A Review on Application of DNA Barcoding Technology for Rapid Molecular Diagnostics of Adulterants in Herbal Medicine.

The rapid molecular diagnostics of adulterants in herbal medicine using DNA barcoding forms the core of this meticulously detailed review, based on two decades of data. With 80% of the world's population using some form of herbal medicine, authentication, quality control, and detection of adulterants warrant DNA barcoding. A combined group of keywords were used for literature review using the PubMed, the ISI Web of Knowledge, Web of Science (WoS), and Google Scholar databases. All the papers (N = 210) returned by the search engines were downloaded and systematically analyzed. Detailed analysis of conventional DNA barcodes were based on retrieved sequences for internal transcribed spacer (ITS) (412,189), rbcL (251,598), matK (210,835), and trnH-psbA (141,846). The utility of databases such as The Barcode of Life Data System (BOLD), NCBI, GenBank, and Medicinal Materials DNA Barcode Database (MMDBD) has been critically examined for the identification of unknown species from known databases. The current review gives an overview of the ratio of adulterated to authentic drugs for some countries along with the state of the art technology currently being used in the identification of adulterated medicines. In this review, efforts were made to systematically analyze and arrange the research and reviews on the basis of technical progress. The review concludes with the future of DNA-based herbal medicine adulteration detection, forecasting the reliance on the metabarcoding technology. DNA barcoding technology for differentiating adulterated herbal medicine.

Development of EST-SSR markers for Pongamia pinnata by transcriptome database mining: cross-species amplification and genetic diversity.

EST-SSR markers were developed from Pongamia pinnata transcriptome libraries. We have successfully utilised EST-SSRs to study the genetic diversity of Indian P. pinnata germplasms and transferability study on legume plants. P. pinnata is a non-edible oil, seed-bearing leguminous tree well known for its multipurpose benefits and acts as a potential source for medicine and biodiesel preparation. Moreover, the plant is not grazable by animal and wildly grown in different agro climatic condition of India. Recently, it is much used in reforestation and rehabilitation of marginal and coal mined land in different part of India. Due to increasing demand for cultivation, understanding of the genetic diversity is important parameter for further breeding and cultivation program. In this investigation, an attempt has been undertaken to develop novel EST-SSR markers by analyzing the assembled transcriptome from previously published Illumina libraries of P. pinnata, which is cross transferrable to legume plants. Twenty EST-SSR markers were developed from oil yielding and secondary metabolite biosynthesis genes. To our knowledge, this is the first EST-SSR marker based genetic diversity study on Indian P. pinnata germplasms. The genetic diversity parameter analysis of P. pinnata showed that the Gangetic plain and Eastern India are highly diverse compared to the Central Deccan and Western germplasms. The lowest genetic diversity in the Western region may be due to the pressure of lower precipitation, high-temperature stress and reduced groundwater availability. Nevertheless, the highest genetic diversity of Gangetic plain and Eastern India may be due to the higher groundwater availability, high precipitation, higher temperature fluctuations and growing by the side of glacier-fed river water. Thus, our study shows the evidence of natural selection on the genetic diversity of P. pinnata germplasms of the Indian subcontinent.

Evaluation of rapid molecular diagnostics for differentiating medicinal Kaempferia species from its adulterants.

Accurate detection of unique herbs is crucial for herbal medicine preparation. Zingiberaceae species, which are important in Ayurvedic medicine of India, are often misidentified in Northeast (NE) Indian herbal markets. Kaempferia galanga (Zingiberaceae) is one of the major components of popular Ayurvedic drugs used for rheumatic diseases (i.e., "Gandha Thailam" and "Rasnairandadi Kashayam"), contusions, fractures, and sprains. In NE India, herbal healers often misidentify plants from the Marantaceae family (e.g., Calathea bachemiana and Maranta leuconeura) as Kaempferia, which leads to adulteration of the medicinal herb. This misidentification of herbs occurs in NE India because Zingiberaceae plant barcoding information is inadequate. As a consequence, herbal medicine is not only therapeutically less effective but may also cause adverse reactions that range from mild to life-threatening. In this study, we used eight barcoding loci to develop "fingerprints" for four Kaempferia species and two species frequently mistaken for Kaempferia. The PCR and sequencing success of the loci matK, rbcL and trnH-psbA were found to be 100%; the combination of matK, rbcL, and trnH-psbA proved to be the ideal locus for discriminating the Kaempferia species from their adulterants because the combined loci showed greater variability than individual loci. This reliable tool was therefore developed in the current study for accurate identification of Kaempferia plants which can effectively resolve identification issues for herbal healers.

Assessment of genetic variation among wild Alpinia nigra (Zingiberaceae) population: an approach based on molecular phylogeny.

Genetic structure was evaluated among wild Alpinia nigra (Gaertn.) B.L. Burtt, populations. The information of genetic relatedness was developed using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and barcoding loci (plastid and mitochondrial). The order (high to low) of Shannon's information index (I) and Nei's gene diversity (h) from the populations was: "IIT Guwahati" > "Amingaon" > "Saraighat". Genetic diversity decreased and genetic differentiation increased among the three populations. We observed no isolation by distance thus lower amount of gene flow was observed. Narrow range of genetic distance among the three populations and appearance of two distinct clusters strengthened the geographical isolation in dendrogram and principal component analysis. No mutation among the three populations was observed for seven plastid loci and two mitochondrial tested suggesting the taxonomic homogeneity. The phylogeny based on nine barcoding loci supported our observation that individuals of IIT Guwahati were partially isolated from the outside populations. Our study will provide a backbone for developing strategies to resist habitat fragmentation of Zingiberaceous plants.

New record of nuclear DNA amounts of some Zingiberaceae species from North east India.

Members of the family Zingiberaceae are important medicinal plants and have great economic significance. Some taxonomic issues are still pending within the family and the genome size estimates of many species are still very scarce. Therefore, studies concerning genome size can provide complementary data that may be useful to characterize the family on whole. Genome size estimate have been used to characterize four Northeast Indian taxa of the family Zingiberaceae occurring in the wild in addition to that of a sacred cultivated species. In this data article we have provided genome size estimates of four species based on flow cytometry for the first time. This data will be valuable for genomic and molecular authentication of these species for all future studies.

Development of flow cytometric protocol for nuclear DNA content estimation and determination of chromosome number in Pongamia pinnata L., a valuable biodiesel plant.

The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays 'CE-777' as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and ß-mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for P. pinnata leaves from different altitudes (37-117 m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root-tip cells and was found to be 2n = 22 corresponding to the diploid level (x = 11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.

Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets.

Pongamia pinnata L. is a multipurpose versatile legume that is well known as a prospective feedstock biodiesel species. However, to date, there has been little genomic research aimed at the exploitation of the biotechnological potential of this species. Genetic characterization of any plant is a challenging task when there is no information about the genome size and organization of the species. Therefore, the genome size of P. pinnata was estimated by flow cytometry with respect to two standards (Zea mays and Pisum sativum), and compared with that of in vitro-raised plants (nodal segment, in vitro-rooted plantlets and acclimatized in vitro plants) to study the potential effect of somaclonal variation on genome size. This method can be used to support the establishment of true-to-type plants to encourage afforestation programs. Modified propidium iodide/hypotonic citrate buffer was used for isolation of the intact nuclei. The 2C DNA value of this species was estimated to be 2.51 ± 0.01 pg. Statistically, there was no significant difference in the DNA content of the in vitro-grown plants and mother plant at α = 0.05. As a result of the low genome size of P. pinnata, a species that has adapted itself to a wide range of edaphic and ecological condition, we can now proceed for its next generation sequencing and genomic diversity studies.

Genetic diversity and relationship of Hedychium from Northeast India as dissected using PCA analysis and hierarchical clustering.

Molecular genetic fingerprints of eleven Hedychium species from Northeast India were developed using PCR based markers. Fifteen inter-simple sequence repeats (ISSRs) and five amplified fragment length polymorphism (AFLP) primers produced 547 polymorphic fragments. Positive correlation (r = 0.46) was observed between the mean genetic similarity and genetic diversity parameters at the inter-species level. AFLP and ISSR markers were able to group the species according to its altitude and intensity of flower aroma. Cophenetic correlation coefficients between the dendrogram and the original similarity matrix were significant for ISSR (r = 0.89) compared to AFLP (r = 0.83) markers. This genetic characterization of Hedychium from Northeast India contributes to the knowledge of genetic structure of the species and can be used to define strategies for their conservation and management.

Ethnomedical uses of Zingiberaceous plants of Northeast India.

AIM OF THE STUDY: Family Zingiberaceae consists of large number of medicinal plants and is well known for its use in ethnomedicine. The objective of this study is to systematically analyse and document the traditional knowledge regarding the use of Zingiberaceous plants for the treatment of various human ailments from NE India, adding information to the valuation of biodiversity and, to forward suggestions for its sustainable use, conservation and for future pharmacological studies. MATERIALS AND METHODS: A survey on the utilization of medicinal plants belonging to Zingibereceae of North-eastern states was carried out by interviewing herbalists followed by collecting plant specimens and identifying the specimen. Ethnobotanical information on traditional plants was catalogued through structured questionnaires in consultations with traditional healers. RESULTS: A total of 34 species were documented belonging to 9 genera of Zingiberaceae for about 25 types of ailments, 67.6% of which were used in curing multiple disorders. Arunachal Pradesh hosts maximum number of Zingiberaceous plant (88%). Rhizomes were found to be the primary plant material as a source for medication and poultices as the predominant mode of preparation. Gastrointestinal conditions (58%) and chest and lungs (41%) related ailments were the main categories for which these plants are used. CONCLUSIONS: The study establishes Zingiberaceae as a medicinal family since 41% of all the available Zingiberaceous plant species in NE were found to possess medicinal value. Some new use of herbs also appeared in this study for the first time.