Juliana Bello Baron Maurer

Effect of the native polysaccharide of cashew-nut tree gum exudate on murine peritoneal macrophage modulatory activities.

The native polysaccharide of cashew-nut tree gum exudate (CNTG) and its arabinogalactan-protein component (CNTG-AGP) were tested by using immuno-stimulant and anti-inflammatory in vitro assays of murine peritoneal macrophage activities. In the assay for immuno-stimulant activity (without previous treatment with lipopolysaccharide; LPS), CNTG increased the production of interleukin (IL)-10 and both CNTG and CNTG-AGP decreased the concentrations of IL6. When the macrophages were incubated in the presence of LPS and CNTG a decrease in the levels of nitric oxide (NO(·)) and IFN-γ was observed. The results could explain the popular use of CNTG as an anti-inflammatory. In addition, CNTG is the main component of the cashew-nut tree gum exudate, which has been considered a versatile polymer with potential pharmaceutical and food industry applications. These data may contribute to the study of the immunomodulation activity of plant polysaccharides, as well as encourage future experiments in the field of cashew-nut tree gum exudate applications.

The involvement of PUMP from mitochondria of Araucaria angustifolia embryogenic cells in response to cold stress

In this study, the responses of plant uncoupling mitochondrial protein (PUMP) and alternative oxidase (AOX) in mitochondria from embryogenic cells of A. angustifolia subjected to cold stress (4°C for 24 h or 48 h) is reported. In the mitochondria of stressed cells, PUMP activity increased by approximately 45% (at 24h and 48 h), which was determined by measuring the oxygen consumption after the addition of linoleic acid and the inhibition by BSA and ATP. PUMP activation was confirmed using transmembrane electrical potential (Δψ) assays. Immunoblot assays showed an increase of PUMP expression by 40% and 150% after 24h and 48 h of cold stress, respectively. AOX activity, measured under conditions similar to those of the PUMP assays, was only slightly increased in the mitochondria from stressed cells (at 24h and 48 h), as demonstrated by oxygen consumption experiments. Cell viability was unaffected by cold stress, indicating that the effects on PUMP and AOX were not caused by cell death. These results show that the main response of this gymnosperm to cold stress is the activation of PUMP, which suggests that this protein may be involved in the control of reactive oxygen species generation, which has been previously associated with this condition.

Arabinogalactan-proteins from cell suspension cultures of Araucaria angustifolia.

Arabinogalactan-proteins (AGPs), found in the culture medium of suspension cells of Araucaria angustifolia grown in plant growth regulator-free and plant growth regulator-containing BM media, BM0 and BM2, respectively, were evaluated quantitatively and qualitatively. The concentrated extracellular fractions (CEFs), obtained from suspension cell cultures grown for 20 days in BM0 and BM2 media yielded two fractions, CEF-0 and CEF-2, respectively. CEF-0 and CEF-2 was submitted to selective precipitation using the beta-glucosyl Yariv reagent (beta-GlcY) to isolate AGPs for structural characterization; this yielded fractions designated CEF-0YPF and CEF-2YPF, respectively. The monosaccharide composition analysis established that samples were composed of Rha, Ara, Gal and uronic acid in a molar ratio 3:37:55:5 (CEF-0YPF) and 1:37:58:4 (CEF-2YPF), although trace amounts (<0.5 mol%) of Xyl were also found. Methylation analysis of CEF-YPF fractions showed similar results for both CEF-0YPF and CEF-2YPF, with non-reducing terminal units of Araf, Arap, Galp, Rhap and Xylp, as well as 3-O-substituted and 5-O-substituted Araf units and 3-O-substituted, 6-O-substituted and 3,6-di-O-substituted Galp units. The amino acid composition analysis established Ser, Ala, and Hyp as major amino acids in both samples. In conclusion, this investigation has shown that CEF-0YPF and CEF-2YPF contain macromolecules having typical AGP characteristics, including a Hyp/Ala/Ser-rich protein moiety, a (1-->3) and/or (1-->6) linked beta-d-galactopyranosyl main chain substituted by Gal, Ara, Rha and Xyl residues, and binding affinity for beta-GlcY and monoclonal anti-AGP antibodies.

Chemical analysis of exopolysaccharide fractions and lipid compounds of the microalga Heterosigma akashiwo grown in vitro

Abstract We report on (i) in vitro growth conditions for different growth stages of Heterosigma akashiwo and (ii) isolation, purification and structural investigation of the exopolysaccharide fraction (EXO). Cell polysaccharide fractions were analyzed for chemical composition, and we performed a comparison of the lipid components between cultures subjected to either a light/dark cycle of 12 h/12 h or 24 h of light. Man, Rha and Gal were the main monosaccharides in the EXO fraction. Following the purification step, the EXO fraction yielded EXO1.0 and EXO1.5 fractions. A linkage analysis of the EXO1.0 fraction indicated the presence of a highly branched polysaccharide with a (1→4)-linked mannan backbone, and analysis of EXO1.5 indicated the presence of a complex rhamnan consisting mainly of 2- and 4-linked Rha units with the non-reducing end of Gal and Rha. In cultures subjected to a 12 h/12 h light/dark cycle, palmitic and myristic acids were the most abundant lipids, and no polyunsaturated acid was detected. However, in cultures subjected to 24 h of light, no myristic acid was detected, and stearic and palmitic acids were the most abundant fatty acids. A polyunsaturated fatty acid and fatty acids with 20 or more carbon atoms also occurred in this fraction.

Atividade ovicida e larvicida do extrato hidroalcoólico de Artemisia annua sobre parasitas gastrintestinais de bovinos

Gastrointestinal parasites cause economic losses to the cattle production, in Brazil and worldwide, mainly caused by Bunostumom sp., Cooperia sp. and Trichostrongylus sp. The aim of this study was to determine the in vitro efficacy of hydroalcoholic extract of Artemisia annua (H.7) against these parasites. The H.7 was produced after 7 days of storage at 4°C and then lyophilized. With this herbal the egg hatch test (EHT) and larval migration inhibition (LMI) were performed,in six replicates with different concentrations (0.78 to 50mg/mL). To analyze the chemistry composition the complete phytochemical screening was done. In EHT efficiency ranged from 94.08±2.58% at the highest concentration to 15.67± 0.97% in the lowest concentration. In LMI test the values ranged from 90.05±0.55% to 4.12±0.46%. Phytochemical tests showed many chemical compounds with anthelmintic properties. The results obtained in biochemical tests together with those found in in vitro tests showed that the extract produced has the potential to combat intestinal nematodes of cattle. Further studies should be conducted to maximize the effectiveness of H.7 and other extractions from A. annua, because it demonstrated excellent results in both experiments.

Assessment of anthelmintic activity and bio-guided chemical analysis of Persea americana seed extracts

The aim of this study was to characterize the extracts and fractions of Persea americana Mill. (Avocado) seeds and to determine the composition and the in vitro anthelmintic activity against third-stage larvae (L3) of Haemonchus contortus. The fresh (F) and dried (H) avocado seeds (PA) were subjected to extraction with hot water (W-PAF, W-PAH), ethanol (E-PAF, E-PAH) or methanol 70% (v/v), and partition with solvents of increasing polarity [n-hexane (H-PAF, H-PAH), chloroform (C-PAF, C-PAH), ethyl acetate (Ea-PAF, Ea-PAH), and n-butanol (B-PAF, B-PAH)], yielding a total of 14 extracts/fractions. After considering the yield, water solubility, and the preliminary results of the larval migration test (LMT), the E-PAF, E-PAH, H-PAF, and H-PAH were selected for further experiments. E-PAH presented an efficiency concentration of 50% (EC50) of 36 μg/mL on the LMT. E-PAH showed the greatest efficiency when its EC50 was compared to the other fractions (E-PAF = 147 μg/mL; H-PAF = 801 μg/mL; H-PAH = 77 μg/mL). After that, the E-PAH was chemically characterized, considering its quantitative polyphenolic and flavonoid contents by colorimetric and chromatographic techniques. E-PAH presented 50, 38, and 24 mg/g of dry matter of total phenol, condensed tannins (CT), and flavonoid contents, respectively. Using high performance liquid chromatography (HPLC) analysis, E-PAH had shown to have epicatechin (4.7 μg/mL), rutin (2.8 μg/mL), and chlorogenic acid (1.4 μg/mL) as its main constituents besides quercetin. These isolated compounds were evaluated using the LMT in order to relate the composition to the anthelmintic activity observed for E-PAH. Quercetin (EC50 = 7.8 μg/mL) and epicatechin (EC50 = 10 μg/mL) presented a higher efficiency than rutin (EC50 = 30 μg/mL). Chlorogenic acid was also tested with the LMT but did not present a significant efficiency. According to the results, the phenolic composition of E-PAH and the EC50 values obtained for the isolated phenols, it can be suggested that, besides the CT content, the presence of epicatechin and rutin contributed to the larvicidal activity of E-PAH. In conclusion, avocado seeds may be used as a source of polyphenols with promising anthelmintic applications.

Avocado leaves: Influence of drying process, thermal incubation, and storage conditions on preservation of polyphenolic compounds and antioxidant activity

ABSTRACT Total phenolic content and antioxidant activity of hydroalcoholic extract of avocado leaves (Persea americana (PA)) were determined by α,α-diphenyl-β-picrylhydrazyl, ∙NO, O2∙–, and ∙OH radical capture and reducing power test. Liquid chromatography coupled to mass spectrometry/electron spray ionization was used to identify 19 types of polyphenols, as flavan-3-ols and flavonol glycosides, in PA. Stability assays were performed to optimize conditions for preserving antioxidant activity and polyphenols during drying, thermal incubation, and storage of the extract. Extracts, obtained from leaves dried at 70°C or higher, showed lower values for total phenolic content and antioxidant activity. Compared with the values of leaves dried at 60°C or lower, these differences were statistically significant (p ≤ 0.05). Incubating PA in solution, at 40°C–100°C for up to 8 h, did not produce significant changes in total phenolic content or antioxidant activity. No statistically significant changes in the total phenolic content or antioxidant activity (p ≤ 0.05) occurred after storage at 4°C and 25°C for up to 10 months. PA was shown to possess diverse polyphenolic compounds with robust antioxidant activity. Precise control of drying temperature was an important parameter for maintaining high levels of phenolic compounds and antioxidant activity of PA.

Modulating Effects of Arabinogalactans from Plant Gum Exudates on Human Complement System

Gum arabic and cashew nut tree gum exudate polysaccharide (CNTG) are plant polysaccharides composed of galactose and arabinose known as arabinogalactans (AGs). Although these fractions are used in food and pharmaceutical industry, cases of allergic reactions were described in clinical reports. As AGs were reported as modulators of the classical (CP) and alternative pathways (AP) of complement system (CS), in the present work, we investigate whether gum arabic and CNTG have an effect on both CS pathways. The complement fixation tests were performed with (CP‐30 and AP‐30) and without pre‐incubation (CP‐0 and AP‐0). For CP‐30, CNTG and gum arabic (833 μg/ml) showed a reduction of 28.0% (P = 0.000174) and 48.5% (P = 0.000143), respectively, on CP‐induced haemolysis. However, no effect was observed for CP‐0 in the CP‐induced haemolysis. For AP‐30, both CNTG and gum arabic (833 μg/ml) showed 87% reduction on the CP‐induced haemolysis, with IC50 values of 100 and 7 μg/ml, respectively. For AP‐0, a reduction of 11.3% for gum arabic and no effect for the CNTG on the CP‐induced haemolysis were observed. These results suggested that gum arabic and CNTG could be acting as activators of the CS. Thus, this effect on the CS, especially on the AP, which accounts for up to 80–90% of total CS activation, indicates that both fractions may be harmful because of their potential pro‐inflammatory action. Considering that CS activation induces inflammatory response, further studies confirming this immunomodulatory effect of these fractions are required to insure their safe use.

Efeito anticoccidiano de extrato hidroalcoólico de Artemisia annua em camas de aves contaminadas com Eimeria sp

The aim of this study was to determine the efficacy of hydroalcoholic extract of Artemisia annua against oocysts of Eimeria sp. in contaminated poultry beds. The extract was produced after 7 days of storage at 4°C, which was used to perform the phytochemical screening; the artemisinin measurement; the total phenolic; antioxidant testing and toxicity test. To test the anticoccidial activity, the birds space composed of shaver trees, were contaminated with 5000 oocysts. Four treatment were formed, in triplicate, were used in different concentrations as G1: 12mg/mL, G2:= 9mg/mL, G3: 6mg/mL, and C-: water. After contamination 800 mL of the herbal at different concentrations were sprayed on the bed and collected, in triplicate, 10 cm2 each site, randomly, at times: 0, 3, 6, 24, 48, and 72 hours after application. In phytochemical analysis, were shown compounds with antiparasitic properties, such as flavonoids and tannins. The herbal contained 59.409±1.47mg/dL artemisinin. The product at a concentration of 12mg.mL-1 showed efficacy from 44.25 to 40.71%. The results of biochemical tests, with the in vitro test showed that the extract has produced high potential for combating Eimeria sp.

Chemical Characterization of Opuntia ficus-indica (L.) Mill. Hydroalcoholic Extract and Its Efficiency against Gastrointestinal Nematodes of Sheep

Opuntia ficus-indica (L.) Mill. is a xerophylous plant that originated in tropical and subtropical America. This plant is popularly known in Brazil as “palma forrageira” (cactus pear) and plays a fundamental role in animal nutrition, mainly in the Northeastern semi-arid region of the country. The plant has several uses since it presents bioactive compounds that confer biological and pharmacological properties. In this context, the cactus pear can also be considered a potential product to combat parasite infections. The objective of this study was to chemically characterize the O. ficus-indica hydroalcoholic extract (OFIEOH) and to determine its efficacy against gastrointestinal parasites using in vitro tests. Initially, the hydroalcoholic extract from cladode peels of O. ficus-indica was produced by maceration for 21 days. For the chemical characterization, colorimetric dosages were performed for carbohydrates, proteins, phenols and condensed tannins. Liquid chromatography coupled to mass spectrometry/electron spray ionization (LC-MS/ESI) was used to characterize the polyphenolic profile of the OFIEOH extract. Fifteen compounds were identified in the OFIEOH extract, such as methyl, glycosylated and aglycone quercetin derivatives and aglycone and glycosylated kaempferol derivatives. Tri-glycosylated methyl quercetin derivatives were the main compounds identified. In vitro egg hatch (EHT) and larval migration tests (LMT) were used in a range of concentrations of OFIEOH from 12.5 to 100 mg/mL for EHT and 12.5 to 200 mg/mL for LMT. In addition, the LMT was used to test ivermectin (IVM) (from 11.4 to 57.1 µM), associated with the inhibitory concentration of 50% (IC50) for OFIEOH. The combination of OFIEOH (12.5 to 200 mg/mL) plus the IC50 of IVM was also tested. The efficacy of OFIEOH alone varied from 19.33 to 90.0% using the EHT. The LMT revealed an efficacy of 5.78 to 77.26% for the extract. Both tests showed a concentration-dependence inhibitory effect. We found a drug-extract antagonistic neutralizing effect when doses of IVM were added to OFIEOH (maximum efficacy of 73.78%), while a positive additive effect was observed when OFIEOH was added to the IC50 of IVM (IC50 of 82.79 for OFIEOH alone against an IC50 of 55.08 of OFIEOH + IVM). The data from this work indicate that OFIEOH alone may be considered as a suitable ecofriendly product to control gastrointestinal parasites of sheep, offering a more holistic approach to improve animal farming and welfare. The drug-extract interaction is also a promising therapeutic alternative, reducing the final dose to the host, with an optimum combination effect.