Wild mushrooms and their mycelia as sources of bioactive compounds: antioxidant, anti-inflammatory and cytotoxic properties

Mushrooms are an important source of natural compounds with acknowledged bioactivity. Pleurotus eryngii (DC.) Quél., in particular, is widely recognized for its organoleptic quality and favorable health effects, being commercially produced in great extent. On the other hand, Suillus bellinii (Inzenga) Watling is an ectomycorrhizal symbiont, whose main properties were only reported in a scarce number of publications. Some current trends point toward using the mycelia and the culture media as potential sources of bioactive compounds, in addition to the fruiting bodies. Accordingly, P. eryngii and S. bellinii were studied for their composition in phenolic acids and sterols, antioxidant capacity (scavenging DPPH radicals, reducing power, β-carotene bleaching inhibition and TBARS formation inhibition), anti-inflammatory effect (by down-regulating LPS-stimulated NO in RAW264.7 cells) and anti-proliferative activity (using MCF-7, NCI-H460, HeLa, HepG2 and PLP2 cell lines). Overall, S. bellinii mycelia showed higher contents of ergosterol and phenolic compounds (which were also detected in higher quantity in its fruiting body) and stronger antioxidant activity than P. eryngii. On the other hand, P. eryngii mycelia showed anti-inflammatory (absent in S. bellinii mycelia) and a cytotoxicity similar (sometimes superior) to its fruiting bodies, in opposition to S. bellinii, whose mycelia presented a decreased anti-proliferative activity. Furthermore, the assayed species showed differences in the growth rate and yielded biomass of their mycelia, which should also be considered in further applications.

HPLC-DAD-ESI/MS phenolic characterization and biological activity of Equisetum giganteum L.

Naturally-occurring phytochemicals have received a pivotal attention in the last years, due to the increasing evidences of biological activities. Equisetum giganteum L., commonly known as “giant horsetail”, is a native plant from Central and South America, being largely used in dietary supplements as diuretic, hemostatic, antiinflammatory and anti-rheumatic agents [1,2]. The aim of the present study was to evaluate the antioxidant (scavenging effects on 2,2-diphenyl-1-picrylhydrazyl radicals- RSA, reducing power- RP, β-carotene bleaching inhibition- CBI and lipid peroxidation inhibition- LPI), anti-inflammatory (inhibition of NO production in lipopolysaccharidestimulated RAW 264.7 macrophages) and cytotoxic (in a panel of four human tumor cell lines: MCF-7- breast adenocarcinoma, NCI-H460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma; and in non-tumor porcine liver primary cells- PLP2) properties of E. giganteum, providing a phytochemical characterization of its extract (ethanol/water, 80:20, v/v), by using highperformance liquid chromatography coupled to diode array detection and electrospray ionisation mass spectrometry (HPLC-DAD–ESI/MS). E. giganteum presented fourteen phenolic compounds, two phenolic acids and twelve flavonol glycoside derivatives, mainly kaempferol derivatives, accounting to 81% of the total phenolic content, being kaempferol-O-glucoside-O-rutinoside, the most abundant molecule (7.6 mg/g extract). The extract exhibited antioxidant (EC50 values = 123, 136, 202 and 57.4 μg/mL for RSA, RP, CBI and LPI, respectively), anti-inflammatory (EC50 value = 239 μg/mL) and cytotoxic (GI50 values = 250, 258, 268 and 239 μg/mL for MCF-7, NCI-H460, HeLa and HepG2, respectively) properties, which were positively correlated with its concentration in phenolic compounds. Furthermore, up to 400 μg/mL, it did not revealed toxicity in non-tumor liver cells. Thus, this study highlights the potential of E. giganteum extracts as rich sources of phenolic compounds that can be used in the food, pharmaceutical and cosmetic fields.

Efeito anti-inflamatório do heparinóide isolado do camarão Litopenaeus vannamei sobre a peritonite aguda

In recent years the heparin has been the subject of several studies that aim to expand its use as a therapeutic agent, due to its ability to modulate the activity of various proteins that play important roles in the regulation of pathophysiological processes. In several experiments and preclinical trials, heparin has demonstrated an anti-inflammatory role. However, its clinical use is limited, due to its strong anticoagulant activity and hemorrhagic complications. For this reason, considerable efforts have been employed in discovery of heparin analogous (heparinoid) with reduced side effects, that retain the anti-inflammatory properties of heparin. In this context, a heparinoid obtained from the head of Litopenaeus vannamei shrimp, which presents a structural similarity to heparin, showed, in previous studies, anti-inflammatory activity in a model of acute peritonitis with reduced anticoagulant effect in vitro and low hemorrhagic activity. Thus, the present work had as objective to evaluate the effect the heparinoid of the cephalothorax of gray shrimp on the acute inflammatory response in different times (3 or 6 hours after the induction of inflammatory stimulus), using the model of acute peritonitis induced in mice. It was also analyzed the HL effect over the activity of elastase, an enzyme involved in leukocyte recruitment. Furthermore to check if the different doses of heparin and heparinoid change the hemostatic balance in vivo, was assessed the effect of these compounds on the plasma clotting time in animals submitted to inflammation. The results show that in 3 hours, all doses of heparinoid were able to prevent efficiently in the acute inflammatory process without any anticoagulant effects, unlike the extrapolation dose of heparin, which has induced a large hemorrhage due its high anticoagulant activity. However, 6 hours after induction of inflammation, only the dosages of 0.1 and 1.0 μg/Kg of heparin and 1.0 μg/Kg of heparinoid kept anti-migratory effect, without changing of the hemostatic balance. These results indicate that the anti-migratory effect of theses compounds depends on the dosage and time of inflammatory stimulus. The HL and heparin were also able to inhibit the activity of the enzyme elastase. The discovery of this bioactive compound in the cephalothorax of shrimps can arouse great interest in biotechnology, since this compound could be useful as a structural model interesting for the development of new therapeutic agents for peritonitis

Anti-nociceptive and anti-inflammatory potentials of fractions from the extract of Tetracarpidium conophorum leaf in rats and mice

Tetracarpidium conophorum (TC) (Euphorbiaceae) is a perennial woody climbing shrub in low bush forest of some parts of West Africa and used among the natives for relief of ailments accompanying pain and inflammation. In this study, the analgesic and anti-inflammatory effects of the methanolic extract (METC) and fractions (ethyl acetate, F1 and n-hexane, F2) of Tetracarpidium conophorum leaf were evaluated in rat and mice. The analgesic activity was evaluated using acetic acid-induced writhing, formalin-induced paw licking and hot plate test models. Carrageenan-induced paw oedema was used to assess anti-inflammatory activity in rats. The mechanism of action of (TC) was explored by the use of naloxone, a non-selective opioid receptor blocker. The highest analgesic effect was observed in F2 extract at 57.21% inhibition and was further studied on various analgesic and anti-inflammatory models in graded doses. F2 significantly inhibited the late phase of formalin-induced paw licking and prolong hot plate latency at 55±1°C. The n-hexane fraction also significantly inhibited carrageenan-induced paw oedema in rats at 100 and 200mg/kg doses significantly (p< 0.001) and reduced paw licking response by 85.08% compared with control. Naloxone, an opioid receptor antagonist, did not significantly affect the changes observed with n-hexane fraction, thus ruling out the possibility of the involvement of opioid receptors in the analgesic actions of Tetracarpidium conophorum. Phytochemical screening showed that the leaf extracts contain alkaloids, tannins, saponins and cardenolides. The investigations showed that Tetracarpidium conophorum possesses significant anti-nociceptive and anti-inflammatory activities that should be explored.

EVALUATION OF THE ANTI-FUNGAL PROPERTIES OF EXTRACTS OF Daniella oliveri

The in vitro anti-fungal activity of leaf and stem bark of Daniella oliveri Rolfe was investigated against selected yeasts and moulds including dermatophytes. Water and methanol were used to extract the powdered leaf and stem bark using cold infusion. Antimicrobial activity was assessed by agar-well diffusion. Phytochemical analysis was carried out using standard procedures. The plant extracts were active against the test organisms at concentrations ranging from 3.125-100 mg/mL. The methanol extracts were more active than the aqueous extracts with the highest inhibition against the yeasts, Candida albicans and Candida krusei (MIC values of 3.125 mg/mL and 6.25 mg/mL respectively). Epidermophyton floccosum and Trichophyton interdigitale were the least inhibited of all the fungal strains. Phytochemical screening revealed the presence of tannins, anthraquinones, flavonoids, cardiac glycosides, alkaloids and saponins. The anti-fungal activity of Daniella oliveri as shown in this study indicates that the plant has the potential of utilisation in the development of chemotherapeutic agents for the treatment of relevant fungal infections.

Anti-inflammatory potential of mushroom extracts and isolated metabolites

Background: In the recent years natural resources are being in focus due to their great potential to be exploited in the discovery/development of novel bioactive compounds and, among them, mushrooms can be highlighted as alternative sources of anti-inflammatory agents. Scope and approach: The present review reports the anti-inflammatory activity of mushroom extracts and of their bioactive metabolites involved in this bioactive action. Additionally the most common assays used to evaluate mushrooms anti-inflammatory activity were also reviewed, including in vitro studies in cell lines, as well as in animal models in vivo. Key findings and conclusions: The anti-inflammatory compounds identified in mushrooms include polysaccharides, terpenes, phenolic acids, steroids, fatty acids and other metabolites. Among them, polysaccharides, terpenoids and phenolic compounds seem to be the most important contributors to the anti-inflammatory activity of mushrooms as demonstrated by numerous studies. However, clinical trials need to be conducted in order to confirm the effectiveness of some of these mushroom compounds namely, inhibitors of NF-κB pathway and of cyclooxygenase related with the expression of many inflammatory mediators.

Biological activity of Pyrrole-Imidazole polyamides in vivo

This thesis focuses on biological activity of pyrrole-imidazole polyamides in vivo. The work presented includes experiments underlining sequence selectivity of these compounds in living cells and potential methods to improve it. A large fraction of this thesis is devoted to activity of Py-Im in murine models of cancer. We investigated the pharmacokinetics and biodistribution of two compounds – targeted to 5'-WGGWCW-3' and 5'-WTWCGW-3' sequences – and characterized their activity by measuring their effects on tumor growth, gene expression in vivo and in tissue culture, and their effects on physiology of tumors. The initial theoretical studies suggested that a large fraction of genomic sites are bound by Py-Im polyamides non-specifically and experimental data shows that the programmed binding sequence is not a sole determinant of the patterns of gene regulation. Despite the likely presence of non-specific effects of Py-Im polyamides in living cells, in vivo administration of Py-Im polyamides resulted in tolerable host toxicity and anti-tumor activity. Py-Im polyamide targeted to Estrogen Receptor Response Element showed downregulation of ER-driven gene expression in tumor cells, while the compound targeted to hypoxia response element reduced vascularization of tumors and their growth rate, induced apoptosis of cells in hypoxic areas and reduced expression of proangiogenic and prometastatic factors. Further studies, showed that polyamides distributed to many of the tested tissues and their FITC-conjugates showed nuclear uptake. The gene expression effects were also present in murine tissues, such as liver and kidneys, indicating a potential for use for Py-Im polyamides in non-cancerous diseases.

Evaluation of the antioxidant activity and antitumor activity of marine invertebrates extracts

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2014

Bioprospection of Petit Verdot grape pomace as a source of anti-inflammatory compounds.

The aim of this study was to evaluate the anti-inflammatory activity of Petit Verdot Extract and hexane, chloroform and ethyl acetate fractions obtained from grape pomace, in addition to identifying active compounds. The PVE and EAF reduced significantly paw edema and neutrophil migration when compared with control groups. The PVE reduced levels of TNF-α and IL1-β in the peritoneal fluid, whereas the EAF did not reduce cytokines significantly. Two hydroxybenzoic acids, two proanthocyanidins, three flavan-3-ol monomers and three anthocyanins were identified in the PVE and EAF by LC-MS/MS. The stilbene transresveratrol was identified only in the EAF. The phenolic compounds were quantified using HPLC-DAD analysis, except for the stilbenes, which were not sensible for the detection by the method. Since PVE and EAF showed significantly anti-inflammatory effects and high concentration of phenolic compounds, we concluded that Petit Verdot pomace could be an interesting source of anti-inflammatory bioactives.

Anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum (Linn)

Purpose: To evaluate the anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum and determine its relevance in the treatment of vibrios infection. Methods: The agar-well diffusion method was used for screening the extracts for their anti-vibrio activity. Broth micro-dilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts. Time-kill assay was used to assess bactericidal and/or bacteriostatic activity. Results: The acetone extract showed activity against 47.5 % (19/40) of the test bacteria, while the aqueous extract had activity against 30 % (12/40). MIC and MBC values range for the acetone extract were 0.625 – 5.0 mg/mL and 2.5 – 10 mg/mL respectively. The range of MIC exhibited by the antibiotic (gentamicin) against the vibrios is 0.002 mg/mL and >0.256 mg/mL. Significant reduction in the bacterial density was at 2 × MIC after a 4 h interaction period, while bacterial density after 6 and 8 h interactions with extract was highly bactericidal. Growth inhibition and efficacy of the crude acetone extract were observed to be both concentration- and time-dependent. Conclusion: The bacteriostatic and bactericidal activities observed for Ocimum gratissimum leaf suggest that the plant is a potential source of bioactive components that may be effective in the treatment of vibrios infections.

Cytotoxic, anti-inflammatory and antioxidant activities of four different extracts of Galega officinalis L (Goat’s rue)

Purpose: To evaluate the cytotoxic, anti-inflammatory and antioxidant activities of four different solvent extracts obtained from the aerial parts of Galega officinalis L Methods: The hexane, DCM, methanol and water extracts of G. officinalis were successively obtained by soxhlet extraction method. The cytotoxic activity of the extracts was assessed against human lung carcinoma (A-549), human colorectal adenocarcinoma (HT-29), human brain glioblastoma (U-87), and colon adenocarcinoma (DLD-1) by Resazurine test. The antioxidant activity of extracts were determined by Folin-Ciocalteau, oxygen radical absorbing capacity (ORAC), and 2’.7’-dichlorofluorescin-diacetate (DCFH-DA) cell-based assay while their anti-inflammatory activity was determined by nitric oxide (NO) assay. Results: DCM extract showed strong cytotoxic activity against lung adenocarcinoma and brain glioblastoma cell lines, with IC50 (concentration inhibiting 50 % of cell growth) values of 11 ± 0.4 and 16 ± 3 μg/mL, respectively. The hexane extract showed moderate anticancer activity against the same cell lines (59 ± 13 and 63 ± 16 μg/mL, respectively). DCM extract also showed significant anti-inflammatory activity, inhibiting NO release by 86.7 % at 40 μg/mL in lipopolysaccharide (LPS) - stimulated murine RAW 264.7 macrophages. Of all test extracts, the methanol extract of G. officinalis showed the highest antioxidant activity with 2.33 ± 0.09 μmol Trolox/mg , 7.10 ± 0.9 g tannic acid equivalent (TAE), and IC50 of 44 ± 4 μg/mL. Conclusion: The findings of this study suggest that DCM extract may possess anticancer effect against lung adenocarcinoma and brain glioblastoma, as well as serve as an anti-inflammatory agent.

Evaluation of a root extract gel from Urtica dioica (Urticaceae) as analgesic and anti-inflammatory therapy in rheumatoid arthritis in mice

Purpose: To develop and characterize an herbal gel prepared from methanol root extract of Urtica dioica (Urticaceae) (Stinging nettle) for the treatment of arthritis in mice. Methods: A methanol root extract from Urtica dioica was prepared, and a gel was then prepared using Carbopol 934. The prepared gel was subjected to various physical tests (color, appearance, pH, texture, viscosity) and in vivo evaluation, including primary skin irritation, analgesic, and anti-inflammatory tests, in arthritic mice and compared with 2 % indomethacin gel, which was used as standard. Results: The prepared herbal gel was of light gray color with a smooth texture. It showed a pH of 7.1 and a viscosity of 21.2 cps. The gel exhibited pseudoplastic rheology, as evidenced by shear thinning with increased shear rate. It was non-irritating to the skin in primary skin irritation test in mice and showed 55.05 % inhibition of paw edema in a carrageenan-induced hind rat paw edema model, comparable to that of the standard gel (53.93 %), after 24 h. The gel showed 58.21 % analgesia, versus 61.19 % analgesia for the indomethacin gel standard in writhing test. Conclusion: The topical gel from methanol root extract of U. dioica may be an efficacious and safe alternative to non-steroidal anti-inflammatory drugs in the treatment of rheumatoid arthritis but this requires further investigations to ascertain its safety and clinical efficacy.

Antitumor activity of doxorubicine-loaded nanoemulsion against Ehrlich ascites carcinoma-bearing mice

Purpose: To evaluate the antitumor activity of doxorubicine (DOX)-loaded nanoemulsion (NE) on Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. Methods: The mice were divided into five groups (n = 20) according to the administered drug. Groups I - V were labeled as negative control (normal), positive control of the untreated EAC bearing mice (EAC control), blank nanoemulsion (BI-NE), DOX-loaded-NE (DOX/LNE) and free DOX (DOX-Sol), respectively. Cardiotoxicity was assessed by measuring changes in body and organ weight, analyzing serum enzymes and lipids, and examining histological changes in heart tissues by light microscopy. In addition, mean survival time (MST), increase in life span (ILS) and survival (S) of the mice were determined. Results: DOX/LNE group reduced levels of serum enzymes and lowered damage to heart tissues relative to DOX-Sol group. The MST of the DOX/LNE group (80 ± 0.0 days) was significantly greater than that for DOX-Sol group (34.6 ± 8.9 days), while ILS of DOX/LNE (265.30 days) was higher than that of DOX-Sol (57.99 days) by 4.6-fold. Conclusion: Administration of DOX/LNE to EAC-bearing mice improves the efficacy of DOX and reduce its side effects on the heart.

Anti-anxiety effect of methanol extract of Pericarpium zanthoxyli using a strychnine-sensitive glycine receptor model

Purpose: To determine if the methanol extract of Pericarpium zanthoxyli exerts anti-anxiety effects and also to explore any probable anti-anxiety mechanism in vivo. Methods: The staircase test, elevated plus maze test, rota-rod treadmill test and convulsions induced by strychnine and picrotoxin on mice were tested to identify potential mechanism of anti-anxiety activity of the plant extract. Results: The plant extract (10 mg/kg, p.o.) significantly reduced rearing numbers in the staircase test while it increased the time spent in the open arms as well as the number of entries to the open arms in the elevated plus maze test, suggesting that it has significant anti-anxiety activity. Furthermore, the extract inhibited strychnine-induced convulsion. However, it had little effect on picrotoxin-induced convulsion, suggesting that its anti-anxiety activity may be linked to strychnine-sensitive glycine receptor and not GABA receptor. Conclusion: These results suggest that the Pericarpium zanthoxyli extract may be beneficial for the control of anxiety.

Antitumor activity of physcion 8-o-β-glucopyranoside against cervical cancer by induction of apoptosis

Purpose: To investigate the antitumor activity of physcion8-O-β-glucopyranoside (PSG) against cervical cancer, as well as its mechanisms. Methods: The anti-proliferative effects of PSG on HeLa cells were determined by CCK-8 assay and the half maximal inhibitory concentration (IC50) values were calculated. Subsequently, a mouse xenograft model of HeLa cell line was established to investigate the antitumor effect of PSG in vivo. Furthermore, cell apoptosis was investigated by fluorescence microscopy via DAPI staining, and other mechanisms were determined by Western blot assay. Results: In vitro, PSG exhibited significant anti-proliferative effect on HeLa cells (p <0.05) in concentration-and time-dependent manners, with an IC50 value of 41.34 μg/mL. In vivo, PSG also had significant anti-tumor activity in nude mouse xenograft model (p < 0.05), inhibiting tumor growth. Furthermore, the results showed that treatment with PSG (20, 40 and 60 μg/mL) for 24 h resulted in significantly increased apoptosis in HeLa cells (p < 0.05). Additionally, Western blot analysis revealed that after exposure to 20, 40 and 60 μg/mL of PSG for 24 h, protein expressions of C-caspase-3, Ccaspase-9 and Bax were markedly up-regulated (p < 0.05) while Bcl-2 was significantly down-regulated (p < 0.05). These results confirmed that PSG inhibited HeLa cell growth by inducing mitochondriamediated apoptosis via up-regulation of caspase-3 and caspase-9 and Bax, and down-regulation of Bcl2. Conclusion: The results demonstrate that PSG possesses notable anti-tumor activity against cervical cancer and that the mechanisms involve induction of apoptosis by mitochondria-mediated signaling pathway.