Chemical composition,antioxidant and macromolecule damage protective effects of Picrorhiza kurroa Royle ex Benth

In the present study, we identified the chemical constituents of 70% hydroalcoholic fraction of Picrorhiza kurroa by LC–ESI–MS/MS which showed the presence of iridoid glucosides such as picroside I, picroside II, picroside III, picroside IV, kutkoside, pikuroside and flavonoids like apocynin and vanillic acid. P. kurroa exhibited DPPH radical scavenging and metal chelating activities with IC₅₀ of 75.16 ± 3.2 and 55.5 ± 4.8 μg/mL and also showed potent reducing power and total antioxidant activities. The extract inhibited macromolecule damage such as H₂O₂ induced plasmid DNA damage and AAPH induced oxidation of bovine serum albumin and lipid peroxidation of rat hepatic tissues.     

Agrobacterium rhizogenes-mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species. CIMAP Publication No.: 2007-28J     

Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.

Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1–8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other media. The pH of the medium played significant role in growth and secondary metabolite production and was found to be highest at pH 6.0 while lowest at pH 3.0 and pH 8.0. To enhance the production of biomass and Picroliv 5 liter working capacity bioreactor was used, 27-fold (324 g FW) higher growth was observed in bioreactor than shake flask and secondary metabolite production was similarly enhanced.     

Free radical scavenging potential of Picrorhiza kurrooa Royle ex Benth

For assessing free radical scavenging potential of P. kurrooa, the antioxidant activity of P. kurrooa extract was studied by lipid peroxidation assay using rat liver homogenate. The extract (1 mg/ml) showed marked protection (up to 66.68%) against peroxidation of liver phospholipids. Besides, reduced glutathione showed very encouraging activity. The extract also exhibited significant scavenging activity. Thus augmenting the wide use of plant in the indigenous system of medicine, which may partly be due to antioxidant and free radical scavening activity of the extract.     

In Vitro Propagation of Endangered Temperate Himalayan Medicinal Herb Picrorhiza kurroa Royle ex Benth using Leaf Explants and Nodal Segments

The present study describes the micropropagation of Picrorhiza kurroa, (commonly known as kutki) an endangered medicinal herb of the temperate Himalayas and a source of hepatoprotective picrosides. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segments and leaf tissue. For shoot regeneration, the hormone combinations kinetin (2.0 mg l^?1) and Kinetin + Indole-3-butyric acid (IBA) (2.0 mg l^?1 + 0.50 mg l^?1) with leaf explant was found superior. Interestingly, the basal MS medium gave 99.94 % response (direct proliferation) with nodal explant. The medium supplemented with IBA (1.0 mg ^?1) was found best for rooting of regenerated shoots. Nodal segments plated on the medium supplemented with TDZ + IBA (0.11 mg ^?1 + 0.50 mg ^?1) formed somatic embryos, however further regeneration could not be achieved. The in vitro raised plantlets were hardened and successfully established in the glass house conditions.     

Online HPLC-DPPH method for antioxidant activity of Picrorhiza kurroa Royle ex Benth. and characterization of kutkoside by ultra-performance LC-electrospray ionization quadrupole time-of-flight mass spectrometry

Picrorhiza kurroa Royle ex Benth., is widely used in the Indian systems of medicine for the treatment of various liver ailments. Since, the role of oxidative stress in the pathogenesis of liver injury has become generally recognized, in present study the free radical scavenging effect of P. kurroa was assessed by on-line HPLC-DPPH and colorimetric DPPH methods. The comparative study on antioxidant activity of P. kurroa extracts by both methods revealed that colorimetric method showed very less free radical scavenging effect while HPLC-DPPH method showed high activity. Further, the kutkoside, an important ingredient of a potent hepatoprotective formulation kutkin/picroliv was investigated for its chemical composition by ultra-performance liquid chromatography coupled with diode array detection/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-DAD/ESI-QTOF-MS). Kutkoside was considered to be a single compound and reported as picroside-II or kutkoside, however, present investigation illustrated that kutkoside is a mixture of iridoid glycosides namely, picroside II, picroside IV and 6-ferulloylcatalpol.     

Habitat distribution modeling of endangered medicinal plant Picrorhiza kurroa (Royle ex Benth) under climate change scenarios in Uttarakhand Himalaya,India

Climate change has been the key factor in changing the alpine vegetation's habitat and causing it to migrate to higher latitudes. The present study aims to model the current and future potential habitat distribution of endangered medicinal plant Picrorhiza kurroa Royle ex Benth in Uttarakhand Himalaya using the maximum entropy (MaxEnt) modeling. We initially select twenty-two environmental variables (bioclimatic + topographic) got from the Fifty-four (54) species occurrence points, which were further reduced to nine variables to prevent multicollinearity. Shared Socioeconomic Pathways (SSP1–2.6 and SSP2–4.5) from the CMIP6 (BCC-CSM2-MR) climate model for the periods 2041–60 and 2061–80 were used to predict the current and future habitat distribution of P. kurroa. Results showed that the precipitation of the driest month (Bio 14; 33.8%), isothermality (Bio 3; 20.2%), mean temperature of warmest quarter (Bio 10; 12.7%), and temperature annual range (Bio 7; 12.2%) were the important bioclimatic variables influencing the habitat of P. kurroa. Overall, there is a decrease in the habitat of P. kurroa under climate change scenarios. The present results may prove insightful for the decision-makers to identify suitable sites in the wild for the further propagation of P. kurroa.     

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 InformationThe online version contains supplementary material available at 10.1007/s12298-021-00972-w.     

Propagation and Conservation of Picrorhiza kurrooa Royle ex Benth.: An Endangered Himalayan Medicinal Herb of High Commercial Value

Picrorhiza kurrooa Royle ex Benth., a high value medicinal herb of alpine Himalaya and a source of hepatoprotective picrosides, is listed as ‘endangered’ due to heavy collection from its natural habitat. The present report deals with successful propagation of this species using both conventional and in vitro techniques. Vegetative propagation was achieved by rooting runner cuttings with indole-3-butyric acid (IBA) or α-naphtheleneacetic acid (NAA) treatment before planting. Nearly 87% rooting success was achieved by treatment of cuttings with 50.0 μM IBA. Seeds were given a presoaking treatment with gibberellic acid (GA₃), 6-benzylaminopurine (BAP) or a combination of both to influence germination. More than 11-fold improvement in germination was recorded in seeds treated with 250.0 μM GA₃. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segment. Multiple shoots were formed following culture for 3 weeks on Murashige and Skoog (MS; 1962. Physiologia Plantarum 15: 473–497) medium containing 1.0 μM BAP. Cent percent rooting success, without basal callus formation, was observed when individual microshoots were placed in MS medium supplemented with IBA. The plantlets raised using conventional as well as tissue culture methods were hardened and successfully established in the experimental field located at 2450 m elevation. In addition, strategies have been discussed to encourage cultivation and in situ conservation of this highly valued medicinal herb so as to reduce pressure on its natural populations.     

Plant regeneration, genetic fidelity, and active ingredient content of encapsulated hairy roots of Picrorhiza kurrooa Royle ex Benth.

Among five hairy root lines of Picrorhiza kurrooa that were established through Agrobacterium rhizogenes, one (H7) was selected for encapsulation due to high accumulation of picrotin and picrotoxinin (8.3 and 47.6 μg/g DW, respectively). Re-grown encapsulated roots induced adventitious shoots with 73 % frequency on MS medium supplemented with 0.1 μM 6-benzylaminopurine, following 6 months of storage at 25 °C. Regenerated plantlets had 85 % survival after 2 months. Regenerants were of similar morphotype having increased leaf number and branched root system as compared to non-transformed plants. The transformed nature of the plants was confirmed through PCR and Southern blot analysis. Genetic fidelity analysis of transformed plants using RAPD and ISSR showed 5.2 and 3.6 % polymorphism, respectively. Phytochemical analysis also showed that picrotin and picrotoxinin content were similar in hairy root line and its regenerants.     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Biosynthesis and accumulation of a medicinal compound,Picroside-I,in cultures of <Emphasis Type=Italic>Picrorhiza kurroa</Emphasis> Royle ex Benth

Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non- detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures.     

Hairy root culture of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> Royle ex Benth.: a promising approach for the production of picrotin and picrotoxinin

Picrorhiza kurroa Royle ex Benth. is an endangered plant producing various compounds of medicinal importance. Hairy roots of P. kurroa were obtained following cocultivation of shoot tip explants with Agrobacterium rhizogenes strains A 4 and PAT 405. Bacterial strain A 4 appeared to be better than the strain PAT 405 in terms of both growth of respective hairy root cultures and secondary metabolite production. The optimal growth of both the hairy root cultures occurred on half-strength semisolid medium with 3% sucrose. Picrotin and picrotoxinin from the roots of wild type field grown plants were compared with 8-week-old hairy root cultures induced by the A 4 and PAT 405 strains of A. rhizogenes. Picrotin and picrotoxinin content were evaluated in hairy root cultures as well as roots of field grown plant of P. kurroa. In terms of the production of picrotin and picrotoxinin, the A 4 induced hairy roots appeared to be a better performer than the PAT 405 induced hairy root cultures. The picrotin and picrotoxinin content was highest in 8-week-old A 4 induced hairy roots (8.8 μg/g DW and 47.1 μg/g DW, respectively). Rapid growth of the hairy roots of P. kurroa with in vitro secondary metabolite production potential may offer an attractive alternative to the exploitation of this endangered plant species.     

Differential biosynthesis and accumulation of picrosides in an endangered medicinal herb Picrorhiza kurroa

Picrorhiza kurroa Royle ex Benth (Family: Scrophulariaceae) is a medicinal herb, mainly found in the North-Western Himalayas. Extensive harvesting for pharmaceutical purposes, lack of organized cultivation and unorganized methods of uprooting the plants because of unawareness has brought an endangered status to this important herb in nature. The medicinal property of this plant is attributed to monoterpenoid picrosides. The influence of developmental status of different growth stages on picrosides content is poorly understood in Picrorhiza kurroa. Picroside-I (P-I) content increased from 0.05 % to 0.76 % in different growth stages of shoots. Significant increase in the contents of P-I (0.15–0.50 %) and Picroside-II (P-II) (0.1–0.45 %) was observed in rhizomes of different developmental stages. Highest amounts of P-I (8.7 %) and P-II (5.3 %) was detected in uppermost part of mature dried rhizomes compared to bottom part with 2.9 % and 2.2 % of P-I and P-II, respectively. P. kurroa grown at high altitude (Sairopa, 4,500 amsl) showed 1.75-folds increase in P-I in leaves whereas exponential increase in the P-I content was detected (0.05–1.7 %) in the leaves of different developmental stages (L1-L5) of P. kurroa grown at lower altitude (Jagatsukh, 1,900 m). Variable amounts of P-I and P-II in different growth and developmental stages of P. kurroa imply importance of selection of plant material (rhizomes and roots). The study undertaken explored the status of metabolites accumulation and biosynthesis in the field grown plants of P. kurroa where not only environmental parameters but different morphogenetic stages of its developmental cycles, different age groups and different parts of plantlets were extensively analysed and estimated for medicinally important picrosides.     

Effects of apocynin, a drug isolated from the roots of Picrorhiza kurroa, on arachidonic acid metabolism.

Apocynin is a constituent of root extracts of the medicinal herb Picrorhiza kurroa and has been shown to possess anti-inflammatory properties. We investigated the effects of apocynin on the production of arachidonic acid-derived inflammatory mediators by guinea pig pulmonary macrophages. Apocynin concentration-dependently inhibited the formation of thromboxane A2, whereas the release of prostaglandins E2 and F2 alpha was stimulated. Apocynin potently inhibited arachidonic acid-induced aggregation of bovine platelets, possibly through inhibition of thromboxane formation. The present results suggest that apocynin might, beside its therapeutic effects in inflammatory conditions when given in a root extract of P. kurroa, also be a valuable tool in the development of new anti-inflammatory or anti-thrombic drugs.     

银条水苏糖提取条件研究

唇形科水苏属植物银条,也称银苗,药食两用,主产于偃师县(市),产量约有95%。本文以偃师产银条为原料,通过单因素、响应面分析法,研究了乙醇提取银条中水苏糖的最佳提取条件。以银条中水苏糖提取率为指标,通过单因素实验确定了银条水苏糖提取条件的参数范围,通过响应面分析法确定了其最佳提取条件。结果表明,最佳提取条件为乙醇浓度60%,液固比10:1,提取温度60℃,提取时间40 min,该条件下银条水苏糖提取率为46.8924%。