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.

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.

Physicochemical Characterization of Candlenut (Aleurites moluccana)-derived Biodiesel Purified with Deed Eutectic Solvents.

A high-oil candlenut (Aleurites moluccana)-derived biodiesel was purified using new generation solvents called Deep eutectic solvents (DESs) and physicochemical properties of the purified biodiesel were assessed according to the accepted international standards for biodiesel (ASTM D6751; EN 14214). Binary and ternary choline chloride-based DESs containing glycerol and ethylene glycol as hydrogen bond donors were synthesized. The results of the investigation showed that the quality of DESs-purified biodiesel generally improved compared with the unpurified biodiesel and fulfilled the accepted standard limits for biodiesel except carbon residue and oxidation stability properties. Choline chloride/ethylene glycol (ChCl/Eg, 1:2 molar ratio) having a DES/biodiesel ratio of 1:1 (DES2-11) has enhanced the Fatty Acid Methyl Ester (FAME) content of unpurified biodiesel from 81.89% to 98.13%, and the oxidation stability from 2.36 h to 3.06 h. 1H NMR examination showed the successful synthesis of DESs; GC-MS analysis of candlenut oil and its biodiesel revealed oil and fatty acid methyl ester compositions, respectively. The similarity in FAME compositions between DESs-purified and unpurified biodiesel as revealed through GC-MS examination suggests the selective removal of impurities and the efficiency of DESs in improving the quality of candlenut-derived biodiesel.

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.

Stable carbon and nitrogen isotopes in kukui (Aleurites moluccanus) endocarp along rainfall and elevation gradients: Archaeological implications.

Stable carbon and nitrogen isotopes are often used to make inferences of past environments and social patterns. We analyze δ 13C and δ 15N values in contemporary kukui (Aleurites moluccanus) endocarp to examine the effects of site environment. Results from across environmental transects on Hawai'i Island show strong patterns for both stable isotopes. For δ 13C a robust linear relationship with elevation is exhibited, strengthened by the inclusion of rainfall. This relationship breaks down at a minimum threshold of annual rainfall, possible relating to physiological responses to drought. For δ 15N, the only significant relationship observed pertains to substrate age. The endocarp from kukui is one of the most readily identified plant remains in the Pacific archaeological records and is often targeted for radiocarbon dating. We discuss the potential implications of our results regarding ancient climate, inferred diets, and habitat composition.

Extraction and composition characterisation of amino acids from tung meal.

The most desirable content of amino acids (AAs) in the extracted products from tung (Aleurites fordii) meal was 93.88%, which was obtained from shelled tung meal at a hydrolysis temperature of 45 °C and a isoelectric precipitation pH value of 4.4. Furthermore, the cytotoxic activity of extracted AAs was also evaluated by MTT assay. Antioxidant activity of extracted AAs was also measured by the DPPH assay. As a result, the high yield of extracted AAs exhibited so low cytotoxic and high antioxidant activity that had the potential use as a functional ingredient.

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.

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.

Tigliane diterpene esters with IFN γ-inducing activity from the leaves of Aleurites fordii.

Bioactivity-guided fractionation on the leaves of Aleurites fordii led to the isolation of a new tigliane diterpene ester, 12-O-hexadecanoyl-7-oxo-5-ene-16-hydroxyphorbol-13-acetate (1) along with four known compounds, 12-O-hexadecanoyl-7-oxo-5-ene-phorbol-13-acetate (2), 12-O-hexadecanoyl-phorbol-13-acetate (3), 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (4), and 12-O-hexadecanoyl-4-deoxy-4α-16-hydroxyphorbol-13-acetate (5). The structures of these compounds were determined by interpretation of NMR (1D and 2D) spectroscopic data and MS data. All the isolates were evaluated for their effects on the induction of IFN-γ in NK92 cells. Compounds 3 and 4 exhibited the most potent responses in IFN-γ induction, comparable to the positive control, phorbol 12-myristate 13-acetate (PMA).

Chemical composition and antioxidant properties of candlenut oil extracted by supercritical CO2.

Candlenut oil was extracted using supercritical CO(2) (SC-CO(2)) with an optimization of parameters, by the response surface methodology. The ground candlenut samples were treated in 2 different ways, that is, dried in either a heat oven (sample moisture content of 2.91%) or dried in a vacuum oven (sample moisture content of 1.98%), before extraction. An untreated sample (moisture content of 4.87%) was used as a control. The maximum percentage of oil was extracted from the heat-oven-dried sample (77.27%), followed by the vacuum-oven-dried sample (74.32%), and the untreated sample (70.12%). At an SC-CO(2) pressure of 48.26 Mpa and 60 min of extraction time, the optimal temperatures for extraction were found to be 76.4 °C, 73.9 °C, and 70.6 °C for the untreated, heat-oven-dried, and vacuum-oven-dried samples, respectively. The heat-oven-dried sample contains the highest percentage of linoleic acid, followed by the untreated and vacuum-oven-dried samples. The antiradical activity of candlenut oil corresponded to an IC(50) value of 30.37 mg/mL.

Accelerated solvent extraction of lignin from Aleurites moluccana (Candlenut) nutshells.

Lignin from candlenut shells was isolated using an ethanol-water accelerated solvent extraction method. Yields (based on Klason lignin) increased from about 14 to 33% as temperature increased from 100 to 195 °C and were also influenced by the amount of aqueous acid used to precipitate lignin from the extraction liquor. These yields were higher than could be obtained using a conventional dioxane-water acidolysis method. The resulting lignin was characterized by IR, 31P NMR, and 1H-13C HMQC NMR spectroscopic techniques. The lignin contained predominantly guaiacyl units, and both the total hydroxyl group content and phenolic hydroxyl group content were high.

Ocular burn: rinsing and healing with ionic marine solutions and vegetable oils.

PURPOSE: We investigated the effects of various rinsing and healing protocols on corneal wound repair and inflammation following alkali burn in rabbits. METHODS: We conducted in vitro, in vivo and ex vivo studies. First, different rinse solutions were tested in vitro after incubation of ocular cells with methanol or NaOH. Cell viability was then assessed using the neutral red test (cytofluorometry). Second, NaOH was applied to rabbit corneas and associations of rinse solutions (NaCl 0.9% or controlled ionization marine solutions) with N-acetylcysteine or vegetable oils (from Calophyllum inophyllum and Aleurites moluccana) were tested in vivo. The regeneration of the corneal epithelium and the infiltration of inflammatory cells were evaluated using in vivo confocal microscopy and ex vivo histological cuts. RESULTS: The association of a controlled ionization marine solution with 10% C. inophyllum oil and 90% A. moluccana oil induced regeneration of the corneal epithelium and a decrease in inflammatory cells. CONCLUSIONS: Irrigation with marine solution followed by treatment with a mixture of C. inophyllum and A. moluccana oils is a promising treatment for ocular burns.

Benefits and side effects of different vegetable oil vectors on apoptosis, oxidative stress, and P2X7 cell death receptor activation.

PURPOSE: Ocular side effects in patients using eye drops may be due to intolerance to the vector used in eye drops. Castor oil is the commonly used lipophilic vector but has been shown to be cytotoxic. Effects on cells of four oils (olive, camelina, Aleurites moluccana, maize) were compared with those of castor oil in human conjunctival cells. METHODS: Human conjunctival cells were incubated with the oils for 15 minutes. After a 24-hour recovery period, cells were tested for viability, proliferation, apoptosis (P2X7 cell death receptor and caspase 3 activation), intracellular redox potential, and reactive oxygen species production. Fatty acid incorporation in cell membranes was also analyzed. In vivo ocular irritation was assessed using the Draize test. RESULTS: Compared to the four other oils, castor oil was shown to induce significant necrosis and P2X7 cell death receptor and caspase 3 activation and to enhance intracellular reactive oxygen species production. Aleurites moluccana and camelina oils were not cytotoxic and increased cell membrane omega-3 fatty acid content. None of the five tested oils showed any in vivo ocular irritation. CONCLUSIONS: The results demonstrated that castor oil exerts cytotoxic effects on conjunctival cells. This cytotoxicity could explain the side effects observed in some patients using eye drops containing castor oil as a vehicle. The lack of cytotoxic effects observed with the four other oils, Aleurites, camelina, maize, and olive, suggest that they could be chosen to replace castor oil in ophthalmic formulations.

The use of chitosan modified with glutaraldehyde and glyoxal as chromatographic support for the separation of flavonoids from Aleurites moluccana leaves.

This paper describes the preparation of chitosan modified with dialdehydes, glutaraldehyde (CH-Glu) and glyoxal (CH-Gly) and its application in the isolation of the flavonoids swertisin and 2"-O-rhamnosylswertisin from A. mollucana. The additional non-polar alkyl groups increase the hydrophobicity of the sorbent. The results show that the separation was mediated by hydrophobic interaction (CH-Glu), as well as hydrogen bonding, between phenolic OH or rhamnosil residues from the flavonoids, and the free amine groups (CH-Gly).

Separation of flavonoids from Aleurites moluccana leaves using chitosan modified with heptaldehyde.

Heptaldehyde-modified chitosan (heptyl-chitosan, CH-Hp) was investigated as adsorbent for chromatograhic separation of the flavonoids from A. moluccana. The amount of 2"-O-rhamnosylswertisin isolated (30.0 mg) was approx. twice as high as swertisin (17.5 mg). The improved surface hydrophobicity effected by the heptyl groups promoted the separation of flavonoids. From the results obtained, CH-Hp seems to be more suitable for separation of glycosylated flavonoids than other flavonoids. Thus, modified chitosan described here can be used for hydrophobic interaction chromatography as sucessfully illustrated with flavonoids.

Recommended methods of fatty acid methylester preparation for conjugated dienes and trienes in food and biological samples.

In this study, we compared three acid-catalyzed methods and three base-catalyzed methods for the methylester preparation of conjugated dienoic fatty acids and conjugated trienoic fatty acids in food and biological samples. Among the six methods examined, the sodium methoxide/methanol (NaOCH3/MeOH) method and the tetramethylguanidine/ methanol (TMG/MeOH) method of methylester preparation from tung oil were most efficient in preventing the artificial isomerization of alpha-eleostearic acid (alpha-ESA; 9c,11t,13t-18:3) to beta-eleostearic acid (beta-ESA: 9t,11t,13t-18:3) and for avoiding the artificial generation of unknown byproducts. Hydrochloric acid/methanol (HCl/MeOH), sulfuric acid/methanol (H2SO4/MeOH) and AOCS (boron trifluoride/methanol (BF3/MeOH)) methods of methylester preparation from tung oil resulted in the breakdown of alpha-ESA due to their long reaction periods and high reaction temperatures. In addition, these three methods did not prevent the generation of beta-ESA. For the methylester preparation of tung oil free fatty acids, the BF3/MeOH method (30 min at room temperature) did not lead to artificial beta-ESA formation or byproducts, while the trimethylsilyldiazomethane (TMSN2CH3) method did form artifacts. For the methylation of conjugated linoleic acid (CLA, free fatty acid), the BF3/ MeOH and TMSN2CH3 methods completely suppressed artificial isomerization of c,t-CLA and t,c-CLA to t,t-CLA. The results indicated that the BF3/MeOH method for free fatty acids is the best method for the methylester preparation of both conjugated dienoic and trienoic fatty acids with respect to preventing artificial isomerization and the formation of byproducts. The BF3/MeOH method was applicable to both food and biological samples.