Sushma Koul

Evaluation of nutritional and antioxidant status of Lepidium latifolium Linn.: a novel phytofood from Ladakh.

Lepidium latifolium Linn. (perennial pepperweed) is one of the preferred phytofoods among cold arid region of Ladakh, India and its leaves contribute significantly to peoples diet. This study was conducted to determine its nutritive value and antioxidant activity. Plant samples from three different locations were selected in the present study. Results showed that this plant is an excellent source of glucosinolates, notably sinigrin that is present in very high amount (∼70–90%). Its value ranged from 149 to 199 µg per g fresh weight. Fatty acid composition analysis showed that its leaves were abundant in unsaturated fatty acids, specifically linolenic acid (18∶3) whose percentage is about 50%. Higher glucose and crude protein along with higher nitrogen to sulfur ratio, supplements the nutritive value of this plant. Based on total phenol, flavanoids, free radical scavenging activity and DNA protective activity showed that this ecotype of perennial pepperweed contains high antioxidant properties. The percentage inhibition for O2 − scavenging activity ranged from 41.3% to 83.9%. Higher content of phenols (26.89 to 50.51 mg gallic acid equivalents per g dry weight) and flavanoids (38.66 to 76.00 mg quercetin equivalents per g dry weight) in leaves could be responsible for the free radical scavenging activity of this plant. Depending upon the location of the plants, variations were observed in different activities. Based on the systematic evaluation in this study, preparations of Lepidium latifolium from Ladakh can be promoted as substitute to dietary requirements.

Inhibition of phosphotidylinositol-3 kinase pathway by a novel naphthol derivative of betulinic acid induces cell cycle arrest and apoptosis in cancer cells of different origin

Betulinic acid (BA) is a pentacyclic triterpenoid natural product reported to inhibit cell growth in a variety of cancers. However, the further clinical development of BA got hampered because of poor solubility and pharmacological properties. Interestingly, this molecule offer several hotspots for structural modifications in order to address its associated issues. In our endeavor, we selected C-3 position for the desirable chemical modification in order to improve its cytotoxic and pharmacological potential and prepared a library of different triazoline derivatives of BA. Among them, we previously reported the identification of a potential molecule, that is, 3{1N(5-hydroxy-naphth-1yl)-1H-1,2,3-triazol-4yl}methyloxy betulinic acid (HBA) with significant inhibition of cancer cell growth and their properties. In the present study, we have shown for the first time that HBA decreased the expression of phosphotidylinositol-3 kinase (PI3K) p110α and p85α and caused significant downregulation of pAKT and of NFκB using human leukemia and breast cancer cells as in vitro models. Further it was revealed that PI3K inhibition by HBA induced cell cycle arrest via effects on different cell cycle regulatory proteins that include CDKis cyclins and pGSK3β. Also, this target-specific inhibition was associated with mitochondrial apoptosis as was reflected by the increased expression of mitochondrial bax, downregulated bcl2 and decreased mitochondrial levels of cytochrome c, together with reactive oxygen species generation and decline in mitochondrial membrane potential. The apoptotic effectors such as caspase 8, caspase 9 and caspase 3 were found to be upregulated besides DNA repair-associated enzyme, that is, PARP cleavage caused cancer cell death. Pharmacodynamic evaluation revealed that both HBA and BA were safe upto the dose of 2000 mg/kg body weight and with acceptable pharmacodynamic parameters. The in vitro data corroborated with in vivo anticancer activity wherein Ehrlich solid tumor showed that HBA as a more potent agent than BA without any body weight loss and mortality.

Conservation and Propagation of High Altitude Medicinal and Aromatic Plant: Hedychium spicatum

The micropropagation of H.spicatum, a medicinal and aromatic plant was investigated as an option for conservation and propagation, as wild populations are fast depleting. The source of raw material is rhizomes of plants that are collected from the wild. There is no planned cultivation of the plant. Multiple shoot cultures were established on MS medium supplemented with BAP and IAA from the pre-existing buds on the rhizome. Prolonged cultivation on the same medium or transfer to hormone free medium induced roots/rhizome formation; liquid medium proved more suitable. Greenhouse hardened plants were transferred to field. A successful protocol with 99% root formation and 80–85.5% field survival has been formulated.

Assessment and characterization of genetic diversity in Withania somnifera (L.) Dunal using RAPD and AFLP markers

Genetic diversity of 23 accessions of Withania somnifera collected from different geographical regions of India was estimated by employing Random Amplification of Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) markers. Eighteen RAPD primers and six AFLP primer combinations revealed 37.82 and 43.94% polymorphism, respectively, among 163 and 286 genetic loci amplified. The AFLP assay revealed higher levels of polymorphism among the tested W. somnifera accessions compared to the RAPD. Mean genetic diversity based on Shannon index ranged from 1.33 (RAPD) to 5.13 (AFLP). Unweighted pair group method based on arithmetic average (UPGMA) analysis was performed on Jaccard’s similarity coefficient matrix. The matrix reveals, two main clusters, wild accessions formed one cluster and the cultivated accessions formed the other. The cultivated accessions are well separated from the wild ones at a low similarity value of 0.3, indicating that cultivated and wild accessions are highly distinct. Morphologically cultivated accessions were also quite distinct from the wild ones and the cluster analysis of RAPD and AFLP fingerprints clearly discriminated the five cultivated accessions of W. somnifera . A strong correlation was observed between morphology and molecular marker systems. Identification of specific markers to wild as well as cultivated accessions is yet another important finding in the present study. Such genetic diversity is useful in facilitating the development of large number of new varieties through hybridization, transfer of useful genes, thus maximizing the use of such available germplasms as genetic resource materials for breeders. The present input, first of its kind in Ashwagandha, will thus assist the marker assisted crop improvement programme. Key words: Withania somnifera, genetic diversity, RAPD, AFLP, polymorphism, Shannon index.

Rapid in vitro propagation of Potentilla fulgens Wall - a Himalayan alpine herb of medicinal value.

An in vitro regeneration system has been developed for Potentilla fulgens, which is an important Himalayan medicinal herb. Axillary shoot proliferation through shoot tip culture has been achieved on Murashige and Skoog (MS) medium containing 1mg l−1 6-benzylaminopurine (BAP) and 1 mg l−1 indole-3-acetic acid (IAA). Continuous production of plantlets with better rate of shoot multiplication and elongation obtained on MS medium supplemented with 1mg l−1 kinetin (Kin) alone or combined with 1mg l−1 α-napthaleneacetic acid (NAA). Established plantlets were successfully transferred to soil in a green house. The procedure ensures 12-fold plantlet production every 6 weeks.

In vitro Propagation and Conservation of Atropa acuminata Royle ex Lindl -An Indigenous Threatened Medicinal Plant

A micropropagation method has been developed for multiplication and conservation of Atropa acominata by induction of axillary shoot proliferation from shoot tips and nodal explants using Murashige and Skoog (MS) medium supplemented with BAP ( 1 mg I-1) and IBA (1 mg I-1). Revised tobacco (RT) medium with IAA (1 mg I-1) was found most suitable for shoot elongation. Rooting was highest on full strength RT medium containing IBA (1 mg I-1). In vitro raised plantlets were hardened and transferred to soil.

Production of β-β,Dimethylacrylshikonin in Callus Cultures of Onosma echioides Var hispidum Clarke

AbstractCallus tissues derived from leaf segments of Onosma echioides var hispidum on three basal media viz. Murashige & Skoog (MS), Gamborg et al (B5) and White’s containing 3% sucrose produce napthaquinone pigments in presence of and NAA. However, β-β,dimethylacrylshikonin synthesis was triggered in dark in undifferentiated parenchyma cells on B5 agar medium containing 1 x10−5 M Kn and 2×10−6 M IBA when proliferated calli after 16 weeks (4th generation) were transferred to it and incubated at 23 ± 1 °C. The pigment biosynthesis increased linearly from 4th to 6th week after a lag of first 3 weeks. Callus grew exponentially after a lag of 2 weeks and diminished from 6th week onward. During 8 weeks of growth, callus grew from 0.8 to 8.2 g and the β-β, dimethylacrylshikonin showed the highest level of 25.41 μg g−1 of fresh tissue. Light microscopic examination of semi-thin sections of pigmented tissue revealed pigment accumlation between the plasma membrane and cell wall and also in the intercellular spaces. Exposure of cultures to white fluorescent light for more than 2 h resulted in complete repression of pigment bosynthesis. The investigations suggest that the regulatory mechanism for the biosynthesis and accumulation of napthaquinone pigment(s) may be similar to that of Lithospermum erythrorhizon. Pigment producing capability of callus cultures of O. echioides var hispidum can be exploited as an alternative raw source for the production of shikonin derivatives.

Assessment of chemical and genetic variability in Tanacetum gracile accessions collected from cold desert of Western Himalaya

Genetic diversity is essential for survival and adaptation of high altitude plants such as those of Tanacetum genus, which are constantly exposed to environmental stress. We collected flowering shoots of ten accessions of Tanacetum gracile Hook.f. & Thomson (Asteraceae) (Tg 1–Tg 10), from different regions of cold desert of Western Himalaya. Chemical profile of the constituents, as inferred from GC–MS, exhibited considerable variability. Percentage yield of essential oil ranged from 0.2 to 0.75% (dry-weight basis) amongst different accessions. Tg 1 and Tg 6 were found to produce high yields of camphor (46%) and lavandulol (41%), respectively. Alpha-phellendrene, alpha-bisabool, p-cymene and chamazulene were the main oil components in other accessions. Genetic variability among the accessions was studied using RAPD markers as well as by sequencing and analyzing nuclear 18S rDNA, and plastid rbcL and matK loci. The polymorphic information content (PIC) of RAPD markers ranged from 0.18 to 0.5 and the analysis clustered the accessions into two major clades. The present study emphasized the importance of survey, collection, and conservation of naturally existing chemotypes of medicinal and aromatic plants, considering their potential use in aroma and pharmaceutical industry.