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.     

Modulation of Picroside-I Biosynthesis in Grown Elicited Shoots of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> In Vitro

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

Tracking dynamics of enzyme activities and their gene expression in <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> with respect to picroside accumulation

Picrosides, the terpenoids synthesized by Picrorhiza kurroa, have ample usage in medicine. Identification of the regulatory enzymes involved in picroside biosynthesis needs to be explored for improving the level of these secondary metabolites. Current efforts are based on the analysis of secondary metabolism in picroside biosynthesis but its interpretation is limited by the lack of information on the involvement of primary metabolic pathways. The present study investigated the connection of primary metabolic enzymes with the picrosides levels in P. kurroa. The results showed changes in the catalytic activities as well as in the gene expression profiles of hexokinase, pyruvate kinase, isocitrate dehydrogenase, malate dehydrogenase, and NADP⁺-malic enzyme in congruence with picroside-I content under different conditions of P. kurroa growth, which indicates the role of these enzymes in the accumulation of picrosides. The significant correlation coefficients (p?<?0.05) observed between gene expression and enzyme activity underline the role of integrative studies for a better understanding of connecting links between metabolic pathways leading to picroside biosynthesis. This is apparently the first report on the involvement of glycolytic and TCA cycle enzymes in the accumulation of picrosides in P. kurroa.     

Optimization of a preparative RP-HPLC method for isolation and purification of picrosides in <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis>

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

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.     

Physiological and genomic evidence supports the role of Serratia quinivorans PKL:12 as a biopriming agent for the biohardening of micropropagated Picrorhiza kurroa plantlets in cold regions

The medicinal herb, Picrorhiza kurroa Royle ex Benth has become endangered because of indiscriminate over-harvesting. Although micropropagation has been attempted for mass propagation of the plant, survival of in vitro plantlets under green house/open field poses a major challenge. Biopriming of micropropagated plantlets with plant growth-promoting rhizobacteria (PGPR) are among the successful methods to combat this problem. Serratia quinivorans PKL:12 was the best-characterized PGPR from rhizospheric soil of P. kurroa as it increased the vegetative growth and survival of the micropropagated plantlets most effectively. Complete genome (5.29 Mb) predicted genes encoding proteins for cold adaptation and plant growth-promoting traits in PKL:12. Antibiotic and biosynthetic gene cluster prediction supported PKL:12 as a potential biocontrol agent. Comparative genomics revealed 226 unique genes with few genes associated with plant growth-promoting potential. Physiological and genomic evidence supports S. quinivorans PKL:12 as a potential agent for bio-hardening of micropropagated P. kurroa plantlets in cold regions.     

Potential of thidiazuron in improved micropropagation of Picrorhiza kurroa– an endangered medicinal herb of alpine Himalaya

The potential of thidiazuron (1-phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea (TDZ) in the micropropagation of Picrorhiza kurroa, a western Himalayan herb was studied. The roots and rhizomes of this plant are rich in medicinally important glycosides i.e., picroside I, picroside II and kutkoside. Nodal segments (2.0–2.5 cm) from plants were pretreated with 0, 0.25, 0.50, 0.75, 1.0 μM TDZ for 7, 15 and 30 days. Maximum shoot multiplication was recorded after 60 days, provided, the nodal segments pretreated with 0.5 μM TDZ for 15 days were transferred to 0.8% agar gelled MS medium containing 3.0% sucrose. The shoots also showed profuse rooting having maximum length. When these plantlets were incubated at 15°C for 10 days and transferred to sand under polyhouse conditions, 100% survival was recorded after one month. In contrast, when the plantlets were not incubated at 15°C, only 86% survival was recorded. While the stomata and chlorophyll content of the tissue culture-raised plantlets treated at 15°C were comparable to that of plants growing in the field, the ones that were not treated at 15°C showed lower number of stomata and chlorophyll content.     

Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation of Picrorhiza kurroa: an endangered medicinal herb of the alpine Himalayas

Picrorhiza kurroa Royle ex. Benth. is a medicinal herb of immense therapeutic value with restricted geographic distribution. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation was developed for this plant. Multiple shoot bud induction was achieved from leaf explants cultured in Gamborg??s B₅ medium containing 3?% (w/v) sucrose, 3?mg/l kinetin and 1?mg/l indole-3-butyric acid. More than 90?% of leaf explants formed shoot buds leading to whole plant regeneration. An Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV3101 harboring binary vector pCAMBIA1302 containing the green fluorescent protein and hygromycin phosphotransferase genes. Leaf explants precultured for 2?d were the most suitable for co-cultivation with Agrobacterium and transformation efficiency was enhanced with 200???M acetosyringone. Putative transformants were selected using media containing 15?mg/l hygromycin. Transformation was verified by detection of the green fluorescent protein using fluorescence microscopy and by polymerase chain reaction. Approximately 56?% of the explants were transformed with an average of 3.4?±?0.4 transgenic plantlets per explant. An efficient regeneration and transformation protocol thus developed enabling a fresh perspective of metabolic engineering in P. kurroa using an Agrobacterium-mediated transformation. This is the first report of direct organogenesis from leaf explants and genetic transformation of P. kurroa.     

ISSR and RAPD based evaluation of genetic fidelity and active ingredient analysis of regenerated plants of Picrorhiza kurroa

Genetic stability and phytochemical analysis of in vitro established plants of Picrorhiza kurroa Royle ex Benth, have been carried out. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of tissue culture products including three adventitious shoots from three calli and 6 months old tissue culture raised plants growing in green house condition with mother plant. Apparent genetic variation was detected in the five types of plant materials. The percentage of polymorphic bands in the RAPD and ISSR analysis were 16.25 and 14.54 %, respectively. The genetic similarity was calculated on the basis of RAPD and ISSR data among the five types of plant materials and were ranged from 0.5 to 1.0 (mean 0.75) and 0.47 to 1.0 (mean 0.73), respectively. The similarity coefficient by both RAPD and ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, but notable differences were observed among three adventitious shoots regenerated from three calli. The phytochemical analysis of tissue culture raised products showed higher secondary metabolite (picrotin and picrotoxinin) content as compare to mother plant. The information gained on genetic stability/variability will be valuable for the large scale propagation and secondary metabolite production of P. kurroa.     

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 partial characterization of chromoprotein from the larval hemolymph of the Japanese oak silkworm (Antheraea yamamai)

A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.     

Catalytic activity with associated and dissociated forms of the yeast hexokinases.

In agreement with previous glucose binding data, kinetic studies of the yeast hexokinases at high protein concentration show that the dimeric forms P-I and P-II require much higher glucose concentrations for half-maximal rates than do the monomeric forms S-I and S-II, but each P form reaches the same V value as the corresponding S form. Raising the temperature from 5 °C to 24 °C causes an apparent dissociation of the P forms, most pronounced in the case of P-I, as evidenced by a lowering of glucose K_M values toward those of the S forms. Similarly, kinetic measurements at low protein concentrations, where the P forms are presumably fully dissociated, give identical glucose K_M values for the P and S forms.