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1.
World J Microbiol Biotechnol ; 36(2): 31, 2020 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-32048066

RESUMO

Biogenic production of nanoparticles is eco-friendly, less expensive method with various medical and biological applications. Nanotechnology along with photodynamic therapy is gaining tremendous importance with enhanced efficacy. The present work was aimed to evaluate methanolic extracts and nanoparticles of two selected plants (Datura suavolens and Verbina tenuisecta) for cytotoxic photodynamic, antioxidant and antimicrobial study. Both extract and silver (5 mM) nanoparticles of Datura plant showed significant activities against bacterial strains. Maximum ZOI of 27.3 ± 1.6 mm was observed with nanoparticles of Datura branches with minimum inhibitory (MIC) value of 32 µg/ml. In case of antifungal and antioxidant assay samples were moderately active. Silver nanoparticles and extracts were effective against rhabdomyosarcoma cell line with lowest IC50 value of 42.5 ± 0.6 µg/ml and percent viability of 25.6 ± 1.3 of Verbena tenuisecta. However, nanoparticles of Datura leaves and branches were more potent with IC50 value of 2.4 ± 0.9 µg/ml and 7.8 ± 1.1 µg/ml respectively. The result of photodynamic study showed that efficacy of photosensitizer was enhanced and percent viability reduced when nanoparticles used as an adjunct. The color change and UV spectra (415‒425 nm) indicated the production of nanoparticles. Fourier transform infrared spectroscopy (FTIR) spectra showed presence of different functional groups e.g., hydroxyl, carbonyl and amino. Nanoparticles are sphenoid in morphology and size ranges between 20-150 nm. Current study showed these silver nanoparticles can be used as cytotoxic agent in photodynamic therapy and can play a critical role to establish medicinal potential of selected plants.


Assuntos
Datura/química , Metanol/farmacologia , Prata/farmacologia , Verbena/química , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Nanopartículas Metálicas , Metanol/química , Metanol/isolamento & purificação , Testes de Sensibilidade Microbiana , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/isolamento & purificação , Fármacos Fotossensibilizantes/farmacologia , Extratos Vegetais/química , Folhas de Planta/química , Prata/química , Prata/isolamento & purificação
2.
J Nat Med ; 74(2): 482-486, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31956959

RESUMO

In our previous study, we found that the methanolic extract of Sanoshashinto () (SHXXTM) exhibited significant vasorelaxant effects in vitro and antihypertensive effects in vivo, and baicalin and berberine were the main antihypertensive constituents in SHXXTM. We also speculated that the baicalin-berberine (BB) combination produced vasorelaxant effects by activating the NO/cGMP pathway, and the BKCa channel and the DAG/PKC/CPI-17 pathway were involved. In this study, we examined the vasorelaxant effects using helical strips of rat aorta pretreated with different activators or inhibitors. The results suggested that the KATP channel and the voltage-dependent Ca2+ channel (VDCC) were also involved in the vasorelaxant effects. Furthermore, we found that SHXXTM and the BB combination reduced left ventricular hypertrophy and altered gut microbiota. Together, the results indicated that Sanoshashinto might have comprehensive effects on ameliorating hypertension.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Metanol/uso terapêutico , Extratos Vegetais/uso terapêutico , Vasodilatadores/uso terapêutico , Animais , Modelos Animais de Doenças , Masculino , Metanol/farmacologia , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Vasodilatadores/farmacologia
3.
Fitoterapia ; 140: 104420, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733344

RESUMO

We report for the first time the isolation of 2-furyl(phenyl)methanol (5) from the chloroform extracts of the Atractylis gummifera roots. A. gummifera is a thistle belonging to the Asteraceae family that produces the ent-kaurane diterpenoid glycoside atractyloside (ATR). ATR (1) was isolated and chemically modified to obtain its aglycone atractyligenin (2) and the methylated derivatives ATR-OMe (3) and genine-OMe (4). The compounds 1-5 were structurally characterised and evaluated against the intracellular amastigote, cultured within macrophages, and the extracellular promastigote of Leishmania donovani, the protozoan parasite responsible for the highly infective disease visceral leishmaniasis, which is fatal if untreated. The 2-furyl(phenyl)methanol 5 exhibited notable activity against the promastigote.


Assuntos
Antiprotozoários/farmacologia , Atractylis/química , Leishmania donovani/efeitos dos fármacos , Metanol/farmacologia , Animais , Antiprotozoários/isolamento & purificação , Itália , Macrófagos/parasitologia , Metanol/análogos & derivados , Metanol/isolamento & purificação , Camundongos Endogâmicos BALB C , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Extratos Vegetais , Rizoma/química
4.
J Helminthol ; 94: e77, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31455460

RESUMO

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.


Assuntos
Annona/química , Anti-Helmínticos/farmacologia , Haemonchus/efeitos dos fármacos , Óvulo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Acetona/farmacologia , Animais , Annona/classificação , Haemonchus/fisiologia , Larva/efeitos dos fármacos , Metanol/farmacologia , Folhas de Planta/química , Polifenóis/farmacologia
5.
DNA Cell Biol ; 38(11): 1338-1345, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31464523

RESUMO

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease, which often affects colon or rectum or both. It is now well recognized that sphingosine kinases-1/sphingosine-1-phosphate (S1P) signaling may have a very significant potential as targets for therapeutic intervention in UC. Compared with the pure dextran sodium sulfate group, administration of PF543 significantly reduced clinical symptoms with less weight loss, diarrhea, and shortening of the colon. The severity of colitis was improved with reduced disease activity index and degree of histological damage in colon. Moreover, treatment with PF543 not only decreased S1P but also inhibited mRNA expression of proinflammatory factors such as interleukin (IL)-1ß and IL-6. This suggests that PF543 might exhibit an anti-inflammatory function against colitis through inhibition of expression of proinflammatory factors.


Assuntos
Colite/patologia , Colo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Metanol/análogos & derivados , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Pirrolidinas/farmacologia , Animais , Colite/sangue , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Lisofosfolipídeos/sangue , Masculino , Metanol/farmacologia , Metanol/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão/efeitos dos fármacos , Pirrolidinas/uso terapêutico , Esfingosina/análogos & derivados , Esfingosina/sangue , Baço/efeitos dos fármacos , Baço/patologia , Especificidade por Substrato
6.
Tuberculosis (Edinb) ; 117: 52-55, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31378268

RESUMO

Pharmacokinetic/pharmacodynamic studies of anti-tuberculosis agents in animal models of tuberculosis are hampered by the frequent necessity to perform sample bioanalysis outside the biosafety level-3 environment. Thus, each specimen has to undergo tedious and time-consuming sample sterilization procedures that may also affect drug stability. Here, we tested treatment of Mycobacterium tuberculosis (Mtb) infected samples with methanol to sterilize samples while preserving drug integrity for further pharmacokinetic/pharmacodynamic evaluations. Tissue samples harvested from Mtb infected mice were homogenized, incubated in methanol, and tested for sterility. Once sterility was confirmed, the samples were used to determine concentrations of the anti-tuberculosis drug spectinamide-1599 in lung homogenates using liquid chromatography coupled with mass spectrometry. The results demonstrate that methanol sterilizes tissue samples harvested from Mtb infected mice without altering the integrity of the drug in the tissue.


Assuntos
Antituberculosos/farmacologia , Metanol/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Manejo de Espécimes/métodos , Esterilização/métodos , Tuberculose/microbiologia , Animais , Antituberculosos/análise , Contagem de Colônia Microbiana , Estudos de Viabilidade , Feminino , Infecção Laboratorial/prevenção & controle , Pulmão/microbiologia , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana/métodos , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/fisiologia , Espectinomicina/análogos & derivados , Espectinomicina/análise , Espectinomicina/farmacologia , Células-Tronco/efeitos dos fármacos
7.
Niger J Physiol Sci ; 34(1): 83-90, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31449276

RESUMO

The present study examines the possible ameliorative effects of the hydromethanolic extract of Citrullus lanatus rind (HECL) on some reproductive function and oxidative indices of the testes in male Wistar rats following administration of nicotine. Twenty male rats were assigned into four groups: Group A to D of five rats each. Group A served as control and received 2ml/kg body weight of 10% extract vehicle; Group B received 1mg/kg body weight of nicotine; Group C were co-administered 1mg/kg body weight nicotine and 500 mg/kg body weight of HECL and Group D received only 500mg/kg body weight of HECL. The drugs and extracts were administered orally to the rats for 42days; blood samples were collected by direct cardiac puncture for determination of serum concentrations of testosterone, Follicle Stimulating Hormone and Luteinizing Hormone. The testes were also harvested for determination of semen parameters: motility, morphology, viability and count and testicular tissue processed for superoxide dismutase and malondialdehyde concentration. Compared to Group A control rats, administration of HECL significantly increased sperm count and reproductive hormone concentrations amongst Group B rats (p<0.05). Treatment with nicotine caused a significant reduction in the levels of all reproductive hormones with significant diminution of some sperm parameters: motility, morphology and viability; and decrease in superoxide dismutase and increase in malondialdehyde concentration amongst Group B rats compared to Group A control rats (p<0.05). Co-administration of HECL with nicotine to Group C rats apparently reversed the effects of nicotine resulting in significant increases in sperm count and the reproductive hormones concentration as compared to Group A control rats (p<0.05). Amongst Group D rats, the extract also caused a significant increase in superoxide dismutase concentration and a significant decrease in malondialdehyde concentration compared with the Group A control rats (p<0.05). The findings suggest that the hydromethanolic extract of Citrullus lanatus rind possibly ameliorates the deleterious effects of nicotine on some reproductive indices in male Wistar rats.


Assuntos
Citrullus , Hormônios Esteroides Gonadais/sangue , Nicotina/toxicidade , Extratos Vegetais/farmacologia , Sêmen/efeitos dos fármacos , Testículo/efeitos dos fármacos , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Hormônios Esteroides Gonadais/antagonistas & inibidores , Masculino , Metanol/farmacologia , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Agonistas Nicotínicos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Sêmen/metabolismo , Testículo/metabolismo , Resultado do Tratamento
8.
Biosci Biotechnol Biochem ; 83(11): 2163-2171, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31272289

RESUMO

Motile bacteria often exhibit chemotaxis toward favorable compounds. However, the diversity of bacteria that are attracted to a given substance is largely unknown. This study aimed to reveal the diversity of bacteria with natural chemotaxis towards methanol. We tried to enrich environmental chemotactic bacteria using a glass capillary that is half-filled with methanol solidified with agarose as a trap ("chemotaxis fishing"). The pilot experiment using methanol-chemotactic Methylobacterium aquaticum strain 22A enriched the cells by 46-fold. The method was then applied to bacterial suspensions from paddy water and plants. Depending on the isolation sources and the methods of motility induction, methylotrophic bacteria were enriched 1.2-330-fold. The fished isolates belong to 32 species in 18 genera, mainly containing Acinetobacter, Methylobacterium and Pseudomonas species. Our chemotaxis fishing unveiled a part of diversity of the bacteria with natural chemotaxis towards methanol.


Assuntos
Bactérias/citologia , Bactérias/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Metanol/farmacologia , Técnicas Microbiológicas/métodos , Plantas/microbiologia
9.
Mol Biol Rep ; 46(5): 5131-5142, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31317456

RESUMO

Medicinal plants can be candidate as a common alternative for cancer treatment according to natural landscaping and native plants in each country. The aim of this study was the evaluations of cytotoxicity, apoptosis, and cell cycle arrest induction by using seven leaves extracts of Catharanthus roseus, Calystegia sepium, Berberis integerrima, Mahonia fortunei, Melia azedarach, Plantago major, Betula pendula and one bulb extract of Narcissus tazetta. Extracts were assessed on three cancer cell lines including MCF-7 breast cancer cells, A431 epidermal cell line, and U87-MG glioma cell line that were compared to HGF-1 as normal cells. According to analysis of MTT, methanolic extract of C. sepium leaves increased significantly the rate of cell death in all cancer cell lines when compared to HGF-1 as normal cells. Among different extracts, methanolic extract of C. roseus leaves and methanolic extract of C. sepium leaves indicated a crucial role in apoptosis of cancer cells according to evidences from MTT assay, cell cycle analysis, and apoptosis assay. Doxorubicin has been used as standard drug to compare with IC50 s of different extracts. In addition, the encapsulation of methanolic and ethanolic extracts in small unilamellar vesicles form (SUV) increased the cytotoxicity on cancer cell lines and normal cells. Our results indicated that different extracts can differently affect the cytotoxicity rate in variety of cancer cell lines.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/genética , Glioblastoma/genética , Plantas Medicinais/química , Neoplasias Cutâneas/genética , Antineoplásicos Fitogênicos/química , Proteínas Reguladoras de Apoptose/genética , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Humanos , Lipossomos , Células MCF-7 , Metanol/química , Metanol/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Neoplasias Cutâneas/tratamento farmacológico
10.
Biopreserv Biobank ; 17(6): 520-529, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31194572

RESUMO

Dinoflagellates of the genus Symbiodinium form symbiotic relationships with corals, other marine invertebrates, and protists; thus, they are considered as important species in coral reef ecosystems. If Symbiodinium could be successfully cryopreserved, the cell bank generated could prove to be a valuable resource for researchers interested in basic biological research of Symbiodinium-invertebrate symbioses. Herein, successful cryopreservation of clade D Symbiodinium was achieved using a two-step freezing protocol. Symbiodinium cells were exposed to cryoprotectants (CPAs) for 30 minutes before being vapor frozen for 20 minutes in liquid nitrogen (LN2); afterward, cells were immediately immersed in LN2 for 2 hours or 10 days. The initial experiment was conducted with the following CPAs at 1, 2, and 3 M concentrations: methanol, dimethyl sulfoxide, glycerol, ethylene glycol (EG), and propylene glycol (PG). It was found that infiltration with 2 M EG and PG yielded cells with the highest percentage viability. Upon thawing, culture of these Symbiodinium was carried out for 2 months in a growth chamber, and cells continued to grow and proliferate over this period. This represents successful cryopreservation of a dominant reef coral symbiont, a feat that will ideally aid in future research of this important lineage of dinoflagellate.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Dinoflagelados/citologia , Animais , Antozoários/parasitologia , Dimetil Sulfóxido/farmacologia , Dinoflagelados/crescimento & desenvolvimento , Etilenoglicol/farmacologia , Glicerol/farmacologia , Metanol/farmacologia , Propilenoglicol/farmacologia , Simbiose , Termotolerância
11.
BMC Biotechnol ; 19(1): 37, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208387

RESUMO

BACKGROUD: Fusaruside is an immunomodulatory fungal sphingolipid which has medical potentials for treating colitis and liver injury, but its poor natural abundance limits its further study. RESULTS: In this study, we described a synthetic biology approach for fusaruside production by engineered Pichia pastoris that was based on polycistronic expression. Two fusaruside biosynthesis genes (Δ3(E)-sd and Δ10(E)-sd), were introduced into P. pastoris to obtain fusaruside producing strain FUS2. To further enhance the yield of fusaruside, three relevant biosynthetic genes (Δ3(E)-sd, Δ10(E)-sd and gcs) were subsequently introduced into P. pastoris to obtain FUS3. All of the biosynthetic genes were successfully co-expressed in FUS2 and FUS3. Compared to that produced by FUS2, fusaruside achieved from FUS3 were slightly increased. In addition, the culture conditions including pH, temperature and methanol concentration were optimized to improve the fusaruside production level. CONCLUSIONS: Here a novel P. pastoris fusaruside production system was developed by introducing the biosynthetic genes linked by 2A peptide gene sequences into a polycistronic expression construct, laying a foundation for further development and application of fusaruside.


Assuntos
Cerebrosídeos/metabolismo , Proteínas Fúngicas/metabolismo , Fatores Imunológicos/metabolismo , Engenharia Metabólica/métodos , Pichia/metabolismo , Cerebrosídeos/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metanol/metabolismo , Metanol/farmacologia , Modelos Químicos , Estrutura Molecular , Pichia/genética , Esfingolipídeos/metabolismo , Temperatura
12.
Biophys J ; 116(5): 755-759, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30777306

RESUMO

Methanol is a common solubilizing agent used to study transmembrane proteins/peptides in biological and synthetic membranes. Using small angle neutron scattering and a strategic contrast-matching scheme, we show that methanol has a major impact on lipid dynamics. Under increasing methanol concentrations, isotopically distinct 1,2-dimyristoyl-sn-glycero-3-phosphocholine large unilamellar vesicle populations exhibit increased mixing. Specifically, 1,2-dimyristoyl-sn-glycero-3-phosphocholine transfer and flip-flop kinetics display linear and exponential rate enhancements, respectively. Ultimately, methanol is capable of influencing the structure-function relationship associated with bilayer composition (e.g., lipid asymmetry). The use of methanol as a carrier solvent, despite better simulating some biological conditions (e.g., antimicrobial attack), can help misconstrue lipid scrambling as the action of proteins or peptides, when in actuality it is a combination of solvent and biological agent. As bilayer compositional stability is crucial to cell survival and protein reconstitution, these results highlight the importance of methanol, and solvents in general, in biomembrane and proteolipid studies.


Assuntos
Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Metanol/farmacologia , Difração de Nêutrons , Espalhamento a Baixo Ângulo , Cinética , Solventes/farmacologia , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
13.
Cryobiology ; 87: 78-85, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30716303

RESUMO

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Oogônios/fisiologia , Células-Tronco de Oogônios/fisiologia , Animais , Carpas , Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Feminino , Congelamento , Metanol/farmacologia , Oogônios/citologia , Ovário/citologia , Propilenoglicol/farmacologia , Sacarose/farmacologia , Trealose/farmacologia
14.
Enzyme Microb Technol ; 122: 55-63, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30638508

RESUMO

Porcine interferon-α (pIFN-α) could be used as the vaccine adjuvant to enhance the antiviral ability of porcine in swine industry. In here, a combinational strategy integrating codon optimization, multiple gene insertion, strong AOX1 promoter, and efficient secretion signal sequence was developed to obtain high-level secreted pIFN-α in Pichia pastoris GS115. The codon optimized pIFN-α shared 76% sequence identity with the original pIFN-α, which was inserted into the P. pastoris genome under AOX1d1-2x201 promoter and MF4I secretion sequence. Our results showed positive correlation between the mRNA and secreted protein levels with the copy numbers of genome-integrated pIFN-α gene in the recombinant P. pastoris strains. The recombinant opt-pIFN-α-6C strain bearing six copies of pIFN-α expression cassette produced the highest extracellular secretion of pIFN-α of 3.2 ± 0.1 mg/mL in shake flask experiment, and 17.0 ± 0.8 mg/mL in a 5 L high-cell-density cultivation after methanol induction of 84 h. The antiviral activity of secreted pIFN-α from the high-cell-density cultivation was determined to be approximately 2.8 ± 0.9 × 109 IU/mL against the vesicular stomatitis virus (VSV) infected Madin-Darby bovine kidney (MDBK) cells. This strategy provided an efficient way to generate recombinant P. pastoris strains in a non-antibiotics-selection manner, which might also give general guidance for the heterologous expression of other proteins in P. pastoris.


Assuntos
Expressão Gênica , Interferon-alfa/biossíntese , Interferon-alfa/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Animais , Antivirais/farmacologia , Reatores Biológicos , Bovinos , Linhagem Celular , Fermentação , Expressão Gênica/efeitos dos fármacos , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Metanol/farmacologia , Pichia/genética , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Suínos , Vesiculovirus/efeitos dos fármacos
15.
Biomed Pharmacother ; 109: 1602-1609, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551414

RESUMO

Calotropis procera, a latex producing plant is known to possess medicinal properties including its beneficial effect in gastrointestinal disorders. The anti-inflammatory effect of its latex in various experimental models is noteworthy and in light of this the present study was carried out with an objective to evaluate its efficacy in ulcerative colitis, an inflammatory condition of the colon. Colitis was induced in rats by acetic acid and the rats were divided into four groups where one group served as experimental control and the other groups were treated with two doses of methanol extract of dried latex of C. procera (MeDL; 50 and 150 mg/kg) and mesalazine (MSZ; 300 mg/kg). The study also included normal control (NC) group for comparison of various parameters related to colon like macroscopic changes, ulcer score, adherent mucus content, weight/length ratio, small intestinal transit, oxidative stress and inflammatory markers, tissue histology and immunoreactivity of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclear factor kappa beta (NFκB) subunit p65. Treatment of colitic rats with MeDL produced a significant reduction in colonic mucosal damage as revealed by macroscopic and microscopic evaluation and normalization of tissue levels of oxidative stress markers and pro-inflammatory mediators. The protection afforded by MeDL was also evident from its restorative effect on tissue histology and expression of COX-2, iNOS and NFκB(p65). This study shows that by targeting oxidative stress and NFκB(p65) mediated pro-inflammatory signaling, the latex of C. procera affords protection in colitis and its effect was comparable to that of mesalazine. This study suggests that latex of C. procera could serve as a promising therapeutic option for treating inflammatory conditions of the colon.


Assuntos
Calotropis , Colite/metabolismo , Colite/prevenção & controle , Mediadores da Inflamação/metabolismo , Látex/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Ácido Acético/toxicidade , Animais , Colite/induzido quimicamente , Modelos Animais de Doenças , Feminino , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Látex/isolamento & purificação , Látex/farmacologia , Masculino , Metanol/farmacologia , Metanol/uso terapêutico , Estresse Oxidativo/fisiologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
16.
Infect Disord Drug Targets ; 19(3): 264-268, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29741144

RESUMO

BACKGROUND: Cystic echinococcosis/ hydatidosis is one of the most important parasitic zoonoses worldwide. Usually, surgery is used for the treatment of active cysts. Due to the risk of cyst rupture during surgery, a protoscolicidal agent is usually injected into the cyst before surgery to prevent secondary cyst formation in case of cyst rupture. Due to the side effects of chemical protoscolicidal agents attempts have been focused on herbal medicine. In this regard, Allium sativum has already been used; however, the protoscolicidal effect of Allium hirtifolium from Allium family has not been investigated, so far. OBJECTIVE: The present study was conducted in order to assess the protoscolicidal effect of methanolic extract of Allium hirtifolium in different concentrations, temperature and exposure times. Cetrimide as a positive control and DMSO as a negative control were also used in this study. METHODS: The viability of protoscoleces was determined by 0.1% eosin staining method. RESULTS: The protoscolicidal effect of Allium hirtifolium on protoscoleces was as follows: The mean percent and standard deviation (SD) of protoscolicidal activity of 50 mg/ml concentration of Allium hirtifolium extract in room temperature at 10, 20, 30, 40 and 50 minutes of exposure was 5.88±2.39, 8.59±4.54, 11.82±5.81, 12.31±6.16and 16.96±8.42 respectively; while at 37°C it was 9.10±5.01, 11.52±7.36, 18.64±4.36, 27.07±6.57 and 38.15±12.80, respectively. CONCLUSION: In conclusion, although Allium hirtifolium from Allium family is similar to Allium sativum; the protoscolicidal effect of its methanolic extract is not similar and less than A. sativum extract.


Assuntos
Echinococcus granulosus/efeitos dos fármacos , Alho/química , Metanol/farmacologia , Extratos Vegetais/farmacologia , Animais , Equinococose/tratamento farmacológico , Fígado/parasitologia , Metanol/química , Extratos Vegetais/química , Ovinos/parasitologia , Doenças dos Ovinos/parasitologia
17.
Anim Reprod Sci ; 200: 7-13, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30473146

RESUMO

The aim of this study was to analyze the effect of 5%, 7.5%, 10%, 12.5% and 15% dimethylsulfoxide (DMSO), methanol and methylglycol alcohols on the cryopreservation of sperm from Steindachneridion scriptum. Male specimens (n = 15) were obtained from Pisciculture and sperm samples were collected by abdominal massage. Post collection the fresh sperm sample was diluted in the Beltsville Thawing Solution and sperm motility was evaluated. Results indicated that the most precise parameters for total and progressive motility were obtained with the use of methylglycol (all concentrations) and 7.5% and 10% methanol (P < 0.05). The motility of the sperm was sustained for the longest time period when 5%, 7.5% and 15% DMSO was used; similar results were also seen for 5% methanol and methylglycol at 7.5%, 10%, 12.5%, and 15% concentration (P < 0.05). With respect to reactive oxygen species it was observed that the production of ROS decreased only in presence of 5% methylglycol but not when DMSO (5%) was used (P < 0.05). Although the use of methanol (12.5%) allowed for a lesser membrane fluidity as compared to DMSO 12.5% (P < 0.05), membrane functional integrity was greater with 10% and 12.5% DMSO (P < 0.05) as compared to 10% methanol or 5% methylglycol (P > 0.05). Additionally, when major mitochondrial functionalities were assessed it was observed that the values obtained with use of 12.5% and 15% DMSO were comparable to all except 5% methyglycol (P < 0.05). In conclusion, the results of the present study indicate that 7.5% methylglycol was the most effective treatment for the cryopreservation of the S. scriptum sperm.


Assuntos
Peixes-Gato , Criopreservação , Crioprotetores/farmacologia , Dimetil Sulfóxido/farmacologia , Metanol/farmacologia , Preservação do Sêmen , Animais , Criopreservação/métodos , Criopreservação/veterinária , Glicóis/farmacologia , Masculino , Análise do Sêmen , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Motilidade Espermática/efeitos dos fármacos
18.
Biotechnol Appl Biochem ; 66(4): 484-493, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26498482

RESUMO

Isoflavonoid representatives such as genistein and daidzein are highly potent anticancer, antibacterial, and antioxidant agents. It have been demonstrated that methylation of flavonoids enhanced the transporting ability, which lead to facilitated absorption and greatly increased bioavailability. In this paper, genetically engineered Escherichia coli was reconstructed by harboring E. coli K12-derived metK encoding S-adenosine-l-methionine (SAM) synthase (accession number: K02129) for enhancement of SAM as a precursor and Streptomyces avermitilis originated SaOMT2 (O-methyltransferase, accession number: NP_823558) for methylation of daidzein and genistein as preferred substrates. The formation of desired products via biotransformation including 4'-O-methyl-genistein and 4'-O-methyl-daidzein was confirmed individually by using chromatographical methods such as high-performance liquid chromatography, liquid chromatography/time-of-flight/mass spectrometry (LC-TOF-MS), and nuclear magnetic resonance (NMR), and NMR (1 H and 13 C). Furthermore, substrates concentration, incubation time, and media parameters were optimized using flask culture. Finally, the most fit conditions were applied for fed-batch fermentation with scale-up to 3 L (working volume) to obtain the maximum yield of the products including 164.25 µM (46.81 mg/L) and 382.50 µM (102.88 mg/L) for 4'-O-methyl genistein and 4'-O-methyl daidzein, respectively. In particular, potent inhibitory activities of those isoflavonoid methoxides against the growth of cancer line (B16F10, AGS, and HepG2) and human umbilical vein endothelial cells were investigated and demonstrated. Taken together, this research work described the production of isoflavonoid-4'-O-methoxides by E. coli engineering, improvement of production, characterization of produced compounds, and preliminary in vitro biological activities of the flavonoids being manufactured.


Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Escherichia coli/metabolismo , Isoflavonas/biossíntese , Isoflavonas/farmacologia , Engenharia Metabólica , Metanol/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Isoflavonas/química , Metanol/química , Metanol/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas
19.
Biotechnol Lett ; 41(2): 273-281, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30542947

RESUMO

OBJECTIVE: The antioxidant activity and protective effect of a methanolic extract obtained from the marine Gram-negative bacterium Novosphingobium sp. PP1Y, isolated from the surface water of a polluted area in the harbour of Pozzuoli (Naples, Italy), was evaluated. RESULTS: The extract was tested in vitro on epithelial colorectal adenocarcinoma cells and in vivo on Caenorhabditis elegans. It showed strong protective activity against oxidative stress, in both experimental systems, by preventing ROS accumulation. In the case of the cells, pre-treatment with methanolic extract was also able to maintain unaltered intracellular GSH levels and phosphorylation levels of mitogen-activated protein kinases p38. Instead, in the case of the worms, the extract was able to modulate the expression levels of stress response genes, by activating the transcription factor skn-1. CONCLUSIONS: From a biotechnological and economical point of view, antioxidants from microorganisms are convenient as they provide a valid alternative to chemical synthesis and respond to the ever-growing market demand for natural antioxidants.


Assuntos
Antioxidantes/isolamento & purificação , Caenorhabditis elegans/metabolismo , Neoplasias Colorretais/metabolismo , Metanol/isolamento & purificação , Sphingomonadaceae/metabolismo , Animais , Antioxidantes/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Metabolômica/métodos , Metanol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Sphingomonadaceae/isolamento & purificação , Fatores de Transcrição/genética , Microbiologia da Água , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
20.
N Biotechnol ; 49: 10-18, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30529567

RESUMO

Laccase-like multicopper oxidases (LMCOs) are a heterogeneous group of oxidases, acting mainly on phenolic compounds and which are widespread among many microorganisms, including Basidiomycetes and Ascomycetes. Here, we report the cloning, heterologous expression, purification and characterization of a novel LMCO from the thermophilic fungus Thermothelomyces thermophila. The 1953 bp lmco gene sequence comprises of 3 exons interrupted by 2 introns and according to the LccED database the translated sequence belongs to superfamily 6 of multicopper oxidases. After removal of the introns, the gene was transformed into Pichia pastoris, under the control of the alcohol oxidase (AOX1) promoter. The heterologous enzyme was purified with an apparent molecular weight of 80 kDa. TtLMCO1 displayed optimum activity at pH 4 and 50 °C and appeared thermostable up to 50 °C. A variety of phenolic compounds were oxidized by TtLMCO1, including standard laccase substrates such as ABTS and 2,6 dimethoxyphenol. The UV/Vis spectrum of purified TtLMCO1 indicates that it belongs to yellow laccase-like oxidases. The enzyme was used for the bioconversion of 2',3,4-trihydroxychalcone to 3',4'-dihydroxy-aurone, a bioactive aurone recently shown to possess inhibitory activity against several isoforms of the histone deacetylase complex (HDAC). Overall, the thermophilic yellow LMCO TtLMCO1 presents a number of superior properties with potential use in industrial biocatalysis.


Assuntos
Chalconas/metabolismo , Lacase/metabolismo , Oxirredutases/metabolismo , Sordariales/enzimologia , Temperatura , Chalconas/química , Sulfato de Cobre/farmacologia , Ciclização , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Metanol/farmacologia , Oxirredução , Pichia/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/metabolismo , Solventes , Espectrofotometria Ultravioleta , Especificidade por Substrato
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