Morphological Changes and Strong Cytotoxicity in Yarrowia lipolytica by Overexpressing Delta-12-Desaturase.

In this study, delta-12 desaturase was overexpressed in Yarrowia lipolytica using the single-copy integrative vector pINA1312 and multicopy integrative vector pINA1292, resulting in the engineered yeast strains 1312-12 and 1292-12, respectively. The content of intracellular linoleic acid (LA) in the 1292-12 strain was much higher than in the 1312-12 strain and the control group. One interesting finding was that the 1292-12 strain showed obvious changes in surface morphology. The 1292-12 colonies were much smaller and smoother, whereas their single cells became much larger compared to the control strain. In addition, the dry cell weight (DCW) of the 1292-12 strain was obviously increased from 8.5 to 12.7 g/L, but the viable cell number sharply decreased from 107 to 105/mL. These results indicated that increased LA content in Yarrowia lipolytica could induce morphological changes or even oxidative stress-dependent cell death. The reactive oxygen species (ROS) and malondialdehyde (MDA) were accumulated in the 1292-12 strain, while the antioxidant activities of intracellular catalase (CAT) and superoxide dismutase (SOD) were significantly decreased by 27.6 and 32.0%, respectively. Furthermore, it was also revealed that these issues could be ameliorated by the exogenous supplementation of vitamin C, fish and colza oil.

Identifying the Anti-MERS-CoV and Anti-HcoV-229E Potential Drugs from the Ginkgo biloba Leaves Extract and Its Eco-Friendly Synthesis of Silver Nanoparticles.

The present study aimed to estimate the antiviral activities of Ginkgo biloba (GB) leaves extract and eco-friendly free silver nanoparticles (Ag NPs) against the MERS-CoV (Middle East respiratory syndrome-coronavirus) and HCoV-229E (human coronavirus 229E), as well as isolation and identification of phytochemicals from GB. Different solvents and high-performance liquid chromatography (HPLC) were used to extract and identify flavonoids and phenolic compounds from GB leaves. The green, silver nanoparticle synthesis was synthesized from GB leaves aqueous extract and investigated for their possible effects as anti-coronaviruses MERS-CoV and HCoV-229E using MTT assay protocol. To verify the synthesis of Ag NPs, several techniques were employed, including X-ray diffraction (XRD), scan, transmission electron microscopy, FT-IR, and UV-visible spectroscopy. The highest contents of flavonoids and phenolic compounds were recorded for acetone, methanol, and ethanol as mixtures with water, in addition to pure water. HPLC flavonoids were detected as apegenin, luteolin, myricetin, and catechin, while HPLC phenolic compounds were pyrogallol, caffeic acid, gallic acid, and ellagic acid. In addition, our results revealed that Ag NPs were produced through the shift from yellow to dark brown. TEM examination of Ag NPs revealed spherical nanoparticles with mean sizes ranging from 5.46 to 19.40 nm and an average particle diameter of 11.81 nm. A UV-visible spectrophotometric investigation revealed an absorption peak at λ max of 441.56 nm. MTT protocol signified the use of GB leaves extract as an anti-coronavirus to be best from Ag NPs because GB extract had moderate anti-MERS-CoV with SI = 8.94, while had promising anti-HCov-229E, with an SI of 21.71. On the other hand, Ag NPs had a mild anti-MERS-CoV with SI = 4.23, and a moderate anti-HCoV-229E, with an SI of 7.51.

Enhancing Genome-Scale Model by Integrative Exometabolome and Transcriptome: Unveiling Carbon Assimilation towards Sphingolipid Biosynthetic Capability of Cordyceps militaris.

Cordyceps militaris is an industrially important fungus, which is often used in Asia as traditional medicine. There has been a published genome-scale metabolic model (GSMM) of C. militaris useful for predicting its growth behaviors; however, lipid metabolism, which plays a vital role in cellular functions, remains incomplete in the GSMM of C. militaris. A comprehensive study on C. militaris was thus performed by enhancing GSMM through integrative analysis of metabolic footprint and transcriptome data. Through the enhanced GSMM of C. militaris (called iPC1469), it contained 1469 genes, 1904 metabolic reactions and 1229 metabolites. After model evaluation, in silico growth simulation results agreed well with the experimental data of the fungal growths on different carbon sources. Beyond the model-driven integrative data analysis, interestingly, we found key metabolic responses in alteration of lipid metabolism in C. militaris upon different carbon sources. The sphingoid bases (e.g., sphinganine, sphingosine, and phytosphingosine) and ceramide were statistically significant accumulated in the xylose culture when compared with other cultures; this study suggests that the sphingolipid biosynthetic capability in C. militaris was dependent on the carbon source assimilated for cell growth; this finding provides a comprehensive basis for the sphingolipid biosynthesis in C. militaris that can help to further redesign its metabolic control for medicinal and functional food applications.

Improved γ-Linolenic Acid Production from Cellulose in Mucor circinelloides via Coexpression of Cellobiohydrolase and Delta-6 Desaturase.

The present study was aimed at facilitating the production of γ-linolenic acid (GLA) from the cellulosic substrate with the engineered oleaginous fungus Mucor circinelloides WJ11. Here, the homologous recombination technology was used to overexpress the cellobiohydrolase (CBH2) derived from Trichoderma longibrachiatum and the original delta-6 fatty acid desaturase (D6) in M. circinelloides to construct genetically engineered strains capable of effectively using cellulose to enhance GLA synthesis. When cultivated in modified K&R medium supplemented with microcrystalline cellulose, the CBH2 and D6 coexpressing strains led to increases in the biomass (up to 12.8 g/L) and lipid yield (up to 3.7 g/L) of 87% and 2.4-fold, respectively, compared to that of the control strain. Notably, when CBH2 and D6 were coexpressed in M. circinelloides, the yield of GLA reached 608 mg/L, which was a dramatic increase of 3.9-fold compared to that of the control strain. This is the first report on promoting the GLA production from the cellulosic substrate via coexpression of CBH2 and delta-6 desaturase. This work provides a theoretical basis for efficient transformation from the cellulosic substrate to functional GLA by CBH2 and D6 coexpressing strains, which might play a positive role in promoting the sustainable development of biological industry.

Efficacy of Ethanolic Extract of Syzygium aromaticum in the Treatment of Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates Associated with Urinary Tract Infections.

Pseudomonas aeruginosa is an organism commonly found in the environment and one of the most common causes of human urinary tract infections in developed and developing countries. The present study aimed to investigate the effect of five medicinal plant extracts on the isolated drug-resistant P. aeruginosa clinical isolates. A total of 100 urine samples were collected from Nagaa Hammadi and Qena General Hospitals and private medical analysis laboratories in Qena governorate, Upper Egypt. Samples were screened for the prevalence of UTI pathogens by biochemical tests, antibiotics sensitivity, detection of virulence, and antibiotic-resistant genes by using multiplex PCR. P. aeruginosa is by far the subdominant causative agent with a percentage of 14%. Clinical isolates were multidrug-resistant, containing bla TEM, bla SHV, toxA, lasB, pslA, and fliC resistant and virulence genes. Based on bioactivity, the ethanolic extract of clove (Syzygium aromaticum) was the most active extract among tested medicinal plants and had the maximum zone of inhibition sized 23 mm against tested bacteria. The results of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) showed a high decrease of inhibition within a concentration range of (10 to 121.25 mg/mL and 20 to 30 mg/mL, respectively). Further, major compounds of oleic acid (27.22%), guanosine (8.91%), indole (6.83%), 1-eicosene (6.30%), and cis-10-nonadecenoic acid (5.37%) were determined among 12 bioactive compounds in the ethanolic extract of S. aromaticum using gas chromatography-mass spectrometry (GC-MS). These results indicated that the ethanolic extract of S. aromaticum is a promising antibacterial agent for further studies aiming to control bacterial infections including MDR bacteria and develop novel therapeutic alternatives for the treatment of UTI.

Hyaluronic acid coating on the surface of curcumin-loaded ZIF-8 nanoparticles for improved breast cancer therapy: An in vitro and in vivo study.

Despite developments in surgery and chemotherapy, effective treatment of breast cancer is still an urgent problem owing to recurrence and metastasis. By combining the advantages of curcumin (Cur), zeolitic imidazolate framework-8 nanoparticles (ZIF-8), and hyaluronic acid (HA) in breast cancer therapy, Cur-loaded and HA-coated ZIF-8 (Cur@ZIF-8@HA) were synthesized using a method based on the pH-dependent solubility of Cur and the electrostatic interactions between zinc ions and carboxyl groups of HA. Cur@ZIF-8 were also prepared as a control group. Comprehensive comparisons of the physicochemical properties and anticancer activities of Cur@ZIF-8@HA and Cur@ZIF-8 were conducted. The results indicated that the degradation of Cur during the synthesis of Cur@ZIF-8 was negligible. The obtained Cur@ZIF-8 and Cur@ZIF-8@HA were truncated cubes with hydrodynamic diameters of 174 and 217 nm, respectively. Cur@ZIF-8@HA possessed better stability during storage in different media, a slower drug release rate under neutral and acidic conditions, and a greater inhibitory effect on breast cancer than Cur@ZIF-8. For 4T1 cells, treatment using Cur@ZIF-8@HA induced more cellular uptake and higher cytotoxicity, accompanied by higher lactate dehydrogenase release, cell cycle arrest in G2/M and S phases, production of reactive oxygen species, and apoptosis. In 4T1 tumor-bearing mice models, Cur@ZIF-8@HA showed a stronger inhibitory effect on tumor growth and pulmonary metastasis. Therefore, Cur@ZIF-8@HA might hold great potential as an agent for the effective therapy of breast cancer.

In Vitro Anticancer Potential of Berberis lycium Royle Extracts against Human Hepatocarcinoma (HepG2) Cells.

Human liver cancer has emerged as a serious health concern in the world, associated with poorly available therapies. The Berberis genus contains vital medicinal plants with miraculous healing properties and a wide range of bioactivities. In this study, different crude extracts of B. lycium Royle were prepared and screened against Human Hepatocarcinoma (HepG2) cell lines. The water/ethanolic extract of B. lycium Royle (BLE) exhibited significant antiproliferative activity against the HepG2 cancer cell line with an IC50 value of 47 µg/mL. The extract decreased the clonogenic potential of HepG2 cells in a dose-dependent manner. It induced apoptotic cell death in HepG2 cells that were confirmed by cytometric analysis and microscopic examination of cellular morphology through DAPI-stained cells. Biochemical evidence of apoptosis came from elevating the intracellular ROS level that was accompanied by the loss of mitochondrial membrane potential. The mechanism of apoptosis was further confirmed by gene expression analysis using RT-qPCR that revealed the decline in Bcl-2 independent of p53 mRNA and a rise in CDK1 while downregulating CDK5, CDK9, and CDK10 mRNA levels at 48 h of BLE treatment. The most active fraction was subjected to HPLC which indicated the presence of berberine (48 µg/mL) and benzoic acid (15.8 µg/mL) as major compounds in BLE and a trace amount of luteolin, rutin, and gallic acid. Our study highlighted the importance of the most active BLE extract as an excellent source of nutraceuticals against Human Hepatocarcinoma that can serve as an herbal natural cure against liver cancer.

Different Classes of Phytohormones Act Synergistically to Enhance the Growth, Lipid and DHA Biosynthetic Capacity of Aurantiochytrium sp. SW1.

In the present study, the impact of eight phytohormones from six different classes on the growth, lipid and docosahexaenoic acid (DHA) biosynthetic capacity of Aurantiochytrium sp. SW1 (SW1) was evaluated. Kinetin (KIN), jasmonic acid (JA) and gibberellic acid (GA) significantly enhanced the growth and DHA production of SW1 by 16%-28% and 66%-84% in comparison to the control, respectively. The synergistic effect of these three phytohormones, evaluated by the response surface methodology (RSM), showed that a combination of 3.6 mg/L GA, 2.0 mg/L KIN and 20.0 mg/L JA further increased the growth and DHA production of SW1 by 16% to 28% and 22% to 36%, respectively, in comparison to the individual supplementation. The synergistic effect of these phytohormones was also shown to be time-dependent, where feeding at 24 h of cultivation led to 15%, 26% and 35% further increments in the biomass, lipid and DHA production in comparison to that of 0 h, respectively. The determination of stress markers, antioxidant enzymes and key enzymes involved in fatty acid biosynthesis aided to elucidate the potential mechanism underlying the improvement of growth and DHA production by SW1 at various times of feeding. Supplementation with the phytohormones at 24 h exhibited the maximum impact on reducing the level of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as augmented the antioxidants (superoxide dismutase and catalase) and key metabolic enzymes involved in lipogenesis (malic, glucose-6-phosphate dehydrogenase and ATP-citrate lyase) in comparison to the control and other time points. This study signifies the potential application of phytohormones for improving the growth, lipid and DHA production in Aurantiochytrium spp.

Antioxidant Potential and the Characterization of Arachis hypogaea Roots.

Arachis hypogaea roots are used as traditional Chinese medicine to treat different ailments, and the present study involves the exploration and comparison of phenolic profile and antioxidant activities (ABTS+ and DPPH assay) of A. hypogaea root extract in different solvents. 70% aqueous acetone and 70% aqueous ethanol were proved to be the best solvents to recover total phenolic compounds, with a yield of 42.59 ± 1.96 and 41.34 ± 0.92 mg/g dry weight of extract, respectively. ABTS+ radical scavenging activity was the highest in 70% aqueous ethanol, while the absolute methanol extract showed the highest DPPH radical scavenging activity (29.50 ± 2.19 µg/mL). Furthermore, phytochemical profiling of 70% acetone extract of A. hypogaea roots was performed by LC-ESI-TOF-MS analysis which in turn indicated the presence of diverse compounds in the A. hypogaea root extract, namely, quinones, stilbenoids, and flavones and flavonoid glucosides.

Evaluation of the Spermatogenic Activity of Polyherbal Formulation in Oligospermic Males.

The therapeutic use of natural herbs is an ancient human civilization act and the numbers of people have reliance on their pharmacological properties and preferred to use the natural herbs. People also use to consume these herbs as supplements to energize, bolster, and eventually enhance sexual ability. Polyherbal formulation (PHF) is one of these herbal amalgams that can be used to treat sexual dysfunction including erectile dysfunction, impotence, ejaculation dysfunction, and hypogonadism. The pilot study was aimed at evaluating the capacity of PHF in enhancing the spermatogenic potential of oligospermic patients. Thirty-six male patients with oligospermia were enrolled and randomized either to treatment (n = 23) with PHF (750 mg/d in three doses for 90 days) or to placebo (n = 13) in the same protocol. The preintervention semen analysis was compared with posttreatment semen analysis. Based on the postintervention semen analysis, patients were advised to undergo either in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) to assess their fertility status. After polyherbal treatment, there was a 256% increase in sperm concentration (9.59 ± 4.37 × 106/mL to 25.61 ± 8.6 × 106/mL; P ≤0.001), 154% increase in semen volume (1.7 ± 0.14 mL to 4.32 ± 0.38 mL; P ≤0.001), and 215% increase in sperm motility (15.43 ± 2.40% to 48.65 ± 5.10%; P ≤ 0.001) on day 90 from baseline. Furthermore, a significant improvement and regulation were also observed in serum hormone levels with PHF treatment as compared to the placebo group. The present study demonstrated the evidence on synergistic spermatogenic effect of PHF as attributed in ayurveda for the treatment of oligospermia leading to infertility.

Microencapsulation and the Characterization of Polyherbal Formulation (PHF) Rich in Natural Polyphenolic Compounds.

Microencapsulation of polyherbal formulation (PHF) extract was carried out by freeze drying method, by employing gum arabic (GA), gelatin (GE), and maltodextrin (MD) with their designated different combinations as encapsulating wall materials. Antioxidant components (i.e., total phenolic contents (TPC), total flavonoids contents (TFC), and total condensed tannins (TCT)), antioxidant activity (i.e., DPPH, β-carotene & ABTS⁺ assays), moisture contents, water activity (aw), solubility, hygroscopicity, glass transition temperature (Tg), particle size, morphology, in vitroα-amylase and α-glucosidase inhibition and bioavailability ratios of the powders were investigated. Amongst all encapsulated products, TB (5% GA & 5% MD) and TC (10% GA) have proven to be the best treatments with respect to the highest preservation of antioxidant components. These treatments also exhibited higher antioxidant potential by DPPH and β-carotene assays and noteworthy for an ABTS⁺ assays. Moreover, the aforesaid treatments also demonstrated lower moisture content, aw, particle size and higher solubility, hygroscopicity and glass transition temperature (Tg). All freeze dried samples showed irregular (asymmetrical) microcrystalline structures. Furthermore, TB and TC also illustrated the highest in vitro anti-diabetic potential due to great potency for inhibiting α-amylase and α-glucosidase activities. In the perspective of bioavailability, TA, TB and TC demonstrated the excellent bioavailability ratios (%). Furthermore, the photochemical profiling of ethanolic extract of PHF was also revealed to find out the bioactive compounds.

Effects of twenty standard amino acids on biochemical constituents, docosahexaenoic acid production and metabolic activity changes of Crypthecodinium cohnii.

The influence of 20 standard amino acids was investigated on growth, lipid accumulation, docosahexaenoic acid (DHA) production and cell biochemical composition of Crypthecodinium cohnii. C. cohnii efficiently utilize organic nitrogen (predominantly threonine and to a lesser extent tyrosine and serine) as compared to inorganic nitrogen (NH4)2SO4. However, No significant effect was observed on major biochemical composition of C. cohnii (lipids, carbohydrates and proteins) under N limitation or supplementation with different N-sources. Key lipogenic enzymes glucose-6-phosphate dehydrogenase, ATP-citrate lyase, fatty acid synthase, malic enzyme, citrate synthase (CS), NAD+ and NADP+ dependent isocitrate dehydrogenase were shown to be vital in lipogenesis of C. cohnii. Our results indicated that the process of lipid accumulation in C. cohnii is growth-associated and does not depend upon the trigger of nitrogen depletion. This unusual behavior would suggest that the metabolism of the cells may not be entirely the same as in other lipid-accumulating microorganisms.

Potential nutritional and antioxidant activity of various solvent extracts from leaves and stem bark of Anisophyllea laurina R. Br ex Sabine used in folk medicine

ABSTRACT Anisophyllea laurina is a plant that has been used in folk medicine to treat malaria, dysentery, diabetes, toothache and various skin diseases. Leaves extract had protein content of 9.68% and a high calcium content of 25084.317 mg/100 g while stem bark extract was found to contain greater amounts of calcium (8560.96 mg/100 g), potassium (7649.47 mg/100 g), magnesium (1462.49 mg/100 g) and iron (973.33 mg/100 g). Palmitic acid, linolenic acid, linoleic acid and oleic acid were the most abundant fatty acids in leaves and stem bark extracts. Furthermore, total phenolic (2382.39 mg GAE /100 g) and total flavonoid (385.79 mg QE/100 g) contents were abundant in stem bark while leaves extract was rich in total tannin content (3466.63 mg CE/100 g). However, both leaves and stem bark contained great amounts of vitamins and amino acids were a good source of antioxidant activities. For the individual polyphenol, stenophyllanin A (45.87 mg/g), casuarinin (24.55 mg/g) and digalloyl-HHDP-glucopyranose isomer (15.63 mg/g) were found to be the major compounds from the leaves whereas procyanidin tetramer (14.89 mg/g, (-)-Epicatechin (12.18 mg/g) and procyanidin trimer (11.25 mg/g) were the most predominant compounds from the stem bark. Additionally, the results revealed a significant and strong correlation between phenolic compounds and antioxidant activities.

Influence of extraction solvents on antioxidant and antimicrobial activities of the pulp and seed of Anisophyllea laurina R. Br. ex Sabine fruits

Objective: To evaluate the influence of extraction solvents on antioxidant and antimi-crobial activities of the pulp and seed of Anisophyllea laurina R. Br. ex Sabine fruits. Methods: The antibacterial activities of pulp and seed extracts were tested by using disk diffusion method against eight bacterial strains and three fungal strains. Total phenolic, flavonoid, monomeric anthocyanin and tannin contents, and antioxidant activities were determined by spectrometric methods. Results: The antioxidant analysis of pulp extract revealed the strong radical scavenging capacity and total phenolic content (4329.66 mg of gallic acid/100 g), while seed extract showed the high antioxidant activity and total tannin content (5326.78 mg catechin equivalent/100 g). Antibacterial and antifungal activities of methanol and ethanol extracts exhibited potent growth inhibitory activity against Aeromonas hydrophila, Bacillus subtilis, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella Typhimurium and Staphylococcus aureus ATCC 6538 with minimum inhibitory concentration values ranged from 125 to 250 mg/mL. However, seed extract had the strongest potential activity against Aspergillus niger and Candida albicans with minimum inhibitory concentration value of 500 mg/mL compared to pulp extract. Conclusions: Our results therefore demonstratedthatethanoland methanol extractionswere more efficient in extracting antioxidants and bioactive compound in pulp and seed. These results support that these plant extracts can be used for the treatment of bacterial infections.

Antibacterial and antifungal activities of various solvent extracts from the leaves and stem bark of Anisophyllea laurina R. Br ex Sabine used as traditional medicine in Guinea.

ETHNOPHARMACOLOGICAL RELEVANCE: Anisophyllea laurina R. Br ex Sabine is a plant that has been used in the folk medicine to treat malaria, dysentery, diabetes and toothache against bacterial infection. Through this study, most likely we are reporting for the first time, its effectiveness as an antibacterial and antifungal agent. AIM OF THE STUDY: To evaluate antibacterial and antifungal activities of various solvent extracts from the leaves and stem bark of A. laurina R. Br ex Sabine. MATERIALS AND METHODS: The various solvent extracts of leaves and stem bark were tested for antibacterial and antifungal activities against eight bacteria strains and three fungal strains using the well diffusion and micro-dilution methods. RESULTS: Antibacterial and antifungal activities of methanol and ethanol extracts were higher than the ethyl acetate and water extract. Leaves extracts had the highest potential activity against bacteria than stem bark extracts, but showed low antifungal activity compared to stem bark extracts. They also exhibited potent growth inhibitory activity against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli ATCC 8739 and Bacillus subtilis with MIC value of 125µg/mL. CONCLUSIONS: The current study confirmed that the leaves and stem bark extracts of A. laurina R. Br ex Sabine proved to be most effective as antibacterial and antifungal activities.

Inhibition of calcium-independent phospholipase A2 suppresses proliferation and tumorigenicity of ovarian carcinoma cells.

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.