Baleshwar Meena

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 β-amyrin have been isolated from the ethyl acetate extract of BUB and structures of these compounds were unraveled by spectroscopic methods. β-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.

ISSR analysis of soap nut (Sapindus mukorossi Gaertn.) genotypes in Western Himalaya (India)

Abstract Members of the genus Sapindus L. (Sapindaceae), commonly known as soap nut, are commercially important plants, that are found both wild and introduced in tropical and sub-tropical regions of the world, predominantly in Indo-Malayasia. Three species, Sapindus emarginatus Vahl, S. mukorossi Gaertn. and S. trifoliatus L., occur in India. In this study, ISSR markers were used to unravel the genetic diversity in soap nut (S. mukorossi) genotypes of Western Himalaya. A total of 54 S. mukorossi genotypes and one accession of Lepisanthes tetraphylla Vahl. as the out-group were considered. ISSR analysis resulted in 258 bands obtained with 20 primers. Out of these, 152 bands were polymorphic revealing 58.91% polymorphism across all genotypes of S. mukorossi. The genetic distance value varied from 0.02 to 0.37 across different genotypes. The results show that ISSR profiles are sufficiently informative and useful to unravel the genetic variations among soap nut plants in the Western Himalayas. The information on genetic diversity of soap nut could further be utilized in the breeding of genetically divergent and geographically isolated populations to obtain better germplasm in terms of yield and quality.

Nucleotide diversity and phylogenetic relationships among Gladiolus cultivars and related taxa of family Iridaceae

The plastid genome regions of two intergenic spacers, psbA–trnH and trnL–trnF, were sequenced to study the nucleotide diversity and phylogenetic relationships among Gladiolus cultivars. Nucleotide diversity of psbA–trnH region was higher than trnL–trnF region of chloroplast. We employed Bayesian, maximum parsimony (MP) and neighbour-joining (NJ) approaches for phylogenetic analysis of Gladiolus and related taxa using combined datasets from chloroplast genome. The psbA–trnH and trnL–trnF intergenic spacers of Gladiolus and related taxa-like Babiana, Chasmanthe, Crocus, Iris, Moraea, Sisyrinchium, Sparaxis and two out group species (Hymenocallis littoralis and Asphodeline lutea) were used in the present investigation. Results showed that subfamily Iridoideae have sister lineage with subfamily Ixioideae and Crocoideae. H. littoralis and A. lutea were separately attached at the base of tree as the diverging Iridaceae relative’s lineage. Present study revealed that psbA–trnH region are useful in addressing questions of phylogenetic relationships among the Gladiolus cultivars, as these intergenic spacers are more variable and have more phylogenetically informative sites than the trnL–trnF spacer, and therefore, are suitable for phylogenetic comparison on a lower taxonomic level. Gladiolus cultivars are extensively used as an ornamental crop and showed high potential in floriculture trade. Gladiolus cultivation still needs to generate new cultivars with stable phenotypes. Moreover, one of the most popular methods for generating new cultivars is hybridization. Hence, information on phylogenetic relationships among cultivars could be useful for hybridization programmes for further improvement of the crop.

Determination of Pentacyclic Triterpenes from Betula utilis by High-Performance Liquid Chromatography and High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy

ABSTRACT A rapid and reliable method was developed and validated for determining betulin and betulinic acid in bark in Betula utilis by high-resolution magic angle spinning 1H nuclear magnetic resonance (HR-MAS NMR) spectroscopy. HR-MAS NMR spectroscopy clearly distinguished the resonances of betulin and betulinic acid in the bark of all accessions of B. utilis. The concentrations of betulin and betulinic acid were calculated and added to the spectra. The determination of the targeted metabolites in chloroform extract of bark of each accession of B. utilis was performed by high-performance liquid chromatography (HPLC). Quantitatively, betulin was present at higher concentrations than betulinic acid in all accessions. The HR-MAS NMR and HPLC results showed that betulin and betulinic acid varied significantly among accessions of B. utilis. Principal component analysis of the NMR and HPLC results provided classification into three metabolic groups in which the betulin concentration was high, moderate, or low. The results show that HR-MAS NMR is rapid for fingerprinting of betulin and betulinic acid in the bark of B. utilis, while minimizing the drawbacks associated with solvent extraction.

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.

Development of ISSR- and RAPD-derived SCAR markers for identification of Gladiolus germplasm

ABSTRACT Gladiolus is an economically important ornamental crop, cultivated for its beautiful flowers throughout the world. The correct genotype identification of plant material is very significant for the floriculture industry. The aim of this study was to develop sequence-characterised amplified region (SCAR) markers from RAPD and ISSR fragments for identification and authentication of Gladiolus germplasm. The SCAR markers developed could be easily employed as valuable tools to identify newly developed Gladiolus cultivars. The SCAR markers, viz. ScG12, ScG34, and ScG36, are specific to the DNA from all 62 Gladiolus cultivars, as they did not amplify the DNA of other taxa of the family Iridaceae, including Iris, Amaryllis, and Narcissus. All three SCAR markers distinguished Gladiolus from other taxa of the family Iridaceae, whereas marker ScAm was specific to the ‘Amethyst’ cultivar. Our results confirmed that this particular SCAR marker distinguished the ‘Amethyst’ cultivar from the other 62 Gladiolus cultivars investigated in the present study. This development of SCAR markers based on RAPD and ISSR markers seems to be the maiden attempt for Gladiolus cultivars.

Biochemical composition of Betula utilis D. Don bark, collected from high altitudes of Indian Himalayas

Betula utilis (family Betulaceae) is an important medicinal plant that grows in high altitudes of Himalayan region of India. The present study is aimed to determine the biochemical composition in the bark of Betula utilis using HPLC, HPTLC and GCMS methods. The optimization of solvent system for maximum extraction yield has been carried out. Total Phenolic Content (TPC), Total Flavonoid Content (TFC) and Antioxidant Activity (AA) were estimated in various solvent extracts. Maximum extractive yield (14.56%), TPC (14.47 mg/g), TFC (20.10 mg/g) in 75% ethanol and scavenging activity (92.89%) were found in 50% ethanol. Pentacyclic triterpenoids (betulin, tupeol, oleanolic acid, beta-sitosterol) were characterized and quantified using HPLC, HPTLC and GCMS found close within limits. Betulin was found maximum in ethanol.

Performance of Quality Protein Maize Under Different Planting Windows

A field experiment was conducted at Forage Research Block, ICAR-NDRI, Karnal during post rainy season of 2014 and 2015 to study the performance of quality protein maize (QPM) cultivars under different planting windows. The treatments consisted of four sowing times viz., Aug. 15, Sept. 5, Sept. 25 and Oct. 15 in main plots and five maize cultivars HQPM-1, HQPM-4, HM-10, African tall and J-1006 in subplots in a split plot design. August 15 sowing recorded the maximum plant height, leaf length, leaf width, number of leaves per plant, green fodder yield (53.49 and 56.17 t/ha) and DM yield (11.39 and 12.03 t/ha) during 2014 and 2015, respectively. The performance of Aug. 15 sown maize in post monsoon season was at par with Sept. 5 sowing, however, further delay in sowing led to a significant (P<0.05) reduction in growth parameters and fodder yield. October 15 sown crop showed reduction (P<0.05) in these growth parameters. The maximum yield was obtained in African tall and the cultivars J-1006, HQPM-1 and HQPM-4 were at par with African tall while HM-10 gave the lowest yield. The levels of CP and NDF were influenced (P<0.05) by planting windows and CP contain increased and NDF decreased with delay in sowing, however, no significant variation in quality parameters was found among the QPM and normal fodder maize cultivars.