Screening of Endophytic Bacteria of Leucojum aestivum 'Gravety Giant' as a Potential Source of Alkaloids and as Antagonist to Some Plant Fungal Pathogens.

Leucojum aestivum is a medicinal plant belonging to the Amaryllidaceae family well known as a producer of alkaloids such as galanthamine and lycorine. However, the endophytic microbes that colonize different plant tissues without causing any damage have not been reported in this plant. Here, we explored the different endophytic bacterial communities isolated from different surface disinfected tissues of L. aestivum 'Gravety giant' and screened bacterial isolates producing alkaloids and their potential use as biocontrol agent against wheat pathogens. For that purpose, endophytic bacteria were isolated from bulbs, roots and shoots of L. aestivum. After taxonomical characterization, these microorganisms were screened for their ability to produce alkaloids using high-performance thin-layer chromatography (HPTLC) and untargeted liquid chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) strategies. We isolated 138 bacteria belonging to four phyla and 42 genera, mainly from roots and shoots. The most abundant genera were Rahnella in shoot, Patulibacter in bulb and Bacillus in roots. Among the different bacterial isolates, the methanolic extracts of Luteibacter rhizovicinus (LaBFB3301) and Commamonas denitrificans (LaBFS2103) slightly delayed the growth of F. graminearum colonies in in vitro dual tests against F. graminearum and M. nivale strains with 15.5% and 19.9% inhibition rates, respectively. These isolates are able to produce an indolic alkaloid tryptophol (C10H11NO, [M + H]+ 162.0913). These endophytic bacteria might be investigated to characterize the plant protection effect and the plant growth promotion effect.

Aloe djiboutiensis: Antioxidant Activity, Molecular Networking-Based Approach and In Vivo Toxicity of This Endemic Species in Djibouti.

For the first time, the study of the antioxidant activity, the characterization of the phytoconstituants, and the evaluation of in vitro and in vivo toxicity of A. djiboutiensis leave and latex are performed. The antioxidant activity of both latex (ADL) and the methanolic extract of leaves (ADM) is determined using 1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) scavenging radical methods and ferric reducing/antioxidant power (FRAP) assay. The phytochemical study of latex is done using Liquid Chromatography-Mass Spectrometry (LC-MS/MS) and a molecular networking-based approach. The evaluation of in vivo toxicity is performed on mice by oral gavage with a suspension of ADL. Our results show that weak antioxidant activity of ADL and ADM in opposition to their high polyphenol, 83.01 mg and 46.4 mg expressed in gallic acid equivalent (GAE)/g of dry weight (DW), respectively, and flavonoid contents 13.12 mg and 4.25 mg expressed in quercetin equivalent (QE)/g dry weight (DW), respectively. Using the Global Natural Products Social Molecular Networking (GNPS) website, nine (9) anthraquinones derivatives, ten (10) chromones derivatives, two (2) flavonols/ chromones isomers are annotated in the molecular network. The treated mice do not display abnormalities in their general physical appearance and biochemistry parameters, compared to the controls. Only glucose and calcium levels are slightly higher in male treated mice compared to the vehicles.

Evaluation of Antiviral, Antibacterial and Antiproliferative Activities of the Endophytic Fungus Curvularia papendorfii, and Isolation of a New Polyhydroxyacid.

An endophytic fungus isolated from Vernonia amygdalina, a medicinal plant from Sudan, was taxonomically characterized as Curvularia papendorfii. Ethyl acetate crude extract of C. papendorfii revealed an important antiviral effect against two viral pathogens, the human coronavirus HCoV 229E and a norovirus surrogate, the feline coronavirus FCV F9. For the last one, 40% of the reduction of the virus-induced cytopathogenic effect at lower multiplicity of infection (MOI) 0.0001 was observed. Selective antibacterial activity was obtained against Staphylococcus sp. (312 µg/mL), and interesting antiproliferative activity with half maximal inhibitory concentration (IC50) value of 21.5 ± 5.9 µg/mL was observed against human breast carcinoma MCF7 cell line. Therefore, C. papendorfii crude extract was further investigated and fractionated. Twenty-two metabolites were identified by gas chromatography coupled to mass spectrometry (GC-MS), and two pure compounds, mannitol and a new polyhydroxyacid, called kheiric acid, were characterized. A combination of spectroscopic methods was used to elucidate the structure of the new aliphatic carboxylic acid: kheiric acid (3,7,11,15-tetrahydroxy-18-hydroxymethyl-14,16,20,22,24-pentamethyl-hexacosa-4E,8E,12E,16,18-pentaenoic acid). Kheiric acid showed an interesting result with a minimum inhibitory concentration (MIC) value of 62.5 µg/mL against meticillin-resistant Staphylococcus aureus (MRSA). Hence, endophytes associated with medicinal plants from Sudan merit more attention, as they could be a treasure of new bioactive compounds.

Evaluation of Antioxidant and Antibacterial Activities, Cytotoxicity of Acacia seyal Del Bark Extracts and Isolated Compounds.

Water extract of Acacia seyal bark is used traditionally by the population in Djibouti for its anti-infectious activity. The evaluation of in vitro antibacterial, antioxidant activities and cytotoxicity as well as chemical characterization of Acacia seyal bark water and methanolic extracts were presented. The water extract has a toxicity against the MRC-5 cells at 256 µg/mL while the methanolic extract has a weak toxicity at the same concentration. The methanolic extract has a strong antioxidant activity with half maximal inhibitory concentration (IC50) of 150 ± 2.2 µg/mL using 1-diphenyl-2-picrylhydrazyl (DPPH) and IC50 of 27 ± 1.3 µg/mL using 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical methods. For ferric reducing/antioxidant power (FRAP) assay, the result is 45.74 ± 5.96 µg Vitamin C Equivalent (VCE)/g of dry weight (DW). The precipitation of tannins from methanol crude extract decreases the MIC from 64 µg/mL to 32 µg/mL against Staphylococcus aureus and Corynebacterium urealyticum. However, the antioxidant activity is higher before tannins precipitation than after (IC50 = 150 µg/mL for methanolic crude extract and 250 µg/mL after tannins precipitation determined by DPPH method). By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, the results showed that the condensed tannins consist of two types of catechin and gallocatechin-based oligomers. The fractionation led to the identification of three pure compounds: two flavanols catechin and epicatechin; one triterpene as lupeol; and a mixture of three steroids and one fatty acid: campesterol, stigmasterol, clionasterol, and oleamide.

Carbohydrates stimulated Amaryllidaceae alkaloids biosynthesis in Leucojum aestivum L. plants cultured in RITA® bioreactor.

BACKGROUND: Leucojum aestivum L. is an important medicinal plant which produces Amaryllidaceae alkaloids, especially galanthamine and lycorine. Research is currently exploring the possibility of producing these alkaloids using biotechnological methods, including in vitro cultures. The biosynthesis of alkaloids may be affected by the types and concentrations of carbohydrate sources used in the medium. In the present investigation we performed such studies on in vitro cultures of L. aestivum with a view to obtaining plant material of good quality, characterized, in particular, by a high content of valuable Amaryllidaceae alkaloids. METHODS: We examined the effects of various types of carbohydrate sources-sucrose, glucose, fructose and maltose-at different concentrations (30, 60 and 90 g/L)-on the quality of L. aestivum plants grown in the RITA® bioreactor. The plants' quality was assessed by their biomass increments, as well by as analysing photosynthetic pigments, endogenous sugar, phenolics and Amaryllidaceae alkaloid content. We also investigated the effect of sugars on the activity of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). RESULTS: The highest biomass increments were observed in plants cultivated in the medium containing 90 g/L sucrose. The highest CAT activity was noted in cultures growing in the medium supplemented with 90 g/L maltose, while the highest POD activity was observed in the presence of 90 g/L fructose and 60 g/L maltose. No differences in SOD activity were observed. Moreover, the sugars did not affect the contents of chlorophyll a and carotenoids, whereas the highest amount of chlorophyll b was recorded in plants growing in the medium with 60 g/L maltose. No statistically significant differences were observed in the contents of endogenous sugars and phenolics in any in vitro conditions. However, the addition of sugar had a decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distribution of alkaloids occurred in plants cultured in the medium containing 60 g/L sucrose. Six Amaryllidaceae alkaloids were detected in the plant tissue. The addition of 30 g/L fructose in the medium resulted in the accumulation of five alkaloids, including ismine, which was not identified in other analysed tissues. The highest concentration of galanthamine was observed in plants cultured in the presence of 30 g/L fructose and 60 g/L sucrose (39.2 and 37.5 µg/g of dry weight (DW), respectively). The plants grown in the medium containing 60 g/L sucrose exhibited the highest lycorine content (1048 µg/g of DW). CONCLUSIONS: The type and concentration of sugar used in the medium have an essential influence on the biosynthesis of Amaryllidaceae alkaloids in L. aestivum plants cultured in a RITA® bioreactor. The results point to an interesting approach for commercial production of galanthamine and lycorine.

Screening of Amaryllidaceae alkaloids in bulbs and tissue cultures of Narcissus papyraceus and four varieties of N. tazetta.

Narcissus spp. are an economically important crop for medicines in relation with the alkaloids production, mainly galanthamine, an acetylcholinesterase inhibitor used for the treatment of Alzheimer's disease. In this article an extensively study of the phytochemistry of both bulbs of different species and varieties of Narcissus grown in Iran and in vitro culture of these plants was investigated. In particular, the Amaryllidaceae alkaloid profile and the galanthamine and lycorine contents in wild bulbs of Narcissus papyraceus (G5) and four varieties of Narcissus tazetta (N. tazetta var. Shahla (G4), N. tazetta var. Shastpar (G1), N. tazetta var. Meskin (G2), N. tazetta var. Panjehgorbei (G3)), growing in Iran are reported. The alkaloid profiles were investigated by GC-MS and LC-MS and the quantitative analysis was performed using GC-MS. In total, thirty alkaloids were identified among them nine alkaloids were observed with the both methods of analysis. The variety Meskin of N. tazetta (G2), showed the highest diversity of alkaloids and the highest content in galanthamine. On this last species (G2) and on N. tazetta var. Shahla (G4), the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram) and naphthalene acetic acid (NAA) at concentrations of 25 and 50 µM were studied on the induction of callus and its capacity to induce organogenesis and alkaloid diversity. All auxins, at the concentrations of 25 and 50 µM, produced calli. Bulblets and roots were formed on calli grown only in the presence of 25 or 50 µM NAA. GC-MS analyses showed the presence of galanthamine and lycorine in calli, roots and bulblets, with all auxins whatever the concentration used while demethylmaritidine and tazettine were found in differentiated tissue cultures cultivated on the medium containing NAA (25 or 50 µM) or in calli initiated with Picloram (50 µM). Precursor 4'-O-methylnorbelladine (MN) of Amaryllidaceae alkaloids feeding was found to significantly improve the accumulation of both galanthamine (82 µg/g DW) and lycorine (1800 µg/g DW) in bulblets of N. tazetta var. Meskin (G2).

Endophytic fungi associated with Sudanese medicinal plants show cytotoxic and antibiotic potential.

In this study, we isolated 15 endophytic fungi from five Sudanese medicinal plants. Each fungal endophytic strain was identified by sequencing of internal transcribed spacer (ITS) regions of rDNA. Ethyl acetate extracts were prepared from each endophyte cultivated in vitro and tested for their respective antibacterial activities and antiproliferative activities against human cancer cells. Antibacterial screening was carried out against two bacterial strains: Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus, by the broth dilution method. Cell viability was evaluated by the MTT procedure after exposure of MCF7 breast cancer cells and HT29 or HCT116 human colon adenocarcinoma cells to each endophytic extract. Of interest, Byssochlamys spectabilis isolated from Euphorbia prostata showed cytotoxicity (IC50 = 1.51 ± 0.2 µg mL(-1)) against MCF7 cells, but had a low effect against HT29 or HCT116 cells (IC50 > 20 µg mL(-1)). Cladosporium cladosporioides 2, isolated from Vernonia amygdalina leaves, showed antiproliferative activities against MCF7 cells (IC50 = 10.5 ± 1.5 µg mL(-1)) only. On the other hand, B. spectabilis and Alternaria sp. extract had antibacterial activities against the S. aureus strain. The findings of this work revealed that endophytic fungi associated with medicinal plants from Sudan could be considered as an attractive source of new therapeutic compounds.

A pilot study of antioxidant potential of endophytic fungi from some Sudanese medicinal plants.

OBJECTIVE: To evaluate the total phenolic content and total antioxidant capacity of ethyl acetate extracts of 21 endophytic fungi isolated from five Sudanese medicinal plants: Calotropis procera, Catharanthus roseus, Euphorbia prostrate, Vernonia amygdalina and Trigonella foenum-graecum. METHODS: Crude extracts of endophytic fungi and their host plants were tested by classical Folin-Ciocalteu colorimetric method to determine the total phenolic content, also total antioxidant capacity was estimated using 1,1-diphenyl-2-picrylhydrazyl free radical scavenging in vitro method. RESULTS: Among the endophytes, endophytic fungus Aspergillus sp. from Trigonella foenum-graecum seeds demonstrated the highest both total phenolic content in term of gallic acid equivalent [(89.9 ± 7.1) mg GAE/g] and antioxidant activity for 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay [IC50: (18.0 ± 0.1) µg/mL]. A high positive linear correlation (R(2) = 0.999 1) was found between total antioxidant capacity and total phenolic content of endophytic fungi isolated from Vernonia amygdalina. CONCLUSIONS: The present study revealed that some endophytic fungi from the five Sudanese medicinal plants could be a potential source of novel natural antioxidant compounds.