Elicitation of Submerged Adventitious Root Cultures of Stevia rebaudiana with Cuscuta reflexa for Production of Biomass and Secondary Metabolites.

Stevia rebaudiana is an important medicinal plant that belongs to the Asteraceae family. The leaves of Stevia rebaudiana are a rich source of many health-promoting agents such as polyphenols, flavonoids, and steviol glycoside, which play a key role in controlling obesity and diabetes. New strategies such as the elicitation of culture media are needed to enhance the productivity of active components. Herein, the Cuscuta reflexa extracts were exploited as elicitors to enhance the productivity of active components. Cuscuta reflexa is one of the parasitic plants that has the ability to elongate very fast and cover the host plant. Consequently, it may be possible that the addition of Cuscuta reflexa extracts to adventitious root cultures (ADR) of Stevia rebaudiana may elongate the root more than control cultures to produce higher quantities of the desired secondary metabolites. Therefore, the main objective of the current study was to investigate the effect of Cuscuta reflexa extract as a biotic elicitor on the biomass accumulation and production of antioxidant secondary metabolite in submerged adventitious root cultures of Stevia rebaudiana. Ten different concentrations of Cuscuta reflexa were added to liquid media containing 0.5 mg/L naphthalene acetic acid (NAA). The growth kinetics of adventitious roots was investigated for a period of 49 days with an interval of 7 days. The maximum biomass accumulation (7.83 g/3 flasks) was observed on medium containing 10 mg/L extract of Cuscuta reflexa on day 49. As the concentration of extract increases in the culture media, the biomass gradually decreases after 49 days of inoculation. In this study, the higher total phenolics content (0.31 mg GAE/g-DW), total flavonoids content (0.22 mg QE/g-DW), and antioxidant activity (85.54%) were observed in 100 mg/L treated cultures. The higher concentration (100 mg/L) of Cuscuta reflexa extract considerably increased the total phenolics content (TPC), total phenolics production (TPP), total flavonoids content (TFC), total flavonoids production (TFP), total polyphenolics content (TPPC), and total polyphenolics production (TPPP). It was concluded that the extract of Cuscuta reflexa moderately improved biomass accumulation but enhanced the synthesis of phenolics, flavonoids, and antioxidant activities. Here, biomass's independent production of secondary metabolites was observed with the addition of extract. The present study will be helpful to scale up adventitious roots culture into a bioreactor for the production of secondary metabolites rather than biomass accumulation in medicinally important Stevia rebaudiana.

Exogenous melatonin trigger biomass accumulation and production of stress enzymes during callogenesis in medicinally important Prunella vulgaris L. (Selfheal).

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.

Effect of different in vitro culture extracts of black pepper (Piper nigrum L.) on toxic metabolites-producing strains.

In the present study, the effect of different in vitro cultures (callus, in vitro shoots) and commercially available peppercorn extract was investigated for its activity against toxic metabolite-producing strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Candida albicans). These in vitro cultures were extracted with ethanol, hexane, and chloroform, and the antipathogenic activity was determined by well-diffusion method. Hexane extract of callus showed 22 mm zone of inhibition against B. cereus, 23 mm against S. aureus, while regenerated shoots and seeds have shown 24.3 and 26 mm zones of inhibition. The ethanolic extracts of regenerated Piper shoots have shown 25 mm activity against S. aureus, 21 mm against B. cereus, and 16 mm in the case of C. albicans in comparison with standard antibiotics. Peppercorn extracts in chloroform and ethanol had shown activities against B. cereus (23.6 mm) and B. subtilis (23.5 mm). During in vitro organogenesis and morphogenesis, cells and tissues produced a comparable phytochemicals profile like mother plant. Morphogenesis is critically controlled by the application of exogenous plant-growth regulators. Such addition alters the hormonal transduction pathways, and cells under in vitro conditions regenerate tissues, which are dependant on the physiological state of cells, and finally enhance the production of secondary metabolites. To the best of our knowledge, this is the first report to compare the antimicrobial potential of in vitro regenerated tissues and peppercorn with standard antibiotics. In conclusion, most of the extracts showed pronounced activities against all the pathogenic microbes. This is a preliminary work, and the minimum inhibitory concentration values needs to be further explored. Regenerated tissues of P. nigrum are a good source of biologically active metabolites for antimicrobial activities, and callus culture presented itself as a good candidate for such activities.

Gas chromatography coupled with mass spectrometric characterization of Curcuma longa: Protection against pathogenic microbes and lipid peroxidation in rat's tissue homogenate.

The present study was designed to investigate the mineral content and antimicrobial activity of Curcuma Longa extracts and its essential oil. We also determined the lipid peroxidation inhibition activity of the ethanolic extract against sodium nitroprusside (SNP) induced thiobarbituric acid reactive species (TBARS) formation in rat's brain, kidney and liver homogenates. Major constituents of essential oil identified by gas chromatography and mass spectrometry (GCMS) were beta-sesquiphellandrene (38.69%), alpha-curcumene (18.44%) and p-mentha-1,4 (8)-diene (16.29%). Atomic absorption spectroscopy (AAS) was used for the quantitative estimation of Calcium (Ca), Magnesium (Mg), Iron (Fe), Copper (Cu), Zinc (Zn), Chromium (Cr), Nickel (Ni) and Manganese (Mn). The extract showed highest Mg (49.4 mg/l) concentration followed by Ca (35.42 mg/l) and Fe (1.27 mg/l). Our data revealed that the ethanolic extract of Curcuma Longa at 1-10 mg/kg significantly inhibited TBARS production in all tested homogenates. Crude extracts and essential oil were tested against three gram positive bacteria i.e. Bacillus subtilis, Bacillus atrophoeus, Staphylococcus aureus, six gram negative bacteria i.e. Escherichia coli, Klebsiella pneumonias, Salmonella typhi, Pseudomonas aeruginosa, Erwinia carotovora, Agrobacterium tumefaciens and one fungal strain namely Candida albicans by disc diffusion assay. Essential oil showed highest anti-microbial activity as compared to the crude extracts. The present study confirms the significant antimicrobial and antioxidant potential of the studied plant, which can be considered as a diet supplement for a variety of oxidative stress induced or infectious diseases.

Antimicrobial activity of different tea varieties available in Pakistan.

In this antimicrobial study, various extracts of Green and Black tea (Camellia sinensis) and Lemon grass (Cymbopogon citrates) were evaluated for antimicrobial activities against six bacterial strains including both human pathogenic bacteria (Escherichia coli, Pseudomonas aeuroginosa, Staphylococcus aureus and Salmonella typhi) and plant pathogenic bacteria (Erwinia carotovora, Agro bacterium tumifaciens) and one fungal strain Candida albicans by disc diffusion susceptibility method. Of human pathogens, P. aeruginosa was most susceptible to all three different tea varieties; though rest of the strains also demonstrated prominent sensitivity. In comparison, black tea extracts were less activities than green tea and lemon grass. However, all the three tea varieties illustrated profound activity against plant pathogenic bacteria. Similarly, when extracts of tea were tested against C. albicans, green tea and lemon grass exhibited significant activity while black tea was mostly inactive.

Antioxidant activity via DPPH, gram-positive and gram-negative antimicrobial potential in edible mushrooms.

Edible mushrooms (EMs) are nutritionally rich source of proteins and essential amino acids. In the present study, the antioxidant activity via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and antimicrobial potential in EMs (Pleurotus ostreatus, Morchella esculenta, P. ostreatus (Black), P. ostreatus (Yellow) and Pleurotus sajor-caju) were investigated. The DPPH radical scavenging activity revealed that the significantly higher activity (66.47%) was observed in Morchella esculenta at a maximum concentration. Similarly, the dose-dependent concentrations (200, 400, 600, 800 and 1000 µg) were also used for other four EMs. Pleurotus ostreatus exhibited 36.13% activity, P. ostreatus (Black (B)) exhibited 30.64%, P. ostreatus (Yellow (Y)) exhibited 40.75% and Pleurotus sajor-caju exhibited 47.39% activity at higher concentrations. Furthermore, the antimicrobial potential were investigated for its toxicity against gram-negative bacterial strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Klebsiella pneumonia, Erwinia carotovora and Agrobacterium tumifaciens), gram-positive bacterial strains (Bacillus subtilis, Bacillus atrophaeus and Staphylococcus aureus) and a fungal strain (Candida albicans) in comparison with standard antibiotics. Antimicrobial screening revealed that the ethanol extract of P. ostreatus was active against all microorganism tested except E. coli. Maximum zone of inhibition (13 mm) was observed against fungus and A. tumifaciens. P. sajor-caju showed best activities (12.5 mm) against B. subtilis, B. atrophaeus and K. pneumonia. P. ostreatus (Y) showed best activities against P. aeroginosa (21.83 mm), B. atrophaeus (20 mm) and C. albicans (21 mm). P. ostreatus (B) exhibited best activities against C. albicans (16 mm) and slightly lower activities against all other microbes except S. typhi. M. esculenta possess maximum activities in terms of inhibition zone against all microorganisms tested except S. typhi.

Melatonin-Induced Stress Enhanced Biomass and Production of High-Value Secondary Cell Products in Submerged Adventitious Root Cultures of Stevia rebaudiana (Bert.).

Stress is one of the important factors that directly or indirectly affects the plant architecture, biochemical pathways, and growth and development. Melatonin (MEL) is an important stress hormone; however, the exogenous addition of melatonin to culture media stimulates the defense mechanism and releases higher quantities of secondary metabolites. In this study, submerged adventitious root cultures (SARCs) of diabetically important Stevia rebaudiana were exposed to variable concentrations (0.5-5.0 mg/L) of MEL in combination with 0.5 mg/L naphthalene acetic acid (NAA) to investigate the biomass accumulation during growth kinetics with 07 days intervals for a period of 56 days. The effects of exogenous MEL on the biosynthesis of stevioside (Stev.), total phenolics content (TPC), total flavonoids content (TFC), total phenolics production (TPP), total flavonoids production (TFP), total polyphenolics content (TPPC), fresh and dry weight (FW & DW), and antioxidant potential were also studied. Most of the SARCs displayed lag, exponential, stationary, and decline phases with variable biomass accumulation. The maximum fresh (236.54 g/L) and dry biomass (28.64 g/L) was observed in SARCs exposed to 3.0 mg/L MEL and 0.5 mg/L NAA. The same combination of MEL and NAA also enhanced the accumulation of TPC (18.96 mg/g-DW), TFC (6.33 mg/g-DW), TPP (271.51 mg/L), TFP (90.64 mg/L), and TPPC (25.29 mg/g-DW). Similarly, the highest stevioside biosynthesis (91.45 mg/g-DW) and antioxidant potential (86.15%) were observed in SARCs exposed to 3.0 mg/L MEL and NAA. Moreover, a strong correlation was observed between the biomass and the contents of phenolics, flavonoids, antioxidants, and stevioside. These results suggest that MEL is one of multidimensional stress hormones that modulate the biosynthetic pathways to release higher quantities of metabolites of interest for various industrial applications.

Correction: Biogenic synthesis of silver nanoparticles using Rubus fruticosus extract and their antibacterial efficacy against Erwinia caratovora and Ralstonia solanacearum phytopathogens.

[This corrects the article DOI: 10.1039/D3RA06723H.].

Biogenic synthesis of silver nanoparticles using Rubus fruticosus extract and their antibacterial efficacy against Erwinia caratovora and Ralstonia solanacearum phytopathogens.

In the current research, we produced green, cost-effective, eco-friendly silver nanoparticles using a single-step approach. Plants are considered highly desirable systems for nanoparticle synthesis because they possess a variety of secondary metabolites with significant reduction potential. In the current research, the dried leaf extract of Rubus fruticosus was utilized as a capping and reducing agent for the fabrication of silver nanoparticles, to prepare reliable biogenic silver nanoparticles and subsequently to investigate their potential against some common phytopathogens. The prepared silver nanoparticles were exploited to quantify the total flavonoid content (TFC), total phenolic content (TPC) and DPPH-based antioxidant activity. Different concentrations of aqueous extracts of plant leaves and silver nitrate (AgNO3) were reacted, and the color change of the reactant mixture confirmed the formation of Rubus fruticosus leaf-mediated silver nanoparticles (RFL-AgNPs). A series of characterization techniques such as UV-vis spectroscopy, transmission electron microscopy, energy dispersive X-ray analysis and X-ray diffraction revealed the successful synthesis of silver nanoparticles. The surface plasmon resonance peak appeared at 449 nm. XRD analysis demonstrated the crystalline nature, EDX confirmed the purity, and TEM demonstrated that the nanoparticles are mostly spherical in form. Furthermore, the biosynthesized nanoparticles were screened for in vitro antibacterial activity, antioxidant activity, and total phenolic and flavonoid content. The nanoparticles were used in different concentrations alone and in combination with plant extracts to inhibit Erwinia caratovora and Ralstonia solanacearum. In high-throughput assays used to inhibit these plant pathogens, the nanoparticles were highly toxic against bacterial pathogens. This study can be exploited for planta assays against phytopathogens utilizing the same formulations for nanoparticle synthesis and to develop potent antibacterial agents to combat plant diseases.

DPPH free radical scavenging activity and phenotypic difference in hepatoprotective plant (Silybum marianum L.).

Silybum marianum L. is medicinally important for its active principle component silymarin. Silymarin regenerates damaged hepatic tissues. On the basis of such regenerative properties, the radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl (DPPH)) of different tissues and the phenotypic difference of the hepatoprotective species, S. marianum L. were evaluated. There was less phenotypic difference in purple and white varieties of S. marianum. Assay of the antioxidant potential of different parts of the plant revealed that significantly higher activity (78.2%) was observed in seeds of the purple flowering plant than seeds of white flowering plant (49%) after different time intervals. Young leaves collected from white flowering plant exhibit 64.8% activity, which is higher than the purple flowering plant (55.1%). Significantly, same activity was observed in mature leaves of white (52%) and purple flowering plants (50%). The main stem collected from both the varieties exhibits similar activity from 50 to 52%. A 67.2% activity was recorded for mature roots of white flowering plant followed by roots of the purple variety (65%). The present study revealed that seeds and roots of both the varieties scavenge and detoxify more DPPH free radicals than other plant parts and can be used as a source of natural antioxidants and food additives.

Identification, characterization, and palynology of high-valued medicinal plants.

High-valued medicinal plants Achillea millefolium, Acorus calamus, Arnebia nobilis, Fumaria indica, Gymnema sylvestre, Origanum vulgare, Paeonia emodi, Peganum harmala, Psoralea corylifolia, Rauwolfia serpentina, and Vetiveria zizanioides were identified with the help of taxonomical markers and investigated for characterization and palynological studies. These parameters are used to analyze their quality, safety, and standardization for their safe use. Botanical description and crude drug description is intended for their quality assurance at the time of collection, commerce stages, manufacturing, and production. For this purpose the detailed morphology was studied and compared with the Flora of Pakistan and other available literatures. Here we reported the pollen grain morphology of Origanum vulgare, Paeonia emodi, Psoralea corylifolia, and Rauwolfia serpentina for the first time. Similarly the crude drug study of Gymnema sylvestre (leaf), Origanum vulgare (aerial parts), Paeonia emodi (tubers), and Peganum harmala (seeds) was also carried out for the first time.

Nutritionally rich biochemical profile in essential oil of various Mentha species and their antimicrobial activities.

The Mentha species of family Lamiaceae are famous for their flavor and are commercially used in many food products worldwide. They are widely used to cure digestive problems as well as a natural source of antioxidants and antimicrobials. In this report, the essential oils (EOs) of five Mentha species, namely Mentha citrata, Mentha x piperita, Mentha pulegium, Mentha spicata, and Mentha suaveolens were extracted and their chemical diversity was investigated through gas chromatography-mass spectroscopy (GC-MS). The differential doses (5, 10, and 15 µl) of EOs were tested for antimicrobial potential against two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), five gram-negative bacteria (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Proteus mirabilis and Klebsiella pneumoniae) and a fungal strain (Candida albicans). The GC-MS results revealed the major components in the EOs were Carvone, Linalool, Hotrienol, Menthol, Isopulegone, Furanone, Piperitone, and Thymol. Moreover, the higher dose (15 µl) of EOs of M. citrata inhibited the growth of S. typhi and C. albicans (35.8 ± 2.4 and 35.2 ± 2.5 mm), M. x piperita inhibited E. coli, B. subtilis, and C. albicans (28.5 ± 3.5, 26.1 ± 2.1, and 25.4 ± 1.1 mm), M. pulegium inhibited K. pneumoniae, B. subtilis, and C. albicans (26.8 ± 1.8, 24.2 ± 2.2, and 25.3 ± 0.9 mm), M. spicata significantly inhibited S. typhi and B. subtilis (35.7 ± 2.7 and 36.3 ± 2.1 mm), and M. suaveolens inhibited K. pneumoniae, C. albicans, and S. typhi (30.8 ± 1.9, 26.9 ± 1.1, and 20.1 ± 0.8 mm) respectively. This study concluded that the EOs of M. citrata was effective against S. typhi and C. albicans. The M. x piperita exhibited strong activities against E. coli, B. subtilis, and C. albicans. Furthermore, the M. pulegium strongly inhibited the growth of K. pneumoniae and C. albicans. The EO of M. spicata was more potent against S. typhi and B. subtilis, while the M. suaveolens was comparatively more effective against S. typhi, K. pneumoniae, and C. albicans. These EOs offer a natural source of antimicrobial agents with high commercial values and social acceptance and could be scale up by food and pharmaceutical industries to control pathogenic diseases.

Antimicrobial efficacy of Mentha piperata-derived biogenic zinc oxide nanoparticles against UTI-resistant pathogens.

Misuse of antibiotics leads to the worldwide spread of antibiotic resistance, which motivates scientists to create new antibiotics. The recurring UTI due to antibiotics-resistant microorganism's challenges scientists globally. The biogenic nanoparticles have the potential to meet the escalating requirements of novel antimicrobial agents. The green synthesis of nanoparticles (NPs) gained more attention due to their reliable applications against resistant microbes. The current study evaluates the biogenic ZnO NPs of Mentha piperata extract against resistant pathogens of urinary tract infections by agar well diffusion assay. The biogenic ZnO NPs revealed comparatively maximum inhibition in comparison to synthetic antibiotics against two bacterial strains (Proteus mirabilis, Pseudomonas aeruginosa) and a fungal strain (Candida albicans).The synthesized biogenic ZnO NPs alone revealed maximum activities than the combination of plant extract (PE) and ZnO NPs, and PE alone. The physiochemical features of ZnO NPs characterized through UV-Vis spectroscopy, FTIR, XRD, SEM, and EDX. The UV-Vis spectroscopy revealed 281.85 nm wavelengths; the XRD pattern revealed the crystalline structure of ZnO NPs. The FTIR analysis revealed the presence of carboxylic and nitro groups, which could be attributed to plant extract. SEM analysis revealed spherical hollow symmetry due to electrostatic forces. The analysis via EDX confirmed the presence of Zn and oxygen in the sample. The physiochemical features of synthesized ZnO NPs provide pivotal information such as quality and effectiveness. The current study revealed excellent dose-dependent antimicrobial activity against the pathogenic isolates from UTI-resistant patients. The higher concentration of ZnONPs interacts with the cell membrane which triggers oxidative burst. They may bind with the enzymes and proteins and brings epigenetic alteration which leads to membrane disruption or cell death.

Free radical scavenging (DPPH) potential in nine Mentha species.

Mentha species are used in every day life in various food items. These species produce valuable secondary metabolites that scavenge toxic free radicals. Toxic free radicals can cause different diseases in the human body. In the present study free radical scavenging potential (1,1-diphenyl-2-picrylhydrazyl scavenging activity) in nine Mentha species were investigated to evaluate and explore new potential sources for natural antioxidants. The activity was performed after different time intervals with incubation period of 30 minutes. The methanolic extracts revealed that significantly higher activity (82%) was observed in Mentha suaveolens, followed by Mentha longifolia (79%), Mentha officinalis (76%) and Mentha piperita, Mentha pulegium, Mentha royleana (75%), respectively. Significantly same activity was observed in Mentha arvensis and Mentha spicata. Lower activity was observed in Mentha citrata (64%). The present study revealed that these species can be used as natural antioxidants.

Potential antimicrobial, antidiabetic, catalytic, antioxidant and ROS/RNS inhibitory activities of Silybum marianum mediated biosynthesized copper oxide nanoparticles.

Use of medicinal plants for the biosynthesis of nanoparticles offers several advantages over other synthesis approaches. Plants contain a variety of bioactive compounds that can participate in reduction and capping of nanoparticles. Plant mediated synthesis has the leverage of cost effectiveness, eco-friendly approach and sustained availability. In the current study Silybum marianum, a medicinally valuable plant rich in silymarin content, is used as a reducing and stabilizing agent for the fabrication of nanoparticles. Biosynthesized CuO-NPs were characterized using High Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Dynamic Light Scattering (DLS) techniques. Characterization revealed that CuO-NPs having a crystalline structure showed spherical morphology with an average size of 15 nm. HPLC analysis demonstrated conjugation of various silymarin components, especially the presence of silybin A (705.06 ± 1.59 mg g-1 DW). CuO-NPs exhibited strong bactericidal potency against clinically important pathogenic bacterial strains e.g. Enterobacter aerogenes and Salmonella typhi with an inhibition zone of 18 ± 1.3 mm and 17 ± 1.2 mm, respectively. Synthesized nanoparticles indicated a dose dependent cytotoxic effect against fibroblast cells exhibiting a percentage cell viability of 83.60 ± 1.505% and 55.1 ± 1.80% at 25 µg mL-1 and 100 µg mL-1 concentration, respectively. Moreover, CuO-NPs displayed higher antioxidant potential in terms of (TAC: 96.9 ± 0.26 µg AAE/mg), (TRP: 68.8 ± 0.35 µg AAE/mg), (DPPH: 55.5 ± 0.62%), (ABTS: 332.34 µM) and a significant value for (FRAP: 215.40 µM). Furthermore, enzyme inhibition assays also exhibited excellent enzyme inhibition potential against α-amylase (35.5 ± 1.54%), urease (78.4 ± 1.26%) and lipase (80.50.91%), respectively. Overall findings indicated that biosynthesized CuO-NPs possess immense in vitro biological and biomedical properties and could be used as a broad-spectrum agent for a wider range of biomedical applications.

Silver and gold nanoparticles induced differential antimicrobial potential in calli cultures of Prunella vulgaris.

BACKGROUND: Prunella vulgaris is medicinally important plant containing high-valued chemical metabolites like Prunellin which belong to family Lamiaceae and it is also known as self-heal. In this research, calli culture were exposed to differential ratios of gold (Au) and silver (Ag) nanoparticles (1:1, 1:2, 1:3, 2:1 and 3:1) along with naphthalene acetic acid (2.0 mg NAA) to investigate its antimicrobial potential. A well diffusion method was used for antimicrobial properties. RESULTS: Here, two concentrations (1 and 2 mg/6 µl) of all treated calli cultures and wild plants were used against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus atrophaeus, Bacillus subtilis, Agrobacterium tumefaciens, Erwinia caratovora and Candida albicans. Dimethyl sulfoxide (DMSO) and antibiotics were used as negative and positive controls. Here, the calli exposed to gold (Au) nanoparticles (NPs) and 2.0 mg naphthalene acetic acid (NAA) displayed the highest activity (25.7 mm) against Salmonella typhi than other extracts, which was considered the most susceptible species, while Agrobacterium tumefaciens and Candida albicans was the most resistance species. A possible mechanism of calli induced nanoparticles was also investigated for cytoplasmic leakage. CONCLUSION: From the above data it is concluded that Prunella vulgaris is medicinally important plant for the development of anti-microbial drugs using nanotechnology and applicable in various pharmaceutical research.

Green and chemically synthesized zinc oxide nanoparticles: effects on in-vitro seedlings and callus cultures of Silybum marianum and evaluation of their antimicrobial and anticancer potential.

Zinc oxide nanoparticles (ZnO-NPs) have been produced by physical and chemical methods. Here, the comparative evaluation of both chemically-synthesised ZnO-NPs (C-ZNPs) and in-vitro cultured S. marianum mediated green-synthesised ZnO-NPs (G-ZNPs) were investigated on seed germination frequency, root and shoot growth, callus induction and biochemical profile of medicinally important plant Silybum marianum. Of all the treatments, callus-mediated ZnO-NPs gave optimum results for seed germination (65%), plantlet's root length (4.3 cm), shoot length (5.3 cm) and fresh and dry weights (220.4 g L-1 and 21.23 g L-1, respectively). Similarly, the accumulation of phenolic (12.3 µg/mg DW) and flavonoid (2.8 µg/mg DW) contents were also enhanced in callus cultures treated with G-ZNPs. We also observed maximum antioxidant activity (99%) in callus cultures treated with G-ZNPs, however, in case of plantlets, these activities were found highest for in-vitro whole plant-mediated ZnO-NPs. Moreover, G-ZNPs also enhanced total protein content (265.32 BSAE/20g FW) in callus cultures. G-ZNPs were further assessed for their effects on several multidrug resistant bacterial strains and human liver carcinoma (HepG2) cells and our findings revealed that callus extracts treated with G-ZNPs show ameliorated antibacterial (highest zone of inhibition (19 mm) against Klebsiella pneumonia) and anticancer (highest cytotoxicity of 64%) activities.

Biosynthesis of antioxidative enzymes and polyphenolics content in calli cultures of Prunella vulgaris L. in response to auxins and cytokinins.

Prunella vulgaris L. is one of the therapeutic herbs containing various polyphenolics, which is used for multiple medicinal purposes. In this study, plant growth regulators (PGRs)-induced calli cultures from seed-derived leaf explants were exploited for the production of stress enzymes and polyphenolics. A growth curve was plotted for each PGR for 49 days period, which showed a distinct lag, log and decline phases. Here, the combination of naphthalene acetic acid (NAA) and 6-benzyleadenine (BA; 0.5 and 2.0 mg l-1) produced maximum fresh (6.32 FW-g/100 ml) and dry biomass (0.75 DW-g/100 ml) in contrast to control. The maximum synthesis of SOD (0.0154 FW-nM/min/mg) was detected on media comprising mixture of NAA and BA (1.5 mg l-1), while POD enzyme (0.366 FW-nM/min/mg) was higher at 0.5 mg l-1 NAA and 2, 4-dichlorophenoxy acetic acid. Further, NAA and BA (1.5 and 2.0 mg l-1) boosted up the synthesis of phenolics (18.83 GAE-mg/g-DW) and flavonoids content (18.05 RE-mg/g-DW) than control. Moreover, NAA of 1.0 and 2.0 mg l-1 were found supportive for maximum antioxidant activity (87.4%) and total protein (716 µg BSAE/mg-DW). This study will contribute in the development of cell culture in fermenter and synthesis of antioxidant secondary metabolites for commercial uses.

Effect of Gibberellic Acid on Production of Biomass, Polyphenolics and Steviol Glycosides in Adventitious Root Cultures of Stevia rebaudiana (Bert.).

In current study, the effect of gibberellic acid was tested for production of biomass, polyphenolics and Steviol glycosides in adventitious root cultures of Stevia rebaudiana. Adventitious cultures were induced from the roots of in vitro grown plantlets on Murashige and Skoog (MS) medium containing combination of gibberellic acid (GA3; 0.5, 1.0, 1.5 and 2.0 mg/L) and naphthalene acetic acid (NAA; 0.5 mg/L). Initially, a known mass of inoculum roots were shifted into suspension media augmented with various GA3 concentrations. The growth behavior of adventitious roots was recorded every 3 days for a period of 30 days. Maximum biomass biosynthesis (13.12 g/flask) was noticed in exponential phase on 27th day in the suspension containing 2.0 mg/L of GA3. Other GA3 concentrations also displayed optimum patterns of biomass accumulation as compared to the control. Adventitious roots were investigated for total phenolic content (TPC) and production (TPP), total flavonoid content (TFC) and production (TFP), and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-based antioxidant potential. Maximum phenolics (TPC 9.84 mg gallic acid equivalent (GAE)/g-dry weight (DW)) and TPP (147.6 mg/L), TFC (5.12 mg Quercitin equivalent (QE)/g-DW) and TFP (76.91 mg/L) were observed in 2.0 mg/L GA3 treated cultures. The same concentration of gibberellic acid enhanced antioxidant activity (77.2%). Furthermore, maximum stevioside (7.13 mg/g-DW), rebaudioside-A (0.27 mg/g-DW) and dulcoside-A (0.001 mg/g-DW) were observed in roots exposed to 2.0 mg/L GA3. This is the first report on the application of GA3 on biomass accumulation and secondary metabolite production in S. rebaudiana. The current study will be helpful to scale up the adventitious root cultures in bioreactors for the production of biomass and pharmaceutically important secondary metabolites.