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Severinia buxifolia (Rutaceae) is often used as a traditional medical plant. The present study was carried out to estimate the effects of solvents (petroleum ether and hexane: ethyl acetate) used in liquid–liquid extraction to total terpenoid content (TTC) and in vitro anti‐inflammatory activity of the extracts obtained from S. buxifolia bark. The results showed that solvent fractionation increased the TTC compared with crude extracts. The hexane: ethyl acetate bark extract fraction (HEF) had the highest TTC (731.48 µg/ml) in comparison with the petroleum ether bark extract fraction (PEF) (564.81 µg/ml) and the crude extract (CE) (184.26 µg/ml). In addition, one of composition of terpenoid of S. buxifolia, namely ursolic acid, was determined by HPLC method from the crude CE and the fractions PEF and HEF: 2.44 μg/g DW, 3.56 μg/g DW and 5.04 μg/g DW, respectively. The samples had an in vitro anti‐inflammatory activity comparable with that of two reference standards (aspirin and indomethacin). Particularly, the HEF fraction had the highest in vitro anti‐inflammatory activity (i.e., albumin denaturation: IC50 = 147.91 μg/mL, heat‐induced hemolysis: IC50 = 159.91 μg/mL, proteinase inhibition: IC50 = 117.72 μg/mL, and lipoxygenase activity: IC50 = 90.45 μg/mL). Besides, the preliminary experiments of this study were conducted to determine the influences of maceration factors (solvent type, temperature, and time) for S. buxifolia bark extract. The TTC ranged from 453.70 to 842.59 mg linalool/g DW, and the extraction yield from 2.40% to 5.120% in all extracts. Based on TTC and EY, the hexane: acetone mixture is recommended as the optimal solvent to obtain the crude bark extract (CE) at 46°C for 24 hr of maceration. Extracts of S. buxifolia bark are a promising source for the treatment of inflammatory diseases. Solvent fractionation was found to increase the TTC compared with crude extracts based on colorimetric assay using linalool as standard reagent. The samples were indicated to have in vitro anti‐inflammatory comparable with two reference standards (aspirin and indomethacin) based on the inhibition of albumin denaturation, proteinase activity, heat‐induced hemolysis, and lipoxygenase assay.

Lignin fractions having different molecular weights and varied chemical structures isolated from kraft lignins of both softwood and hardwood via a sequential solvent fractionation technique were incorporated into a tunicate cellulose nanofibers (CNF)—starch mixture to prepare 100% bio-based composite films. The aim was to investigate the impact of lignin structural diversity on film performance. It was confirmed that lignin’s distribution in the films was dependent on the polarity of solvents used for fractionation (acetone > methanol > ethanol > ethyl acetate) and influenced the optical properties of the films. The –OH group content and molecular weight of lignin were positively related to film density. In general, the addition of lignin fractions led to decrease in thermal stability and increase in Young’s modulus of the composite films. The modulus of the films was found to decrease as the molecular weight of lignin increased, and a higher amount of carboxyl and phenolic –OH groups in the lignin fraction resulted in films with higher stiffness. The thermal analysis showed higher char content formation for lignin-containing films in a nitrogen atmosphere with increased molecular weight. In an oxygen atmosphere, the phenol content, saturated side chains and short chain structures of lignin had impacts on the maximum decomposition temperature of the films, confirming the relationship between the chemical structure of lignin and thermo-oxidative stability of the corresponding film. This study addresses the importance of lignin diversities on composite film performance, which could be helpful for tailoring lignin’s applications in bio-based materials based on their specific characteristics.

The present study investigates the effect of Ulva lactuca extract as seed-priming agent for tomato plants under optimal and salinity stress conditions. The aims of this experiment were to assess the effect of seed priming using Ulva lactuca extract in alleviating the salinity stress tomato plants were subjected to, and to find out the possible mechanism of actions behind such a positive effect via means of fractionation of the crude extract and characterization. Salinity application decreased the plant biomass and altered different physiological traits of tomato. However, the application of Ulva lactuca methanol extract (ME) and its fractions (residual fraction (RF), chloroform fraction (CF), butanol fraction (BF), and hexane fraction (HF)) at 1 mg·mL−1 as seed priming substances attenuated the negative effects of salinity on tomato seedlings. Under salinity stress conditions, RF application increased the tomato fresh weight; while ME, RF, and HF treatments significantly decreased the hydrogen peroxide (H2O2) concentration and antioxidant activity in tomato plants. The biochemical analyses of Ulva lactuca extract and fractions showed that the RF recorded the highest concentration of glycine betaine, while the ME was the part with the highest concentrations of total phenols and soluble sugars. This suggests that these compounds might play a key role in the mechanism by which seaweed extracts mitigate salinity stress on plants.

A series of ethylene/1-octene copolymers synthesized with diamine-bis(phenolate) complexes activated with Al(iBu)3/[Ph3C][B(C6F5)4] were subjected to preparative temperature rising elution fractionation (TREF). The complexes used differed in the type of metallic center (Zr or Ti) or the amine donor in the pendant arm of the ligand (NMe2 or NiPr2). The obtained fractions were then characterized via FTIR, DSC and GPC methods. It was found that all the copolymers had very broad chemical composition distributions, and the most heterogeneous was the copolymer produced by the titanium complex bearing a ligand with the NiPr2 donor group. The difference in the comonomer incorporation into the macromolecules of the fractions was as high as 8.3 mol%. The melting temperature and molecular weight of the fractions changed nearly linearly with the increased elution temperature. Copolymers produced by zirconium catalysts were also fractionated by molecular weight using the solvent/non-solvent technique with subsequent analysis of the fractions. It was shown that the fractions have a similar composition, low molecular weight distribution and very broad comonomer distribution. Therefore, the comonomer content in the fractions was not a function of the molecular weight as was observed for the copolymers synthesized with the Ziegler–Natta catalysts.

Background Many studies have revealed that bioactive compounds for different indications are not extracted from plants with water, the only extractant practically available to rural communities. We compared the acaricidal activity of acetone extracts of 13 species used traditionally to protect cattle against ticks. We also investigated if the extraction of biologically active compounds against Rhipicephalus (Boophilus) decoloratus ticks could be enhanced by adding a liquid soap that is locally available to smallholder farmers. Methods A total of 13 plant species selected based on reported traditional use in Zimbabwe, were dried and finely ground before extraction with water, or water plus a surfactant, or acetone. The adapted Shaw Larval Immersion Test (SLIT) method was used to determine the activity of acetone and crude water extracts with or without liquid soap against the tick larvae. The activity of four fractions of crude acetone extracts (extracted using solvents of different polarity), of the most active plant species, Maerua edulis (tuber and leaf) was also compared to identify the most active fraction. Results Aqueous plant extracts were not toxic to ticks, but the addition of 1% liquid soap as a surfactant increased mortality of the R. (B) decoloratus larvae significantly. With the Maerua edulis tuber extract, the efficacy of the 1% liquid soap was comparable to that of the amitraz based commercial synthetic acaricide. The use of acetone as an extractant, also increased the mortality of the tick larvae in all the plant species. With M. edulis (tuber and leaf), Monadenium lugardae and Kleinia sp. acetone extracts, the activity was comparable to that of the positive control (a commercially available amitraz-based synthetic acaricide). The non-polar fractions of the acetone extract of leaf and tuber of M. edulis caused up to 100% mortality. This indicates that non-polar to intermediate polarity compounds are responsible for the acaricidal activity. Conclusion Organic solvents such as acetone extracted active compounds but water did not. By adding commonly available dishwashing soap to water active compounds were extracted leading to a high acaricidal activity of the plant extracts. In some cases, it was as active as non-polar extracts and a synthetic commercial acaricide (positive control). This approach makes it possible for the smallholder farmers and traditional healers to extract biologically active compounds from plants by using water.

Eurycoma longifolia is a popular folk medicine in South East Asia. This study was focused on saccharide-containing compounds including saponins, mainly because of their medical potentials. Different organic solvents such as ethyl acetate, butanol, and chloroform were used to fractionate the phytochemical groups, which were consequently precipitated in cold acetone. Solvent fractionation was found to increase the total saponin content based on colorimetric assay using vanillin and sulfuric acid. Ethyl acetate fraction and its precipitate were showed to have the highest crude saponins after acetone precipitation. The samples were shown to have anti-proliferative activity comparable with tamoxifen (IC50 = 110.6 µg/mL) against human breast cancer cells. The anti-proliferative activities of the samples were significantly improved from crude extract (IC50 = 616.3 µg/mL) to ethyl acetate fraction (IC50 = 185.4 µg/mL) and its precipitate (IC50 = 153.4 µg/mL). LC-DAD-MS/MS analysis revealed that the saccharide-containing compounds such as m/z 497, 610, 723, 836, and 949 were abundant in the samples, and they could be ionized in negative ion mode. The compounds consisted of 226 amu monomers with UV-absorbing property at 254 nm, and were tentatively identified as formylated hexoses. To conclude, solvent fractionation and acetone precipitation could produce saccharide-containing compounds including saponins with higher anti-proliferative activity than crude extract against MCF-7 cells. This is the first study to use non-toxic solvents for fractionation of bioactive compounds from highly complex plant extract of E. longifolia.

Bacterial polyhydroxyalkanoates (PHAs) are promising bio-based biodegradable polyesters. It was recently reported that novel PHA block copolymers composed of (R)-3-hydroxybutyrate (3HB) and (R)-2-hydroxybutyrate (2HB) were synthesized by Escherichia coli expressing PhaCAR, a chimeric enzyme of PHA synthases derived from Aeromonas caviae and Ralstonia eutropha. In this study, the sequence-regulating PhaCAR was applied in the natural PHA-producing bacterium, R. eutropha. During the investigation, (R/S)-2HB was found to exhibit strong growth inhibitory effects on the cells of R. eutropha. This was probably due to formation of excess 2-ketobutyrate (2KB) from (R/S)-2HB and the consequent l-valine depletion caused by dominant l-isoleucine synthesis attributed to the excess 2KB. Deletion analyses for genes of lactate dehydrogenase homologs identified cytochrome-dependent d-lactate dehydrogenase (Dld) and [Fe-S] protein-dependent l-lactate dehydrogenase as the enzymes responsible for sensitivity to (R)-2HB and (S)-2HB, respectively. The engineered R. eutropha strain (phaCAR+, ldhACd-hadACd+ encoding clostridial (R)-2-hydroxyisocaproate dehydrogenase and (R)-2-hydoroxyisocaproate CoA transferase, ∆dld) synthesized PHA containing 10 mol% of 2HB when cultivated on glucose with addition of sodium (RS)-2HB, and the 2HB composition in PHA increased up to 35 mol% by overexpression phaCAR. The solvent fractionation and NMR analyses showed that the resulting PHAs were most likely to be block polymers consisting of P(3HB-co-3HV) and P(2HB) segments, suggesting that PhaCAR functions as the sequence-regulating PHA synthase independently from genetic and metabolic backgrounds of the host cell.Key points(R/S)-2-hydroxubutyrates (2HB) caused l-valine deletion in Ralstonia eutropha(R)- and (S)-lactate/2HB dehydrogenases functional in R. eutropha were identifiedThe engineered R. eutropha synthesized block copolymers of 2HB-containing polyhydroxyalkanoates on glucose and 2HB

Iturin, a structurally cyclic heptapeptides with a number of homologous derivatives has broad-spectrum antimicrobial and antibiofilm properties. The present study elucidates the structure and antimicrobial efficacy of iturin C 3 biosurfactant (BS) produced by the endophytic bacterium Bacillus sp. LLB-04. Bacillus sp. LLB-04 was isolated from the leaves of hemiparasite Scurrula parasitica L. during the winter season. The biosurfactant was produced in a static batch culture of 120 h using Nutrient Broth (NB) medium and was extracted by a series of solvent systems. The BS was purified by solvent fractionation and preparative High-Performance Liquid Chromatography (HPLC) method, and then it was structurally characterized through various analytical methods. Its antimicrobial and antibiofilm activities were tested against 0, to 18 h old topical and food-borne pathogens. Furthermore, the cellular aggregation and bacterial cell adhesion on polystyrene and urethral catheters were checked at the Biofilm inhibitory concentration (BIC). The cell line toxicity of BS (0–1.568 mg/ml) was tested against the human embryonic lung tissue L-132 and human alveolar epithelial cancer cell line, and the in silico mode of action was studied using standard methods. From the spectroscopic studies of 96 h culture harvested BS revealed that Bacillus sp. LLB-04 (GenBank Accession No.: MF037706) produced the BS as iturin C 3 . The BS had broad-spectrum antimicrobial with minimum inhibitory concentration (MIC) values ranging from 0.1 to 1.6 mg/ml and an average biofilm inhibition concentration (BIC) of 0.8–1.6 mg/ml in 18 h old cells where biofilm formation was inhibited up to 46.4 times at 1.6 mg/ml concentration. It could also destabilize 40–48 h old preformed biofilm and had a synergistic response with streptomycin ( Bacillus subtilis MTCC 411, Escherichia coli MTCC 730), ciprofloxacin ( B. subtilis MTCC 411, E. coli MTCC 730), and vancomycin ( Staphylococcus epidermidis MTCC 3086, B. subtilis MTCC 411). It had antiproliferative activity (0.1–0.8 mg/ml) on cancer cell lines. In-silico protein–ligand interactions predicted that it could interact with different membrane proteins of topical and food-borne pathogens. Thus, the study revealed for the first time that the endophytic Bacillus sp. could be exploited for large-scale production of iturin C 3 that could be used in combating biofilm formation and cellular adhesion of topical and food-borne pathogens. Graphical abstract

Lignin fractionation aims at dividing the original lignin material into more uniform fractions that have the qualities desired for further processing of the material. Solvent fractionation is a versatile method that produces a soluble and an insoluble fraction with varying compositions, including molecular weight distribution, polydispersity (PDI), and phenolic hydroxyl (OHph) content. These properties are key factors in the further downstream applicability of lignin. Kraft, organosolv, and hydrolysis lignins were fractionated using multiple solvents and their water solutions with a single-stage fractionation protocol. The produced fractions were analyzed for their molecular size distribution and OHph content. Soluble fractions were discovered with desirable qualities, including a narrow size distribution (lowest PDI 1.4) and accumulation of OHph groups (up to 3.05 mmolOH/glignin). Three fractionation solvents, i.e., pure isopropanol, pure ethanol, and 30% γ-valerolactone, were found to produce solvent-specific soluble fractions that had relatively identical characteristic Mw, Mn, and PDI values regardless of the initial lignin material. This is crucially important new data that is beneficial for robust lignin applicability at industrial scale. Other important observations were made regarding water-alcohol solutions, as the increase in initial solid content resulted in changes in the composition of the fractions produced. Further applicability of the obtained fractions is examined in this paper. Patterns as well as differences between the three lignin materials were observed in their dissolution and the resulting compositions of the soluble fractions. This wide comparable dataset of industrially relevant solvents and lignin materials provides significant insight into the possibilities of lignin downstream processing.

The extraction and characterization of defined and carboxyl-rich oligomeric lignin fragments with narrow molecular weight distribution is presented herein. With regard to the well-known pulp bleaching process, oxidative lignin depolymerization was investigated using hydrogen peroxide in an aqueous alkaline solution (i.e., at T = 318 K, t = 1 h) and subsequent selective fractionation with a 10/90 (v/v) acetone/water mixture. While the weight average molecular weight (MW) of lignin in comparison to the starting material was reduced by 82% after oxidation (T = 318 K, t = 1 h, clignin = 40 g L−1, cH2O2 = 80 g L−1, cNaOH = 2 mol L−1) and subsequent solvent fractionation (T = 298 K, t = 18 h, ccleavage product = 20 g L−1), the carboxyl group (–COOH) content increased from 1.29 mmol g−1 up to 2.66 mmol g−1. Finally, the successful scale-up of this whole process to 3 L scale led to gram amounts (14% yield) of oligomeric lignin fragments with a MW of 1607 g mol−1, a number average molecular weight (MN) of 646 g mol−1, a narrow polydispersity index of 3.0, and a high –COOH content of 2.96 mmol g−1. Application of these oligomeric lignin fragments in epoxy resins or as adsorbents is conceivable without further functionalization.