Dietary flaxseed oil and vitamin E improve semen quality via propionic acid metabolism.

Introduction: Flaxseed oil (FO) and vitamin E (VE) both have antioxidant effects on sperm. The present study investigated the effects of dietary supplementation with FO and/or VE on semen quality. Methods: 16 fertile Simmental bulls were selected and randomly divided into 4 groups (n = 4): the control group (control diet), FO group (control diet containing 24 g/kg FO), VE group (control diet containing 150 mg/kg VE) and FOVE group (control diet containing 150 mg/kg VE and 24 g/kg FO), and the trial lasted 10 weeks. Results: The results showed that the addition of FO independently can increase sperm motion parameters, the levels of catalase (CAT), glutathione peroxidase (GSH-Px), testosterone (T) and estradiol (E2), while reduce oxidative stress in seminal plasma (P < 0.05). Supplement of VE independently can increased the motility, motility parameters, CAT and superoxide dismutase (SOD) levels, and reduce oxidative stress in seminal plasma (P < 0.05). There was an interaction effect of FO × VE on motility and reactive oxygen species (ROS), while GSH-Px and ROS were affected by week × VE 2-way interaction, levels of T and E2 were also affected by the dietary FO × week interaction (P < 0.05). The triple interaction effects of FO, VE and week were significant for malondialdehyde (MDA) (P < 0.05). Compared with the control group, sperm from the FOVE group had a significantly higher in vitro fertilization (IVF) rate, and subsequent embryos had increased developmental ability with reduced ROS levels at the eight-cell stage, then increased adenosine triphosphate (ATP) content and gene expression levels of CAT, CDX2, Nanog, and SOD at the blastocyst stage (P < 0.05). Metabolomic and transcriptomic results indicated that dietary supplementation of FO and VE increased the expression of the metabolite aconitic acid, as well as the expression of ABAT and AHDHA genes. Conclusion: With in-silico analysis, it can be concluded that the effects of dietary FO and VE on improving semen quality and embryo development may be related to increased aconitic acid via the ABAT and AHDHA genes involved in the propionic acid metabolism pathway.

An integrated strategy to study the combination mechanisms of Bupleurum chinense DC and Paeonia lactiflora Pall for treating depression based on correlation analysis between serum chemical components profiles and endogenous metabolites profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Bupleurum chinense DC-Paeonia lactiflora Pall (BCD-PLP) is a common clinical herb pair in traditional Chinese medicine (TCM) prescriptions commonly used to treat depression. However, its combination mechanisms with its anti-depressive effects remain highly unclear. AIM OF THE STUDY: Here, an effective strategy has been developed to study the combination mechanisms of Bupleurum chinense DC (BCD) and Paeonia lactiflora Pall (PLP) by integrating serum pharmacochemistry analysis, metabolomics technology, and molecular docking technology. MATERIALS AND METHODS: First, the depression model rats were replicated by the chronic unpredictable mild stress (CUMS) procedure, and the difference in the chemical composition in vivo before and after the combination of BCD and PLP was analyzed by integrating background subtraction and multivariate statistical analysis techniques. Then, UPLC/HRMS-based serum metabolomics was performed to analyze the synergistic effect on metabolite regulation before and after the combination of BCD and PLP. Further, the correlation analysis between the differential exogenous chemical components and the differential endogenous metabolites before and after the combination was employed to dissect the combination mechanisms from a global perspective of combining metabolomics and serum pharmacochemistry. Finally, the molecular docking between the differential chemical components and the key metabolic enzymes was applied to verify the regulatory effect of the differential exogenous chemical components on the differential endogenous metabolites. RESULTS: The serum pharmacochemistry analysis results demonstrated that the combination of BCD and PLP could significantly affect the content of 10 components in BCD (including 5 prototype components were significantly decreased and 5 metabolites were significantly increased) and 8 components in PLP (including 4 prototype components and 3 metabolites were significantly increased, 1 metabolite was significantly decreased), which indicated that the combination could enhance BCD prototype components' metabolism and the absorption of the PLP prototype components. Besides, metabolomics results indicated that the BCD-PLP herb pair group significantly reversed more metabolites (8) than BCD and PLP single herb group (5 & 4) and has a stronger regulatory effect on metabolite disorders caused by CUMS. Furthermore, the correlation analysis results suggested that saikogenin F and saikogenin G were significantly positively correlated with the endogenous metabolite itaconate, an endogenous anti-inflammatory metabolite; and benzoic acid was significantly positively correlated with D-serine, an endogenous metabolite with an antidepressant effect. Finally, the molecular docking results further confirmed that the combination of BCD and PLP could affect the activities of cis-aconitic acid decarboxylase and D-amino acid oxidase by increasing the in vivo concentration of saikogenin F and benzoic acid, which further enhances its anti-inflammatory activity and anti-depressive effect. CONCLUSIONS: In this study, an effective strategy has been developed to study the combination mechanisms of BCD and PLP by integrating serum pharmacochemistry analysis, multivariate statistical analysis, metabolomics technology, and molecular docking technology. Based on this strategy, the present study indicated that the combination of BCD and PLP could affect the activities of cis-aconitic acid decarboxylase and D-amino acid oxidase by increasing the concentration of saikogenin F and benzoic acid in vivo, which further enhances its anti-depressive effect. In short, this strategy will provide a reliable method for elucidating the herb-herb compatibility mechanism of TCM.

Insecticidal potential of extracts, fractions, and molecules of Aconitum heterophyllum Wall ex. Royle against aphid Aphis craccivora Koch (Hemiptera: Aphididae).

BACKGROUND: Aphis craccivora is the major sap-sucking pest of leguminous crops and vector of plant viruses that cause damage to plants and reduce yield. Indiscriminate and nonjudicious use of synthetic insecticides led to resistance development and harmful to environment. Therefore, it is important to discover plant-based lead(s) which can replace synthetic insecticides. In the current study the residual toxicity of extracts, fractions, and isolated compounds of Aconitum heterophyllum were evaluated against A. craccivora to identify lead(s) for further development of botanical formulation. RESULTS: In residual contact assay, ethanolic (LC50  = 2837.17 mg L-1 ) and aqueous methanolic extracts (LC50  = 2971.59 mg L-1 ) were effective against A. craccivora. Among fractions, the n-butanol fraction of the aqueous methanolic extract (LC50  = 986.96 mg L-1 ) was found to be most effective, followed by the ethyl acetate fraction of the ethanolic extract (LC50  = 1037.52 mg L-1 ) and the n-hexane fraction of both extracts (LC50  = 1113.85 to 1233.11 mg L-1 ). Among pure molecules, aconitic acid was found to be the most effective (68% mortality; LC50  = 1313.19 mg L-1 ) and was on a par with azadirachtin 0.15% EC (66% mortality; LC50  = 1921.10 mg L-1 ). Furthermore, from the effect of ethanoic extract on detoxification enzyme inhibition in A. craccivora we concluded that the target site of action of this extract in A. craccivora might be glutathione S-transferase. CONCLUSIONS: The parent extract/fractions of A. heterophylum showed promising activity against A. craccivora. Among phytoconstituents of the active extract and fractions, aconitic acid was found to be on a par with azadirachtin 0.15% EC. © 2022 Society of Chemical Industry.

Metabolic engineering of Schizosaccharomyces pombe for itaconic acid production.

The economical production of value-added chemicals from renewable biomass is a promising aspect of producing a sustainable economy. Itaconic acid (IA) is a high value-added compound that is expected to be an alternative to petroleum-based chemicals. In this study, we developed a metabolic engineering strategy for the large-scale production of IA from glucose using the fission yeast Schizosaccharomyces pombe. Heterologous expression of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus, which encodes cis-aconitate decarboxylase in the cytosol, led to the production of 0.132 g/L of IA. We demonstrated that mitochondrial localization of CAD enhanced the production of IA. To prevent the leakage of carbon flux from the TCA cycle, we generated a strain in which the endogenous malate exporter, citrate lyase, and citrate transporter genes were disrupted. A titer of 1.110 g/L of IA was obtained from a culture of this strain started with 50 g/L of glucose. By culturing the multiple mutant strain at increased cell density, we succeeded in enhancing the IA production to 1.555 g/L. The metabolic engineering strategies presented in this study have the potential to improve the titer of the biosynthesis of derivatives of intermediates of the TCA cycle.

cis-Aconitic Acid, a Constituent of Echinodorus grandiflorus Leaves, Inhibits Antigen-Induced Arthritis and Gout in Mice.

cis-Aconitic acid is a constituent from the leaves of Echinodorus grandiflorus, a medicinal plant traditionally used in Brazil to treat inflammatory conditions, including arthritic diseases. The present study aimed to investigate the anti-arthritic effect of cis-aconitic acid in murine models of antigen-induced arthritis and monosodium urate-induced gout. The possible underlying mechanisms of action was evaluated in THP-1 macrophages. Oral treatment with cis-aconitic acid (10, 30, and 90 mg/kg) reduced leukocyte accumulation in the joint cavity and C-X-C motif chemokine ligand 1 and IL-1ß levels in periarticular tissue. cis-Aconitic acid treatment reduced joint inflammation in tissue sections of antigen-induced arthritis mice and these effects were associated with decreased mechanical hypernociception. Administration of cis-aconitic acid (30 mg/kg p. o.) also reduced leukocyte accumulation in the joint cavity after the injection of monosodium urate crystals. cis-Aconitic acid reduced in vitro the release of TNF-α and phosphorylation of IκBα in lipopolysaccharide-stimulated THP-1 macrophages, suggesting that inhibition of nuclear factor kappa B activation was an underlying mechanism of cis-aconitic acid-induced anti-inflammatory effects. In conclusion, cis-aconitic acid has significant anti-inflammatory effects in antigen-induced arthritis and monosodium urate-induced arthritis in mice, suggesting its potential for the treatment of inflammatory diseases of the joint in humans. Additionally, our findings suggest that this compound may contribute to the anti-inflammatory effect previously reported for E. grandiflorus extracts.

Encapsulation of trans-aconitic acid in mucoadhesive microspheres prolongs the anti-inflammatory effect in LPS-induced acute arthritis.

trans-Aconitic acid (TAA) is the main constituent of the leaves from the medicinal plant Echinodorus grandiflorus, used to treat different inflammatory diseases. TAA induces a potent but short-lasting biological response, credited to its high polarity and unfavorable pharmacokinetics. Here we developed, characterized and evaluated the anti-inflammatory activity of mucoadhesive microspheres loaded with TAA. Seven batches of mucoadhesive microspheres were prepared by the emulsification/solvent evaporation method, employing different proportions of TAA and Carbopol 934 or/and hydroxypropylmethylcellulose. All batches were characterized for their particle medium size, polydispersity index and entrapment percentage. The batch coded F3c showed highest entrapment percentage and was characterized by infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermogravimetric analyses (TGA) and zeta potential. The anti-inflammatory activity of F3c was assessed in a model of acute arthritis induced by injection of LPS in the knee joint of Swiss mice. The granulometric analyses indicated heterogeneous size distribution for F3c. SEM characterization indicated microspheres with slightly irregular shape and rough surface. Results from ATR-FTIR and thermal analyses (DSC and TGA) pointed out absence of incompatibility between the components of the formulation; thermal events related to the constituents were isolated and randomly located, suggesting amorphous distribution of TAA in the formulation matrix. The zeta potential of the formulations varied from -30 to -34 mV, which may contribute to good stability. When given orally to mice, F3c induced a prolonged anti-inflammatory response by reducing total cell count and neutrophilic accumulation in the joint cavity even when given 48 and 36 h before the stimulus, respectively, in comparison to free TAA (up to 24 and 6 h, respectively). Therefore, the encapsulation of TAA in mucoadhesive microspheres provided its sustained release, indicating that this drug delivery system is a potential agent to treat inflammatory diseases by regulating cell influx.

Esterification of trans-aconitic acid improves its anti-inflammatory activity in LPS-induced acute arthritis.

trans-Aconitic acid (TAA) is an abundant constituent in the leaves of Echinodorus grandiflorus, a medicinal plant used to treat rheumatoid arthritis in Brazil. Esterification was explored as a strategy to increase lipophilicity and biopharmaceutical properties of TAA, a highly polar tricarboxylic acid. We herein report the synthesis of TAA esters via Fischer esterification with ethanol, n-butanol and n-octanol. The reaction kinetics was investigated to produce mono-, di- and tri- derivatives. Mono- and diesters of TAA were obtained as a mixture of positional isomers, whereas the triesters were recovered as pure compounds. The obtained esters were screened in a model of acute arthritis induced by the injection of LPS in the knee joint of Swiss mice. The diesters were the most active compounds, regardless of the alcohol employed in the reaction, whereas bioactivity of the derivatives improved by increasing the length of the aliphatic chain of the alcohol employed in esterification. In general, the esters showed higher potency than TAA. When administered orally to mice at doses of 0.017-172.3 µmol/Kg, the diethyl, di-n-butyl and di-n-octyl esters of TAA reduced the cellular infiltration into the knee joint, especially of neutrophils. The study identified diesters of TAA as potential useful derivatives for the management of rheumatoid arthritis and other inflammatory diseases.

Co-delivery of docetaxel and curcumin prodrug via dual-targeted nanoparticles with synergistic antitumor activity against prostate cancer.

PURPOSE: Combination therapy is increasingly used as a primary cancer treatment regimen. In this report, we designed EGFR peptide decorated nanoparticles (NPs) to co-deliver docetaxel (DTX) and pH sensitive curcumin (CUR) prodrug for the treatment of prostate cancer. RESULTS: EGFR peptide (GE11) targeted, pH sensitive, DTX and CUR prodrug NPs (GE11-DTX-CUR NPs) had an average diameter of 167nm and a zeta potential of -37.5mV. The particle size of the NPs was adequately maintained in serum and a sustained drug release pattern was observed. Improved inhibition of cancer cell and tumor tissue growth was shown in the GE11-DTX-CUR NPs group compared to the other groups. CONCLUSION: It can be summarized that DTX and CUR prodrug could be delivered into tumor cells simultaneously by the GE 11 targeting and the EPR effect of NPs. The resulting GE11-DTX-CUR NPs is a promising system for the synergistic antitumor treatment of prostate cancer.

Contribution of trans-aconitic acid to DPPH scavenging ability in different media.

The antioxidant properties of trans-aconitic acid (TAA) alone or in the presence of usual antioxidants were assessed by DPPH assay. The IC50 value equal to 70mM was very high compared to usual antioxidants (vitamin C and trolox). A joint experimental/theoretical study suggested that hydrogen atom abstraction in TAA by DPPH was located on -CH2- methylene bridge because the corresponding radical was more stabilized than COO(·) and CC(·) radicals. In combination with antioxidants (vitamin C, gallic acid, caffeic acid, trolox), synergy or additivity effects were noticed. The magnitude of the synergistic effect varied between 1.06 and 1.24 depending on the type and concentration of antioxidant for a concentration of TAA equal to 22.3mM. Especially, the addition of TAA at a concentration below 32mM to a solution containing 20µM of vitamin C had a synergy effect. Beyond this concentration, TAA showed an additive effect.

Graphene oxide-incorporated pH-responsive folate-albumin-photosensitizer nanocomplex as image-guided dual therapeutics.

The objective of this study was to develop an active-targeted, pH-responsive albumin-photosensitizer-incorporated graphene oxide nanocomplex as an image-guided theranostic agent for dual therapies. Herein, bovine serum albumin (BSA)-cis-aconityl pheophorbide-a (c-PheoA) conjugate was complexed with graphene oxide (GO) at ratios of 1:1, 1:0.5, and 1:0.1 with the mean hydrodynamic diameter of the resulting complex being 100-200nm. Further, with the 1:0.5 ratio, we developed a folate-BSA-c-PheoA conjugate:GO complex incorporated free PheoA (PheoA+GO:FA-BSA-c-PheoA NC) with a mean hydrodynamic diameter of 182.0±33.2nm. The release study showed that the photosensitizer from the nanocomplex was released rapidly at pH5.5 compared to that at pH7.4 when incubated for 24h. Cellular uptake results showed that the PheoA+GO:FA-BSA-c-PheoA NCs was readily taken up by B16F10 and MCF7 cancer cells. In vitro phototoxicity results showed that PheoA+GO:FA-BSA-c-PheoA NC has a higher efficacy against cancer cells than free PheoA, thereby demonstrating the synergistic effect of PS and GO in response to a single laser of 670nm. In vivo and ex vivo bioimaging results showed that fluorescence signals of higher intensity were observed in the tumor area of mice treated with PheoA+GO:FA-BSA-c-PheoA NC than those in the tumor of mice treated with free PheoA, thereby suggesting that the targeted nanocomplex selectively accumulated in the tumor area compared to free PheoA. Through antitumor study, PheoA+GO:FA-BSA-c-PheoA NC showed a synergistic effect in tumor-bearing mice by a single 671nm laser treatment. These results demonstrate that our prepared PheoA+GO:FA-BSA-c-PheoA NC can be used as a theranostic agent in phototherapies and for the photodiagnosis of cancer.

Cis-aconitic anhydride ethyl ester and phenolic compounds from the seeds of Alisma orientale.

From the seeds of Alisma orientale, cis-aconitic anhydride ethyl ester and cis-2,4,5-trihydroxycinnamic acid were isolated, together with nine known phenolic compounds and a megastigmane sesquiterpene. All compounds are reported for the first time from Alisma species.

Antiedematogenic activity and phytochemical composition of preparations from Echinodorus grandiflorus leaves.

The leaves of Echinodorus grandiflorus (Alismataceae) are traditionally used in Brazil to treat inflammatory conditions. The aim of the present study was to evaluate the antidematogenic activity of crude aqueous, dichloromethane and hydroethanolic extracts from E. grandiflorus leaves using the carrageenan-induced paw edema model in mice, along with of fractions enriched in diterpenes, flavonoids and hydroxycinnamoyltartaric acids (HCTA). Significant inhibitions of paw edema were elicited by the 50% and 70% EtOH extracts (1000 mg/kg, p.o.), as well as by the fractions enriched in diterpenes (70-420 mg/kg, p.o.) and flavonoids (7.2-36 mg/kg, p.o.). Isovitexin, isoorientin, trans-aconitic and chicoric acids were identified in all extracts by HPLC analysis. Trans-aconitic acid itself exhibited significant antiedematogenic effect (270 mg/kg, p.o.). The biological activity correlated positively with the contents of flavonoids and diterpenes, but negatively with HCTA concentrations, demonstrating the participation of the two classes of compounds in the antiedematogenic activity of E. grandiflorus.

Chemical interactions of Brachiaria plantaginea with Commelina bengalensis and Acanthospermum hispidum in soybean cropping systems.

Previous results obtained in soybean-wheat rotations under no-tillage conditions showed reductions in the seedbank of the weed species Commelina benghalensis, but no alteration in the seedbank of Acanthospermum hispidum in areas infested with Brachiaria plantaginea. Analyses of the soluble fraction of B. plantaginea indicated the predominance of aconitic acid (AA) among the aliphatic acids and ferulic acid (FA) among the phenolic acids. Laboratory bioassays using C. benghalensis and A. hispidum were carried out to evaluate phytotoxic effects of pure organic acid solutions and dilute extracts of B. plantaginea on seed germination, root development, and fungal germination. Solutions of AA and FA were prepared at 0.25, 0.50, and 1.0 mM. Extracts of B. plantaginea were diluted to obtain concentrations of AA similar to those in the prepared solutions. Seeds were sown on 0.5% agar (containing AA, FA, or diluted extract) in plastic-covered receptacles and maintained in a germination chamber for 10 days. AA and FA solutions and the B. plantaginea extract reduced germination and root length, mainly of C. benghalensis. AA also stimulated the development of endophytic fungi (Fusarium solani), which had complementary adverse effects on C. benghalensis germination. FA and AA may play important roles in reducing the seedbank of some weed species, acting directly on germination and development and, indirectly, by stimulating endophytic fungi that alter germination.

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate.

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.