Thymol-enriched extract from Thymus vulgaris L leaves: Green extraction processes and antiaggregant effects on human platelets.

This work focuses on the selection and the optimization of an efficient green-extraction method, used to recover a thymol-enriched extract from thyme (Thymus vulgaris L), as well as the evaluation of the inhibitory effect of this latter on the human platelet aggregation. Different innovative extraction techniques, namely bead milling extraction, ultrasound and microwave assisted extraction, were tested for their ability to recover a high added value extract from thyme. Among all tested eco-extraction techniques, microwave extraction (MAE) was the best method in term of its extraction yield (20.84% ± 0.51), thymol concentration (731.71 mg/g) and total phenolic (23.53 ± 1.83 mg (GAE)/g of extract) and flavonoid (6.22 ± 0.35 mg of QE/g of extract) contents. Moreover, thyme extract obtained by microwave assisted extraction (TMAE) showed the most active antioxidant effect comparing to the other tested extracts. Based on these results, TMAE was chosen to be evaluated for its antiplatelet effect. Thereby, arachidonic acid, collagen and ADP were used to induce the platelet aggregation on human platelet rich plasma taken from healthy controls and results revealed that TMAE strongly inhibited the induced platelet aggregation. Indeed, TMAE exhibited potent antiaggregant activity by inhibiting platelet activation, secretion and aggregation. Additionally, cytotoxicity assay on normal HEK-293 cells showed that TMAE has no cytotoxic effect even at high concentration (8 mg/ml) and can further be taken up to various biomedical applications mainly in the prevention of cardiovascular diseases.

Biocontrol and plant growth-promoting potentiality of bacteria isolated from compost extract.

The use of compost extracts is steadily increasing, offering an attractive way for plant growth enhancement and disease management replacing chemical pesticides. In this study, potential mechanisms involved in plant growth promotion and suppressive activity against fungal diseases, of a compost extract produced from poultry manure/olive husk compost, were investigated. Results of physico-chemical and microbiological investigations showed high ability to reduce Fusarium oxysporum, Alternaria alternata, Aspergillus niger and Botrytis cinerea growth. The suppressive ability detected using confrontation test and the phytostimulatory effect tested on tomato seeds were related mainly to its microbial population content. Among 150 bacterial strains, isolated from the compost extract, 13 isolates showed antifungal activity against the four tested plant pathogenic fungi. Their identification based on 16S rRNA gene sequence revealed they belonged to different species of the genus Bacillus, Alcaligenes, Providencia and Ochrobactrum. When tested for their ability to produce cell wall degradation enzymes using specific media, the majority of the 13 isolates were shown to synthesize proteases, lipases and glucanases. Similarly, the best part of them showed positive reaction for plant growth promoting substances liberation, biosurfactant production and biofilm formation. In vivo tests were carried out using tomato seeds and fruits and proved that 92% of strains improved tomato plants vigor indexes when compared to the control and 6 among them were able to reduce decay severity caused by B. cinerea over 50%. Principal component analysis showed an important correlation between in vitro and in vivo potentialities and that Bacillus siamensis CEBZ11 strain was statistically the most effective strain in protecting tomato plants from gray mould disease. This study revealed the selected strains would be useful for plant pathogenic fungi control and plant growth promotion.

Emex spinosa (L.) Campd. ethyl acetate fractions effects on inflammation and oxidative stress markers in carrageenan induced paw oedema in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Emex spinosa (L.) Campd. (E. spinosa) locally known as "hillaioua" has always been used in folk medicine for the treatment of inflammation and pain. It is still being exploited by pharmaceutical companies for its potential remedial effects. AIM OF THE STUDY: In this study, the effects of E. spinosa (L.) Campd. against acute inflammation, pain and oxidative damage were evaluated. MATERIALS AND METHODS: Total phenols and flavonoids were evaluated. Anti-inflammatory and analgesic activities the E. spinosa ethyl acetate fractions of the aerial (Es EtOAc-AP) and underground (Es EtOAc-R) parts were assessed on carrageenan-induced paw oedema (100 mg/kg BW) and acetic acid-induced writhing response (50, 100 and 150 mg/kg BW), respectively. The E. spinosa fractions effects on oxidative stress markers and inflammatory parameters were determined. Gas chromatography-mass spectrometry (GC-MS) analysis was performed to identify various chemical components. RESULTS: The ethyl acetate fractions were shown to be the most active thanks to their phenolic and flavonoid contents richness. Intraperitoneal administration of E. spinosa ethyl acetate fractions at 100 mg/kg BW, one hour before carrageenan injection, significantly inhibited the oedema formation by 89.31% and 97.7% for the aerial and underground parts respectively when compared to the reference drug "dexamethasone" (51.9%). Besides, a significant increase (p ≤ 0.001) of the dermal antioxidant enzymes (the superoxide dismutase (SOD)), catalase (CAT) and glutathione peroxidase (GPx) was observed five hours after carrageenan administration. The best restoration was obtained with Es EtOAc-R (82.04%, 93.55% and 93.55% respectively for SOD, CAT and GPx activities). Moreover, EtOAc-fractions treated mice proved their ability to restore both of CRP and fibrinogen (p < 0.001). In addition, E. spinosa EtOAc-fractions attenuated abdominal contractions (p < 0.05) by 71.69% and 82.41% for the aerial part and roots respectively at 150 mg/kg BW against 100% for dichlofenac sodium used as standard drug. The phytochemical analysis of Es EtOAc-AP and Es EtOAc-R by GC-MS may explain the obtained results. The analysis of the fractions demonstrated the presence of palmitic and linoleic acids known for their anti-inflammatory and analgesic capacities. CONCLUSIONS: These findings explain the traditional use of E. spinosa in folk medicine and suggest that E. spinosa fractions could be a promising herbal drug.

Combinational Effect of Rumex tingitanus (Polygonaceae) Hexane Extract and Bacillus thuringiensis δ-Endotoxin against Spodoptera littoralis (Lepidoptera: Noctuidae).

The increasing insect resistance against Bacillus thuringiensis delta-endotoxins is a serious problem which makes it urgent to look for new eco-friendly strategies. Combining these toxins with other biomolecules is one of the promising strategies against insect pests. In this work, we evaluated the bioinsecticidal potential of Rumex tingitanus extracts and B. thuringiensis strain BLB250 against Spodoptera littoralis (Lepidoptera: Noctuidae) larvae. The chemical composition of the hexane extract, the most active fraction, was analyzed to validate the correlation between chemical composition and biological activity. Among the tested extracts, only the hexanic extract showed toxicity against first and second instar larvae with LC50 of 2.56 and 2.95 mg g-1, respectively. The Bacillus thuringiensis BLB250 delta-endotoxins showed toxicity with an LC50 of 56.3 µg g-1. Therefore, the investigated combinational effect of BLB250 delta-endotoxins and R. tingitanus hexane extract proved significant synergistic effect against S. littoralis larvae. The GC-MS analysis of R. tingitanus hexane extract showed the richness of this extract in phytosterols such as ß and γ-sitosterol (48.91%), campesterol (6.43%), and ß-amyrin (8.92%) which are known for their insecticidal activity. This novel finding highlights the potential use of this combination against insect pests to prevent the appearance of resistance problems.

Effects of Juniperus phoenicea Hydroalcoholic Extract on Inflammatory Mediators and Oxidative Stress Markers in Carrageenan-Induced Paw Oedema in Mice.

Juniperus phoenicea (J. phoenicea) is a wild tree belonging to the Cupressaceae family, commonly used for the treatment of several disorders. This study aimed to evaluate the potential protective effects of J. phoenicea hydroethanolic extract (EtOH-H2OE) against oxidation, acute inflammation, and pain in mice models. For the purpose, chemical compounds of J. phoenicea EtOH-H2OE were also analyzed by GC-MS. The J. phoenicea EtOH-H2OE showed a potent antioxidant activity in vitro, thanks to its richness in phenolic and flavonoid compounds. Mice treated with EtOH-H2OE (100 mg/kg BW) showed reduced paw oedema formation and decreased malondialdehyde (MDA) content. The evaluation of antioxidant enzyme activities in paw oedema tissue after five hours of carrageenan induction showed a significant increase (P < 0.05). Inflammatory biomarkers explorations of J. phoenicea EtOH-H2OE-treated mice showed a restoration of the studied parameters to near-normal values. Furthermore, EtOH-H2OE of J. phoenicea produced a significant reduction of the number of abdominal writhes (P < 0.05) in a dose-dependent way. Phytochemical analysis of the J. phoenicea EtOH-H2OE by GC-MS showed the presence of hexadecanoic and stearic acids known as anti-inflammatory and analgesic compounds. Our investigation provided evidence that J. phoenicea EtOH-H2OE can effectively reduce the inflammation and pain in mice models.

Antioxidant, Hepatoprotective, and Antidepression Effects of Rumex tingitanus Extracts and Identification of a Novel Bioactive Compound.

Over the last few decades, Rumex species have been recognized as a promising source of new compounds with numerous pharmacological activities. Therefore, the antioxidant activity of Rumex tingitanus (R. tingitanus) leaves extracts was evaluated in vitro and then confirmed in vivo as well as the antidepressant-like and toxicological effects of the extracts. The ethyl acetate fraction (Rt EtOAcF) followed by hydroalcoholic extract (Rt EtOH-H2O) showed a remarkable in vitro antioxidant activity. The hydroalcoholic extract (Rt EtOH-H2O) showed significant hepatoprotective activity against carbon tetrachloride- (CCl4-) induced liver toxicity which is seen from inhibition of the malondialdehyde (MDA) accumulation and enhancement of the liver antioxidant enzymes activities. The Rt EtOH-H2O and Rt EtOAcF extracts were able to reduce the immobility time in mice and then elicited a significant antidepressant-like effect. The ethyl acetate fraction (Rt EtOAcF) was purified and resulted in the identification of a new antioxidant component called 4'-p-acetylcoumaroyl luteolin. The Rt EtOAcF and the 4'-p-acetylcoumaroyl luteolin revealed a strong antioxidant activity using DPPH test with IC50 of 11.7 ± 0.2 and 20.74 ± 0.6 µg/ml, respectively, and AAI of 3.39 and 1.92 better than that of BHT, used as control.

Exploring the Urtica dioica Leaves Hemostatic and Wound-Healing Potential.

The present paper investigated the efficiency of Urtica dioica (U. dioica) on hemostatic and wound healing activities. U. dioica leaf extracts were evaluated for their antibacterial and antioxidant effects as well as their flavonoid and polyphenol content. The hydroethanolic extract (EtOH-H2OE), showing the most potent antibacterial and antioxidant activities in vitro, thanks to its flavonoid and polyphenol richness, was selected for hemostatic and wound healing evaluation. Twenty-four rats completing full-thickness wounds were split into four groups. The wounds were topically treated with saline solution, glycerol, "CICAFLORA," and U. dioica EtOH-H2OE (50 µL/mm2) until day 11. The wound healing effect was assessed by macroscopic, histological, and biochemical parameters. Rats treated with EtOH-H2OE showed fast wound closure (92.39%) compared to the control animals (60.91%) on the 11th day of wounding (P < 0.01). Histopathological and biochemical explorations showed full epidermal regeneration and an improvement of the hydroxyproline content in the U. dioica EtOH-H2OE treated rats. Analysis of fatty acids and sterols by GC-MS showed the presence of unsaturated fatty acids and a high concentration of lupeol known for their involvement in reepithelialization. These results prove the efficiency of U. dioica EtOH-H2OE in wound healing and supported its traditional use.

Antimicrobial activity and bioguided fractionation of Rumex tingitanus extracts for meat preservation.

This study was undertaken to investigate the antibacterial and antifungal activities of Rumex tingitanus leaves extracts as well as the identification of bioactive components and their performance in meat preservation. Total phenolics and flavonoids showed the highest content of phenolics and flavonoids in the ethyl acetate fraction (Rt EtOAcF). For antimicrobial efficacy, leaves extract and derived fraction were tested for their capacity to inhibit bacterial and fungal proliferation in vitro and in vivo. The ethyl acetate fraction showed the most potent antibacterial and antifungal activities compared to the others extracts. Thus, the efficacy of this extract to inhibit the proliferation of Listeria monocytogenes in minced beef meat model was examined. This fraction eradicates the L. monocytogenes population in meat in a concentration- and time-dependent manner. A bio-guided purification of the Rt EtOAc fraction resulted in the isolation of the compound responsible for the observed antimicrobial activity. This compound was identified as luteolin by analysis of spectroscopic data. CHEMICAL COMPOUNDS ISOLATED IN THIS ARTICLE: Luteolin (PubChem CID: 5280445); p-iodonitrotetrazolium chloride (PubChem CID: 64957); Amphotericin B (PubChem CID: 5280965); Gentamicin and (PubChem CID: 6419933); Hexane (PubChem CID: 8058); Methanol (PubChem CID: 887); Ethanol (PubChem CID: 702); Dimethylsulfoxide (PubChem CID: 679); Quercetin (PubChem CID: 5280343); Gallic acid (PubChem CID: 370).

Antagonist effects of Bacillus spp. strains against Fusarium graminearum for protection of durum wheat (Triticum turgidum L. subsp. durum).

Bacillus species are attractive due to their potential use in the biological control of fungal diseases. Bacillus amyloliquefaciens strain BLB369, Bacillus subtilis strain BLB277, and Paenibacillus polymyxa strain BLB267 were isolated and identified using biochemical and molecular (16S rDNA, gyrA, and rpoB) approaches. They could produce, respectively, (iturin and surfactin), (surfactin and fengycin), and (fusaricidin and polymyxin) exhibiting broad spectrum against several phytopathogenic fungi. In vivo examination of wheat seed germination, plant height, phenolic compounds, chlorophyll, and carotenoid contents proved the efficiency of the bacterial cells and the secreted antagonist activities to protect Tunisian durum wheat (Triticum turgidum L. subsp. durum) cultivar Om Rabiia against F. graminearum fungus. Application of single bacterial culture medium, particularly that of B. amyloliquefaciens, showed better protection than combinations of various culture media. The tertiary combination of B. amyloliquefaciens, B. subtilis, and P. polymyxa bacterial cells led to the highest protection rate which could be due to strains synergistic or complementary effects. Hence, combination of compatible biocontrol agents could be a strategic approach to control plant diseases.

Toxin stability improvement and toxicity increase against dipteran and lepidopteran larvae of Bacillus thuringiensis crystal protein Cry2Aa.

BACKGROUND: Bacillus thuringiensis δ-endotoxins are the most widely used biopesticides for controlling economically important crop pests and disease vectors. Improving their efficacy is of great benefit. Here, an improvement in Cry2Aa δ-endotoxin toxicity was attempted via a cry gene over expression system using P20 from B. thuringiensis israelensis. RESULTS: The coexpression of Cry2Aa with P20 resulted in a seven fold increase in its production yield in B. thuringiensis. Generated crystals proved to be significantly more toxic (505.207 µg g-1 , 1.99 mg L-1 and 1.49 mg L-1 ) than the P20-lacking control (720.78 µg g-1 , 705.69 mg L-1 and 508.51 mg L-1 ) against Ephestia kuehniella, Aedes aegypti and Culex pipiens larvae respectively. In vitro, processing experiments revealed a P20-mediated protection of Cry2Aa against degradation under larval gut conditions. Thus, P20 could promote the maintenance of a tightly packaged conformation of Cry2Aa toxins in the larval midgut upon correct activation and binding to its membrane receptors. CONCLUSION: Based on their resistance against excessive proteolysis, Cry2Aa δ-endotoxins, produced in the presence of P20, could be considered as a successful control agent for E. kuehniella and an effective alternative for mosquito control, implying its possible exploitation in pest management programmes. © 2016 Society of Chemical Industry.

Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato.

The aim of this study is to evaluate the efficacy of the strain Bacillus subtilis V26, a local isolate from the Tunisian soil, to control potato black scurf caused by Rhizoctonia solani. The in vitro antifungal activity of V26 significantly inhibited R. solani growth compared to the untreated control. Microscopic observations revealed that V26 caused considerable morphological deformations of the fungal hyphae such as vacuolation, protoplast leakage and mycelia crack. The most effective control was achieved when strain V26 was applied 24h prior to inoculation (protective activity) in potato slices. The antagonistic bacterium V26 induced significant suppression of root canker and black scurf tuber colonization compared to untreated controls with a decrease in incidence disease of 63% and 81%, respectively, and promoted plant growth under greenhouse conditions on potato plants. Therefore, B. subtilis V26 has a great potential to be commercialized as a biocontrol agent against R. solani on potato crops.

Histopathological and combinatorial effects of the metalloprotease InhA1 and Cry proteins of Bacillus thuringiensis against Spodoptera littoralis.

The zinc metalloprotease (InhA) of Bacillus thuringiensis specifically hydrolyzes cecropins and attacins, two antibacterial peptides in the immune hemolymph of insects, leading to a high resistance of the bacteria to the humoral defense system of its host. In the present study, the inhA gene of B. thuringiensis strain BUPM28 was cloned and the nucleotide sequence analysis revealed that it was identical to that of B. thuringiensis 8010. The expressed InhA1 protein in Escherichia coli showed toxicity to neonate Spodoptera littoralis larvae with a LC50 of 2.07±0.72µg/cm(2). Study of the effect of combining Cry proteins with InhA1 showed that one improves the toxicity of the other one against S. littoralis. Investigation of the histopathological effect of this metalloprotease showed an extensive damage of S. littoralis epithelium tissue. These results provide an insight to the use of InhA as supplement to Cry toxins to improve the efficacy of B. thuringiensis formulations and to overcome possible resistance problems.