General and genetic toxicology studies of Aleurites moluccana (L.) Willd. seeds in vitro and in vivo assays.

ETHNOPHARMACOLOGY RELEVANCE: Aleurites moluccana is popularly used for the diseases like ulcers, fever, headache, asthma, conjunctivitis, gonorrhea, inflammation, hepatitis, and rheumatism. The seed, also known as "noz da Índia", has been popularly consumed for weight loss purposes but reports of toxicity have been associated with its ingestion. In the literature, there are not enough studies to elucidate its toxicology, so evaluating the general and genetic toxicological of A. moluccana seeds can provide data to ensure their intake. AIM OF THE STUDY: The objective of the present study was to elucidate the oral toxicity, mutagenicity, genotoxicity and cytotoxicity of A. moluccana seeds in vitro and in vivo assays. MATERIALS AND METHODS: The chemical composition of the aqueous extract of A. moluccana seeds (AEAMS) was analyzed in relation to phenolic compounds, tannins, flavonoids and fatty acid. For the in vitro assays, the cytotoxic potential was assessed by the MTS assay whereas the mutagenic potential was assessed by the Ames test. For in vivo assays, was conducted an acute oral toxicity study, with "Up-and-Down Procedure" and repeated dose toxicity with "Repeated Dose 28-Day Oral Toxicity". To assess genetic damage, mutagenic potential was assessed by the micronucleus test whereas the polychromatic erythrocyte/normochromatic erythrocyte ratio was obtained with bone marrow cells to determine the cytotoxic potential and genotoxic potential was assessed by the comet assay using peripheral blood cells. RESULTS: AEAMS did not show cytotoxic and mutagenic potential in vitro. No clinical signs of toxicity were observed in animals after the acute oral toxicity test, suggesting that the LD50 of aqueous extract of A. moluccana seeds > 2000 mg/kg in a single dose by intragastric route. However, in toxicity at repeated doses for 28 days, the doses initially established (250; 500 and 750 mg/kg/day by intragastric route) caused mortality in the animals and the reestablished doses (25, 50 and 100 mg/kg/day by intragastric route) showed no changes in parameters or clinical signs attributed to toxicity. Furthermore, AEAMS also did not show mutagenic, genotoxic and cytotoxic potential in vivo. CONCLUSIONS: AEAMS did not show cytotoxic, genotoxic and mutagenic potential in vitro and in vivo. And although the AEAMS has an LD50 > 2000 mg/kg, and does not have physiological, biochemical, hematological, histopathological changes or clinical signs related to toxicity when administered in low concentrations and for a short period, in high concentrations and continued use caused toxicity and mortality in Wistar rats. In order to obtain complementary results, is recommended highly that further mid and long-term toxicological studies are investigated, and in no-rodent specie.

Phytochemical Analysis and Antinociceptive Properties of Hydroalcoholic Extracts of Aleurites moluccanus Bark.

Aleurites moluccanus is used in folk medicine to treat many diseases including pain and inflammatory processes in general. Considering the potential of the leaf extract, evidenced in a previous study, the present study investigates the antinociceptive and anti-inflammatory properties of the hydroethanolic extract of A. moluccanus bark and isolated compounds in animal models of pain. The antinociceptive and anti-inflammatory activities of A. moluccanus bark were evaluated through hyperalgesia induced by carrageenan, PGE2, cytokines, bradykinin, epinephrine, Freund's complete adjuvant, and lipopolysaccharide. Five compounds were isolated from the dichloromethane bark extract: acetyl aleuritolic acid, atraric acid, spruceanol, (5ß,10α)-12-hydroxy-13-methoxy-8,11,13-podocarpatrien-3-one and sonderianol. To optimize the extraction conditions, ethanol 50, 70, and 90°GL were used as extracting solvent, in a 1 : 20 (w/v) drug : solvent ratio, under stirring at room temperature for 4 h. The extracts were named AMC50, AMC70, and AMC90, respectively. These extracts were administered to mice (250 mg/kg, p. o.) with reduced mechanical hyperalgesia activity in the carrageenan test. Of these, AMC90 showed the best results. Pure (5ß,10α)-12-hydroxy-13-methoxy-8,11,13-podocarpatrien-3-one showed a beneficial effect for up to 48 hours after the administration of carrageenan, while acetyl aleuritolic acid was effective only in the first hour. AMC90 was able to reverse the analgesia induced only by prostaglandin E2 and tumor necrosis factor. We also induced hyperalgesia using the lipopolysaccharide and Freund's complete adjuvant models, with positive results. These results support the antinociceptive and anti-inflammatory activity of A. moluccanus bark extract. The observed effects are partly due to the presence of acetyl aleuritolic acid, atraric acid, and (5ß,10α)-12-hydroxy-13-methoxy-8,11,13-podocarpatrien-3-one.

Vernicia fordii (Hemsl.) Airy Shaw extract stimulates insulin secretion in pancreatic ß-cells and improves insulin sensitivity in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernicia fordii (Hemsl.) Airy Shaw (V. fordii) is also known as the tung tree and its leaves and fruit are used as an oriental treatment for dyspepsia, edema, and skin diseases, which are known as diabetic complications. AIM OF THE STUDY: In this study, we aimed to investigate the methanolic extract (VF5) of the leaves of V. fordii as an insulin secretagogue and its probable mechanism and verify the effect in HFD-fed mice. MATERIALS AND METHODS: The insulin secretagogue activity of different doses of VF5 (0.1, 0.3 and 1.0 µg/ml) was assessed using in vitro insulin secretion assay and confirmed the anti-diabetic effect in mice fed HFD for 4 weeks with different doses of VF5 (10, 20 and 50 mg/kg oral) for another 6 weeks. Glbenclamide (30 mg/kg, oral) was used as positive control drug. The possible mechanisms were evaluated by using Gö6983 (10 µM), U73122 (10 µM) and nifedipine (10 µM). The major constituents of VF5 were analyzed by UPLC-QToF-MS and 1H and 13C NMR spectroscopy. RESULTS: UPLC-QToF-MS and NMR spectroscopy analysis indicated that one of the main active components of VF5 was tigliane-diterpene esters. VF5 functioned as an insulin secretagogue and enhanced mitochondria respiration and insulin homeostasis. We confirmed that VF5 preserved the ß-cell and reduced the ß-cell expansion which caused by metabolic stress under HFD. The antidiabetic role of VF5 in HFD fed mice was assessed by glucose tolerance test (GTT) and insulin tolerance test (ITT), fasting plasma insulin level, fasting blood glucose level, AKT signal in peripheral tissue in the absence of toxic effects. Mechanistically, insulinotropic effect of VF5 was mediated by activation of PKCα via intracellular Ca2+ influx and enhanced mitochondria function. CONCLUSION: VF5 exhibits potent insulin secretagogue function and improves insulin sensitivity and protection of pancreatic ß-cells from metabolic stress without toxicity. Taken together, our study suggests that VF5 could be potentially used for treating diabetes and metabolic diseases through improving ß-cell function.

Weight loss supplement causing acute heart block in a child.

A 16-year-old male was admitted to the paediatric ICU with acute onset of vomiting, somnolence, and chest pain, and electrocardiogram showing 2nd degree heart block after ingesting an Aleurites moluccana (Candlenut) seed as a herbal weight loss supplement. Electrocardiogram showed progressively worsening heart block with down-sloping of the ST segments, resembling digoxin toxicity. After 2 days of ICU observation, his symptoms began to improve and eventually resolved. The side effects of herbal supplements are often unknown but by analysing cases such as these, physicians can develop a better understanding of these substances to help guide management.

Aleurites moluccanus and its main active constituent, the flavonoid 2″-O-rhamnosylswertisin, in experimental model of rheumatoid arthritis.

ETHNOPHARMMACOLOGICAL RELEVANCE: Aleurites moluccana is used in folk medicine to treat pain, fever, asthma, hepatitis, gastric ulcer and inflammatory process in general, and the nut oil had been topically applied to treat arthritis and other joint pain, however the seeds are classified as toxic for oral use. AIM: Faced with the need for new alternative to treat the symptoms and modify rheumatoid arthritis (RA) the aim of this work was to evaluate the effects of A. moluccanus' leaves dried extract in rats and mice submitted to complete Freund adjuvant (CFA)-induced RA. MATERIAL AND METHODS: Wistar Rats and Swiss mice were submitted to CFA-induced RA in the right hindpaw. They received A. moluccanus extract (orally; p.o.), dexamethasone (subcutaneously), 2″-O-rhamnosylswertisin (p.o.) or vehicle (p.o.), from the 14th day after the CFA injection for up to 8 days. The mechanical hypersensitivity was evaluated using the von Frey filaments and the paw-oedema was measured using a plethysmometer. The rats' injected hindpaw was used to perform the histological analysis. RESULTS: A. moluccanus was able to significantly reduce the mechanical hypersensitivity in both ipsi- and contralateral hindpaws of mice injected with CFA, in a dose dependent manner. Furthermore, the paw-oedema was progressively reduced by A. moluccanus. Similar results were obtained for the positive-control drug dexamethasone and the isolated compound 2″-O-rhamnosylswertisin. Besides the effects mentioned above, the extract was also effective to repair the joint damage in CFA-induced RA rats, including reduction of fibrosis, cartilage degradation and bone erosion scores. CONCLUSION: These results together with the literature data reinforce the anti-hypersensitivity and anti-inflammatory activity of A. moluccanus extract. Part of the observed effects is due to the presence of the compound 2″-O-rhamnosylswertisin. The fact that the extract acted as a disease modifier point this herbal product as a promisor and safe tool to treat RA and other associated chronic diseases.

Tung Tree (Vernicia fordii) Genome Provides A Resource for Understanding Genome Evolution and Improved Oil Production.

Tung tree (Vernicia fordii) is an economically important woody oil plant that produces tung oil rich in eleostearic acid. Here, we report a high-quality chromosome-scale genome sequence of tung tree. The genome sequence was assembled by combining Illumina short reads, Pacific Biosciences single-molecule real-time long reads, and Hi-C sequencing data. The size of tung tree genome is 1.12 Gb, with 28,422 predicted genes and over 73% repeat sequences. The V. fordii underwent an ancient genome triplication event shared by core eudicots but no further whole-genome duplication in the subsequent ca. 34.55 million years of evolutionary history of the tung tree lineage. Insertion time analysis revealed that repeat-driven genome expansion might have arisen as a result of long-standing long terminal repeat retrotransposon bursts and lack of efficient DNA deletion mechanisms. The genome harbors 88 resistance genes encoding nucleotide-binding sites; 17 of these genes may be involved in early-infection stage of Fusarium wilt resistance. Further, 651 oil-related genes were identified, 88 of which are predicted to be directly involved in tung oil biosynthesis. Relatively few phosphoenolpyruvate carboxykinase genes, and synergistic effects between transcription factors and oil biosynthesis-related genes might contribute to the high oil content of tung seed. The tung tree genome constitutes a valuable resource for understanding genome evolution, as well as for molecular breeding and genetic improvements for oil production.

Molecular mechanism of the extended oil accumulation phase contributing to the high seed oil content for the genotype of tung tree (Vernicia fordii).

BACKGROUND: Oil from seeds of the tung tree (Vernicia fordii) has unique drying properties that are industrially important. We found that the extended oil accumulation period was related to the high seed oil content at maturity among tung tree population. In order to understand the molecular mechanism underlying the high oil content in tung tree seed, Tree H and L were adopted for the further investigation, with seed oil content of about 70 and 45%, respectively. We compared the transcriptomic changes of seed at various times during oil accumulation between the two trees. RESULTS: Transcriptomes analysis revealed that many genes involved in glycolysis, fatty acid synthesis, and tri-acyl glyceride assembly still kept high expression in the late period of seed oil accumulation for Tree H only. Many genes in fatty acid degradation pathway were largely up regulated in the late period of seed oil accumulation for Tree L only. Four transcription factors related to fatty acid biosynthesis had different expression pattern in the seed oil accumulation period for the two trees. WRI1 was down regulated and kept the low expression in the late period of seed oil accumulation for the two trees. PII, LEC1 and LEC1-LIKE extended the high expression in the late period of seed oil accumulation in Tree H only. CONCLUSIONS: The continued accumulation of oil in the late period of seed oil accumulation for Tree H was associated with relatively high expression of the relevant genes in glycolysis, fatty acid synthesis and tri-acyl glyceride assembly. PII, LEC1, and LEC1-LIKE rather than WRI1 should play an important role in the oil continual accumulation in the late period of seed oil accumulation in Tree H. This study provides novel insight into the variation in seed oil content and informs plant breeding strategies to maximize oil yield.

Production of Biodiesel from Candlenut Oil Using a Two-step Co-solvent Method and Evaluation of Its Gaseous Emissions.

Candlenut oil (CNO) is a potentially new feedstock for biodiesel (BDF) production. In this paper, a two-step co-solvent method for BDF production from CNO was examined. Firstly, esterification of free fatty acids (FFAs) (7 wt%) present in CNO was carried out using a co-solvent of acetonitrile (30 wt%) and H2SO4 as a catalyst. The content of FFAs was reduced to 0.8 wt% in 1 h at 65°C. Subsequent transesterification of the crude oil produced was carried out using a co-solvent of acetone (20 wt%) and 1 wt% potassium hydroxide (KOH). Ester content of 99.3% was obtained at 40°C in 45 min. The water content in BDF was 0.023% upon purification using vacuum distillation at 5 kPa. The components of CNO BDF were characterized using a Fourier-transform infrared spectrometry and gas chromatography-flame ionization detector. The physicochemical properties of BDF satisfied the ASTM D6751-02 standard. The gaseous exhaust emissions from the diesel engine upon combustion of the BDF blends (B0-B100) with petrodiesel were examined. The emissions of carbon monoxide and hydrocarbons were clearly lower, but that of nitrogen oxides was higher in comparison to those from petro-diesel.

Proteomic Analysis of Tung Tree (Vernicia fordii) Oilseeds during the Developmental Stages.

The tung tree (Vernicia fordii), a non-model woody plant belonging to the Euphorbiaceae family, is a promising economic plant due to the high content of a novel high-value oil in its seeds. Many metabolic pathways are active during seed development. Oil (triacylglycerols (TAGs)) accumulates in oil bodies distributed in the endosperm cells of tung tree seeds. The relationship between oil bodies and oil content during tung tree seed development was analyzed using ultrastructural observations, which confirmed that oil accumulation was correlated with the volumes and numbers of oil bodies in the endosperm cells during three different developmental stages. For a deeper understanding of seed development, we carried out proteomic analyses. At least 144 proteins were differentially expressed during three different developmental stages. A total of 76 proteins were successfully identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF/MS/MS). These proteins were grouped into 11 classes according to their functions. The major groups of differentially expressed proteins were associated with energy metabolism (25%), fatty acid metabolism (15.79%) and defense (14.47%). These results strongly suggested that a very high percentage of gene expression in seed development is dedicated to the synthesis and accumulation of TAGs.

Molecular cloning and expression analysis of a novel BCCP subunit gene from Aleurites moluccana.

Aleurites moluccana L. is grown as a roadside tree in southern China and the oil content of its seed is higher than other oil plants, such as Jatropha curcas and Camellia oleifera. A. moluccana is considered a promising energy plant because its seed oil could be used to produce biodiesel and bio-jet fuel. In addition, the bark, leaves, and kernels of A. moluccana have various medical and commercial uses. Here, a novel gene coding the biotin carboxyl carrier protein subunit (BCCP) was cloned from A. moluccana L. using the homology cloning method combined with rapid amplification of cDNA end (RACE) technology. The isolated full-length cDNA sequence (designated AM-accB) was 1188 bp, containing a 795-bp open reading frame coding for 265 amino acids. The deduced amino acid sequence of AM-accB contained a biotinylated domain located between amino acids 190 and 263. A. moluccana BCCP shows high identity at the amino acid level to its homologues in other higher plants, such as Vernicia fordii, J. curcas, and Ricinus communis (86, 77, and 70%, respectively), which all contain conserved domains for ACCase activity. The expression of the AM-accB gene during the middle stage of development and maturation in A. moluccana seeds was higher than that in early and later stages. The expression pattern of the AM-accB gene is very similar to that of the oil accumulation rate.

Topical anti-inflammatory activity of semisolid containing standardized Aleurites moluccana L. Willd (Euphorbiaceae) leaves extract.

ETHNOPHARMMACOLOGICAL RELEVANCE: Aleurites moluccana is a medicinal plant popularly used to treat pain, fever, asthma, hepatitis, gastric ulcer and inflammatory process in general. Recently, pre-clinical studies have demonstrated that the dry extract obtained from A. moluccana leaves was effective as analgesic, anti-inflammatory and wound healing. AIM: The present study has aimed to evaluate the mechanisms involved in the topical anti-inflammatory effects of the semisolid containing 10mg/g of A. moluccana dried extract. MATERIAL AND METHODS: Ear edema induced by croton oil (2.5%) in mice was used throughout the study. The level of cytokines tumour necrosis factor (TNF) and interleukine-1ß (IL-1ß) and chemokine keratinocyte chemoattractant (CXCL1/KC), and neutrophil migration were quantified. The histological analysis has also been performed. RESULTS: The topical treatment with the semisolid was able to significantly inhibite the ear edema (35.77±7.35%). This effect was accompanied by the reduction of leukocyte migration, as well as TNF (53.75±12.96%), IL-1ß (38.36±5.92%), and CXCL1/KC (62.29±11.65%) decreased levels. CONCLUSIONS: This study demonstrated for the first time the mechanisms involved in the topical anti-inflammatory effect presented by the semisolid containing A. moluccana dried extract pointing as the main mechanism is the reduction in the leukocyte migration and consequently resulting in diminished levels of cytokins and chemokines, indicating this herbal product as a promissor anti-inflammatory phytomedicine to treat skin inflammatory diseases.

[Changes of inherent immune response and acquired immune response in the tung tissue and the intestinal tissue of ulcerative colitis rats and the intervention of Chinese compound: an experimental research].

OBJECTIVE: To explore Chinese medical theory of Fei and Dachang being interior-exteriorly correlated by observing changes of inherent immune response and acquired immune response in the lung tissue and the intestinal tissue of ulcerative colitis (UC) model rats and the intervention of Chinese compounds (CM). METHODS: Seventy rats were randomly divided into 5 groups, i.e., the normal control group (n = 10), the model group (n = 15), the treatment 1 group (n = 15, treated from Fei), the treatment 2 group (n = 15, treated from the intestine), and the Western medicine (WM) group [n = 15, treated with Sulfasalazine (SASP). Except those in the normal control group, the UC rat model was prepared by allergizing colon mucosa combined with TNBS-alcohol (50%) enema, and then intervened by medication (treated with CM complex prescription of treatment from lung, CM complex prescription of treatment from intestine, and SASP). After intragastric administration for 4 weeks, rats were sacrificed and samples taken. The expression of tumor necrosis factor α (TNF-α) and IL-8 contents in the lung tissue, the intestinal tissue, and the serum were detected by radioimmunoassay. Serum MedCAM-1 contents were detected using ELISA. Changes of the expression of Toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), neutrophil migration inhibition factor (MIF), mucosal addressin cell adhesion molecule-1 (MadCAM-1) mRNA in the lung tissue and the intestinal tissue were detected by real time PCR. RESULTS: Compared with the normal control group, the expression levels of TNF-α, TLR4 mRNA, IL-8, MIF mR- NA, and MadCAM-1 mRNA obviously increased in the model group (P < 0.01). Compared with the model group, the expression levels of TNF-α, TLR4 mRNA, IL-8, MIF mRNA, and MadCAM-1 mRNA obviously decreased in the treatment 1 and 2 groups (P < 0.01). The expression of MadCAM-1 mRNA in the intestinal tissue was obviously higher in the model group than in the normal control group (P < 0.01), while the expressions of TNF-α and NF-κB mRNA was obviously lower in the model group than in the normal control group (P < 0.05, P < 0.01). Compared with the model group, the expression of MadCAM-1 mRNA all significantly deceased in each treatment group (P < 0.05, P < 0.01). Serum TNF-α contents were higher in the model group than in the normal control group (P < 0.05). Compared with the model group, serum TNF-α contents could be lowered in the treatment 1 and 2 groups (P < 0.05, P < 0.01). CONCLUSIONS: The main mechanisms of the intestinal injury in this UC model might be related with activation of acquired immune response, accompanied with lowered functions of inherent immune response. The main mechanisms of the lung injury in this UC model might be related acquired immune response and inherent immune response. Treatment from Fei and treatment from Dachang both could obviously improve the immunodissonance of Fei and Dachang, indicating the special relation between the lung tissue and the intestinal tissue, thus providing experimental evidence for Chinese medical theory of Fei and Dachang being interior-exteriorly correlated.

Changes of inherent immune response and acquired immune response in the tung tissue and the intestinal tissue of ulcerative colitis rats and the intervention of Chinese compound: an experimental research / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore Chinese medical theory of Fei and Dachang being interior-exteriorly correlated by observing changes of inherent immune response and acquired immune response in the lung tissue and the intestinal tissue of ulcerative colitis (UC) model rats and the intervention of Chinese compounds (CM).</p><p><b>METHODS</b>Seventy rats were randomly divided into 5 groups, i.e., the normal control group (n = 10), the model group (n = 15), the treatment 1 group (n = 15, treated from Fei), the treatment 2 group (n = 15, treated from the intestine), and the Western medicine (WM) group [n = 15, treated with Sulfasalazine (SASP). Except those in the normal control group, the UC rat model was prepared by allergizing colon mucosa combined with TNBS-alcohol (50%) enema, and then intervened by medication (treated with CM complex prescription of treatment from lung, CM complex prescription of treatment from intestine, and SASP). After intragastric administration for 4 weeks, rats were sacrificed and samples taken. The expression of tumor necrosis factor α (TNF-α) and IL-8 contents in the lung tissue, the intestinal tissue, and the serum were detected by radioimmunoassay. Serum MedCAM-1 contents were detected using ELISA. Changes of the expression of Toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), neutrophil migration inhibition factor (MIF), mucosal addressin cell adhesion molecule-1 (MadCAM-1) mRNA in the lung tissue and the intestinal tissue were detected by real time PCR.</p><p><b>RESULTS</b>Compared with the normal control group, the expression levels of TNF-α, TLR4 mRNA, IL-8, MIF mR- NA, and MadCAM-1 mRNA obviously increased in the model group (P < 0.01). Compared with the model group, the expression levels of TNF-α, TLR4 mRNA, IL-8, MIF mRNA, and MadCAM-1 mRNA obviously decreased in the treatment 1 and 2 groups (P < 0.01). The expression of MadCAM-1 mRNA in the intestinal tissue was obviously higher in the model group than in the normal control group (P < 0.01), while the expressions of TNF-α and NF-κB mRNA was obviously lower in the model group than in the normal control group (P < 0.05, P < 0.01). Compared with the model group, the expression of MadCAM-1 mRNA all significantly deceased in each treatment group (P < 0.05, P < 0.01). Serum TNF-α contents were higher in the model group than in the normal control group (P < 0.05). Compared with the model group, serum TNF-α contents could be lowered in the treatment 1 and 2 groups (P < 0.05, P < 0.01).</p><p><b>CONCLUSIONS</b>The main mechanisms of the intestinal injury in this UC model might be related with activation of acquired immune response, accompanied with lowered functions of inherent immune response. The main mechanisms of the lung injury in this UC model might be related acquired immune response and inherent immune response. Treatment from Fei and treatment from Dachang both could obviously improve the immunodissonance of Fei and Dachang, indicating the special relation between the lung tissue and the intestinal tissue, thus providing experimental evidence for Chinese medical theory of Fei and Dachang being interior-exteriorly correlated.</p>

Contribution of α,ß-Amyrenone to the Anti-Inflammatory and Antihypersensitivity Effects of Aleurites moluccana (L.) Willd.

The aim of the study was to analyze the constituents of the dichloromethane fraction obtained from A. moluccana and also to evaluate the anti-inflammatory and antinociceptive properties of α,ß-amyrenone isolated from A. moluccana in mice. The dichloromethane fraction was evaluated by gas chromatography and submitted to purification. The mixture of α,ß-amyrenone was isolated and then evaluated using the carrageenan-induced paw-oedema or pleurisy and CFA-induced arthritis models in mice. Five triterpenes, α,ß-amyrenone, glutinol, and α,ß-amyrin were isolated from dichloromethane fraction of A. moluccana leaf extract. The mixture of α,ß-amyrenone, dosed orally, was able to reduce mechanical hypersensitivity and paw-oedema induced by carrageenan, interfering with neutrophil migration. Similar results were observed in the carrageenan-induced pleurisy model. Repeated administration of the compounds was also effective in reducing the mechanical sensitization and oedema developed in the arthritis model induced by CFA. In conclusion, the results demonstrate that α,ß-amyrenone interferes in both acute and chronic inflammatory processes. We can infer that these effects involve, at least in part, a reduction in the neutrophil migration. Therefore, it seems reasonable to suggest that α,ß-amyrenone could represent a new therapeutic tool for the management of painful and inflammatory diseases, especially those presenting a chronic profile.

Development of a high-throughput assay for measuring phospholipase A activity using synthetic 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine coated on microtiter plates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen phospholipase A (PLA) activities. With the aim of developing a convenient, specific, sensitive, and continuous new ultraviolet (UV) spectrophotometric assay for PLA, we have synthesized a specific glycerophosphatidylcholine (PC) esterified at the sn-1 and sn-2 positions, with α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the glycerophospholipids harboring it. This coated PC film cannot be desorbed by the various buffers used during PLA assays. Following the action of PLA at the oil-water interface, α-eleostearic acid is freed and desorbed from the film and then solubilized with ß-cyclodextrin. The UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water-soluble state. The PLA activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of absorption at 272 nm, which was found to be linear with time and proportional to the amount of added PLA. This continuous high-throughput PLA assay could be used to screen new PLA and/or PLA inhibitors present in various biological samples.

Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii).

Triacylglycerols (TAG) are the major molecules of energy storage in eukaryotes. TAG are packed in subcellular structures called oil bodies or lipid droplets. Oleosins (OLE) are the major proteins in plant oil bodies. Multiple isoforms of OLE are present in plants such as tung tree (Vernicia fordii), whose seeds are rich in novel TAG with a wide range of industrial applications. The objectives of this study were to identify OLE genes, classify OLE proteins and analyze OLE gene expression in tung trees. We identified five tung tree OLE genes coding for small hydrophobic proteins. Genome-wide phylogenetic analysis and multiple sequence alignment demonstrated that the five tung OLE genes represented the five OLE subfamilies and all contained the "proline knot" motif (PX5SPX3P) shared among 65 OLE from 19 tree species, including the sequenced genomes of Prunus persica (peach), Populus trichocarpa (poplar), Ricinus communis (castor bean), Theobroma cacao (cacao) and Vitis vinifera (grapevine). Tung OLE1, OLE2 and OLE3 belong to the S type and OLE4 and OLE5 belong to the SM type of Arabidopsis OLE. TaqMan and SYBR Green qPCR methods were used to study the differential expression of OLE genes in tung tree tissues. Expression results demonstrated that 1) All five OLE genes were expressed in developing tung seeds, leaves and flowers; 2) OLE mRNA levels were much higher in seeds than leaves or flowers; 3) OLE1, OLE2 and OLE3 genes were expressed in tung seeds at much higher levels than OLE4 and OLE5 genes; 4) OLE mRNA levels rapidly increased during seed development; and 5) OLE gene expression was well-coordinated with tung oil accumulation in the seeds. These results suggest that tung OLE genes 1-3 probably play major roles in tung oil accumulation and/or oil body development. Therefore, they might be preferred targets for tung oil engineering in transgenic plants.

Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues.

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.

Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates.

We have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the Aleurites fordii seed oil which contains α-eleostearic acid (9,11,13,cis,trans,trans-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, α-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 °C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of α-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL(-1) of pure Thermomyces lanuginosus or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples.

Antinociceptive, anti-inflammatory and wound healing features in animal models treated with a semisolid herbal medicine based on Aleurites moluccana L. Willd. Euforbiaceae standardized leaf extract: semisolid herbal.

ETHNOPHARMACOLOGICAL RELEVANCE: Aleurites moluccana L. (Willd) Euforbiaceae is a native tree of Indonesia and India that has become acclimatized and well-adapted to the South and Southwest of Brazil. It is commonly used in traditional medicine to treat pain, fever, inflammation, asthma, hepatitis, headache, gastric ulcer, cuts, skin sores and other ailments. The oral antinociceptive effects of standardized 70:30 (v/v) ethanol:water spray dried extract of A. moluccana leaf, as well as its flavonoids 2"-O-rhamnosylswertisin (I) and swertisin (II), have previously been reported. AIM: The aim of this study was to develop a stable and effective semisolid herbal medicine for topical use in the treatment of pain, inflammation and wound healing, containing 0.5 and 1.0% of standardized dried extract of A. moluccana. MATERIALS AND METHODS: The chemical markers I and II were assayed by HPLC-UV analysis after extraction by matrix solid dispersion phase (MSDP) followed analytical validation as ICH Guidelines. The semisolid preparations of Hostacerin CG(®) vehicle containing 0.5 and 1.0% of dried extract of A. moluccana were submitted to stability studies (180 day of accelerated and long-term studies). The phytomedicine semisolid was analysed in croton oil-induced ear oedema model in mice, in the healing process, using the excisional wound model in rats, and to prevent mechanical sensitization following plantar incision in rats in the postoperative model of pain. RESULTS: The MSDP method showed average recovery of 101.6 and 105.7% for I and II, respectively, with good precision (RSD<2.0%) and selectivity, without interference of the excipients. The formulations were approved in the stability studies, maintaining conformity after 180 day of accelerated and long-term studies, with variation<10% in the analytical parameters. The phytomedicine reduced the ear oedema in 37.6±5.7% and 64.8±6.2%, for 0.5 and 1.0% of dried extract, respectively. The formulation also accelerated the healing process by up to 50.8±4.1% and 46.0±4.0% at 0.5 and 1.0% of extract, respectively, and both amounts were capable of preventing the development of mechanical sensitization following plantar incision in rats. CONCLUSIONS: The MSDP followed by HPLC-UV analytical method was appropriate for the quality control of the topical phytomedicine based on A. moluccana. The formulation developed at 0.5 and 1.0% of A. moluccana dried extract proved to be effective as an analgesic, anti-inflammatory and wound healing in the pre-clinical studies, which is in agreement with the ethnopharmacological data.

Aleurites moluccana and its main active ingredient, the flavonoid 2″-O-rhamnosylswertisin, have promising antinociceptive effects in experimental models of hypersensitivity in mice.

This study investigated the antinociceptive effect of Aleurites moluccana dried extract (DE; 125 to 500 mg/kg, p.o.) and the isolated flavonoid 2″-O-rhamnosylswertisin (5 to 50.6 µmol/kg, p.o.) using different models of long-lasting inflammatory and neuropathic pain in mice. Attempts were made to analyse the mechanisms through which A. moluccana exerted its effects. A. moluccana DE inhibited complete Freund's adjuvant (CFA)-induced mechanical nociception. It was also evidenced by a reduction of sensitization in the contralateral hindpaw. The extract reversed the mechanical hypersensitivity of partial ligation of sciatic nerve (PLSN)-treated animals, similar to gabapentin. In PLSN model, the opioid, dopaminergic and oxidonitrergic pathways were involved in the A. moluccana DE antinociceptive effects. A single dose of 2″-O-rhamnosylswertisin inhibited the carrageenan- and CFA-induced mechanical nociception. Furthermore, the compound caused expressive antinociception in PLSN-mice, with inhibition value greater than obtained with gabapentin. Oral treatment with the extract or the isolated compound attenuated the neutrophil migration and IL-1ß levels following carrageenan injection. Of note, A. moluccana DE did not interfere with thermal sensitivity in healthy mice. The absence of side effects, including interference in locomotor activity, motor performance in animals treated with the extract, showed excellent potential for the therapeutic use of this medicinal plant in treating persistent pain in humans.