Elicitation of silver nanoparticles enhanced the secondary metabolites and pharmacological activities in cell suspension cultures of bitter gourd.

This study describes the influence of bio-synthesized silver nanoparticles (AgNPs) on phytochemicals and their pharmacological activities in the cell suspension cultures (CSC) of bitter gourd. To standardize the effect of sucrose, plant growth regulators, medium, AgNPs and growth kinetics for the biomass and bioactive compounds accumulation in CSC of bitter gourd. The medium comprising MS salts, sucrose (30 g/L) with 2,4-D (1.0 mg/L) and TDZ (0.1 mg/L) at 28 days of CSC was appropriate for biomass and bioactive compound accumulation. The contents of silver, malondialdehyde and hydrogen peroxide were highly elevated in AgNPs (10 mg/L)-elicited CSC when compared with non-elicited CSC. AgNPs (5 mg/L) elicited CSC extracts had significantly enhanced the production of total phenolic (3.5 ± 0.2 mg/g), and flavonoid (2.5 ± 0.06 mg/g) contents than in the control CSC extracts (2.5 ± 0.1 and 1.6 ± 0.05 mg/g). AgNPs (5 mg/L) elicited CSC showed a higher amount of flavonols (1822.37 µg/g), hydroxybenzoic (1713.40 µg/g) and hydroxycinnamic (1080.10 µg/g) acids than the control CSC (1199, 1394.42 and 944.52 µg/g, respectively). Because of these metabolic changes, the pharmacological activities (antioxidant, antidiabetic, antibacterial, antifungal and anticancer) were high in the AgNPs (5 mg/L)-elicited CSC extracts in bitter gourd. The study suggested the effectiveness of elicitation process in enhancing the accumulation of phenolic compounds and pharmacological activities. AgNPs-elicited CSC offered an effective and favorable in vitro method to improve the production of bioactive compounds for potential uses in pharmaceutical industries.

Influence of silver nanoparticles on the enhancement and transcriptional changes of glucosinolates and phenolic compounds in genetically transformed root cultures of Brassica rapa ssp. rapa.

Glucosinolates (GSLs) and phenolic compounds (PCs) are biologically active and involved in the defense reaction of plants; these compounds have a beneficial effect on human health. In this study, we described the influence of biologically synthesized silver nanoparticles (Ag NPs) to enhance the phytochemicals (GSLs and PCs), their transcription levels, and their biological activities in genetically transformed root cultures (hairy root cultures) of Brassica rapa. The concentrations of silver and reactive oxygen species (malondialdehyde and hydrogen peroxide) were highly elevated in the Ag NP-elicited hairy roots (HRs). Glucosinolates (glucoallysin, glucobrassicanapin, sinigrin, progoitrin, gluconapin, 4-methoxyglucobrassicin, 4-hydroxyglucobrassicin, glucobrassicin, neoglucobrassicin, and gluconasturtiin) and their transcripts (MYB34, MYB51, MYB28, and MYB29) were significantly enhanced in the Ag NP-elicited HRs. Moreover, the phenolic compounds (flavonols, hydroxybenzoic, and hydroxycinnamic acids) were significantly enriched in the Ag NP-elicited HRs. Total phenolic and flavonoid concentrations and their transcripts (PAL, CHI, and FLS) were higher in the Ag NP-elicited HRs than in the non-elicited HRs. Additionally, biological (antioxidant, antimicrobial, and anticancer) activities were significantly higher in the Ag NP-elicited HRs than in the non-elicited HRs. The Ag NP-elicited HR cultures offered an efficient and promising in vitro method to increase the production of health-promoting bioactive compounds, which may be useful in nutraceutical and pharmaceutical industries.

Jasmonic and salicylic acids enhanced phytochemical production and biological activities in cell suspension cultures of spine gourd (Momordica dioica Roxb).

In vitro cell suspension culture was established for the production of commercially valuable phytochemicals in Momordica dioica. The influence of elicitors in jasmonic acid (JA) and salicylic acid (SA) increased their effect on phytochemical production and biomass accumulation in M. dioica. The results indicate that compared with non-elicited cultures, JA- and SA-elicited cell suspension cultures had significantly enhanced phenolic, flavonoid, and carotenoid production, as well as antioxidant, antimicrobial, and antiproliferative activities. Furthermore, elicited cultures produced 22 phenolic compounds, such as flavonols, hydroxycinnamic acids, and hydroxybenzoic acids. Greater biomass production, phytochemical accumulation, and biological activity occurred in JA- than in SA-elicited cell cultures. This study is the first to successfully establish M. dioica cell suspension cultures for the production of phenolic compounds and carotenoids, as well as for biomass accumulation.

Enhanced Production of Anthraquinones and Phenolic Compounds and Biological Activities in the Cell Suspension Cultures of Polygonum multiflorum.

Anthraquinones (AQs) and phenolic compounds are important phytochemicals that are biosynthesized in cell suspension cultures of Polygonum multiflorum. We wanted to optimize the effects of plant growth regulators (PGRs), media, sucrose, l-glutamine, jasmonic acid (JA), and salicylic acid (SA) for the production of phytochemicals and biomass accumulation in a cell suspension culture of P. multiflorum. The medium containing Murashige and Skoog (MS) salts and 4% sucrose supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L thidiazuron, and 100 µM l-glutamine at 28 days of cell suspension culture was suitable for biomass accumulation and AQ production. Maximum biomass accumulation (12.5 and 12.35 g fresh mass (FM); 3 and 2.93 g dry mass (DM)) and AQ production (emodin 295.20 and 282 mg/g DM; physcion 421.55 and 410.25 mg/g DM) were observed using 100 µM JA and SA, respectively. JA- and SA-elicited cell cultures showed several-fold higher biomass accumulation and AQ production than the control cell cultures. Furthermore, the cell suspension cultures effectively produced 23 phenolic compounds, such as flavonols and hydroxycinnamic and hydroxybenzoic acid derivatives. PGR-, JA-, and SA-elicited cell cultures produced a higher amount of AQs and phenolic compounds. Because of these metabolic changes, the antioxidant, antimicrobial, and anticancer activities were high in the PGR-, JA-, and SA-elicited cell cultures. The results showed that the elicitors (JA and SA) induced the enhancement of biomass accumulation and phytochemical (AQs and phenolic compounds) production as well as biological activities in the cell suspension cultures of P. multiflorum. This optimized protocol can be developed for large-scale biomass accumulation and production of phytochemicals (AQs and phenolic compounds) from cell suspension cultures, and the phytochemicals can be used for various biological activities.

Production of glucosinolates, phenolic compounds and associated gene expression profiles of hairy root cultures in turnip (Brassica rapa ssp. rapa).

Turnip (Brassica rapa ssp. rapa) is an important vegetable crop producing glucosinolates (GSLs) and phenolic compounds. The GSLs, phenolic compound contents and transcript levels in hairy root cultures, as well as their antioxidant, antimicrobial and anticancer activity were studied in turnip. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and reverse transcription-PCR. GSLs levels (glucoallysin, glucobrassicanapin, gluconasturtiin, glucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin and 4-hydroxyglucobrassicin) and their gene expression levels (BrMYB28, BrMYB29, BrMYB34, BrMYB51, BrMYB122, CYP79 and CYP83) significantly increased in hairy roots compared with that in non-transformed roots. Furthermore, hairy roots efficiently produced several important individual phenolic compounds (flavonols, hydroxybenzoic and hydroxycinnamic acids). Colorimetric analysis revealed that the highest levels of total phenol, flavonoid contents, and their gene expression levels (PAL, CHI and FLS) in hairy roots than non-transformed roots. Our study provides beneficial information on the molecular and physiological active processes that are associated with the phytochemical content and biosynthetic gene expression in turnip. Moreover, antioxidant activity, as measured by DPPH scavenging activity, reducing potential, phosphomolybdenum and ferrous ion chelating ability assays was significantly higher in hairy roots. Hairy root extracts exhibited higher antimicrobial activity against bacterial and fungal species. The extract of hairy roots showed inhibition of human breast and colon cancer cell lines.

Induction of hairy roots by Agrobacterium rhizogenes-mediated transformation of spine gourd (Momordica dioica Roxb. ex. willd) for the assessment of phenolic compounds and biological activities.

An efficient protocol for hairy root induction of spine gourd (Momordica dioica) was established using Agrobacterium rhizogenes (KCTC 2703). This study evaluates the phenolic compound production, antioxidant and antimicrobial (antibacterial, antifungal and antiviral) activities of transgenic hairy root cultures in M. dioica . Hairy roots were induced from leaves, petiole, and internodal explants. Molecular analysis of PCR and gene sequencing using specific primers of rolC and aux1 revealed T-DNA integration in the hairy root clones and RT-PCR analysis confirmed the expression of hairy root inducible genes (rolC and aux1). The greatest biomass accumulation of hairy roots on MS liquid medium supplemented with 3% sucrose was observed at 22 days. Ultra-HPLC was used to compare the individual phenolic compound contents of transgenic and non-transgenic roots. Moreover, transgenic hairy roots efficiently produced several phenolic compounds, such as flavonols, hydroxycinnamic acid and hydroxybenzoic acid derivatives. The total phenolic, flavonoid contents and biological (antioxidant, antibacterial, antifungal and antiviral) activities were higher in hairy roots compared to non-transformed roots. These results demonstrate the greater potentiality of M. dioica hairy root cultures for the production of valuable phenolic compounds and for studies of their biological activity.

Elicitation Enhanced the Production of Phenolic Compounds and Biological Activities in Hairy Root Cultures of Bitter melon ( Momordica charantia L.)

ABSTRACT Momordica charantia (Cucurbitaceae) is an important vegetable and also medicinal crop which produces the bioactive compounds for various biological activities with potential uses in human health. The present investigation relates to elicitors of jasmonic acid (JA) and salicylic acid (SA) to enhance biomass accumulation and phenolic compound production in hairy root cultures of M. charantia. Hairy root cultures were elicited with JA and SA at 0, 25, 50 and 100 μM concentrations respectively. The adding of elicitation to the hairy root cultures on the 15th day of culture and the roots were harvested on day 25. Cultures supplemented with 100 μM JA and SA enhanced the phenolic compounds significantly compared to that of non-elicited hairy root cultures. The biomass of hairy root culture significantly increased by SA whereas decreased in JA elicitation at 100 μM. JA and SA-elicited hairy root cultures significantly produced a higher amount of phenolic compounds (12811.23 and 11939.37µg/g), total phenolic (4.1 and 3.7 mg/g) and flavonoid (3.5 and 3.2 mg/g) contents than non-elicited hairy root cultures (10964.25 µg/g, 2.8 and 2.5 mg/g). JA and SA-elicited hairy root cultures were significantly higher antioxidant activity of DPPH (84 and 78%), reducing potential (0.53 and 0.48), phosphomolybdenum (3.6 and 3.2 mg/g) and ferrous ion chelating assays (80 and 74%) than non-elicited hairy root cultures. The higher antimicrobial and anticancer activity were exhibited in JA and SA-elicited than non-elicited hairy root cultures. This protocol can be developed for the production of phenolic compounds from JA and SA-elicited hairy root cultures.

Evaluation of polyphenol composition and biological activities of two samples from summer and winter seasons of Ligularia fischeri var. Spiciformis Nakai.

Phenolic contents, antioxidant and antimicrobial activities were determined by two samples from summer (June) and winter (December) seasons of Ligularia fischeri var. spiciformis Nakai. A total of 24 phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) analysis. Myricetin (1964.35 and 1829.12 µg/g) was the most dominant flavonol compared to quercetin and kaempferol. Salicylic acid (222.80 and 215.25 µg/g) was the most important phenolic compound compared to pyrogallol, caffeic acid, gentisic acid, o-coumaric acid, gallic acid, protocatechuic acid and ferulic acid in summer (June) and winter (December) seasons. Phenolic contents and antioxidant capacities were estimated for the various solvent extracts (petroleum ether, butanol, ethyl acetate, methanol and water). Ethyl acetate extract exhibited the highest phenolic (332.64 and 299.44 mg/g gallic acid equivalent) and flavonoid contents (5.72 and 5.29 mg/g quercetin equivalent) and also the strongest antioxidant activity in summer and winter seasons. Due to these metabolic variations, the antioxidant and antimicrobial activities were increased with summer seasons compared to winter seasons. Our study shows that the samples collected in June had higher phenolic compounds, stronger antioxidative and antimicrobial activity than the samples of L. fischeri leaf extracts collected in December.

Growth and replication of infectious bursal disease virus in the DF-1 cell line and chicken embryo fibroblasts.

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. To determine a suitable cell line for IBDV infection, replication, and growth kinetics of the virus, DF-1 cells and chicken embryo fibroblasts (CEF) were used. The population doubling per day (Pd/D) was found to be higher in DF-1 as compared to CEF cells. A suitable time of infection (TOI) was established for increased production of virus and greater infectivity titers. The DF-1 and CEF cells were found to be susceptible to infection by producing marked cytopathic effects (CPEs), and the growth curves of IBDV in DF-1 and CEF cells were evaluated by infectivity assay using tissue culture infectious dose (TCID50). The cytopathic effects of the virus in DF-1 and CEF cells were found to be similar, but higher viral titers were detected in the DF-1 cells as compared to CEF. Thus the DF-1 cell line had a higher growth potential and infectivity, which will be of advantage in vaccine production.