Estimation of genetic diversity and population genetic structure in Gymnema sylvestre (Retz.) R. Br. ex Schult. populations using DAMD and ISSR markers.

BACKGROUND: Gymnema sylvestre (Retz.) R. Br. ex Schult. is a well-known medicinal plant against diabetes in India. There is as such no organized cultivation in India, and the plant is still being collected from the wild for their therapeutic uses. It is, therefore, important to estimate the genetic diversity and population genetic structure of G. sylvestre to ascertain the genetically diverse germplasm. The present study, therefore, was undertaken to analyze the genetic variability in 118 accessions belonging to 11 wild populations of G. sylvestre using directed amplification of minisatellite-region DNA (DAMD) and inter simple sequence repeats (ISSR). RESULTS: The present genetic analyses of 11 populations with 25 markers (8 DAMD and 17 ISSR) revealed significant genetic diversity (H = 0.26, I = 0.40, PPL = 80.89%) at a species level, while the average genetic diversity at the population level was low. Among the 11 populations studied, PCH and UTK populations showed maximum genetic diversity, followed by KNR and AMB, while TEL population revealed the lowest genetic diversity. AMOVA and Gst values (0.18) revealed that most of the genetic variations are found within populations and very less among populations, and higher gene flow (Nm = 2.29) was found to be responsible for the genetic homogenization of the populations. The clustering pattern resulting from the UPGMA dendrogram was in congruence with STRUCTURE and PCoA, segregating all the 11 populations into two main genetic clusters: cluster I (populations of North and Central India) and cluster II (populations of South India). The clustering patterns obtained from all three statistical methods indicate that the genetic structure in G. sylvestre populations corresponds to the geographical diversity of the populations and represents a strong genetic structure. CONCLUSION: The genetically diverse populations identified during the present study could be a potential genetic resource for further prospecting and conserving this important plant resource.

De Novo Hybrid Assembled Draft Genome of Commiphora wightii (Arnott) Bhandari Reveals Key Enzymes Involved in Phytosterol Biosynthesis.

Genome sequence and identification of specific genes involved in the targeted secondary metabolite biosynthesis are two essential requirements for the improvement of any medicinal plant. Commiphora wightii (Arnott) Bhandari (family: Burseraceae), a medicinal plant native to Western India, produces a phytosterol guggulsterone, which is useful for treating atherosclerosis, arthritis, high cholesterol, acne, and obesity. For enhanced guggulsterone yield, key genes involved in its biosynthesis pathway need to be predicted, for which the genome sequence of the species is a pre-requisite. Therefore, we assembled the first-ever hybrid draft genome of C. wightii with a genome size of 1.03 Gb and 107,221 contigs using Illumina and PacBio platforms. The N50 and L50 values in this assembled genome were ~74 Kb and 3486 bp, respectively with a guanine-cytosine (GC) content of 35.6% and 98.7%. The Benchmarking Universal Single Copy Ortholog (BUSCO) value indicated good integrity of assembly. Analysis predicted the presence of 31,187 genes and 342.35 Mb repeat elements in the genome. The comparative genome analysis of C. wightii with relevant orthogroups predicted a few key genes associated with phytosterol biosynthesis and secondary metabolism pathways. The assembled draft genome and the predicted genes should help the future variety development program with improved guggulsterone contents in C. wightii.

Molecular analyses of genetic variability in the populations of Bergenia ciliata in Indian Himalayan Region (IHR).

Bergenia ciliata is an important medicinal plant species of Indian Himalayan Region (IHR). Genetic variability and population genetic structure of B. ciliata sampled from IHR was studied using two single primer amplification reaction (SPAR) methods (DAMD: Directed Amplification of Minisatellite region DNA; ISSR: Inter Simple Sequence Repeats). To provide a reasonable scientific basis for management and conservation of B. ciliata populations in IHR, genetic diversity analysis of 11 populations with 24 SPAR markers (15 ISSR and 9 DAMD) revealed significantly high level of (90.03%) polymorphism at species level. However, genetic variability was low at population level and KUL and BWS populations showed maximum while SHM population revealed least genetic diversity among the 11 populations. Analysis of molecular variance revealed highest percentage of variation (73%) within populations, followed by 17% among populations and least (10%) among the Himalayan regions. Clustering pattern obtained from UPGMA dendrogram was supported by STRUCTURE and principal coordinate analysis, segregating all the 11 natural populations of B. ciliata into two genetic clusters: Eastern and Western Himalayan populations. The clustering patterns of all the three statistical methods indicated that populations of B. ciliata have structured in response to the local micro-climates of the habitats in IHR, and therefore, it can be concluded that genetic variability is in congruence with the geographical diversity.

Major bioactive phenolics in Bergenia species from the Indian Himalayan region: Method development, validation and quantitative estimation using UHPLC-QqQLIT-MS/MS.

Bergenia species are important medicinal plants used in indigenous systems of medicine for their antilithiatic and diuretic properties. An ultra-high performance liquid chromatography coupled to hybrid linear ion trap triple quadrupole mass spectrometry (UHPLC-QqQLIT-MS/MS) method has been developed and validated for the estimation of quantitative variation of eight major bioactive phenolics in the rhizomes (150 samples) of four species of this herb, Bergenia (B. ciliata, B. ligulata, B. purpurascens and B. stracheyi). Chromatographic separation was obtained on a Waters ACQUITY UPLCTM BEH (ethylene bridged hybrid) C18 column with a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile under a gradient elution manner. A hybrid linear ion trap triple quadrupole mass spectrometer was operated in negative electrospray ionization mode with multiple reactions monitoring for detection and quantification of the eight compounds. The validated method demonstrated good linearity (r2 ≥ 0.9991), precision (RSD ≤ 1.87%) and accuracy (95.16-102.11%, RSD ≤ 1.83%) for all reference analytes. The quantitative results revealed that B. ligulata contains the highest amount of the major active marker-bergenin. The results also suggest that sensitive UHPLC-QqQLIT-MS/MS method, a sensitive, accurate and convenient one, could be helpful in identification of potential accession(s), rapid quality control and establishing authenticity of Bergenia species as raw material for pharmaceutical industries.

Isolation, Characterization and Anticancer Potential of Cytotoxic Triterpenes from Betula utilis Bark.

Betula utilis, also known as Himalayan silver birch has been used as a traditional medicine for many health ailments like inflammatation, HIV, renal and bladder disorders as well as many cancers from ages. Here, we performed bio-guided fractionation of Betula utilis Bark (BUB), in which it was extracted in methanol and fractionated with hexane, ethyl acetate, chloroform, n-butanol and water. All six fractions were evaluated for their in-vitro anticancer activity in nine different cancer cell lines and ethyl acetate fraction was found to be one of the most potent fractions in terms of inducing cytotoxic activity against various cancer cell lines. By utilizing column chromatography, six triterpenes namely betulin, betulinic acid, lupeol, ursolic acid (UA), oleanolic acid and ß-amyrin have been isolated from the ethyl acetate extract of BUB and structures of these compounds were unraveled by spectroscopic methods. ß-amyrin and UA were isolated for the first time from Betula utilis. Isolated triterpenes were tested for in-vitro cytotoxic activity against six different cancer cell lines where UA was found to be selective for breast cancer cells over non-tumorigenic breast epithelial cells (MCF 10A). Tumor cell selective apoptotic action of UA was mainly attributed due to the activation of extrinsic apoptosis pathway via up regulation of DR4, DR5 and PARP cleavage in MCF-7 cells over non-tumorigenic MCF-10A cells. Moreover, UA mediated intracellular ROS generation and mitochondrial membrane potential disruption also play a key role for its anti cancer effect. UA also inhibits breast cancer migration. Altogether, we discovered novel source of UA having potent tumor cell specific cytotoxic property, indicating its therapeutic potential against breast cancer.

Composition and in vitro cytotoxic activities of essential oil of Hedychium spicatum from different geographical regions of western Himalaya by principal components analysis.

The rhizome of Hedychium spicatum has been widely used in traditional medicines. The present study deals with the evaluation of the cytotoxic potential of rhizome essential oils from four different regions of the Western Himalaya (India) along with comparative correlation analysis to characterise the bioactive cytotoxic component. The essential oils were coded as MHS-1, MHS-2, MHS-3 and MHS-4, and characterised using GC-FID and GC-MS. The main volatile compounds identified were 1,8-cineol, eudesmol, cubenol, spathulenol and α-cadinol. In vitro cytotoxic activities were assessed against human cancer cell lines such as, the lung (A549), colon (DLD-1, SW 620), breast (MCF-7, MDA-MB-231), head and neck (FaDu), and cervix (HeLa). MHS-4 is significantly active in comparison to other samples against all cancer cell lines. Sample MHS-4 has major proportion of monoterpene alcohol mainly 1,8-cineol. Principal components analysis was performed for the experimental results and all four samples were clustered according to their percentage inhibition at different doses.

Essential oil composition of aerial parts of Angelica glauca growing wild in North-West Himalaya (India).

Fresh aerial parts of Angelica glauca, growing wild in Kashmir valley in higher Himalaya (Jammu and Kashmir, India), collected at flowering stage from different locations, on hydro-distillation provided a refreshing light pale coloured essential oil with characteristic floral woody flavour. The oil was found to be a complex mixture of mono- and sesquiterpenes and 34 compounds accounting for nearly 97.4% of the oil were characterized with the help of capillary GC, GC-MS, and NMR. Major compounds of the oil were characterized as alpha-phellandrene (13.5%), trans-carveol (12.0%), beta-pinene (11.7%), thujene (7.5%), beta-caryophyllene oxide (7.2%), beta-caryophyllene (7.0%), gamma-terpinene (6.7%), nerolidol (6.5%), beta-bisabolene (5.2%) and germacrene D (4.5%). It is the first report to exploit the essential oil from Himalayan A. glauca herb collected at flowering stage.