ITS1) were retrieved and analysed. Then the most suitable barcode was assessed for discrimination of T. crispa from closely related species by HRM analysis and used in authentication of commercial products. RESULTS: The ITS1 barcode was found to be the suitable primer as melting data from the HRM assay proved to be capable of distinguishing T. crispa from its related species. The developed protocol was then employed to authenticate medicinal products in powdered form. HRM analysis of all tested samples here revealed that five out of eight products contained not only the indicated species T. crispa but also other Tinospora, that have a high level of morphological similarity. CONCLUSION: Misrepresentation, poor packaging and inappropriate labeling of the tested medicinal herbal products are thought to be the reason of the results here. Using Bar-HRM with the ITS marker lead to success in authenticating the tested herbal products.

Study of Some Zanthoxylum Species by Chemical and DNA Analysis Approaches.

The authentication and traceability of spices is a major concern for industrials and consumers. We focused on species from Zanthoxylum genera which are used for many different applications by local populations and also for trading as spices (dried pericarps or whole fruits). In this case, literature gives contradictory data about botanical names, and commercial labelling is often confusing. We studied commercial fruits pericarps extracts obtained by supercritical CO2 and analyzed them by GC/MS. The very complex volatile and semi volatile fractions composition of each extract is described. The barcoding method including molecular biology and phylogenetic analyses was also developed in order to check the commercial botanical identification of the raw material. This is a robust method to identify species in berries samples. We used one genetic marker to identify two Rutaceae clusters, including several species of Zanthoxylum genus. These results indicate that Fagara and Zanthoxylum groups could be considered as two different genera. Combination of chemical analysis and DNA analysis provides an original approach to increase chemical and botanical Zanthoxylum genus knowledge.

Assessing product adulteration of Eurycoma longifolia (Tongkat Ali) herbal medicinal product using DNA barcoding and HPLC analysis.

CONTEXT: Eurycoma longifolia Jack (Simaroubaceae) commonly known as Tongkat Ali is one of the most important plants in Malaysia. The plant extracts (particularly roots) are widely used for the treatment of cough and fever besides having antimalarial, antidiabetic, anticancer and aphrodisiac activities. OBJECTIVES: This study assesses the extent of adulteration of E. longifolia herbal medicinal products (HMPs) using DNA barcoding validated by HPLC analysis. MATERIALS AND METHODS: Chloroplastic rbcL and nuclear ITS2 barcode regions were used in the present study. The sequences generated from E. longifolia HMPs were compared to sequences in the GenBank using MEGABLAST to verify their taxonomic identity. These results were verified by neighbor-joining tree analysis in which branches of unknown specimen are compared to the reference sequences established from this study and other retrieved from the GenBank. The HMPs were also analysed using HPLC analysis for the presence of eurycomanone bioactive marker. RESULTS: Identification using DNA barcoding revealed that 37% of the tested HMPs were authentic while 27% were adulterated with the ITS2 barcode region proven to be the ideal marker. The validation of the authenticity using HPLC analysis showed a situation in which a species which was identified as authentic was found not to contain the expected chemical compound. DISCUSSION AND CONCLUSIONS: DNA barcoding should be used as the first screening step for testing of HMPs raw materials. However, integration of DNA barcoding with HPLC analysis will help to provide detailed knowledge about the safety and efficacy of the HMPs.

The anemonin content of four different Ranunculus species.

The Ranunculus species are poorly known as medicinal plants. They have potential toxicity given by the ranunculin and its enzymatic degradation compounds: protoanemonin and anemonin. This paper aims to evaluate the anemonin content of four species: R. bulbosus, R. ficaria, R. sardous and R. sceleratus. The evaluation was performed by TLC and HPLC. There were evaluated two types of extracts hydroalcoholic (HA) and glycerol-ethanol (GE). The most concentrated extract in anemonin was found to be the R. sardous aerial part HA extract: 2.66 mg/ml. The lowest anemonin content is in R. sceleratus: 0.13-0.19 mg/ml. In R. bulbosus aerial part the anemonin content is less than the used HPLC method detection limits (7.68 mg/ml). In all cases the GE extracts are less concentrated in anemonin, being more safely for human administration.

Assessment of Genetic and Chemical Variability in Curcumae Longae Rhizoma (Curcuma longa) Based on DNA Barcoding Markers and HPLC Fingerprints.

Curcumae Longae Rhizoma (Curcuma longa L.) is an important traditional Chinese medicine with multiple beneficial effects. To elucidate the genetic and chemical differences among Curcumae Longae Rhizoma samples, three DNA barcoding markers (rbcL, matK, and ITS-LSU D1/D3) and HPLC fingerprinting were employed in this study. The discriminatory power of rbcL and matK was low, as they only detected one sequence type that showed 100% similarity with more than 20 congeneric species in the Barcode of Life Data Systems (BOLD) database. In contrast, ITS-LSU D1/D3 showed sufficient discriminatory power to precisely identify all of the market samples as C. longa L. in a BLAST search as well as differentiate each sample based on 2-10 ITS-LSU D1/D3 haplotypes with intragenomic variability (mean p-distance: 0.7%, range: 0-2.6%; mean number of differences: 9.6 sites, range: 0-38 sites). HPLC fingerprinting of 13 commercial samples showed a similarity that ranged from 0.769 to 0.996, indicating that the sample quality varied. A cluster analysis based on 5 common peak areas from the HPLC chromatogram resulted in two groups. Group I included 9 samples with a relatively high chemical content, and group II contained 4 samples with a low chemical content. A Mantel test revealed a low correlation (r=0.1721, p=0.047) between genetic and chemical differences. Our findings suggest that the integrated approach of ITS-LSU D1/D3 DNA barcoding and HPLC fingerprinting provides a comprehensive, precise, and convenient method to clarify the genetic and chemical differences in Curcumae Longae Rhizoma.

Antioxidant and Proteinase Inhibitory Activities of Selected Poppy (Papaver somniferum L.) Genotypes.

Poppy seeds (Papaver somniferum L.) belong to tasty food ingredients however, they should be considered also as valuable source of biologically active compounds. Content of selected metabolites, antioxidant and proteinase inhibitory activities were analyzed in vitro in extracts from seeds of fifteen poppy genotypes. Considerable variation in all parameters was detected within the set of analyzed poppy genotypes. The genotype Major expressed the highest antioxidant activity determined by all four methodological approaches (DPPH, ABTS, FRAP, RP). The genotype MS 423 exhibited the highest inhibitory activities against trypsin, thrombin and collagenase. Very specific position among all had the genotype Redy. Its grain extract reached significantly high levels in 9 out of 14 measured parameters (TPC, TFC, TTC, TAC, FRAP, RP, inhibitory activities against trypsin, thrombin, collagenase). Edible grains of poppy are valuable source of natural compounds which may be beneficial in pathological states associated with oxidative stress or increased proteinase activities.

Common ragweed (Ambrosia artemisiifolia L.): allergenicity and molecular characterization of pollen after plant exposure to elevated NO2.

Ragweed pollen is the main cause of allergenic diseases in Northern America, and the weed has become a spreading neophyte in Europe. Climate change and air pollution are speculated to affect the allergenic potential of pollen. The objective of this study was to investigate the effects of NO2 , a major air pollutant, under controlled conditions, on the allergenicity of ragweed pollen. Ragweed was exposed to different levels of NO2 throughout the entire growing season, and its pollen further analysed. Spectroscopic analysis showed increased outer cell wall polymers and decreased amounts of pectin. Proteome studies using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry indicated increased amounts of several Amb a 1 isoforms and of another allergen with great homology to enolase Hev b 9 from rubber tree. Analysis of protein S-nitrosylation identified nitrosylated proteins in pollen from both conditions, including Amb a 1 isoforms. However, elevated NO2 significantly enhanced the overall nitrosylation. Finally, we demonstrated increased overall pollen allergenicity by immunoblotting using ragweed antisera, showing a significantly higher allergenicity for Amb a 1. The data highlight a direct influence of elevated NO2 on the increased allergenicity of ragweed pollen and a direct correlation with an increased risk for human health.

DNA barcoding and NMR spectroscopy-based assessment of species adulteration in the raw herbal trade of Saraca asoca (Roxb.) Willd, an important medicinal plant.

Saraca asoca (Roxb.) Willd, commonly known as "Asoka" or "Ashoka," is one of the most important medicinal plants used in raw herbal trade in India. The bark extracts of the tree are used in the treatment of leucorrhea and other uterine disorders besides also having anti-inflammatory, anti-bacterial, anti-pyretic, anti-helminthic, and analgesic activity. The indiscriminate and rampant extraction of the wood to meet the ever-increasing market demand has led to a sharp decline in naturally occurring populations of the species in the country. Consequently, the species has recently been classified as "vulnerable" by the International Union for Conservation of Nature (IUCN). Increasing deforestation and increasing demand for this medicinal plant have resulted in a limited supply and suspected widespread adulteration of the species in the raw herbal trade market. Adulteration is a serious concern due to: (i) reduction in the efficacy of this traditional medicine, (ii) considerable health risk to consumers, and (iii) fraudulent product substitution that impacts the economy for the Natural Health Product (NHP) Industry and consumers. In this paper, we provide the first attempt to assess the extent of adulteration in the raw herbal trade of S. asoca using DNA barcoding validated by NMR spectroscopic techniques. Analyzing market samples drawn from 25 shops, mostly from peninsular India, we show that more than 80 % of the samples were spurious, representing plant material from at least 7 different families. This is the first comprehensive and large-scale study to demonstrate the widespread adulteration of market samples of S. asoca in India. These results pose grave implications for the use of raw herbal drugs, such as that of S. asoca, on consumer health and safety. Based on these findings, we argue for a strong and robust regulatory framework to be put in place, which would ensure the quality of raw herbal trade products and reassure consumer confidence in indigenous medicinal systems. Graphical Abstract DNA barcoding and NMR spectroscopy-based assessment of adulteration in Saraca asoca.

Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine.

BACKGROUND: Ayurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets. METHODS: We have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18). RESULTS: Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %). CONCLUSIONS: The present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.