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.

Global uses of traditional herbs for hepatic diseases and other pharmacological actions: A comprehensive review.

Hepatocellular carcinoma (HCC) is the 7th most common cancer and the 3rd leading cause of cancer-related death worldwide. It is resistant to the majority of chemotherapeutics and has a dismal prognosis. Hepatocellular carcinoma is a prevalent complication of chronic liver disease (CLD) in India. Primary liver cancer is the 6th most common cancer worldwide and the 4th most prevalent cause of cancer-related death. In 2018, it affected 841,000 people and caused 782,000 deaths around the world. Thus, research into the tumor cycle and its prevention through suitable herbal (Unani/Ayurvedic) medication is critical for reducing the impact of primary liver cancer. Treatment options for end-stage liver cancer are limited, necessitating costly liver transplantation, which is unavailable in most countries. Here, we present the results of a comprehensive literature survey to determine the benefits of using various herbs with liver protective and antioxidant properties. This information will be useful to researchers working on liver carcinoma and free radical scavenging, both of which are important in curbing potential carcinogens.

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.

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.

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.

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.

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation.

Picrorhiza kurroa is a medicinally important, high altitude perennial herb, endemic to the Himalayas. It possesses strong hepato-protective bioactivity that is contributed by two iridoid picroside compounds viz Picroside-I (P-I) and Picroside-II (P-II). Commercially, many P. kurroa based hepato-stimulatory Ayurvedic drug brands that use different proportions of P-I and P-II are available in the market. To identify genetically heterozygous and high yielding genotypes for multiplication, sustained use and conservation, it is essential to assess genetic and phytochemical diversity and understand the population structure of P. kurroa. In the present study, isolation and HPLC based quantification of picrosides P-I and P-II and molecular DNA fingerprinting using RAPD, AFLP and ISSR markers have been undertaken in 124 and 91 genotypes, respectively. The analyzed samples were collected from 10 natural P. kurroa Himalayan populations spread across four states (Jammu & Kashmir, Sikkim, Uttarakhand and Himachal Pradesh) of India. Genotypes used in this study covered around 1000 km geographical area of the total Indian Himalayan habitat range of P. kurroa. Significant quantitative variation ranging from 0.01 per cent to 4.15% for P-I, and from 0.01% to 3.18% in P-II picroside was observed in the analyzed samples. Three molecular DNA markers, RAPD (22 primers), ISSR (15 primers) and AFLP (07 primer combinations) also revealed a high level of genetic variation. The percentage polymorphism and effective number of alleles for RAPD, ISSR and AFLP analysis varied from 83.5%, 80.6% and 72.1%; 1.5722, 1.5787 and 1.5665, respectively. Further, the rate of gene flow (Nm) between populations was moderate for RAPD (0.8434), and AFLP (0.9882) and comparatively higher for ISSR (1.6093). Fst values were observed to be 0.56, 0.33, and 0.51 for RAPD, ISSR and AFLP markers, respectively. These values suggest that most of the observed genetic variation resided within populations. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian based STRUCTURE grouped all the analyzed accessions into largely region-wise clusters and showed some inter-mixing between the populations, indicating the existence of distinct gene pools with limited gene flow/exchange. The present study has revealed a high level of genetic diversity in the analyzed populations. The analysis has resulted in identification of genetically diverse and high picrosides containing P. kurroa genotypes from Sainj, Dayara, Tungnath, Furkia, Parsuthach, Arampatri, Manvarsar, Kedarnath, Thangu and Temza in the Indian Himalayan region. The inferences generated in this study can be used to devise future resource management and conservation strategies in P. kurroa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00972-w.

[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.

Metabolic signatures provide novel insights to Picrorhiza kurroa adaptation along the altitude in Himalayan region.

INTRODUCTION: Along the altitude, environmental conditions vary significantly that might influence plant performance and distribution. Adaptation to these changing conditions is a complex biological process that involves reprogramming of genes, proteins and metabolites. The metabolic response of medicinal plants along the altitude has been less explored yet. OBJECTIVES: In the present study, we investigated the adaptation strategies of Picrorhiza kurroa Royle ex Benth. along the altitude in organ specific manner using metabolomic approach. METHODS: Picrorhiza kurroa plants at flowering stage were randomly sampled from three altitudes viz. 3400, 3800 and 4100 masl in the Himalayan region. Leaf, root and rhizome were used for LC-MS based non-targeted metabolite profiling and targeted analysis of sugars, amino acids, picrosides and their corresponding phenolic acids. RESULTS: A total of 220, primary and secondary metabolites (SMs) were identified (p < 0.05) representing an extensive inventory of metabolites and their spatial distribution in P. kurroa. Differential accumulation of metabolites suggests source-sink carbon partitioning, occurrence of partial TCA cycle, ascorbate metabolism, purine catabolism and salvage route, pyrimidine synthesis, lipid alteration besides gibberellins and cytokinin inhibition might be an adaptive strategy to alpine environmental stress along the altitude. Further, marked differences of organ and altitude specific SMs reflect alteration in secondary metabolic pathways. Significant accumulation of picrosides suggests their probable role in P. kurroa adaptation. CONCLUSION: This study provides a platform that would be useful in deciphering the role of metabolites considered to be involved in plant adaptation.

Comparative transcriptome analysis in different tissues of a medicinal herb, Picrorhiza kurroa pinpoints transcription factors regulating picrosides biosynthesis.

Transcriptional regulation of picrosides biosynthesis, the iridoid glycosides of an endangered medicinal herb, Picrorhiza kurroa, is completely unknown. P. kurroa plants obtained from natural habitat accumulate higher picrosides than in-vitro cultured plants, which necessitates identification of transcription factors (TFs) regulating their differential biosynthesis. The current study investigates complete spectrum of different TF classes in P. kurroa transcriptomes and discerns their association with picrosides biosynthesis. Transcriptomes of differential picroside-I content shoots and picroside-II content roots were mined for seven classes of TFs implicated in secondary metabolism regulation in plants. Key TFs were identified through in silico transcript abundance and qPCR analysis was performed to confirm transcript levels of TFs under study in differential content tissues and genotypes. Promoter regions of key picrosides biosynthetic pathway genes were explored to hypothesize which TFs can possibly regulate target genes. A total of 131, 137, 107, 82 and 101 transcripts encoding different TFs families were identified in PKS-25, PKS-15, PKSS, PKR-25 and PKSR transcriptomes, respectively. ERF-18, bHLH-104, NAC-25, 32, 94 and SUF-4 showed elevated expression in roots (up to 37 folds) and shoots (up to 195 folds) of plants obtained from natural habitat, indicating their role as activators of picrosides biosynthesis whereas, elevated expression of WRKY-17, 40, 71 and MYB-4 in low picrosides content conditions suggested their down-regulatory role. In silico analysis of key picrosides biosynthetic pathway gene promoter regions revealed binding domains for ERF-18, NAC-25, WRKY-40 and MYB-4. Identification of candidate TFs contributing towards picrosides biosynthesis is a pre-requisite for designing appropriate metabolic engineering strategies aimed at enhancing picrosides content in vitro and in vivo.

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

KEY MESSAGE: 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.

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-1ß (IL-1ß), 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.

An endophyte of Picrorhiza kurroa Royle ex. Benth, producing menthol, phenylethyl alcohol and 3-hydroxypropionic acid, and other volatile organic compounds.

An endophytic fungus, PR4 was found in nature associated with the rhizome of Picrorhiza kurroa, a high altitude medicinal plant of Kashmir Himalayas. The fungus was found to inhibit the growth of several phyto-pathogens by virtue of its volatile organic compounds (VOCs). Molecular phylogeny, based on its ITS1-5.8S-ITS2 ribosomal gene sequence, revealed the identity of the fungus as Phomopsis/Diaporthe sp. This endophyte was found to produce a unique array of VOCs, particularly, menthol, phenylethyl alcohol, (+)-isomenthol, ß-phellandrene, ß-bisabolene, limonene, 3-pentanone and 1-pentanol. The purification of compounds from the culture broth of PR4 led to the isolation of 3-hydroxypropionic acid (3-HPA) as a major metabolite. This is the first report of a fungal culture producing a combination of biologically and industrially important metabolites­menthol, phenylethyl alcohol, and 3-HPA. The investigation into the monoterpene biosynthetic pathway of PR4 led to the partial characterization of isopiperitenone reductase (ipr) gene, which seems to be significantly distinct from the plant homologue. The biosynthesis of plant-like-metabolites, such as menthol, is of significant academic and industrial significance. This study indicates that PR4 is a potential candidate for upscaling of menthol, phenylethyl alcohol, and 3-HPA, as well as for understanding the menthol/monoterpene biosynthetic pathway in fungi.

Androsin alleviates non-alcoholic fatty liver disease by activating autophagy and attenuating de novo lipogenesis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease with therapeutic options on the horizon. Picrorhiza kurroa, enriched with iridoid glycosides like picroside I and picroside II is known for its hepatoprotective activity and anti-inflammatory properties. Androsin, the other phytochemical present in P. kurroa has been shown to have anti-inflammatory and anti-asthmatic properties. However, its role in NAFLD is yet to be investigated. PURPOSE: This study aims to identify the potent hepatoprotective agent from P. kurroa that can attenuate NAFLD in HFrD-fed ApoE-/- mice, and elucidate the underlying mechanisms governing its effects. METHODS: Classical purification methods were used to isolate seven compounds, including picroside I, picroside II and androsin from the roots of P. kurroa. NAFLD-induced ApoE-/- mice were administered orally with either picroside I, picroside II, or androsin for 7 weeks. Animals were scanned non-invasively by ultrasonography at 1st and 14th week. Gross histomorphometry was examined by HE and Sirius red staining. mRNA transcript and protein profile associated with autophagy, lipogenesis, inflammation, and fibrosis was done through RT-PCR and Western blot analysis. RESULTS: In-vitro and in-vivo studies revealed that among the seven evaluated compounds, androsin shows the most potent in-vitro activity. Oral dosing of androsin (10 mg/kg) protected the liver against HFrD-induced NAFLD in ApoE-/- mice model. Biochemical analysis revealed a reduction in ALT and AST enzymes and a significant reduction in cholesterol levels. Hepatocyte ballooning, hepatic lipid deposition, inflammation, and fibrosis were reduced. Androsin treatment significantly reduced fibrosis (α-SMA, collagens, TGF-ß) and inflammation (ILs, TNF-α, NFκB) in ApoE-/- mice. Mechanistically, androsin activated AMPKα and down-regulated the expression of SREBP-1c, resulting in ameliorating hepatic lipogenesis. CONCLUSION: Our results support autophagy as one of the therapeutic strategies to reduce steatosis and hepatic damage. We found that androsin treatment significantly ameliorated hepatic steatosis, serum lipid levels, and hepatic injury in ApoE-/- induced by HFrD. Androsin administration mitigated lipogenesis by inhibiting SREBP1c/FASN pathway and activating autophagy through AMPKα/PI3K/Beclin1/LC3 pathway.

A randomized, placebo controlled, double blinded, parallel group clinical study to evaluate the efficacy and safety of AEV01 along with standard care for elderly patients with mild COVID-19.

BACKGROUND: While several drugs are in the pipeline for treatment of various grades of COVID-19 disease, none of them have shown promise until now. Medicinal plants are crucial in developing and developed countries for their primary and basic health needs owing to better tolerability, superior compatibility with human body and having lesser side effects. OBJECTIVE: To test adjuvant effect of AEV01, a patented herbal extract of kutki (Picrorhiza kurroa), in mild COVID19 in elderly patients at risk of complications and aging immunity. METHODS: A prospective, phase 3, randomized, multicentric, placebo controlled double blinded parallel group interventional trial was conducted in elderly patients aged above 50 years diagnosed as COVID-19 with mild symptoms. 70 patients were randomized into two groups to receive AEV01 capsule (100 mg) and placebo respectively thrice daily for 30 days along with standard care of treatment as per national (WHO/ICMR) guidelines in both the groups. Clinical improvement timelines and corresponding scores using 8-point ordinal scale and NEWS were assessed for efficacy and safety of AEV01 in COVID-19 patients. RESULTS: AEV01 group showed a significant improvement (p = 0.0001) in the normalization of the SpO2 rate and relief from cough. There was also significant difference in time to recovery, with patients in the AEV01 drug treated group recovering in 4.5 days as compared to Placebo in 9.1 days. ESR, LDH, serum ferritin, Neutrophil-Lymphocyte ratio (NLR), TNF- alpha, IL-6 and CD4 cell counts, which are considered as prognostic markers in COVID-19 infected patients, showed a significant improvement in patients treated with AEV01. The AEV01 drug treated group showed significant clinical improvement in the 8-point ordinal scale severity rating and National Early Warning Score (NEW) Score from day 3 onwards which continued up to end of study. CONCLUSION: AEV01 has shown a significant improvement in clinical and laboratory parameters. There was also reduction in the progression of the COVID-19 disease in unvaccinated elderly patients from mild to moderate when treated at an earlier stage.

A proposed biosynthetic pathway of picrosides linked through the detection of biochemical intermediates in the endangered medicinal herb Picrorhiza kurroa.

INTRODUCTION: Picrorhiza kurroa Royle ex Benth is an important medicinal herb used in the preparation of several herbal drug formulations due to the presence of picroside-I (P-I) and picroside-II (P-II) along with other iridoid-glucosides derivatives. OBJECTIVE: The endangered status of P. kurroa coupled with lack of information on biosynthesis of P-I and P-II necessitate deciphering the biosynthetic pathway for picrosides. METHODS: LC with electrospray ionisation (ESI) and quadrupole time of flight combined with MS/MS was used to detect intermediates and assemble the picrosides biosynthetic pathway in P. kurroa. RESULTS: The presence of catalpol and aucubin, the major backbone structures of picrosides, along with intermediate metabolites boschnaloside, bartsioside and mussaenosidic acid, was confirmed in ESI negative mode with pseudomolecular ion peaks, that is, m/z 361, m/z 343, m/z 345, m/z 329 and m/z 375 ions and their fragmentation patterns. CONCLUSION: The picrosides biosynthetic pathway is expected to provide a reliable platform towards understanding the molecular components (genes/enzymes) of P-I and P-II biosynthesis in P. kurroa for their eventual utilisation in various applications.

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.

Picrorhiza kurroa, Royle ex Benth:Traditional uses, phytopharmacology, and translational potential in therapy of fatty liver disease.

Picrorhiza kurroa Royle ex Benth, Kutki (P.kurroa) is an important medicinal plant, traditionally recommended and used in Ayurveda for millennia, with certain cautions. There has been a significant revival of keen interest in its pharmacology, pharmacognosy, and phytochemistry for the last few decades. The evidence of its hepatoprotective activity, in experimental and clinical studies, accelerated the correlation of the specific phytochemical constituents of P.kurroa with precise pharmacological activities. Iridoid glycosides, particularly picrosides, emerged as the active molecules. For effective translation of traditional remedies into modern therapy, value addition by mechanistic understanding of molecular actions, drug targets, the degrees of efficacy and safety as well as convenient dosage forms is needed. Reverse pharmacology approach and phytopharmaceutical drug category facilitate such a translation. The present review illustrates how a potential translation of traditional practices of using P.kurroa into a phytochemically standardized, clinically targeted natural product for global unmet medical needs viz. Fatty liver disease can be attained.

Evaluation of Ayush-64 (a Polyherbal Formulation) and Its Ingredients in the Syrian Hamster Model for SARS-CoV-2 Infection Reveals the Preventative Potential of Alstonia scholaris.

In the current study, we evaluated the efficacy of Ayush-64 (A64), a polyherbal formulation containing Alstonia scholaris (L.) R. Br. (A. scholaris), Caesalpinia crista L. (C. crista), Picrorhiza kurroa Royle ex Benth (P. kurroa), and Swertia chirata (Roxb.) H. Karst. (S. chirata) against COVID-19 in a Syrian hamster infection model. Preventative use of A64 resulted in the late-phase recovery of body weight loss in severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected hamsters, suppression of pro-inflammatory cytokines, and blunted pulmonary pathology. In addition, we also investigated the efficacy of individual ingredients of A64, viz., A. scholaris, C. crista, P. kurroa, and S. chirata, in the hamster model. The hamster challenge data showed robust anti-viral and immunomodulatory potential in A. scholaris, followed by P. kurroa. However, C. crista and S. chirata of A64 showed prominent immunomodulatory potential without limiting the lung viral load. In order to better understand the immunomodulatory potential of these herbal extracts, we used an in vitro assay of helper T cell differentiation and found that A. scholaris mediated a more profound suppression of Th1, Th2, and Th17 cell differentiation as compared to A64 and other ingredients. Taken together, our animal study data identifies the ameliorative potential of A64 in mitigating coronavirus disease-19 (COVID-19) pulmonary pathology. A. scholaris, a constituent extract of A64, showed relatively higher anti-viral and immunomodulatory potential against COVID-19. The present study warrants further investigations to identify the active pharmaceutical ingredients of A. scholaris for further studies.

A Comparative Study on Clinical Evaluation of the Hypolipidemic Effects of Allium sativum, Trigonella foenum-graecum, Commiphora mukul, Picrorhiza kurroa, and Piper nigrum: A Pilot Study.

Background Cardiovascular disease is a leading cause of morbidity and mortality. Therefore, it is essential to prevent cardiovascular diseases by correcting modifiable risk factors such as lowering lipid levels, lowering blood pressure, improving eating habits, giving up smoking, etc. The present study assessed the efficacy of herbal preparation containing Allium sativum (A. sativum), Commiphora mukul (C. mukul), and Trigonella foenum-graecum (T. foenum-graecum) in patients with hyperlipidemia. Methodology Patients were given extracts of A. sativum 350 mg, T. foenum-graecum 350 mg, C. mukul 200 mg, Picrorhiza kurroa (P. kurroa) 200 mg, and Piper nigrum (P. nigrum) 5 mg. Unichem Laboratories, Mumbai, provided placebo tablets similar in shape and size to herbal tablets. Patients were assessed for compliance, and a complete lipid profile was done at DO, D15, D46, D76, and D106. In addition, total cholesterol and high-density lipoprotein-cholesterol (HDL-C) serum triglyceride were estimated by the respective methods throughout the study. Results The weight of the patients remained stable, the mean weight before being 65.42 ± 8.35 kg and after completion of the study being 65.42 ± 8.35 kg. There were no changes in the ECG during or after the drug therapy in any of the patients. Group A comprised nine patients, and group B had ten patients. Serum creatinine (mg %) was 0.94 and 0.95, fasting blood sugar mg (%) was 111.05 and 99.63, and postprandial blood sugar (mg %) was 150.89 and 147.94 on pre-treatment and post-treatment, respectively. The mean serum triglyceride levels in group A were 271.11, 261.11, 293.89, 167.22, and 128.89, and serum HDL- C levels were 46.11, 46.11, 54.44, 52.22, and 54.44. Serum triglyceride levels in group B were 268, 268.5, 202, 171, and 116, and serum HDL- C levels were 48.5, 48, 50, 50, and 53.5 on day 0, 15, 46, 76, and 106, respectively. A significant reduction in total cholesterol levels was observed on D46, D76, and D106, with a maximum reduction on D76 (25.36%). Similarly, a reduction in serum triglyceride was also observed on D46, D76, and D106, with a maximum reduction on D106 (52.02%). A significant difference was observed (P <0.05). There was also a significant reduction of low-density lipoprotein cholesterol (LDL-C) on D46, D76, and D106, with the maximum reduction on D76 (28.79%). There was a significant rise of HDL-C on D46 and D106, with a maximum rise on D106 (15.41%). A significant difference was observed (P <0.05). Conclusion The study drugs are safe and efficacious in reducing the total cholesterol, serum triglycerides, LDL-C levels, and increasing HDL-C levels.