Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures.

BACKGROUND: One of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah. METHODS: The elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control. RESULTS: Elicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3'-hydroxylase (F3'H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds. CONCLUSIONS: This study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.

Agrobacterium rhizogenes mediated transformation of Ficus carica L. for the efficient production of secondary metabolites.

BACKGROUND: Ficus carica L., an ancient source of food and medicines, is rich in valuable nutritional and secondary compounds with antioxidant, antimicrobial, and anticancer effects. The present study is the first attempt to examine hairy root (HR) induction of F. carica (Sabz and Siah) by inoculating the 3-week-old shoots and leaves with different strains of Agrobacterium rhizogenes and also to investigate methyl jasmonate (MeJA) elicitation of HRs to produce a fast and high-yield production method for secondary metabolites. RESULTS: The maximum transformation rate (100%) was achieved by inoculating the shoots with Agrobacterium rhizogenes strain A7. Siah HRs elicited with 100 and 200 µmol L-1 MeJA and Sabz HRs with 100 µmol L-1 MeJA showed the highest total phenolic content. The highest flavonoid content was 3.935 mg QE g-1 DW in Siah HRs treated with 200 µmol L-1 MeJA and 2.762 mg QE g-1 DW in Sabz HRs treated with 300 µmol L-1 MeJA. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) value of HRs were affected by MeJA treatments. Methyl jasmonate elicitation also significantly enhanced the content of six phenolic acids (gallic acid, caffeic acid, chlorogenic acid, coumaric acid, rosmarinic acid, and cinnamic acid) and three flavonoids (rutin, quercetin, and apigenin). Thymol, a monoterpene phenol, was the main HR compound detected in gas chromatography mass spectrometry (GC-MS) analysis of the essential oils. CONCLUSION: Induction of HRs and elicitation of F. carica HRs by MeJA resulted in a significant increase in the production of important phenolic compounds and a significant increase in antioxidant capacity. © 2020 Society of Chemical Industry.

Anti-Leishmania major activity of Calotropis procera extract by increasing ROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions.

BACKGROUND: Leishmaniasis, caused by protozoan parasites of the genus Leishmania, is a neglected tropical disease with 700,000 to 1,000,000 global new cases annually. Adverse effects associated with expense, long-term treatment and drug resistance have made conventional therapies unfavorable, encouraging the search for alternative drugs based on plant products. In this study, the effect of Calotropis procera (Asclepiadaceae) extract against viability of promastigotes and amastigotes of Leishmania major was evaluated in vitro. METHODS: The extract from the leaves of C. procera seedlings was prepared using a methanol maceration method. The colorimetric cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth-inhibitory effect of the extract on promastigotes. The level of reactive oxygen species (ROS) in promastigote cultures was determined after treatment with the extract using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method and compared with untreated cultures (control). After exposure to the extract the expression levels of tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) genes were determined and compared to control in peripheral blood mononuclear cells (PBMCs) infected with L. major. RESULTS: Based on the MTT assay, the C. procera extract significantly reduced the proliferation of L. major promastigotes with IC50 values of 377.28 and 222.44 µg/mL for 24 and 72 h, respectively (p < 0.01). After treatment with 222.44 and 377.28 µg/mL of C. procera extract, ROS production in L. major promastigote cultures increased 1.2- to 1.65-fold and 2- to 4-fold compared to the control, respectively (p < 0.05). C. procera extract induced significant increases in gene expression of TNF-α (2.76-14.83 fold), IFN-γ (25.63-threefold) and iNOS (16.32-3.97 fold) in infected PBMCs compared to control (p < 0.01). CONCLUSIONS: On the basis of its anti-leishmanial activity, C. procera can be considered as a promising new plant source for the potential treatment of leishmaniasis.

The Effect of Curcumin on the Expression of INFγ, TNF-α, and iNOS Genes in PBMCs Infected with Leishmania major [MRHO/IR/75/ER].

BACKGROUND: Leishmaniasis, caused by the Leishmania parasite, is one of the most important tropical neglected diseases. The urgent search for effective, inexpensive, and preferably herbal anti-leishmanial agents, is needed. OBJECTIVE: Curcumin is a natural polyphenolic compound derived from turmeric that is well known for its antioxidant, anti-inflammatory, anti-tumor, and anti-cancer activity. METHODS: The present work evaluates the anti-leishmanial [Leishmania major] activity of curcumin. The infected PBMCs were treated with curcumin. The ROS level at 6, 12, 24 h and gene expression levels at 24, 48, and 72 h of PBMCs after treatment with curcumin were determined. RESULTS: Based on the results, the curcumin concentrations of 268 µM [24 h] and 181.2 µM [72 h] were defined as IC50 against L. major promastigotes. Treatment of L. major infected-peripheral blood mononuclear cells [PBMCs] with IC50 concentrations of curcumin, depending on exposure time, significantly induced the reactive oxygen species [ROS] generation and increased the expression levels of interferongamma [IFN-γ], tumor necrosis factor-alpha [TNF-α], and nitric oxide synthase [iNOS] genes. CONCLUSION: These findings suggest the potential of curcumin against Leishmaniasis.

Piriformospora indica based elicitation for overproduction of phenolic compounds by hairy root cultures of Ficus carica.

Ficus carica L. is an important source of phenolic and flavonoid compounds with valuable pharmaceutical application across various diseases. The current study was carried out to investigate the influence of Piriformospora indica elicitation on growth, production of phenolic compounds, antioxidant capacity, and expression level of flavonoid biosynthetic pathway genes in hairy root (HR) cultures of F. carica. The maximum improvement in accumulation of phenolic compounds was observed when HR culture of Ficus carica L. was exposed to 2% culture filtrate of P. indica for 72 h: gallic acid (80.5- fold), caffeic acid (26.2-fold), coumaric acid (4.5-fold), and cinnamic acid (60.1-fold), apigenin (27.6-fold) and rutin (5.7-fold). While the highest levels of chlorogenic acid (4.9-fold) and quercetin flavonoid (8.8-fold) were obtained after 48 h elicitation with culture filtrate and cell extract of P. indica at 6% (v/v), respectively. The analysis of biosynthetic genes revealed that the exposure to fungal elicitors resulted in up-regulation of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT) and MYB3 transcription factor. This study shows the potential of P. indica as an efficacious elicitor for enhancing the secondary metabolites production by F. carica HRs.