Source: https://sciforum.net/profile/296514/timeline
Timestamp: 2019-04-24 20:03:52+00:00

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Fagonia indica is valuable for its multiple uses such as anticancer, antimicrobial and antioxidant throughout the world. Various biotechnological approaches need to be employed for the sustainable production of plant biomass and its phytochemical content. Elicitation has been shown to be a very potent tool for enhanced production of secondary metabolites in different in vitro cultures. The current study involves the application of various concentrations of chitosan and salicylic acid as elicitors in callus cultures keeping Thiadizuron induced callus as a control. The main aim was to enhance the accumulation of biomass and secondary metabolite contents. The results show that maximum biomass; Fresh weight (FW: 38.0 g/100 mL) and secondary metabolites [Phenolic content (16.9 μgGAE/mg; Flavonoid content (2.2 μgQE/mg)] were observed in CHT treated cultures. The SA treated calli also showed better accumulation of biomass (FW: 37.5 g/100 mL) and phytochemicals [Phenolic content (12.29 μgGAE/mg); Flavonoid content (1.73 μgQE/mg)] as compared with control. The antioxidant potential was found in correlation with the production of PC/FC and found higher in CHT (94.3%) cultures than SA (90.2%) and control. For instance, the antioxidative enzyme activities [peroxidase (POD: 4.41) and superoxide dismutase (SOD: 0.42 nM/min/mg FW)] were found optimum in CHT treated samples. HPLC analyses revealed significant accumulation (5.5 ± 0.004 μg/mg DW) and (5.2 ± 0.001 μg/mg DW) of pharmacologically active components in CHT and SA treated samples, respectively. The results showed that both elicitors have potential to enhance the biomass accumulation and polyphenols in callus cultures of Fagonia indica.
Ocimum basilicum L. (Purple basil) is a source of biologically active antioxidant compounds, particularly phenolic acids and anthocyanins. In this study, we have developed a valuable protocol for the establishment of in vitro callus cultures of O. basilicum and culture conditions for the enhanced production of distinct classes of phenylpropanoid metabolites such as hydroxycinnamic acid derivatives (caffeic acid, chicoric acid, rosmarinic acid) and anthocyanins (cyanidin and peonidin). Callus cultures were established by culturing leaf explants on Murashige and Skoog medium augmented with different concentrations of plant growth regulators (PGRs) [thidiazuron (TDZ), α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP)] either alone or in combination with 1.0 mg/l NAA. Among all the above mentioned PGRs, NAA at 2.5 mg/l led to the highest biomass accumulation (23.2 g/l DW), along with total phenolic (TPP; 210.7 mg/l) and flavonoid (TFP; 196.4 mg/l) production, respectively. HPLC analysis confirmed the differential accumulation of phenolic acid [caffeic acid (44.67mg/g DW), rosmarinic acid (52.22 mg/g DW) and chicoric acid (43.89mg/g DW)] and anthocyanins [cyanidin (16.39mg/g DW) and peonidin (10.77mg/g DW)] as a function of the PGRs treatment. The highest in vitro antioxidant activity, was determined with the ORAC assay as compared to the FRAP assay, suggesting the prominence of the HAT over the ET-based mechanism for the antioxidant action of callus extracts. Furthermore, in vivo results illustrated the protective action of the callus extract to limit the deleterious effects of UV-induced oxidative stress, ROS/RNS production and membrane integrity in yeast cell culture. Altogether, these results clearly demonstrated the great potential of in vitro callus of O. basilicum as a source of human health-promoting antioxidant phytochemicals.
Isodon rugosus (Wall. ex Benth.) Codd accumulates large amounts of phenolics and pentacyclic triterpenes. The present study deals with the in vitro callus induction from stem and leaf explants of I. rugosus under various plant growth regulators (PGRs) for the production of antioxidant and anti-ageing compounds. Among all the tested PGRs, thidiazuron (TDZ) used alone or in conjunction with α-napthalene acetic acid (NAA) induced highest callogenesis in stem-derived explants, as compared to leaf-derived explants. Stem-derived callus culture displayed maximum total phenolic content and antioxidant activity under optimum hormonal combination (3.0 mg/L TDZ + 1.0 mg/L NAA). HPLC analysis revealed the presence of plectranthoic acid (373.92 µg/g DW), oleanolic acid (287.58 µg/g DW), betulinic acid (90.51 µg/g DW), caffeic acid (91.71 µg/g DW), and rosmarinic acid (1732.61 µg/g DW). Complete antioxidant and anti-aging potential of extracts with very contrasting phytochemical profiles were investigated. Correlation analyses revealed rosmarinic acid as the main contributor for antioxidant activity and anti-aging hyaluronidase, advance glycation end-products inhibitions and SIRT1 activation, whereas, pentacyclic triterpenoids were correlated with elastase, collagenase, and tyrosinase inhibitions. Altogether, these results clearly evidenced the great valorization potential of I. rugosus calli for the production of antioxidant and anti-aging bioactive extracts for cosmetic applications.
Ocimum basilicum is a medicinal plant with multiple health benefits including cardiovascular, cancer and diabetics. In the present study, the influence of light emitting diodes (LEDs) was investigated on the accumulation of biologically active ingredients in callus cultures of Ocimum basilicum. Among the various tested treatments optimum levels of Total phenolic content (TPC) was noted in callus culture grown under blue lights as compared to control, while maximum accumulation of Total flavonoid content (TFC) was noted in callus culture grown under red light as compared to control. HPLC analyses showed that highest concentrations of Rosmarinic acid (96.0 mg/g DW) and Eugenol (0.273 mg/g DW) were accumulated in blue light which was 2.46 and 2.25 times greater than control (39.0 mg/g DW, 0.171 mg/g DW), respectively. Chicoric acid (81.40 mg/g DW) optimum accumulation was noted in callus grown under the continuous white light, which was almost 4.52 times greater than control. Anthocyanins content were also analyzed, the highest amount of Peonidin (0.127 mg/g DW) and cyanidin (0.1216 mg/g DW) were found in callus culture grown under red light. These findings suggest that application of LED's is a promising strategy for enhancing production of biologically active ingredients in callus cultures Ocimum basilicum.
Fagonia indica is one of the commercially vital medicinal plant species. It is well-known for biosynthesis of anticancer phenolics and flavonoids metabolites. The plant has been exploited for in vitro studies and production of vital phytochemicals, however, the synergistic effects of melatonin and lights remains to be investigated. In current study, we have evaluated the synergistic effects of melatonin and different light emitting diodes (LEDs) in callus cultures of F. indica. Both, light and melatonin play vital role in physiological and biochemical processes of plant cell. The highest Fresh weight (FW: 320 g/L) and Dry weight (DW: 20 g/L) was recorded in cultures under white LEDs. Optimum total phenolics content (11.3 μg GAE/mg), total flavonoids content (4.02 μg QAE/mg) and Free radical scavenging activity (97%) was found in cultures grown under white LED and melatonin. Furthermore, cultures maintained under white light were also found with highest levels of phenolic and flavonoids production (total phenolic production; 226.9 μg GAE/mg, Total flavonoid production; 81 μg QAE/mg) than other LED-grown cultures. However, the antioxidant enzymes; Superoxide dismutase (SOD: 0.53 nM/min/mg FW) and Peroxidase (POD:1.18 nM/min/mg FW) were found optimum in cultures grown under blue LED. The HPLC data showed that enhanced total production of metabolites was recorded in cultures under white LED (6.765 μg/mg DW) than other lights and control. The findings of this study comprehend the role of melatonin and influence of light quality on biomass accumulation and production of phytochemicals in callus cultures of F. indica.
Tumor necrosis factor alpha (TNF-α) is a multifunctional cytokine that acts as a central biological mediator for critical immune functions, including inflammation, infection, and antitumor responses. It plays pivotal role in auto-immune diseases like rheumatoid arthritis (RA). The synthetic antibodies etanercept, infliximab, and adalimumab are approved drugs for the treatment of inflammatory diseases bind to TNF-α directly, preventing its association with the tumor necrosis factor receptor (TNFR). These biologics causes serious side effects such as triggering an autoimmune anti-antibody response or the weakening of the body's immune defenses. Therefore, alternative small-molecule based therapies for TNF-α inhibition is a hot topic both in academia and industry. Most of small-molecule inhibitors reported in the literature target TNF-α, indirectly. In this study, combined in silico approaches have been applied to better understand the important direct interactions between TNF-α and small inhibitors. Our effort executed with the extensive literature review to select the compounds that inhibit TNF-α. High-throughput structure-based and ligand-based virtual screening methods are applied to identify TNF-α inhibitors from 3 different small molecule databases (∼256.000 molecules from Otava drug-like green chemical collection, ∼500.000 molecules from Otava Tangible database, ∼2.500.000 Enamine small molecule database) and ∼240.000 molecules from ZINC natural products libraries. Moreover, therapeutic activity prediction, as well as pharmacokinetic and toxicity profiles are also investigated using MetaCore/MetaDrug platform which is based on a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism and toxicity information, uses binary QSAR models. Particular therapeutic activity and toxic effect predictions are based on the ChemTree ability to correlate structural descriptors to that property using recursive partitioning algorithm. Molecular Dynamics (MD) simulations were also performed for selected hits to investigate their detailed structural and dynamical analysis beyond docking studies. As a result, at least one hit from each database were identified as novel TNF-α inhibitors after comprehensive virtual screening, multiple docking, e-Pharmacophore modeling (structure-based pharmacophore modeling), MD simulations, and MetaCore/MetaDrug analysis. Identified hits show predicted promising anti-arthritic activity and no toxicity.
Plant manipulation by herbivores requires fine-tuned reprogramming of host metabolism, mediated by effector molecules delivered by the parasite into its host. While plant galls may represent the epitome of plant manipulation, secretomes of gall-inducers and their impact on host-plants have been rarely studied. We characterized, with transcriptomic and enzymatic approaches, salivary glands and saliva of a gall-inducing aphid, Phloeomyzus passerinii. Early responses to aphid saliva of plant genes belonging to different metabolic and signaling pathways were assessed in vivo, with poplar protoplasts, and in planta, in a heterologous Arabidopsis system. Several effectors potentially interfering with plant signaling have been identified, including binding proteins, oxidoreductases, and phosphatidylinositol phosphate kinases. Compatible interactions between protoplasts of a susceptible poplar genotype and the saliva of P. passerinii led to an overall downregulation of defense-related genes while an upregulation was observed during both incompatible interactions, with a resistant poplar genotype, and non-host interactions, with the saliva of Myzus persicae, an aphid which does not feed on poplars. Compatible interactions affected both auxin transport and homeostasis potentially leading to an intracellular accumulation of auxin, which was further supported by in planta assays. Our results support the hypothesis that effectors interfere with downstream signaling and phytohormone pathways.
Chemogenic silver nanoparticles enhance lignans and neolignans in cell suspension cultures of Linum usitatissimum L.
Cell suspension culture of Linum usitatissimum is a great source of the novel and multipurpose medicinal compounds lignans and neolignans. Conventional culturing practices usually result in low yield of plant secondary metabolites; therefore, we conceived a successful mechanism to elicit production of lignans and neolignans in cell suspension cultures, simply, by addition of chemogenic Ag-NPs into the culture medium. A three stage feeding strategy (day 10, 10 and 15, and 10 and 20, respectively, after inoculation) spanning the log growth phase (day 10–20), was implemented to elicit cell suspension cultures of Linum usitatissimum. Though enhancing effects of Ag-NPs were observed at each stage, feeding Ag-NPs at day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Furthermore, a linear trend in accumulation of lignans and neolignans was observed throughout log phase as compared to control, wherein growth non-associated trend in biosynthesis of these metabolites was observed. Optimum production of both lignans and neolignans occurred on day 20 of culture; a ten fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, five fold increase in dehydrodiconiferyl alcohol glucoside and 1.75 fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively.
This study describes ZnO NPs biosynthesis using leaf extracts of Verbena officinalis and Verbena tenuisecta. The extracts serve as natural reducing, capping and stabilization facilitators. Plant extracts phytochemical analysis, revealed that V. officinalis showed higher total phenolic and flavonoid content (22.12 and 6.38 mg g −1 DW) as compared to V. tennuisecta (12.18 and 2.7 mg g −1 DW). ZnO NPs were characterised by ultraviolet–visible spectroscopy, Fourier transform infrared, X-ray diffraction, scanning electron microscope, transmission electron microscopy (TEM) and energy dispersive X-ray. TEM analysis of ZnO NPs reveals rod and flower shapes and were in the range of 65–75 and 14–31 nm, for V. tenuisecta and V. officinalis, respectively. Bio-potential of ZnO NPs was examined through their leishmanicidal potential against Leishmania tropica. ZnO NPs showed potent leishmanicidal activity with 250 µg ml−1 being the most potent concentration. V. officinalis mediated ZnO NPs showed more potent leishmanicidal activity compared to V. tenuisecta mediated ZnO NPs due to their smaller size and increased phenolics doped onto its surface. These results can be a step forward towards the development of novel compounds that can efficiently replace the current medication schemes for leishmaniasis treatment.
Elicitation is a very feasible strategy to enhance important secondary metabolites, such as phenolic compounds, flavonoids, and terpenoids in plant cell cultures. In this report, we studied the effects of different carbohydrate sources on biomass and secondary metabolism of Fagonia indica callus cultures. The results showed that disaccharides, especially sucrose, are favorable for biomass accumulation when applied in higher concentration. Maximum biomass (Fresh weight = 52.05 g/100 mL and dry weight = 2.72 g/100 mL) was recorded in callus cultures raised in vitro in 5% sucrose. Maltose-supplemented callus cultures (5%) also responded with higher biomass (fresh weight = 43.75 g/100 mL and Dry weight 2.715 g/100 mL). Considering the production of secondary metabolites, 3% glucose produced the highest total phenolic content (TPC) in callus cultures (1.677 mg GAE/g DW) followed by fructose (1.625 mg GAE/g DW). The high-performance liquid chromatography data showed that a higher concentration of carbohydrates in the media elicited higher quantities of important phenolic compounds. It is concluded that the antioxidant potential of callus cultures is directly related to the secretion of phenolic compounds because 3% glucose-treated callus cultures gave the highest 82.11% antioxidant activity. The total chlorophyll content was found to decrease with the increasing concentration of carbohydrates. In conclusion, maltose, as a source of carbohydrate in F. indica callus cultures elicits the production of secondary metabolites including Gallic acid, Caffeic acid, Salicylic Acid, Quercetin, Myricetin, and Ellagic acid.
Linum usitatissimum is a source of pharmacologically active lignans and neolignans. An effective protocol has been established for the enhanced biosynthesis of lignans and neolignans in cell cultures of Linum usitatissimum by using chitosan addition. Gene expression analysis of monoligols (PAL, CCR and CAD), lignans (DIR, PLR and UGT) and neolignans (PCBER) biosynthetic genes by RT-qPCR as well as monolignol biosynthetic PAL, CCR and CAD enzyme activities evidenced a stimulation following chitosan treatment. Validated reverse phase high-performance liquid chromatography coupled to diode array detection was used to quantify secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG), dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) showed that chitosan treated cell cultures had better accumulation of these metabolites. Maximum enhancements of 7.3-fold (28 mg/g DW) occurred for LDG, 3.5-fold (58.85 mg/g DW) in DCG and while the least enhancement of 2-fold (18.42 mg/g DW) for SDG was observed in 10 mg/L chitosan treated cell cultures than to controls. Furthermore, same concentration of chitosan also resulted in 1.3-fold increase in antioxidant activity. Compared to the lignans and neolignans accumulations observed in wild type and RNAi-PLR transgenic flaxseeds, chitosan-treated cell cultures appeared to be a very effective production system for these compounds.
Nelumbo nucifera Gaertn. has been used as an important ingredient for traditional medicines since ancient times, especially in Asian countries. Nowadays, many new or unknown phytochemical compounds from N. nucifera are still being discovered. Most of the current research about pharmacological activity focus on nuciferine, many other alkaloids, phenolic compounds, etc. However, there is no current review emphasizing on flavonoids, which is one of the potent secondary metabolites of this species and its pharmacological activities. Therefore, following a taxonomic description, we aim to illustrate and update the diversity of flavonoid phytochemical compounds from N. nucifera, the comparative analysis of flavonoid compositions and contents in various organs. The uses of this species in traditional medicine and the main pharmacological activities such as antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, anti-angiogenic and anti-cancer activities are also illustrated in this works.
A selective acylation protocol using cerium chloride (CeCl3) as catalyst was applied to functionalize silybinin (1), a natural antioxidant flavonolignan from milk thistle fruit, in order to increase its solubility in lipophilic media while retaining its strong antioxidant activity. The selective esterification of 1 at the position 3-OH with a palmitate acyl chain leading to the formation of the 3-O-palmitoyl-silybin (2) was confirmed by both mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analyses. The antioxidant activity of 1 was at least retained and even increased with the CUPRAC assay designed to estimate the antioxidant activity of both hydrophilic and lipophilic compounds. Finally, the 3-O-palmitoylation of 1, resulting in the formation of 2, also increased its anti-lipoperoxidant activity (i.e., inhibition of conjugated diene production) in two different lipophilic media (bulk oil and o/w emulsion) subjected to accelerated storage test.
The multipurpose plant species Linum usitatissimum famous for producing linen fibre and containing valuable pharmacologically active polyphenols, has rarely been tested for it's in vitro biosynthesis potential of lignans and neolignans. The current study aims at the synergistic effects of mineral nutrients variation and different photoperiod treatments on growth kinetics and biomass accumulation in in vitro cultures of Linum usitatissimum. Both nutrient quality and quantity affected growth patterns, as cultures established on Gamborg B5 medium had comparatively long exponential phase compared to Murashige and Skoog medium, while growth was slow but steady until last phases of the culture on Schenk and Hildebrandt medium. Similarly, we observed that boron deficiency and nitrogen limitation in culture medium (Gamborg B5 medium) enhanced callus biomass (fresh weight 413 g/l and dry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside 6.33 and lariciresinol diglucoside 5.22 mg/g dry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans (dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) in continuous dark after 4th week of culture. Conversely, maximum flavonoids production occurred at both Murashige and Skoog, Schenk and Hildebrandt media (both media types contain comparatively higher boron and nitrogen content) in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter. This study opens new dimension for optimizing growing conditions and evaluating underlying mechanisms in biosynthesis of lignans and neolignans in in vitro cultures of Linum usitatissimum.
Tumor necrosis factor alpha (TNFα) is a homotrimer protein that plays a pivotal role for critical immune functions, including infection, inflammation and antitumor responses. It also plays a primary role in autoimmune diseases like rheumatoid arthritis (RA). So far, only biological therapeutics like infliximab, etanercept, and adalimumab are available as treatment of inflammatory diseases. They directly bind to TNFα and interrupt its binding to its receptor protein tumor necrosis factor receptor (TNFR). However, they may also cause serious side effects such as activating an autoimmune anti-antibody response or the weakening of the body's immune defenses. Thus, small molecule-based therapies can be considered as alternative methods. In this study, a novel method is applied to develop energetically optimized, structure-based pharmacophore models for rapid in silico drug screening. Fragment-based docking results were used in the construction of an universal e-pharmacophore model development. The developed model is then used for screening of small-molecule library Specs-screening compounds (Specs-SC) which includes more than 200.000 drug-like molecules. In another approach, binary QSAR-based models were used to screen Specs-SC, as well as Specs-natural products (NP) which has around 750 compounds, and a library of drugs registered or approved for use in humans NIH's NCGC pharmaceutical collection (NPC) which has around 7500 molecules. The MetaCore/MetaDrug platform was used for binary QSAR models for therapeutic activity prediction as well as pharmacokinetic and toxicity profile predictions of screening molecules. This platform is constructed based on a manually curated database of molecular interactions, molecular pathways, gene-disease associations, chemical metabolism, and toxicity information. Molecular docking and molecular dynamics (MD) simulations were performed for the selected hit molecules. As target protein, both homodimer and homotrimer forms of TNFα were considered. The screening results showed that indinavir and medroxalol from NPC chemical library and a set of compounds (AT-057/43115940, AP-970/42897107, AK-968/41925665, AI-204/31679053, AN-648/41666950, AN-698/42006940) from Specs-SC database were identified as safe and active direct inhibitors of TNFα.
LED-enhanced biosynthesis of biologically active ingredients in callus cultures of Ocimum basilicum.
Ocimum basilicum is a medicinal plant with multiple health benefits including cardiovascular, cancer and diabetics. In the present study, the influences of light emitting diodes (LEDs) were investigated on the accumulation of biologically active ingredients in callus cultures of Ocimum basilicum. Among the various tested treatments optimum levels of Total phenolic content (TPC) was noted in callus culture grown under blue lights as compared to control, while maximum accumulation of Total flavonoid content (TFC) was noted in callus culture grown under red light as compared to control. HPLC analyses showed that highest concentrations of Rosmarinic acid (96.0 mg/g DW) and Eugenol (0.273 mg/g DW) were accumulated in blue light which was 2.46 and 2.25 times greater than control (39.0 mg/g DW, 0.171 mg/g DW), respectively. Chicoric acid (81.40 mg/g DW) optimum accumulation was noted in callus grown under the continuous white light, which was almost 4.52 times greater than control. Anthocyanins content were also analyzed, the highest amount of Peonidin (0.127 mg/g DW) and cyanidin (0.1216 mg/g DW) were found in callus culture grown under red light. These findings suggest that application of LED's is a promising strategy for enhancing production of biologically active ingredients in callus cultures Ocimum basilicum.
Yeast-extract improved biosynthesis of lignans and neolignans in cell suspension cultures of Linum usitatissimum L.
Lignans and neolignans are important biologically active ingredients (BAIs) biosynthesized by Linum usitatissimum. These BAIs have multi-dimensional effects against cancer, diabetes and cardio vascular diseases. In this study, yeast extract (YE) was employed as an elicitor to evaluate its effects on dynamics of biomass, BAIs and antioxidant activities in L. usitatissimum cell cultures. During preliminary experiments, flax cultures were grown on different concentrations of YE (0–1000 mg/L), and 200 mg/L YE was found to be optimum to enhance several biochemical parameters in these cell cultures. A two-fold increase in fresh (FW) and dry weight (DW) over the control was observed in cultures grown on MS medium supplemented with 200 mg/L YE. Similarly, total phenolic (TPC; 16 mg/g DW) and flavonoids content (TFC; 5.1 mg/g DW) were also positively affected by YE (200 mg/L). Stimulatory effects of YE on biosynthesis of lignans and neolignans was also noted. Thus, 200 mg/L of YE enhanced biosynthesis of secoisolariciresinol diglucoside (SDG; 3.36-fold or 10.1 mg/g DW), lariciresinol diglucoside (LDG; 1.3-fold or 11.0 mg/g DW) and dehydrodiconiferyl alcohol glucoside (DCG; 4.26-fold or 21.3 mg/g DW) in L. usitatissimum cell cultures with respect to controls. This elicitation strategy could be scaled up for production of commercially feasible levels of these precious metabolites by cell cultures of Linum.
Manipulation in the light regimes combined with the effects of plant growth regulators (PGRs) and elicitors through plant cell culture technology is a promising strategy for enhancing the yield of medicinally important secondary metabolites. In this study, the effects of interplay between PGRs, elicitors and light regimes on cell cultures of F. indica have been investigated. The results showed that callus cultures resulted in maximum biomass formation (13.2 g/L) when incubated on solid MS (Murashige and Skoog) medium containing 1.0 mg/L BA under continuous light for 4 weeks. Among the other PGRs, compared with the auxins such as 2,4-D, and IBA, TDZ resulted in higher biomass accumulation (12.1 g/L). Elicitors (Me-J and PAA) resulted in a lower growth response, when compared with cytokinins and a higher response than auxins under all the light regimes on solid MS media. However, in liquid media no significant increase in biomass was observed in response to the combined effects of PGRs and photoperiod regimes. Further, the highest phenolic content (TPC = 6.8 mg) and flavonoid content (TFC = 5.2 mg) were detected in the dark-grown cell cultures raised in vitro at 0.5 mg/L Me-J treated. The highest antioxidant activity (88%) was recorded in the dark-grown cell cultures harvested from LOG phase of the growth cycle supplemented with 0.5 mg/L Me-J. Furthermore, BA resulted in considerable flavonoids production (TFC = 4.7 mg) in the cell cultures grown under continuous light. However, overall dark treatment and elicitation with Me-J resulted in the optimum metabolic response in terms of secondary metabolites accumulation in cell suspension cultures of F. indica.
Eclipta alba (False daisy) is an important medicinal plant with well-known antihepatotoxic activity. However, no previous in vitro studies are available for its callus culture for increased production of antioxidant secondary metabolites. Herein, we maintained a competent protocol for callus culture of E. alba using stem and leaf explants grown on MS medium containing various concentrations of thidiazuron, 6-benzylaminopurine (BAP) either alone or in association with α-naphthalene acetic acid (NAA). Among all the applied plant growth regulators, BAP along with NAA resulted in maximal dry biomass of 18.0 and 13.8 g/l for stem and leaf explants, respectively. Furthermore, the highest production of phenolics (375.7 mg/l for stem-associated callus and 298 mg/l for leaf-associated callus) and flavonoids (62.0 and 52.3 mg/l for stem- and leaf-associated callus, respectively) were found to be present in optimized callus culture. Antioxidant activity was also elucidated for both stem and leaf derived calli. The highest antioxidant activities (~ 93.5%) were witnessed for stem and leaf associated calli at set concentrations of 3.0 mg/l BAP + 1.0 mg/l NAA and 4.0 mg/l BAP, respectively. High-performance liquid chromatography analyses revealed optimum accumulation of coumarin (1.98 mg/g DW) and wedelolactone (49.63 mg/g DW) in leaf associated callus and desmethylwedelolactone (69.96 mg/g DW), β-amyrin (0.8179 mg/g DW) and eclalbatin (0.3202 mg/g DW) in stem associated callus at optimized concentration.
Microbial contamination is the major cause of economic losses in commercial and scientific plant tissue culture laboratories. For successful micropropagation, it is important to control contamination during in vitro cultures. The present study was designed to isolate, identify and eradicate endophytic contaminants from in vitro cultures of medicinally important plant Fagonia indica. A total of eight distinct bacterial isolates from in vitro grown plantlets of F. indica were selected based on analysis of colony morphology. The endophytic bacterial contaminants identified at the species level through 16S rRNA sequence analysis were Enterobacter xiangfangensis, Bacillus vallismortis, Bacillus tequilensis, Terribacillus halophilus, Pantoea dispersa, Serratia marcescens subsp. Sakuensis, Staphylococcus epidermidis and Bacillus atrophaeus. It was observed that almost 60% of seedlings were contaminated with Bacillus sp. and out of those, Bacillus tequelensis contributed to most infections (70% out of the Bacillus infections). The other most frequently occurring bacteria were Bacillus vallismortis, Terribacillus halophilus and Serratia marcescens subsp. sakuensis. Furthermore, the addition of antimicrobials to the media either completely inhibited or drastically decreased the growth of endophytic bacteria as compared to the control in which 92% of the plantlets were contaminated with these endophytes. Nine different antibiotics (rifampicin, teicoplanin, gentamicin, vancomycin, ciprofloxacin, tobramycin, tetracycline, doxycycline and ampicillin) were tested for their activity against the identified endophytes. Antibiotics such as ciprofloxacin and tobramycin showed a good response and inhibited the growth of all the bacterial isolates at low doses compared to the other antibiotics. Tobramycin was the most effective as it inhibited the growth of five of the bacterial isolates at a dosage as low as 4 mg/L. In case of tetracycline (16 mg/L) and doxycycline (64 mg/L), the contamination frequency in plantlets was 25.6 and 45%, respectively. It is, therefore, important to search for more endophytes, causing adverse effects during in vitro cultures and should devise a feasible anti-microbial strategy for controlling such contamination.
Silybum marianum L. (Milk thistle) is one of the most extensively studied medicinal herbs with well-known hepatoprotective activity. Light is considered as a key abiotic elicitor influencing several physiological processes in plants, including the biosynthesis of secondary metabolites. In this study, we investigated the influence of light quality on morphological and biochemical aspects in in vitro grown leaf-derived callus cultures of S. marianum. Combination of 6-benzylaminopurine (BAP 2.5 mg/l) and α-naphthalene acetic acid (NAA 1.0 mg/l) resulted in optimum callogenic response (97%) when placed under cool-white light with 16 h light and 8 h dark. Red light significantly increased the total phenolic content (TPC), total flavonoid content (TFC), antioxidant and superoxide dismutase (SOD) activities while highest peroxidase (POD) activity was recorded for the dark grown cultures, followed by green light grown cultures. HPLC analysis revealed enhanced total silymarin content under red light (18.67 mg/g DW), which was almost double than control (9.17 mg/g DW). Individually, the level of silychristin, isosilychristin, silydianin, silybin A and silybin B were found greatest under red light, whereas green spectrum resulted in highest accumulation of isosilybin A and isosilybin B. Conversely, the amount of taxifolin was found maximum under continuous white light (0.480 mg/g DW) which was almost 8-fold greater than control (0.063 mg/g DW). A positive correlation was found between the TPC, TFC and antioxidant activities. This study will assist in comprehending the influence of light quality on production of valuable secondary metabolites in in vitro cultures of S. marianum L.
The objective of the current study was to monitor the variations caused by the application of exogenous melatonin on growth kinetics and production of stress enzymes in Prunella vulgaris. Leaf and petiole explants were used for callogenesis. These explants were inoculated on Murashige and Skoog media containing various concentrations of melatonin alone or in combination with 2.0 mg/l naphthalene acetic acid. Herein, a maximum of 3.18-g/100 ml fresh biomass accumulation was observed on day 35 during log phase of growth kinetics at 1.0 mg/l melatonin concentration from leaf explants. While 0.5 and 1.0 mg/l melatonin enhanced the biomass accumulation from petiole explants. Moreover, the synergistic combination of melatonin and naphthalene acetic acid also promoted growth from leaf and petiole explants. Leaf derived callus cultures treated with 1.0 mg/l melatonin induced the production of total protein content (90.47 μg BSAE/mg FW) and protease activity (4.77 U/g FW). While the calli obtained from petiole explants have shown highest content of total protein (160.8 μg BSAE/mg FW) and protease activity (5.35 U/g FW) on media containing 0.5 mg/l melatonin. Similarly, 0.5 mg/l melatonin enhanced superoxide dismutase (3.011 nM/min/mg FW) and peroxidase (1.73 nM/min/mg FW) enzymes from leaf derived callus cultures. The combination of 1.0 and 1.5 mg/l naphthalene acetic acid enhanced content of total protein and protease activity in leaf and petiole derived cultures. These results suggested that the application of melatonin play a positive role in biomass accumulation and production of stress enzymes in P. vulgaris.
Melatonin as plant growth regulator induces differential effects on metabolites that are responsible for reduction, capping and stabilization of zinc oxide nanoparticles. Phytochemical analysis of callus cultures was performed and results were compared with callus cultures supplemented with other plant growth regulators (α-napthalene acetic acid, 2,4-dichlorophenoxy acetic acid and thidiazuron). Highest total phenolic and flavonoid content [42.23 mg of gallic acid equivalent (GAE) g−1 DW and 36.4 mg of (quercetin equivalent) g−1 DW, respectively] were recorded at melatonin (1.0 µM) + NAA (13.5 µM). ZnONPs were synthesized from NAA (13.5 µM) and melatonin (1.0 µM) + NAA (13.5 µM)-induced calli extracts separately and characterized via X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). FTIR analysis confirmed the presence of phenolics and flavonoids that were mainly found responsible for reduction and capping of ZnONPs. SEM analysis showed triangular shaped ZnONPs synthesized from melatonin + NAA callus extract and these NPs were more dispersed as compared to the spherical-agglomerates of ZnONPs synthesized from NAA-mediated callus extract. Melatonin + NAA callus extract-mediated ZnONPs (having smaller size) were more potent against multiple drug resistant bacterial strains, e.g. Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by producing zone of inhibitions 17 ± 0.76 mm,10 ± 0.57 mm and 13 ± 0.54 mm, respectively.
A number of approaches can be implemented to ensure plant-based material authentication for cosmetic applications. Doing this requires knowledge and data dealing with botany, molecular biology, and analytical chemistry, the main techniques of which are described here. A comprehensive and critical view of the methods is provided with comments as well as examples of their application domains.
Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress. Dirigent proteins (DIRs) were discovered during 8-8′ lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (−)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8′ linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (−)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.
Rotavirus A species (RVA) is the leading cause of severe diarrhea among children in both developed and developing countries. Among different RVA G types, humans are most commonly infected with G1, G2, G3, G4 and G9. During 2003–2004, G3 rotavirus termed as “new variant G3” emerged in Japan that later disseminated to multiple countries across the world. Although G3 rotaviruses are now commonly detected globally, they have been rarely reported from Pakistan. We investigated the genetic diversity of G3 strains responsible RVA gastroenteritis in children hospitalized in Rawalpindi, Pakistan during 2014. G3P (18.3%; n = 24) was detected as the most common genotype causing majority of infections in children less than 06 months. Phylogenetic analysis of Pakistani G3 strains showed high amino acid similarity to “new variant G3” and G3 strains reported from China, Russia, USA, Japan, Belgium and Hungary during 2007–2012. Pakistani G3 strains belonged to lineage 3 within sub-lineage 3d, containing an extra N-linked glycosylation site compared to the G3 strain of RotaTeqTM. To our knowledge, this is the first report on the molecular epidemiology of G3 rotavirus strains from Pakistan and calls for immediate response measures to introduce RV vaccine in the routine immunization program of the country on priority.
Fruits of Silybum marianum (L.) Gaernt are the main source of taxifolin derived flavonolignans. Together, these molecules constitute a mixture called silymarin with many useful applications for cosmetic and pharmaceutic industries. Here, a validated method for the separation of the silymarin constituents has been developed to ensure precision and accuracy in their quantification. Each compound was separated with a high reproducibility. Precision and repeatability of the quantification method were validated according to the AOAC recommendations. The method was then applied to study the natural variability of wild accessions of S. marianum. Analysis of the variation in the fruits composition of these 12 accessions from Pakistan evidenced a huge natural diversity. Correlation analysis suggested a synergistic action of the different flavonolignans to reach the maximal antioxidant activity, as determined by cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays. Principal component analysis (PCA) separated the 12 accessions into three distinct groups that were differing from their silymarin contents, whereas hierarchical clustering analysis (HCA) evidenced strong variations in their silymarin composition, leading to the identification of new silybin-rich chemotypes. These results proved that the present method allows for an efficient separation and quantification of the main flavonolignans with potent antioxidant activities.
Breast cancer is one of the most commonly diagnosed cancers around the globe and accounts for a large proportion of fatalities in women.
As a part of strategy to control diarrheal diseases, World Health Organization (WHO) recommends to include rotavirus vaccines in national immunization programs. Sentinel surveillance networks have been established to monitor rotavirus disease burden and genotype distribution in both pre and post vaccine era in many countries. Unfortunately, due to lack of proper surveillance programs, data on rotavirus disease burden and genotype distribution from Pakistan is scarce. We investigated 502 stool samples from children (<5years) hospitalized due to gastroenteritis in Rawalpindi, Pakistan during 2014 for the presence of group A rotavirus (RVA) and its genotypic diversity. Among 147 ELISA positive samples, 131 were successfully genotyped for RVA. Common G types detected were G1 (23.6%), followed by G3 (22.9%), G12 (19.8%), G2 (19.08%) and G9 (9.9%). The most common P-type was P (41.2%), followed by P (29%) and P (28.24%). G3P (17.55%) was the most prevalent genotype combination followed by G12P (16.7%), G2P (15.2%) and G1P (14.5%). Mixed infection of rotavirus G-P types was also observed in 6% of samples. Phylogenetic analysis of VP7 and VP4 genes of Pakistani strains showed that G1, G2, G9 and P, P, P were closely related to strains circulating worldwide as well as previously reported strains from Pakistan. Pakistani G12P strains NIH-BBH-3981 and NIH-BBH-4003 belonged to lineage 3 cluster 3a along with strains from USA and Italy whereas G12P strains NIH-BBH-3978, NIH-BBH-4052 and NIH-BBH-4444 were closely related to strains from Italy, Thailand, United Kingdom and with previously reported G12 strains from Pakistan within lineage 3 cluster 3b. This pre-vaccination data supports the need for RVA vaccine inclusion at our national level and will be helpful in assessing the effect of vaccination on RVA genotype diversity due to vaccine selection pressure once post-vaccination data becomes available.
From last decade, there has been progressive improvement in computational drug designing. Several diseases are being cured from different plant extracts and products. Rheumatoid Arthritis (RA) is the most shared disease among auto-inflammatory diseases. Tumour necrosis factor (TNF)-α is associated with RA pathway and has adverse effects. Extensive literature review showed that plant species under study (Cannabis sativa, Prunella vulgaris and Withania somnifera) possess anti-inflammatory, anti-arthritic and anti-rheumatic properties. 13 anti-inflammatory compounds were characterised and filtered out from medicinal plant species and analysed for RA by targeting TNF-α through in silico analyses. By using ligand based pharmacophore generation approach and virtual screening against natural products libraries we retrieved twenty unique molecules that displayed utmost binding affinity, least binding energies and effective drug properties. The docking analyses revealed that Ala-22, Glu-23, Ser-65, Gln-67, Tyr-141, Leu-142, Asp-143, Phe-144 and Ala-145 were critical interacting residues for receptor-ligand interactions. It is proposed that the RA patients should use reported compounds for the prescription of RA by targeting TNF-α. This report is opening new dimensions for designing innovative therapeutic targets to cure RA.

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