Picrorhiza Kurroa Research Articles

Pharmacokinetics and comparative metabolic profiling of iridoid enriched fraction of Picrorhiza kurroa – An Ayurvedic Herb

Ethno-pharmacological relevancePicrosides I, II and apocynin are the main active principles present in the roots and rhizomes of Picrorhiza kurroa Royle ex. Benth (Kutki). Ethno-medicinally, the plant is used for the treatment of liver, upper respiratory tract disorders and dyspepsia, since long in Ayurveda. Aim of the studyThis study attempts to determine the pharmacokinetic profile of picrosides I, II and apocynin in rats after oral administration of iridoid enriched fraction (IRF) and to recognize the pattern of its metabolites as such in IRF and in plasma. Materials and methodsA simple, precise, specific and sensitive RP-HPLC method was developed for simultaneous quantification of picrosides I, II and apocynin in rat plasma and in plant extract. Acetonitrile (ACN) and water was used as a solvent system with a gradient elution for pharmacokinetic studies using HPLC-PDA (Flow rate: 1.0mL/min) and metabolic profiling through UPLC-MS (Flow rate: 0.5mL/min) in selected reaction monitoring. A comparative study was performed in order to recognize the pattern and fate of metabolites in rat plasma up to 24h after single oral administration of IRF. ResultsDeveloped method produced more than 85% recovery of the targeted metabolites in rat plasma. The content of picrosides I, II and apocynin in IRF were found 5.7%, 18.3% and 27.3% w/w, respectively. The mean plasma concentration versus time profiles of picroside I, II and apocynin resulted in peak plasma concentration (Cmax) 244.9, 104.6 and 504.2ng/mL with half-life (t1/2) 14, 8 and 6h, respectively. Other pharmacokinetic parameters such as time to reach Cmax (tmax), area under curve (AUC), absorption (ka) and elimination (ke) constant, volume of distribution (Vd) were also determined. Pattern recognition analysis showed fate of 18 metabolites in rat plasma up to 24h out of 26 present in IRF. ConclusionThe information gained from this study postulates the basic pharmacokinetic profiling of picroside I, II and apocynin as well as fate of other metabolites after oral administration of IRF, demonstrating scientific basis of its traditional use in Ayurveda.

Exogenous feeding of immediate precursors reveals synergistic effect on picroside-I biosynthesis in shoot cultures of Picrorhiza kurroa Royle ex Benth.

In the current study, we asked how the supply of immediate biosynthetic precursors i.e. cinnamic acid (CA) and catalpol (CAT) influences the synthesis of picroside-I (P-I) in shoot cultures of P. kurroa. Our results revealed that only CA and CA+CAT stimulated P-I production with 1.6-fold and 4.2-fold, respectively at 2.5 mg/100 mL concentration treatment. Interestingly, feeding CA+CAT not only directed flux towards p-Coumaric acid (p-CA) production but also appeared to trigger the metabolic flux through both shikimate/phenylpropanoid and iridoid pathways by utilizing more of CA and CAT for P-I biosynthesis. However, a deficiency in the supply of either the iridoid or the phenylpropanoid precursor limits flux through the respective pathways as reflected by feedback inhibition effect on PAL and decreased transcripts expressions of rate limiting enzymes (DAHPS, CM, PAL, GS and G10H). It also appears that addition of CA alone directed flux towards both p-CA and P-I production. Based on precursor feeding and metabolic fluxes, a current hypothesis is that precursors from both the iridoid and shikimate/phenylpropanoid pathways are a flux limitation for P-I production in shoot cultures of P. kurroa plants. This work thus sets a stage for future endeavour to elevate production of P-I in cultured plant cells.

In vitro – In vivo metabolism and pharmacokinetics of picroside I and II using LC-ESI-MS method

Picroside I and II, iridoid glycosides, are the major active markers of roots and rhizomes of Picrorhiza kurroa (family: Scrophulariaceae). The rhizomes of P. kurroa have been traditionally used to treat worms, constipation, low fever, scorpion sting, asthma and ailments affecting the liver. Various Ayurvedic and herbal preparations are available in the market which contains P. kurroa e.g. Arogyavadhini vati, Tiktadi kwath, Picrolax capsules and suspension. These preparations are used without any significant pharmacokinetics data. Previously, we have reported that oral bioavailability of picroside I and II is low. Most of the iridoid glycosides are primarily metabolized by intestinal microbial flora. So, it is necessary to determine the metabolic profile of picroside I and II and check the correlation with lower bioavailability. Therefore, this study was designed to check metabolic (in vitro and in vivo) profile along with pharmacokinetic profile of picroside I and II. For this, a sensitive and selective LC-ESI-MS method was developed and validated for simultaneous determination of picroside I and II in rat plasma. Chromatographic separations were performed on C18 column. The mobile phase consisted of acetonitrile: 10 mM ammonium acetate buffer [90:10 v/v], pH 3.5. In-vitro Metabolic study was performed on rat liver microsomes and primary hepatocytes. In-vivo pharmacokinetic and metabolic profile of picroside I and II was generated after oral administration of Kutkin (mixture of picroside I and II) to Sprague-Dawley rats. Various pharmacokinetic parameters viz. Cmax, Tmax, AUC(0−t) were determined. In metabolic study, eight metabolites of picroside I and six metabolites of picroside II were identified in vitro, out of which four metabolites for each picroside I and picroside II were identified in vivo.

Anti-inflammatory Effect of Picrorhiza kurroa in Experimental Models of Inflammation.

Picrorhiza kurroa is an important medicinal plant in the Ayurvedic system of medicine. The root and rhizome of this plant are used for the treatment of various liver and inflammatory conditions. In the present study, we sought to investigate the anti-inflammatory activity of P. kurroa rhizome extract against carrageenan-induced paw edema and cotton pellet implantation-induced granuloma formation in rats. In addition, its immunomodulatory activity was evaluated in Complete Freund's Adjuvant-induced stimulation of a peritoneal macrophage model and lipopolysaccharide-stimulated RAW 264.7 murine macrophages. Pretreatment with P. kurroa rhizome extract inhibited carrageenan-induced paw edema and cotton pellet-induced granuloma formation in a dose-dependent manner. This was associated with reduced levels of inflammatory cytokines (TNF-α, IL-1β, IL-6) accompanied with increased anti-inflammatory cytokine (IL-10) in the serum and peritoneal macrophages. Additionally, P. kurroa rhizome extract inhibited inflammatory TNF-receptor 1 and cyclooxygenase-2 in Complete Freund's Adjuvant-induced activated peritoneal macrophages. Furthermore, P. kurroa rhizome extract treatment significantly inhibited iNOS and suppressed the activation of NF-κB through inhibition of its phosphorylation and by blocking the activation of IκB kinase alpha in lipopolysaccharide-stimulated RAW264.7 macrophages. Taken together, these results suggest that P. kurroa has anti-inflammatory activity that is mediated through the suppression of macrophage-derived cytokine and mediators via suppression of NF-κB signaling.

EN

Picrorhiza kurroa Royle ex. Benth is an endangered and important medicinal plant of alpine Himalayas. Developing an efficient protocol for its mass multiplication is essential to meet the requirements of pharmaceutical industries and also in conservation of this plant under its natural habitat. Present study was undertaken to develop a protocol for in vitro mass multiplication of P. kurroa. Result of study revealed that highest frequency of shoot regeneration was achieved on Murashige and Skoog's basal medium supplemented with 1.0mg/l BAP, 0.5 mg/l Kn and 1.0mg/l GA3, while the best rooting was observed in MS medium supplemented with 2.5 mg/l IBA. MS medium supplemented with 3.0 mg/l 2,4-D resulted in highest frequency of embryogenic callus. Callus inoculated on MS media supplemented with BAP and IAA resulted in both shoot and root formation while the callus on MS media supplemented with NAA and IBA resulted only root formation. The somatic embryos were established from callus on MS medium supplemented 2.5 mg/l 2,4- D after four weeks. MS medium containing 1.0 mg/l BAP and 1.0 mg/l GA3 resulted into shoots from well developed somatic embryos. This protocol will provide a system for the germplasm conservation in P. kurroa by multiplication and regeneration of true to type plants.

Discerning picroside-I biosynthesis via molecular dissection of in vitro shoot regeneration in Picrorhiza kurroa.

Expression analysis of primary and secondary metabolic pathways genes vis-à-vis shoot regeneration revealed developmental regulation of picroside-I biosynthesis in Picrorhiza kurroa. Picroside-I (P-I) is an important iridoid glycoside used in several herbal formulations for treatment of various disorders. P-I is synthesized in shoots of Picrorhiza kurroa and Picrorhiza scrophulariiflora. Current study reports on understanding P-I biosynthesis in different morphogenetic stages, viz. plant segment (PS), callus initiation (CI), callus mass (CM), shoot primordia (SP), multiple shoots (MS) and fully developed (FD) stages of P. kurroa. Expression analysis of genes involved in primary and secondary metabolism revealed that genes encoding HMGR, PMK, DXPS, ISPE, GS, G10H, DAHPS and PAL enzymes of MVA, MEP, iridoid and shikimate/phenylpropanoid pathways showed significant modulation of expression in SP, MS and FD stages in congruence with P-I content compared to CM stage. While HK, PK, ICDH, MDH and G6PDH showed high expression in MS and FD stages of P. kurroa, RBA, HisK and CytO showed high expression with progress in regeneration of shoots. Quantitative expression analysis of secondary metabolism genes at two temperatures revealed that 7 genes HMGR, PMK, DXPS, GS, G10H, DAHPS and PAL showed high transcript abundance (32-87-folds) in FD stage derived from leaf and root segments at 15°C compared to 25°C in P. kurroa. Further screening of these genes at species level showed high expression pattern in P. kurroa (6-19-folds) vis-à-vis P. scrophulariiflora that was in corroboration with P-I content. Therefore, current study revealed developmental regulation of P-I biosynthesis in P. kurroa which would be useful in designing a suitable genetic intervention study by targeting these genes for enhancing P-I production.

Allelopathic Performance of Medicinal Plants on Traditional Oilseed and Pulse Crop of Central Himalaya, India

The present study was aimed to determine the allelopathic effect of aqueous extract of above ground (AG) and below ground (BG) part of four medicinal plants (MAPs) viz., Picrorhiza kurroa, Asperagus racemosus, Ocimum sanctum and Valeriana wallichii on germination and seedling growth of some traditional food and oilseed crops. The aqueous extract of AG and BG part of the studied MAPs at 2 % significantly reduced the germination, plumule and radicle growth of the selected pulse and oil seed crops in bioassays. The results of the present experiments revealed that MAPs have inhibitory effect on food and oilseed crops owing an occurrence of allelochemicals.

Modulation of Picroside-I Biosynthesis in Grown Elicited Shoots of Picrorhiza kurroa In Vitro

Elicitors are considered as biostimulants for growth improvement and enhancement of secondary metabolite content. To date, only seaweed extract (SWE) powder has been studied for its effect on picroside-I (P-I) production in in vitro grown Picrorhiza kurroa plants. However, little is known at the molecular level about P-I production in P. kurroa plants upon SWE treatment. Here, we investigated the relative effects of supplying different elicitors including methyl jasmonate (MeJa), sodium nitroprusside (SNP), and abscisic acid (ABA) with SWE on plant growth and P-I production in addition to their effects at the molecular level reflecting the metabolic status of P-I biosynthesis. Our results indicated that only SWE, ABA, and SNP stimulated P-I production by 2.60-, 2.01-, and 1.35-fold, respectively, whereas MeJa decreased P-I content. Interestingly, SWE modulated all four integrating secondary metabolic pathways, covering almost all critical steps in the methylerythritol phosphate (MEP), mevalonate (MVA), iridoid, and phenylpropanoid pathways to stimulate P-I biosynthesis. SNP targeted the MVA/MEP pathways in conjunction with the iridoid pathway, whereas ABA modulated the phenylpropanoid pathway to increase the P-I content in P. kurroa. This is apparently the first report on treatment of different elicitors in in vitro grown P. kurroa plants for eliciting P-I content and exploring the role of different elicitors at the molecular level.

Comprehensive Chemical Profiling of Picrorhiza kurroa Royle ex Benth Using NMR, HPTLC and LC-MS/MS Techniques.

Picrorhiza kurroa is an important herb in Indian medicine and contains cucurbitacins, flavonoids, phenolics, iridoid-glucoside and their derivatives as active constituents for the treatment of indigestion, fever, hepatitis, cancer, liver and respiratory diseases. Extensive use of P. kurroa needs detailed analysis and recognition of chemical diversity, is of great importance to evaluate their role as quality control markers. In the present study, comprehensive metabolic profiling of crude extracts of leaves and rhizomes of P. kurroa was carried out using NMR, HPTLC and LC-MS/MS. Primary and secondary metabolites were unambiguously identified along with a new report of monoterpenic glycoside (1-β-D-glucopyranosyl)-8-hydroxy-3,7-dimethyl-oct-2(E),6(E)-dienoate) in P. Kurroa. Significant qualitative differences with respect to the secondary metabolites were noticed between the leaves and rhizomes tissues. Leaves contained more cucurbitacins and flavonoids while iridoids were present more in rhizomes. The comprehensive chemical profiling is expected to give an idea of chemical diversity and quality of P. kurroa, for their ultimate utilisation in various applications.

First Report of Leaf Spot on Medicinal Herb Picrorhiza kurroa (Royle ex. Benth) Caused by Alternaria tenuissima in India

First Report of Leaf Spot on Medicinal Herb <i>Picrorhiza kurroa</i> (Royle ex. Benth) Caused by <i>Alternaria tenuissima</i> in India

Phytochemical studies for quantitative estimation of iridoid glycosides in Picrorhiza kurroa Royle.

Background Picrorhiza kurroa Royle commonly known as ‘Kutki or Kutaki’ is an important medicinal plant in Ayurvedic system of medicine and has traditionally been used to treat disorders of the liver and upper respiratory tract. The plant is the principle source of iridoid glycosides, picrosides I, II and kutkoside used in various herbal drug formulations mainly as strong hepatoprotective and immune-modulatory compound. The species has become endangered to near extinction due to the unregulated collection from the wild, slower plant growth and ecological destruction of natural habitats. There is a severe shortage of plant material, while the market demand is ever increasing. Hence, it is very important to apply a simple and precise analytical method to determine and validate the concentration of the major bioactive constituents in different populations of this plant species for development of a high yielding chemotype for large scale production and its commercial exploitation on scientific lines.ResultsThis study assessed and validated a fast and reliable chromatography method for the determination of picroside-I and picroside-II in different populations of this priortized medicinal plant species. Separation and resolution of picrosides was carried out on a reversed phase (C-18) column by using a mobile phase of methanol and water (40:60 v/v). The detection of picrosides was carried out at 270 nm. The average levels of these two major marker compounds in all the seven accessions showed significant quantitative variation (ANOVA, p < 0.05) between mean levels of marker compounds and their accumulation in different parts of the plant viz. roots, rhizomes and leaves. The highest content of pk-I was found in the accession from Gurez altitude (3750 masl) while the highest content of pk-II was found in accession from Keller (Shopian) altitude (3300 masl) demonstrate that picroside accusation is directly correlated with altitudinal variation. The method was validated in terms of linearity, accuracy and precision (within- and between-assay variation).ConclusionA simple chromatographic method with the ability to separate both the major chemical constituents effectively in different herbal extracts of P. kurroa and other related species has been standardized and validated, which is more suitable for regular and normal analysis of picrosides in different herbal formulations. The paper accomplish that picroside concentration in different samples showed significant variation based on altitude and other agroclimatic factors, which can be useful in the selection and collection of superior genotypes with higher concentration of these marker compounds.

Single dose oral toxicity study of &lt;i&gt;Picrorhiza kurroa&lt;/i&gt; rhizome extract in Wistar rats

Picrorhiza kurroa is a well-known ayurvedic or herbal medicine which is used very commonly in the treatment of various diseases. Therefore, we studied the oral toxicity of Picrorhiza kurroa rhizome extract in rats. A single high dose of the extract at 2000 mg/kg body weight was tested on Wistar rats. Mortality/viability and clinical signs were recorded on test day 0 (prior to administration), 7, 14 and at death. All animals appeared normal from day one to throughout the experimental procedure. Picrorhiza kurroa rhizome extract is non-toxic to rats and helped in weight gain with LD50 > 2000 mg/kg body weight. Oral administration of Picrorhiza kurroa is not connected with any toxicologically significant effects and the data could provide satisfactory preclinical evidence of safety to launch a clinical trial on a standardized formulation of the plant extracts. Language: en

Efficient in vitro establishment and multiplication of Picrorhiza kurroa Royle ex Benth through in vitro raised seedlings

The aim of present investigation was to develop a highly reproducible and well documented protocol for in vitro germination and multiplication of highly valuable medicinal herb, Picrorhiza kurroa via callus mediated organogenesis using the axenic epicotyls originated from in vitro germinated seedlings. Highest germination percentage (88.6) was observed in stratified seeds (4°C for 5 days) treated with 100 mg/l GA3 and maintained at 25 ±1°C under continuous light conditions. Out of different combinations of plant growth regulators tested, Murashige and Skoog (MS) medium supplemented with 1.5 mg/l α naphthalene acetic acid (NAA)+0.75 mg/l kinetin (Kn)+0.75 mg/l 6-benzyladenine (BAP) was found best for callus induction and further proliferation. Addition of gibberellic acid (GA3) at 1.0 mg/l concentration in hormonal combination of 2.0 mg/l Kn and 0.75 mg/l indole-3-acetic acid (IAA) was found to be optimum to produce highest shooting response (95%) with an average number of 11.40 shoots per calli. The rooting was optimized using 0.5 mg/l IAA with 85% response. The plantlets with well-developed roots were hardened and were successfully acclimatized in field conditions. This protocol may offer the potential to substantially reduce the pressure of commercial need of this valuable herb on wild stock.

Safety evaluation of Picrorhiza kurroa rhizome extract by bacterial reverse mutation test

Safety evaluation of Picrorhiza kurroa rhizome extract by bacterial reverse mutation test

NGS Transcriptomes and Enzyme Inhibitors Unravel Complexity of Picrosides Biosynthesis in Picrorhiza kurroa Royle ex. Benth

Picrorhiza kurroa is an important medicinal herb valued for iridoid glycosides, Picroside-I (P-I) and Picroside-II (P-II), which have several pharmacological activities. Genetic interventions for developing a picroside production platform would require knowledge on biosynthetic pathway and key control points, which does not exist as of today. The current study reports that geranyl pyrophosphate (GPP) moiety is mainly contributed by the non-mevalonate (MEP) route, which is further modified to P-I and P-II through phenylpropanoid and iridoid pathways, in total consisting of 41 and 35 enzymatic steps, respectively. The role of the MEP pathway was ascertained through enzyme inhibitors fosmidomycin and mevinolin along with importance of other integrating pathways using glyphosate, aminooxy acetic acid (AOA) and actinomycin D, which overall resulted in 17%-92% inhibition of P-I accumulation. Retrieval of gene sequences for enzymatic steps from NGS transcriptomes and their expression analysis vis-à-vis picrosides content in different tissues/organs showed elevated transcripts for twenty genes, which were further shortlisted to seven key genes, ISPD, DXPS, ISPE, PMK, 2HFD, EPSPS and SK, on the basis of expression analysis between high versus low picrosides content strains of P. kurroa so as to eliminate tissue type/ developmental variations in picrosides contents. The higher expression of the majority of the MEP pathway genes (ISPD, DXPS and ISPE), coupled with higher inhibition of DXPR enzyme by fosmidomycin, suggested that the MEP route contributed to the biosynthesis of P-I in P. kurroa. The outcome of the study is expected to be useful in designing a suitable genetic intervention strategy towards enhanced production of picrosides. Possible key genes contributing to picroside biosynthesis have been identified with potential implications in molecular breeding and metabolic engineering of P. kurroa.

Optimization of a preparative RP-HPLC method for isolation and purification of picrosides in Picrorhiza kurroa

Preparative reversed phase high pressure liquid chromatography (prep-RP-HPLC) coupled with photodiode array (PDA) and evaporative light scattering (ELSD) detectors was employed to isolate picrosides present in Picrorhiza kurroa Royle ex Benth. A binary gradient method (water and acetonitrile) was optimized on Water Spherisorb S10 ODS2 20 mm × 250 mm Semiprep Column with a 20 mL/min flow rate at ambient temperature with linear binary gradient conditions; at 0 min 15 % acetonitrile hold for 15 min; 15 to 22 % acetonitrile in next 2 min, hold for 13 min; 22 to 15 % acetonitrile in 5 min hold for 5 min to equilibrate column for next injection. The picroside-I and picroside-II fractions were 98.6 and 99.7 % pure with 13.9 mg and 9.8 mg yield per 200 mg of crude extract, respectively from mature dry rhizomes. Structures of isolated iridoids were confirmed with UV scan, 1H-NMR and direct infusion ESI-Q-TOF-MS/MS data.

Picrorhiza kurroa Inhibits Experimental Arthritis Through Inhibition of Pro-inflammatory Cytokines, Angiogenesis and MMPs.

The present study investigates the anti-arthritic activity of Picrorhiza kurroa (PK), on formaldehyde and adjuvant-induced arthritis (AIA) in rat. Administration of Picrorhiza kurroa rhizome extract (PKRE) significantly inhibited joint inflammation in both animal models. In AIA-induced arthritic rat, treatment with PKRE considerably decreased synovial expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor receptor-1 (TNF-R1) and vascular endothelial growth factor as compared with control. The anti-arthritic activity was found to be well substantiated with significant suppression of oxidative and inflammatory markers as there was decreased malonaldehyde, Nitric oxide, tumor necrosis factor alpha levels accompanied with increased glutathione and superoxide dismutase, catalase activities. Additionally, PKRE significantly inhibited the expression of degrading enzymes, matrix metalloproteinases-3 and matrix metalloproteinases-9 in AIA-induced arthritic rat. Histopathology of paw tissue displayed decreased inflammatory cell infiltration as compared with control. Taken together, these results demonstrated the anti-arthritic activity of PKRE against experimental arthritis, and the underlying mechanism behind this efficacy might be mediated by inhibition of inflammatory mediators and angiogenesis, improvement of the synovium redox status and decreased expression of matrix metalloproteinases.

Use of Piriformospora indica as Potential Biological Hardening Agent for Endangered Micropropagated Picrorhiza kurroa Royel ex Benth

The rich plant biodiversity of Himalayan region is vanishing at an alarming rate due to urbanization and indiscriminate collection of important plants at a large scale from the wild for trade. Overexploitation of Picrorhiza kurroa, an alpine Himalayan medicinal plant for extraction of its active compounds has led to the reduction of its natural population. In vitro regeneration offers a noteworthy solution for the propagation of endangered medicinal plants. Successful acclimatization of in vitro plants with fungal endophyte Piriformospora indica reduces the mortality rate of in vitro plantlets due to transplantation shock. Root colonization with P. indica renders growth promotion and better adaptation of a wide range of plants. Present study focuses on the use of P. indica as a biological hardening agent for increasing the percentage survival of in vitro shoots. Survival of fungus treated biohardened in vitro plants was increased by 1.3 folds. Root number in 4 weeks old P. indica treated biohardened in vitro plants enhanced by 1.25 fold and facilitated its establishment under greenhouse conditions.

Mining simple sequence repeats inPicrorhiza kurroatranscriptomes for assessing genetic diversity among accessions varying for picrosides contents

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 ofP. kurroafrom 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 fromP. kurroatranscriptomes. Out of 361 SSR primers tested on 26 accessions, 35 primers yielded polymorphic profiles. Overall low genetic diversity was observed inP. kurroaaccessions. 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.

An insight into conflux of metabolic traffic leading to picroside-I biosynthesis by tracking molecular time course changes in a medicinal herb, Picrorhiza kurroa

Picroside-I (P-I) is a member of the iridoid glycosides family of natural products, which are used to treat liver disorders. The growing medicinal need for this benign compound has stimulated the present study to identify genes important for the biosynthesis of P-I. In this study, molecular screens have been generated using gene expression patterns obtained by quantitative RT-PCR which have extended the knowledge of genes associated with P-I biosynthesis. A total of 13 genes encoding the rate limiting enzymes of different pathways, were analyzed by qRT-PCR in Picrorhiza kurroa shoots collected at 0, 10, 20, 30 and 40 days. The results showed that five of the genes (HK, DXPS, ISPD, HMGR and PMK) are supposed to be essential for P-I biosynthesis up to 20 days while DAHPS and G-10-H, in conjunction might assist P-I biosynthesis between 20 and 30 days of P. kurroa growth. This is apparently the first report on the molecular aspects of different pathways integrated in P-I biosynthesis. Moreover, principal component analysis prediction also corroborated the genes selection by identifying genes signatures for different samples (collected at different time intervals) and supported the link between samples and gene expression patterns. Overall, this study capitalizes on dynamic gene expression patterns obtained in response to the P-I stimuli under different stages of P. kurroa growth which are likely to define the regulatory steps in P-I biosynthesis.