Picrorhiza kurroa, has become an endangered medicinal herb due to excessive utilization, therefore it necessitates the understanding of biology and molecular basis of major chemical constituents i.e. Picroside-I (P-I) and Picroside-II (P-II). Estimation of P-I and P-II in different tissues of P. kurroa showed that shoots contain only P-I whereas P-II is present only in roots. Differential conditions with varying concentrations of P-I (0-27 μg/mg) and P-II (0-4 μg/mg) were selected. Four genes of MEP pathway; DXPS, ISPD, ISPE, MECPS and one gene of MVA pathway PMK showed elevated levels of transcripts in shoots (57-166 folds) and stolons (5-15 folds) with P-I contents 0-27 μg/mg and 2.9-19.7 μg/mg, respectively. Further HDS and DXPR genes of MEP pathway showed higher expression ~9-12 folds in roots having P-II (0-4 μg/mg). The expression of ISPH and ISPE was also high ~5 folds in roots accumulating P-II. GDPS was the only gene with high transcript level in roots (9 folds) and shoots (20 folds). Differential biosynthesis and accumulation of picrosides would assist in regulating quality of plant material for herbal drug formulations.

The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.

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Picrorhiza kurroa (family Scrophulariaceae) is a high value medicinal herb used in herbal drug formulations like picroliv, picrolax, etc. It has been collected recklessly from its natural habitat posing endangered status for its existence, thereby necessitating characterization of genetic diversity for its sustainable utilization and conservation. In this study, picrosides content and genetic profiles of 26 accessions of P. kurroa from different locations in north-western Himalayas have been analysed. Picroside-I (P-I) content ranged from 0.37 to 2.7% in fresh shoots whereas total picrosides content (P-I+P-II) ranged from 3.7 to 10.9% in dry rhizomes. High picrosides content accession PKS-1 was identified, both for shoots and rhizomes. To study genetic diversity and correlate picrosides content with their genetic factors, genetic profiling was done using simple sequence repeats (SSRs) identified from P. kurroa transcriptomes. Out of 361 SSR primers tested on 26 accessions, 35 primers yielded polymorphic profiles. Overall low genetic diversity was observed in P. kurroa accessions. The highest polymorphism information content (PIC) of 0.55 was given by SSR marker PKSTS-P9 (TGGTG)4. Mean allele number was 2.97. Mean observed and expected heterozygosities were 0.597 and 0.452, respectively. Among all accessions Nei's genetic diversity (H) was 0.39 and Shannon's information index (I) was 0.58. Cluster analysis of STRUCTURE was comparable to DARWIN. Microsatellite markers would be helpful in the development of DNA diagnostics for the authentication of quality plant material as well as planning a genetic improvement strategy.