Endomicrobiome of in vitro and natural plants deciphering the endophytes-associated secondary metabolite biosynthesis in Picrorhiza kurrooa, a Himalayan medicinal herb.

IMPORTANCE: Picrorhiza kurrooa is a major source of picrosides, potent hepatoprotective molecules. Due to the ever-increasing demands, overexploitation has caused an extensive decline in its population in the wild and placed it in the endangered plants' category. At present plant in-vitro systems are widely used for the sustainable generation of P. kurrooa plants, and also for the conservation of other commercially important, rare, endangered, and threatened plant species. Furthermore, the in-vitro-generated plants had reduced content of therapeutic secondary metabolites compared to their wild counterparts, and the reason behind, not well-explored. Here, we revealed the loss of plant-associated endophytic communities during in-vitro propagation of P. kurrooa plants which also correlated to in-planta secondary metabolite biosynthesis. Therefore, this study emphasized to consider the essential role of plant-associated endophytic communities in in-vitro practices which may be the possible reason for reduced secondary metabolites in in-vitro plants.

Simultaneous Determination of Five Iridoids of Picrorhiza scrophulariiflora in Rat Plasma Using UHPLC-ESI-MS/MS.

In this study, we developed an ultra-performance liquid chromatography-electrospray tandem quadrupole mass spectrometry (UHPLC-ESI-MS/MS) method to simultaneously determine Picroside-I, Picroside-II, Picroside-III, minecoside, and sweroside in rat plasma. The chromatographic column was an ACQUITY UHPLC® BEH Amide Column (2.1 × 100 mm, 1.7 µm; Waters, MA, USA), column temperature 40 °C. The mobile phase was 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile solution. The flow rate was 0.4 mL/min. Multiple reaction monitoring (MRM) and negative ion modes were adopted. The results showed that the calibration curves of five compounds in plasma showed good linearity (r > 0.9911) over the studied dose range. The lower limits of quantification (LLOQ) for Picroside-I, Picroside-II, Picroside-III, minecoside, and sweroside were 6.876, 5.193, 5.040, 1.260, and 4.527 ng/mL, respectively. The intra-day and inter-day precision were <15%. The matrix effects ranged from 95.77 to 101.9%. The Tmax were 1.1 ± 0.2, 1.1 ± 0.1, 0.8 ± 0.1, 1.0 ± 0.2, and 2.1 ± 0.1 h. This study will be useful in understanding the behavior of drugs in the body and the body's effect on drugs. It also offers theoretical underpinnings and highlights the importance of clinical applications and creating novel drugs.

Hepatoprotective Principles from the Rhizomes of Picrorhiza kurroa.

A methanol extract of rhizomes of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae) showed hepatoprotective effects against D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. We had previously isolated 46 compounds, including several types of iridoid glycosides, phenylethanoid glycosides, and aromatics, etc., from the extract. Among them, picroside II, androsin, and 4-hydroxy-3-methoxyacetophenone exhibited active hepatoprotective effects at doses of 50-100 mg/kg, per os (p.o.) To characterize the mechanisms of action of these isolates and to clarify the structural requirements of phenylethanoid glycosides for their hepatoprotective effects, their effects were assessed in in vitro studies on (i) D-GalN-induced cytotoxicity in mouse primary hepatocytes, (ii) LPS-induced nitric oxide (NO) production in mouse peritoneal macrophages, and (iii) tumor necrosis factor-α (TNF-α)-induced cytotoxicity in L929 cells. These isolates decreased the cytotoxicity caused by D-GalN without inhibiting LPS-induced macrophage activation and also reduced the sensitivity of hepatocytes to TNF-α. In addition, the structural requirements of phenylethanoids for the protective effects of D-GalN-induced cytotoxicity in mouse primary hepatocytes were evaluated.

Network Pharmacology Study of Bioactive Components and Molecular Mechanisms of the Glycoside Fraction from Picrorhiza scrophulariiflora Against Experimental Colitis.

Purpose: To explore the potential mechanism of glycosidic fraction of Picrorhiza scrophulariiflora Pennell (GPS) extract for the treatment of colitis using UPLC-QTOF-MS analysis, network pharmacology and experimental research. Methods: The active components of GPS extract were identified by UPLC-QTOF-MS analysis and extracted their targets from the databases, which was used for network pharmacology analysis. Kyoto Encyclopedia of genes and genomes (KEGG) pathway analysis was performed to discover potential therapeutic mechanisms, and the network pharmacology results were then validated by in vivo and in vitro experiments. Results: The results showed that GPS extract significantly alleviated the clinical signs of colitis, including body weight, disease activity index, colon shortening, and colon tissue damage, and inhibited the transcription and production of colonic IL-1ß and IL-6 in DSS-induced colitis mice. In vitro, GPS extract also significantly suppressed nitric oxide (NO) production, iNOS expression, IL-1ß and IL-6 transcription of LPS-activated RAW 264.7 cells. Network pharmacology integrated with experimental validation identified that GPS extract significantly suppressed Akt, p38, ERK, and JNK phosphorylation in vivo and in vitro, and luteolin, apocynin, caffeic acid, caffeic acid methyl ester, luteoloside, picroside II, aucubin, cinnamic acid, vanillic acid, and sweroside were the main components responsible for the anti-inflammatory effect of GPS. These findings demonstrate that the potential anti-inflammatory effect of GPS extract against colitis is achieved through suppressing PI3K/Akt and MAPK pathways, and that the abovementioned active components mainly exerted its anti-inflammatory effect. Conclusion: The therapeutic effect of GPS extract on colitis is related to PI3K/Akt and MAPK pathways, which is a promising remedy for colitis therapy.

Picroside III, an Active Ingredient of Picrorhiza scrophulariiflora, Ameliorates Experimental Colitis by Protecting Intestinal Barrier Integrity.

This study aims to explore the protective effects of Picroside III, an active ingredient of Picrorhiza scrophulariiflora, on the intestinal epithelial barrier in tumor necrosis factor-α (TNF-α) induced Caco-2 cells and dextran sulfate sodium (DSS) induced colitis in mice. Results show that Picroside III significantly alleviated clinical signs of colitis including body weight loss, disease activity index increase, colon shortening, and colon tissue damage. It also increased claudin-3, ZO-1 and occludin expressions and decreased claudin-2 expression in the colon tissues of mice with colitis. In vitro, Picroside III also significantly promoted wound healing, decreased the permeability of cell monolayer, upregulated the expressions of claudin-3, ZO-1 and occludin and downregulated the expression of claudin-2 in TNF-α treated Caco-2 cells. Mechanism studies show that Picroside III significantly promoted AMP-activated protein kinase (AMPK) phosphorylation in vitro and in vivo, and blockade with AMPK could significantly attenuate the upregulation of Picroside III in ZO-1 and occludin expressions and the downregulation of claudin-2 expression in TNF-α treated Caco-2 cells. In conclusion, this study demonstrates that Picroside III attenuated DSS-induced colitis by promoting colonic mucosal wound healing and epithelial barrier function recovery via the activation of AMPK.

ABC transporters mined through comparative transcriptomics associate with organ-specific accumulation of picrosides in a medicinal herb, Picrorhiza kurroa.

Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the preparation of several hepatoprotective herbal drugs. These compounds accumulate in stolons/rhizomes; however, biosynthesized in different organs, viz., picroside-I in shoots and picroside-II in roots. As of today, no information exists on what transporters are transporting these metabolites from shoots and roots to the final storage organ, stolon, which ultimately transforms into rhizome. The ATP-binding cassette (ABC) transporters are reported to transport majority of secondary metabolites, including terpenoids in plants, therefore, we mined P. kurroa transcriptomes to identify and shortlist potential candidates. A total of 99 ABC transporter-encoding transcripts were identified in 3 differential transcriptomes, PKSS (shoots), PKSTS (stolons), and PKSR (roots) of P. kurroa, based on in silico comparative analysis and transcript abundance. 15 of these transcripts were further validated for their association using qRT-PCR in shoots, roots and stolon tissues in P. kurroa accessions varying for picroside-I and picroside-II contents. Organ-specific expression analysis revealed that PkABCA1, PkABCG1, and PkABCB5 had comparatively elevated expression in shoots; PkABCB2 and PkABCC2 in roots; PkABCB3 and PkABCC1 in stolon tissues of P. kurroa. Co-expression network analysis using ABC genes as hubs further unravelled important interactions with additional components of biosynthetic machinery. Our study has provided leads, first to our knowledge as of today, on putative ABC transporters possibly involved in long distance and local transport of picrosides in P. kurroa organs, thus opening avenues for designing a suitable genetic intervention strategy.

Pharmacological and Clinical Efficacy of Picrorhiza kurroa and Its Secondary Metabolites: A Comprehensive Review.

Traditional remedies for the treatment of various ailments are gaining popularity. Traditionally, one of the most valuable therapeutic herbs has been Picrorhiza kurroa Royle ex Benth. Traditional and folk uses of P. kurroa include chronic constipation, skin-related problems, burning sensation, chronic reoccurring fever, jaundice, heart problems, breathing, digestion, allergy, tuberculosis, blood-related problems, prediabetes and obesity, laxative, cholagogue, and liver stimulatory. Phytoconstituents such as glycosides, alkaloids, cucurbitacins, iridoids, phenolics, and terpenes in P. kurroa have shown promising pharmacological potential. In order to uncover novel compounds that may cure chronic illnesses, such as cardiovascular, diabetes, cancer, respiratory, and hepatoprotective diseases, the screening of P. kurroa is essential. This study comprehensively evaluated the ethnopharmacological efficacy, phytochemistry, pharmacological activity, dose, and toxicity of P. kurroa. This review provides comprehensive insights into this traditional medication for future research and therapeutic application. The purpose of this review article was to determine the pharmacological effects of P. kurroa on a variety of disorders. P. kurroa may be a natural alternative to the standard treatment for eradicating newly evolving diseases. This study is intended as a resource for future fundamental and clinical investigations.

Biological Activity of Picrorhiza kurroa: A Source of Potential Antimicrobial Compounds against Yersinia enterocolitica.

Yersiniosis, caused by Yersinia enterocolitica, is the third most rampant zoonotic disease in Europe; the pathogen shows high antibiotic resistance. Herbs have multiple anti-microbial components that reduce microorganism resistance. Therefore, an extract of Picrorhiza kurroa (P. kurroa) was evaluated for potential antimicrobial activity. We report that the ethanolic extract of P. kurroa showed effective antimicrobial activity (zone of inhibition: 29.8 mm, Minimum inhibitory concentration (MIC): 2.45 mg/mL, minimum bactericidal concentration (MBC): 2.4 mg/mL) against Yersinia enterocolitica. Potential bioactive compounds from P. kurroa were identified using LC-MS, namely, cerberidol, annonidine A, benzyl formate, picroside-1, and furcatoside A. P. kurroa showed effective antimicrobial potential in skim milk at different pH, acidity, and water activity levels. P. kurroa affected the physiology of Yersinia enterocolitica and reduced the number of live cells. Yersinia enterocolitica, when incubated with P. kurroa extract, showed lower toxin production. Picroside-1 was isolated and showed higher antimicrobial potential in comparison to the standard antibiotic. Picroside-1 lysed the Yersinia enterocolitica cells, as observed under scanning electron microscopy. Docking revealed that picroside-1 (ligand) showed both hydrophilic and hydrophobic interactions with the dihydrofolate reductase (DHFR) protein of Yersinia enterocolitica and that DHFR is a possible drug target. The high activity and natural origin of Picroside-1 justify its potential as a possible drug candidate for Yersinia enterocolitica.

Kutkin, iridoid glycosides enriched fraction of Picrorrhiza kurroa promotes insulin sensitivity and enhances glucose uptake by activating PI3K/Akt signaling in 3T3-L1 adipocytes.

BACKGROUND: Therapeutic failure and drug resistance are common sequelae to insulin resistance associated with type 2 diabetes mellitus (T2DM). Consequently, there is an unmet need of alternative strategies to overcome insulin resistance associated complications. PURPOSE: To demonstrate whether Kutkin (KT), iridoid glycoside enriched fraction of Picrorhiza kurroa extract (PKE) has potential to increase the insulin sensitivity vis à vis glucose uptake in differentiated adipocytes. METHODS: Molecular interaction of KT phytoconstituents, picroside-I (P-I) & picroside- II (P-II) with peroxisome proliferator-activated receptor gamma (PPARγ), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were analyzed in silico. Cellular viability and adipogenesis were determined by following 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium bromide (MTT) assay and Oil Red-O staining. Further, ELISA kit based triglycerides and diacylglycerol-O-Acyltransferase-1 (DGAT1) were assessed in differentiated adipocytes. ELISA based determination were performed to check the levels of adiponectin and tumor necrosis factor alpha (TNF-α). However, Flow cytometry and immunofluorescence based assays were employed to measure the glucose uptake and glucose transporter 4 (glut4) expression in differentiated adipocytes, respectively. Further to explore the targeted signaling axis, mRNA expression levels of PPARγ, CCAAT/enhancer binding protein α (CEBPα), and glut4 were determined using qRT-PCR and insulin receptor substrate-1 (IRS-1), Insulin receptor substrate-2 (IRS-2), PI3K/Akt, AS160, glut4 followed by protein validation using immunoblotting in differentiated adipocytes. RESULTS: In silico analysis revealed the binding affinities of major constituents of KT (P-I& P-II) with PPARγ/PI3K/Akt. The enhanced intracellular accumulation of triglycerides with concomitant activation of PPARγ and C/EBPα in KT treated differentiated adipocytes indicates augmentation of adipogenesis in a concentration-dependent manner. Additionally, at cellular level, KT upregulated the expression of DAGT1, and decreases fatty acid synthase (FAS), and lipoprotein lipase (LPL), further affirmed improvement in lipid milieu. It was also observed that KT upregulated the levels of adiponectin and reduced TNFα expression, thus improving the secretory functions of adipocytes along with enhanced insulin sensitivity. Furthermore, KT significantly promoted insulin mediated glucose uptake by increasing glut4 translocation to the membrane via PI3/Akt signaling cascade. The results were further validated using PI3K specific inhibitor, wortmannin and findings revealed that KT treatment significantly enhanced the expression and activation of p-PI3K/PI3K and p-Akt/Akt even in case of treatment with PI3K inhibitor wortmannin alone and co-treatment with KT in differentiated adipocytes and affirmed that KT as activator of PI3K/Akt axis in the presence of inhibitor as well. CONCLUSION: Collectively, KT fraction of PKE showed anti-diabetic effects by enhancing glucose uptake in differentiated adipocytes via activation of PI3K/Akt signaling cascade. Therefore, KT may be used as a promising novel natural therapeutic agent for managing T2DMand to the best of our knowledge, this is the first report, showing the efficacy and potential molecular mechanism of KT in enhancing insulin sensitivity and glucose uptake in differentiated adipocytes.

Capturing acyltransferase(s) transforming final step in the biosynthesis of a major Iridoid Glycoside, (Picroside-II) in a Himalayan Medicinal Herb, Picrorhiza kurroa.

BACKGROUND: Picrorhiza kurroa has been reported as an age-old ayurvedic hepato-protection to treat hepatic disorders due to the presence of iridoids such as picroside-II (P-II), picroside-I, and kutkoside. The acylation of catalpol and vanilloyl coenzyme A by acyltransferases (ATs) is critical step in P-II biosynthesis. Since accumulation of P-II occurs only in roots, rhizomes and stolons in comparison to leaves uprooting of this critically endangered herb has been the only source of this compound. Recently, we reported that P-II acylation likely happen in roots, while stolons serve as the vital P-II storage compartment. Therefore, developing an alternate engineered platform for P-II biosynthesis require identification of P-II specific AT/s. METHODS AND RESULTS: In that direction, egg-NOG function annotated 815 ATs from de novo RNA sequencing of tissue culture based 'shoots-only' system and nursery grown shoots, roots, and stolons varying in P-II content, were cross-compared in silico to arrive at ATs sequences unique and/or common to stolons and roots. Verification for organ and accession-wise upregulation in gene expression of these ATs by qRT-PCR has shortlisted six putative 'P-II-forming' ATs. Further, six-frame translation, ab initio protein structure modelling and protein-ligand molecular docking of these ATs signified one MBOAT domain containing AT with preferential binding to the vanillic acid CoA thiol ester as well as with P-II, implying that this could be potential AT decorating final structure of P-II. CONCLUSIONS: Organ-wise comparative transcriptome mining coupled with reverse transcription real time qRT-PCR and protein-ligand docking led to the identification of an acyltransferases, contributing to the final structure of P-II.

GC-MS Metabolomics profiling and HR-APCI-MS characterization of potential anticancer compounds and antimicrobial activities of extracts from Picrorhiza kurroa roots.

The present study explores pharmacological potential and phytochemicals profiling of Picrorhiza kurroa extracts against mammalian cancer cell lines and pathogenic microbes. Bioactive extracts from roots of Picrorhiza kurroa were recovered in the methanol, 50% aqueous dichloromethane (50 : 50 v/v) and n-hexane. Antimicrobial activity of the bioactive extracts was assessed against selected strains of bacteria and pathogenic fungi. Aqueous dichloromethane extract showed highest zone of growth inhibition (39.06 ± 1.0 mm) towards Staphylococcus aureus bacteria while methanolic extract showed the lowest inhibition (6.3 ± 4.1 mm) to Escherichia coli bacteria. The tested extracts such as methanol and aqueous dichloromethane exhibited higher inhibition antifungal activity against Aspergillus flavus compared to Fusarium oxysporum. As far as cytotoxicity (MTT assay) of the tested extracts is concerned, n-hexane and aqueous dichloromethane extracts were found to be very active against all cancer cell lines (breast cancer MCF7, MDA-MB-231, SKBR3 and ovarian cancer SKOV3). A preliminary phytochemicals profiling was performed in extracts using GC-MS. Several fractions of active extract were separated with HPLC and analyzed using High Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry (HR-APCI-MS). Two purified compounds (Dihydromikanolide and 1,3-Dicyclohexyl-4-(cyclohexylimino)-2-(cyclohexylethylamino)-3,4-dihydro-1,3-diazetium) were further evaluated for their anticancer activity against ovarian cancer cell line. Our findings depict that all the tested extracts showed considerable anticancer potential through cell viability assays. The purified compound 1 - Dihydromikanolide from methanolic extract was found to be active against ovarian cancer cells and can be explored as a promising nutra-pharmaceutical candidate against ovarian cancer. However, further studies exploring the molecular pathways and in vivo testing are required.

[Advances in research on chemical composition of Picrorhiza scrophulariiflora and P. kurroa and their biological activities].

At present, 141 compounds have been isolated from Picrorhiza scrophulariiflora and P. kurroa of the Scrophulariaceae plants, including 46 iridoid glycosides, 29 tetracyclic triterpenoids, 25 phenylpropanoids, and 11 phenylethanoid glycosides. Pharmacological studies have demonstrated that they have liver-, heart-, brain-, kidney-, and nerve cells-protecting effects as well as anti-tumor, anti-inflammatory, anti-bacterial, anti-asthma, anti-diabetic, immunomodulatory, and blood lipid-lowering activities. This article reviews the chemical components and pharmacological activities of P. scrophulariiflora and P. kurroa, aiming to provide a basis for the in-depth research, development, and utilization of the two plants.

In-depth assembly of organ and development dissected Picrorhiza kurroa proteome map using mass spectrometry.

BACKGROUND: Picrorhiza kurroa Royle ex Benth. being a rich source of phytochemicals, is a promising high altitude medicinal herb of Himalaya. The medicinal potential is attributed to picrosides i.e. iridoid glycosides, which synthesized in organ-specific manner through highly complex pathways. Here, we present a large-scale proteome reference map of P. kurroa, consisting of four morphologically differentiated organs and two developmental stages. RESULTS: We were able to identify 5186 protein accessions (FDR < 1%) providing a deep coverage of protein abundance array, spanning around six orders of magnitude. Most of the identified proteins are associated with metabolic processes, response to abiotic stimuli and cellular processes. Organ specific sub-proteomes highlights organ specialized functions that would offer insights to explore tissue profile for specific protein classes. With reference to P. kurroa development, vegetative phase is enriched with growth related processes, however generative phase harvests more energy in secondary metabolic pathways. Furthermore, stress-responsive proteins, RNA binding proteins (RBPs) and post-translational modifications (PTMs), particularly phosphorylation and ADP-ribosylation play an important role in P. kurroa adaptation to alpine environment. The proteins involved in the synthesis of secondary metabolites are well represented in P. kurroa proteome. The phytochemical analysis revealed that marker compounds were highly accumulated in rhizome and overall, during the late stage of development. CONCLUSIONS: This report represents first extensive proteomic description of organ and developmental dissected P. kurroa, providing a platform for future studies related to stress tolerance and medical applications.

Green Synthesis, Characterization and Antimicrobial Activities of Copper Nanoparticles from the Rhizomes Extract of Picrorhiza kurroa.

BACKGROUND: Green synthesized nanoparticles from the solvent extract of various plant parts show better biological activities as compared to parent solvent plant extract. Traditionally rhizomes of Picrorhiza kurroa are used to cure various diseases like diarrhea, fever, jaundice, eye infection, skin problems, asthma, arthritis, cancer, diabetes, gastrointestinal problems. OBJECTIVE: The present study describes the synthesis of copper nanoparticles from a hydroethanolic extract of P. kurroa rhizomes (CuNPs-Pk) and their evaluation for antimicrobial activities against gram-negative, gram-positive bacterial, and fungal strains. METHODS: The solution of copper sulfate and hydroethanolic extract of rhizomes of P. kurroa was mixed with help of a magnetic stirrer at 60°C temperature for 1 h. The blue color of CuSO4.5H2O changed to brownish-black colored copper nanoparticles within 10 minutes. These nanoparticles were centrifuged at 4000 rpm for 20 min, washed with ethanol, followed by deionized water, dried, and were characterized by Ultra violet-visible (UV-Vis) absorption spectra, Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (TEM). Different concentrations of hydroethanolic extract of Picrorhiza kurroa rhizomes (HEEPk), CuNPs-Pk and copper oxide nanoparticles (bare CuO) ranging from 100-400 ppm had been studied against selected bacterial and fungal strains by using the well plate diffusion method. Ciprofloxin and fluconazole were used as standard and Dimethyl sulfoxide (DMSO) as a control for selected strains. RESULTS: The UV-Vis spectral studies confirmed the surface plasmon resonance of green-synthesized CuNPs-Pk. The particle size was found to be 275-285 nm. FTIR analysis of biosynthesis nanoparticles conformed the presence of various functional groups (flavonoids, glycosides, tannins, phenols). SEM and TEM of biosynthesized nanoparticles have predicted their spherical shape and their size (20-40 nm) and these particles have shown effective antimicrobial activities against selected pathogenic organisms viz. Escherichia coli, Staphylococcus aureus, and Aspergillus niger than that of HEEPk and bare CuO. CONCLUSION: The CuNPs-Pk shows effective antimicrobial activities against bacterial and fungal pathogens as compared to HEEPk and bare CuO.

Evaluating Peptides of Picrorhiza kurroa and Their Inhibitory Potential against ACE, DPP-IV, and Oxidative Stress.

Picrorhiza kurroa Royle ex Benth. is a high-altitude plant having great medicinal value. However, its medicinal value at the peptide level is still unknown, which limits its utility in the development of peptide-based therapeutics. Here, we identify 65 peptides fromP. kurroa hydrolysate. Sequence analysis suggests that one novel bioactive peptide, ASGLCPEEAVPRR (BP1), has antioxidant potential and shows angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. The molecular docking study showed that BP1 has a lower binding energy and strong affinity toward active pockets of ACE and DPP-IV, which explains its higher ACE [IC50 = 59.90 ± 9.52 µg/mL (43.40 µM)] and DPP-IV [IC50 = 3.04 ± 0.26 µg/mL (2.2 µM)] inhibitory activities. BP1 protects HEK293 cells from H2O2-induced oxidative damage by inhibiting intracellular reactive oxygen species (ROS) and malondialdehyde accumulation and activating the intrinsic antioxidant defense system. Additionally, phase-contrast microscopy studies revealed that pre-treatment of BP1 to HEK293 cells before exposure to H2O2 retains the normal morphology and blocks apoptosis. Furthermore, it also suppresses ROS-induced mitochondrial apoptosis via restoring the mitochondrial membrane potential (ΔΨm) and inhibiting caspase 3/7 activity. Therefore, BP1 has antioxidant potential and ACE and DPP-IV inhibitory activities that could be used for peptide-based formulation(s) in pharmaceuticals to treat diabetes, cardiovascular diseases, and other diseases associated with ROS.

The first draft genome of Picrorhiza kurrooa, an endangered medicinal herb from Himalayas.

Picrorhiza kurrooa is an endangered medicinal herb which is distributed across the Himalayan region at an altitude between 3000-5000 m above mean sea level. The medicinal properties of P. kurrooa are attributed to monoterpenoid picrosides present in leaf, rhizome and root of the plant. However, no genomic information is currently available for P. kurrooa, which limits our understanding about its molecular systems and associated responses. The present study brings the first assembled draft genome of P. kurrooa by using 227 Gb of raw data generated by Illumina and PacBio RS II sequencing platforms. The assembled genome has a size of n = ~ 1.7 Gb with 12,924 scaffolds. Four pronged assembly quality validations studies, including experimentally reported ESTs mapping and directed sequencing of the assembled contigs, confirmed high reliability of the assembly. About 76% of the genome is covered by complex repeats alone. Annotation revealed 24,798 protein coding and 9789 non-coding genes. Using the assembled genome, a total of 710 miRNAs were discovered, many of which were found responsible for molecular response against temperature changes. The miRNAs and targets were validated experimentally. The availability of draft genome sequence will aid in genetic improvement and conservation of P. kurrooa. Also, this study provided an efficient approach for assembling complex genomes while dealing with repeats when regular assemblers failed to progress due to repeats.

Transcriptome profiling reveal key hub genes in co-expression networks involved in Iridoid glycosides biosynthetic machinery in Picrorhiza kurroa.

Picrorhiza kurroa is a medicinal herb rich in hepatoprotective iridoid glycosides, picroside-I (P-I) and picroside-II (P-II). The biosynthetic machinery of picrosides is poorly understood, therefore, 'no-direction' gene co-expression networks were used to extract linked/closed and separated interactions in terpenoid glycosides-specific sub-networks. Transcriptomes generated from different organs, varying for P-I and P-II contents such as shoots grown at 15 and 25 °C and nursery-grown shoots, stolons, and roots resulted in 47,726, 44,958, 40,117, 66,979, and 55,578 annotated transcripts, respectively. Occurrence of 2810 ± 136 nodes and 15,626 ± 696 edges in these networks indicated intense, co-expressed, closed loop interactions. Either deregulation/inhibition of abscisic acid (ABA) biosynthesis/signaling or constitutive degradation of ABA resulted in organ-specific accumulation of P-I and P-II. Biosynthesis, condensation and glucosylation of isoprene units may occur in shoots, roots or stolons; but addition of phenylpropanoid moiety and further modification/s of the iridoid backbone occurs mainly inside vacuoles in roots.

Complexity of gene paralogues resolved in biosynthetic pathway of hepatoprotective iridoid glycosides in a medicinal herb, Picrorhiza kurroa through differential NGS transcriptomes.

Picrorhiza kurroa is a medicinal herb with diverse pharmacological applications due to the presence of iridoid glycosides, picroside-I (P-I), and picroside-II (P-II), among others. Any genetic improvement in this medicinal herb can only be undertaken if the biosynthetic pathway genes are correctly identified. Our previous studies have deciphered biosynthetic pathways for P-I and P-II, however, the occurrence of multiple copies of genes has been a stumbling block in their usage. Therefore, a methodological strategy was designed to identify and prioritize paralogues of pathway genes associated with contents of P-I and P-II. We used differential transcriptomes varying for P-I and P-II contents in different tissues of P. kurroa. All transcripts for a particular pathway gene were identified, clustered based on multiple sequence alignment to notify as a representative of the same gene (≥ 99% sequence identity) or a paralogue of the same gene. Further, individual paralogues were tested for their expression level via qRT-PCR in tissue-specific manner. In total 44 paralogues in 14 key genes have been identified out of which 19 gene paralogues showed the highest expression pattern via qRT-PCR. Overall analysis shortlisted 6 gene paralogues, PKHMGR3, PKPAL2, PKDXPS1, PK4CL2, PKG10H2 and PKIS2 that might be playing role in the biosynthesis of P-I and P-II, however, their functional analysis need to be further validated either through gene silencing or over-expression. The usefulness of this approach can be expanded to other non-model plant species for which transcriptome resources have been generated.

Advances in Ethnobotany, Synthetic Phytochemistry and Pharmacology of Endangered Herb Picrorhiza kurroa (Kutki): A Comprehensive Review (2010-2020).

Picrorhiza kurroa Royle ex Benth. (Family: Plantaginaceae) is a well-recognized Ayurvedic herb. It is commonly called "Kutki" or "Kurro" and 'Indian gentian'. Iridoid glycosides are the plant's bioactive constituents accountable for the bitter taste and medicinal properties of the plant. The iridoid glycosides such as picrosides and other active metabolites of the plant exhibit many pharmacological activities like hepatoprotective, antioxidant, anti-inflammatory, anticancer, immunomodulator, anti-ulcerative colitis, antimicrobial, etc. This review aims to provide updated information on the ethnobotany, synthetic phytochemistry, pharmacological potential, safety and toxicology of P. kurroa and its active metabolites. Indiscriminate exploitation, ecological destruction of natural habitats, slower plant growth and unawareness regarding cultivation and uprooting of plants have brought kutki an endangered status. Therefore, various techniques used for the conservation and production of bioactive metabolites from P. kurroa have also been reported. Information on the plant has been collected from Science Direct, Google Scholar, PubMed, Scopus using 'Picrorhiza kurroa', 'Picroside-', 'Picroside-II', 'Picroliv', 'Immunomodulator' keywords. All studies on ethnobotany, phytochemistry and pharmacology of plant from 2010- 2020 were comprised in this review article. The possible directions for future research have also been outlined briefly in this review article.

An in vitro mechanistic approach towards understanding the distinct pathways regulating insulin resistance and adipogenesis by apocynin.

Adipogenesis is a cascade of processes that entail the differentiation of fibroblasts into mature adipocytes, which results in the accumulation of triglycerides in the adipose cells due to high dietary supplements. This physiological condition increases the risk of type 2 diabetes. Apocynin (4-hydroxy-3-methoxyacetophenone), an organic compound from the root extracts of the medicinal herb Picrorhiza kurroa, has been used in various experimental studies. The current study focuses on deciphering the cellular and molecular mechanisms interlinking obesity and diabetes by validating the various key targets involved in insulin signaling and adipogenesis. Apocynin exhibited enhanced glucose uptake and decreased lipid accumulation in the adipocytes. Furthermore, the expression of molecular markers involved in the insulin signaling pathway, such as IRTK, IRS-1, PI3K, GLUT-4, and the adipogenic pathway, such as PPAR α, adiponectin, C/EBP-α and SREBP1C, by qPCR supported our hypothesis largely. Apocynin mimicked insulin in the insulin-signaling pathway by showing equivalent gene expression. It ameliorated adipogenesis by downregulating the key markers in the adipogenic pathway. Corroborating the hypothesis that Apocynin is antihyperlipidemic in nature, it reduced the expression of PPARα and adiponectin. These results substantiate that Apocynin exerts anti-diabetic and anti-adipogenic effects by regulating resistin and antioxidant enzyme levels in vitro.