Biomimetic transport of simple olive biophenol and analogues through model biological membranes by differential scanning calorimetry.

The different interactions of p-hydroxybenzoic acid (1), a simple biophenol (BP) found in olives and their food products, and its substitute analogues, benzoic (2), anisic (3), and toluic (4) acids, with a model membrane represented by dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) was studied by differential scanning calorimetry (DSC). The influence of their different lipophilic character on transfer and absorption processes through an aqueous medium into a lipid bilayer was also investigated. DSC experiments allowed monitoring of the interaction of BP with biomembranes by considering the effects exerted on the thermotropic behavior of DMPC multilamellar and unilamellar vesicles at different pHs (4 and 7.4). The examined compounds affect the transition temperature (T(m)) of phospholipid vesicles, causing a shift toward lower values, which is modulated by the molecular fraction entering into the lipid bilayer, as well as by their molecular interaction with the lipids. Kinetic calorimetric measurements were performed on suspensions of blank liposomes immediately after being added to fixed weighed amounts of powdered compounds and after increasing incubation periods at 37 degrees C. T(m) shifts, due to molecular dissolution and transfer of the compounds into the membrane surface occurring during the incubation time, were compared with those determined by a fixed molar fraction of free compounds directly dispersed in the membrane. The results show that the kinetic process, involved in molecular release, transfer through aqueous medium, and uptake by the model membrane surface, is influenced by lipophilicity as well as by pH, acting on the acid solubility and membrane disorder, allowing us to gather useful information on the BP intake process of olive derived foodstuffs.     

Differences between coumaric and cinnamic acids in membrane permeation as evidenced by time-dependent calorimetry.

We carried out an investigation by differential scanning calorimetry (DSC) on the effect of two structurally similar bioactive plant phenols, cinnamic and p-coumaric acids, on the phase transition of model membranes constituted by dimyristoylphosphatidylcholine (DMPC) vesicles. The aim was to evaluate how pH and molecular substituents influence liposolubility and thereby modify vesicle permeability. A change in permeability would result in modifications to the phase transition for DMPC liposomes such that the transition temperature would be lowered and the enthalpy changes should be little affected. Evidence of differences in permeability was obtained by comparing the effects exerted by the two compounds left in touch with unilamellar and multilamellar vesicles, at two different pHs (4 and 7.4). While the p-coumaric acid was unable to appreciably modify the thermotropic behavior of the model membrane, the cinnamic acid interacted with lipid vesicles at both pH's, even if at acidic pH the effect was greater than neutral. It can be hypothesized that the interaction between the cinnamic acid and the lipidic layers is due to the lack of a hydroxyl group. This interaction is enhanced by an acidic pH, where the carboxylic acid is in a protonated form.     

Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionization and electrospray ionization liquid chromatography-mass spectrometry

Improved analytical techniques for bitter limonoids in citrus and citrus juices can expedite the evaluation of freeze-induced citrus damage for citrus growers and juice quality for citrus juice producers. Microbore normal-phase and reverse-phase chromatography coupled to a mass spectrometer operating in a positive ion atmospheric pressure chemical ionization and electrospray ionization modes were found to be rapid, selective, and sensitive methods for the analysis of the bitter limonoids limonin and nomilin in citrus juices. Analysis was performed on a chloroform extract of citrus juice to which an internal standard was added. The methods are capable of detecting citrus limonoids in citrus juice in the 60-200 picogram range and quantifying citrus juice limonoids in concentrations as low as 120 picograms. An accurate "total limonoid bitterness" in citrus juice, as represented by the combined occurrence of limonin and nomilin, is easily determined by these methods.     

Interaction of resveratrol and its trimethyl and triacetyl derivatives with biomembrane models studied by differential scanning calorimetry

The interaction of resveratrol (trans-3,5,4'-trihydroxystilbene) and two of its derivatives (3,5,4'-tri-O-methylresveratrol and 3,5,4'-tri-O-triacetylresveratrol) with biomembrane models, represented by dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV), has been studied by differential scanning calorimetry (DSC). The analysis of MLV prepared in the presence of increasing molar fraction of such compounds has been carried out to reveal their maximum interaction with biomembrane models. The results from these studies have been compared with kinetic experiments results, in order to detect the entity and rate of compound absorption by the biomembrane models. The findings indicate that the compounds affected the thermotropic properties of DMPC MLV by suppressing the pretransition peak and broadening the DMPC main phase transition calorimetric peak and shifting it to lower temperatures. The order of effectiveness found was resveratrol > trimethylresveratrol > triacetylresveratrol. The kinetic experiments reveal that in an aqueous medium the absorption of resveratrol by the biomembranes models is allowed, whereas the absorption of its derivatives is hindered; in contrast when a lipophilic medium is employed, all three compounds are easily absorbed.     

Evaluation of the antioxidant capacity of limonin, nomilin, and limonin glucoside

The antioxidant capacity (AOC) of three representative citrus limonoids, limonin, nomilin, and limonin glucoside, was examined by the oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), beta-carotene-linoleic acid bleaching, and 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging assays. Pure compounds and proper negative (cinnamic acid) and positive (2,6-di-tert-butyl-4-methylphenol (BHT) and ascorbic acid) controls were used to remove any ambiguity in interpreting results. In all cases, limonin and nomilin gave results equivalent to those of cinnamic acid, indicating that they do not possess any inherent AOC and should not be considered antioxidants. Similar results were observed for limonin glucoside, with the exception of an anomalous result obtained from the beta-carotene-linoleic acid bleaching assay. Limonin glucoside was deemed not to be an antioxidant on the basis of the three unequivocal assays.     

Biomimesis of linolenic acid transport through model lipidic membranes by differential scanning calorimetry

Multienoic fatty acids, such as linolenic acid, show their ability to interact with and to penetrate into model biomembranes by biomimetic experiments performed to support the absorption route followed by n-3 fatty acid in cells. The thermotropic behavior of model biomembranes, that is, dimyristoylphosphatidylcholine multilamellar or unilamellar vesicles, interacting with linolenic acid was investigated by differential scanning calorimetry. When dispersed in liposomes during their preparation, the examined biomolecule was found to interact with the phospholipid bilayers by modifying the gel to liquid-crystal phase transition of lipid vesicles; this modification is a function of the fatty acid concentration. Calorimetric analysis was also performed on samples obtained by leaving the pure n-3 acid in contact with lipid aqueous dispersions (multilamellar or unilamellar vesicles) and then examining the thermotropic behavior of these systems for increasing incubation times at temperatures higher than the transitional lipid temperature. Linolenic acid (LNA) was able to migrate through the aqueous medium and successively to interact with the vesicle surface and to penetrate into the model membranes, following a flip-flop mechanism, with a faster and higher effect for unilamellar vesicles, caused by the larger lipid surface exposed, compared to the multilamellar ones, although due to the lipophilic nature of LNA, such a transfer is hindered by the aqueous medium. The relevance of the medium in LNA absorption has been well clarified by other biomimetic transfer experiments, which showed the LNA transfer from loaded multilamellar vesicles to empty vesicles. Taken together, the present findings support the hypothesis of a passive n-3 acid transport as the main route of absorption into cell membranes.     

Citrus limonoids and their semisynthetic derivatives as antifeedant agents against Spodoptera frugiperda larvae. A structure-activity relationship study

The antifeedant activity of Citrus-derived limonoids limonin (1), nomilin (2), and obacunone (3) and their semisynthetic derivatives 4-26 was evaluated against a commercially important pest, Spodoptera frugiperda. Simple chemical conversions were carried out on the natural limonoids obtained from seeds of Citrus limon. These conversions focused on functional groups considered to be important for the biological activity, namely the C-7 carbonyl and the furan ring. In particular, reduction at C-7 afforded the related alcohols, and from these their acetates, oximes, and methoximes were prepared. Hydrogenation of the furan ring was also performed on limonin and obacunone. The known antifeedant properties of the Citrus limonoids are confirmed. Comparison with previously reported data shows that insect species vary in their behavioral responses to these structural modifications. Highly significant antifeedant activity (P < 0.01) for two natural (1 and 3) and three semisynthetic limonoids (4, 8, and 10) was observed against S. frugiperda.     

Bioavailability of citrus limonoids in humans

This study utilizes liquid chromatography/mass spectrometry (LC-MS) to analyze the plasma of four groups of four healthy male and female subjects administered high doses of pure limonin glucoside (0.25-2.0 g in 200 mL of buffered water) for the presence of limonin to establish the absorption, metabolism, and bioavailability of citrus limonoids to humans. The plasma analysis revealed increasing amounts of limonin associated with increasing doses of limonin glucoside among the subject groups in mean maximum concentration amounts ranging from 1.74 to 5.27 nmol/L. A high degree of variability in the analyzed limonin concentration was observed within the subject groups. The mean time to maximum concentration was 6 h. A second compound with MS/MS characteristics identical to limonin was detected in amounts up to 5.13 nmol/L and is hypothesized to be a limonin epimer. Poststudy health evaluation established no ill effects among study subjects consuming high doses of limonin glucoside.     

Interaction of antioxidant biobased epicatechin conjugates with biomembrane models

(-)-Epicatechin conjugates with sulfur-containing moieties are strong free radical scavengers with cell-protecting activities, which may be in part modulated by their capacity to bind to biological membranes. We present here a study of the interaction of these conjugates with membrane models such as multilamellar vesicles and a phospholipid-coated silica column (immobilized artificial membrane), monitored by differential scanning calorimetry and high-performance liquid chromatography, respectively. The nonpolyphenolic moiety significantly influenced the membrane behavior of the whole molecules. Bulky and hydrophobic conjugates clearly interacted with the phospholipids and may have a tendency to penetrate into the hydrophobic core of the vesicles. In contrast, the smaller cationic 4beta-(2-aminoethylthio)epicatechin may be located at the outer interface of the lipid membrane. The outcomes from both experimental set-ups were in good agreement. The differences detected in the biological activities of the conjugates may be explained in part by their tendency to penetrate the cell membrane.     

Antioxidant activity of citrus limonoids, flavonoids, and coumarins

A variety of in vitro models such as beta-carotene-linoleic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH), superoxide, and hamster low-density lipoprotein (LDL) were used to measure the antioxidant activity of 11 citrus bioactive compounds. The compounds tested included two limonoids, limonin (Lim) and limonin 17-beta-D-glucopyranoside (LG); eight flavonoids, apigenin (Api), scutellarein (Scu), kaempferol (Kae), rutin trihydrate (Rut), neohesperidin (Neh), neoeriocitrin (Nee), naringenin (Ngn), and naringin(Ng); and a coumarin (bergapten). The above compounds were tested at concentration of 10 microM in all four methods. It was found that Lim, LG, and Ber inhibited <7%, whereas Scu, Kae, and Rut inhibited 51.3%, 47.0%, and 44.4%, respectively, using the beta-carotene-linoleate model system. Lim, LG, Rut, Scu, Nee, and Kae showed 0.5% 0.25%, 32.2%, 18.3%, 17.2%, and 12.2%, respectively, free radical scavenging activity using the DPPH method. In the superoxide model, Lim, LG, and Ber inhibited the production of superoxide radicals by 2.5-10%, while the flavonoids such as Rut, Scu, Nee, and Neh inhibited superoxide formation by 64.1%, 52.1%, 48.3%, and 37.7%, respectively. However, LG did not inhibit LDL oxidation in the hamster LDL model. But, Lim and Ber offered some protection against LDL oxidation, increasing lag time to 345 min (3-fold) and 160 min (33% increase), respectively, while both Rut and Nee increased lag time to 2800 min (23-fold). Scu and Kae increased lag time to 2140 min (18-fold) and 1879 min (15.7-fold), respectively. In general, it seems that flavonoids, which contain a chromanol ring system, had stronger antioxidant activity as compared to limonoids and bergapten, which lack the hydroxy groups. The present study confirmed that several structural features were linked to the strong antioxidant activity of flavonoids. This is the first report on the antioxidant activity of limonin, limonin glucoside, and neoeriocitrin.     

6beta-hydroxygedunin from Azadirachta indica. Its potentiation effects with some non-azadirachtin limonoids in neem against lepidopteran larvae

The biological activity of 6beta-hydroxygedunin isolated from Azadirachta indica A. Juss. was assessed using the gram pod borer, Helicoverpa armigera (Hubner), and Asian armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), alone and in combination with other limonoids, gedunin, salannin, nimbinene, and azadirachtin. The compound exhibited growth inhibitory activity in artificial diet bioassays, with 24.2 and 21.5 ppm, respectively, inhibiting growth by 50%. This efficacy was higher in comparison to gedunin (EC(50) = 50.8 and 40.4 ppm), salannin (EC(50) = 74.5 and 72.0 ppm), and nimbinene (EC(50) = 391.4 and 404.5 ppm). Azadirachtin, however, remained the most active neem allelochemical against both insect species. Nutritional assays clearly demonstrated that, though relative consumption and growth rates of fourth instar larvae were reduced, gedunin-type compounds induced physiological toxicity, evident by reduced efficiency of conversion of ingested food (ECI) in feeding experiments. Salannin and nimbinene, on the contrary, induced concentration-dependent feeding deterrence only. In feeding experiments, combinations of the compounds revealed that when azadirachtin was present in a mixture, EC(50) values did not deviate from the individual efficacy of azadirachtin (0.26 and 0.21 ppm, respectively) against H. armigera and S. litura larvae. However, a combination without azadirachtin did show a potentiation effect with potent EC(50) values among structurally different molecules, i.e., when salannin or nimbinene was combined with 6beta-hydroxygedunin or gedunin rather than structurally similar salannin + nimbinene or 6beta-hydroxygedunin + gedunin. Obviously, azadirachtin being the most active compound in neem is not synergized or influenced by any other limonoid, but other non-azadirachtin limonoids were more potent in specific combinations vis-à-vis the structural chemistry of the compound. It is obvious from the present study that potentiation among non-azadirachtin limonoids having explicitly two different modes of action, such as feeding deterrence and physiological toxicity, may be playing a significant role in the potentiation effect.     

柠檬籽粒中柠檬苦素离子液体双水相提取体系的优化与抗氧化活性分析

为更好地开发和利用柠檬籽粒中的功能性成分柠檬苦素,本试验研究了离子液体双水相对柠檬苦素提取的最优体系,并通过响应面分析法对最优体系的提取工艺进行了优化。结果表明,1.50 mol·L^-1 [TMG][Cl]/1.35 mol·L^-1 NaH₂PO₄离子液体双水相萃取体系中柠檬苦素的提取量最高,萃取率达97.59%;响应面优化试验得出最优工艺参数:提取时间90.74 min、乙醇浓度59.40%、提取温度74.22℃、液料比18.08∶1条件下,预测柠檬苦素最大提取量为12.126 mg·g^-1FW,校正工艺下柠檬苦素实际提取量为12.381 mg·g^-1 FW,方差分析显示两数值差异不显著,表明有关柠檬苦素提取量的四元二次方程模型预测准确度高。此外,柠檬苦素可有效清除活性氧自由基并抑制不饱和脂肪酸的过氧化,具有较强的抗氧化活性。本研究结果为柠檬资源的充分开发和实际应用提供了理论基础。     

Spectrophotometric evidence for the solubilization site of betalain pigments in membrane biomimetic systems

The solubilization site of two betalain pigments, namely, betanin and indicaxantin, into l-alpha-dipalmitoyl-phosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) vesicles was investigated by a spectrophotometric study. Pigment absorbance was monitored by varying phospholipid concentration, at a constant temperature that was varied in a range including the main phase transition temperature (Tm) of the relevant phospholipid bilayer. Maximum betanin absorption increased with the increase of DPPC concentration within the entire temperature range, reaching a plateau. The binding constant (Kb) of the pigment, calculated according to a pseudo-two-phase model, varied with the temperature, indicating that betanin is located at the hydrophobic interior of the bilayer. Other measurements of binding of betanin to DMPC and of indicaxanthin to either DPPC or DMPC vesicles ruled out that these compounds were solubilized in the hydrophobic interior of these bilayers.     

Probing the interaction of polyphenols with lipid bilayers by solid-state NMR spectroscopy

Polyphenols are bioactive natural products that appear to act against a wide range of pathologies. Mechanisms of activity have not been established, but recent studies have suggested that some polyphenols bind to membranes. This study examined the interaction between lipid bilayers and three structurally diverse polyphenols. It was hypothesized that features of the polyphenols such as polarity, molecular size, molecular geometry, and number and arrangement of phenol hydroxyl groups would determine the tendency to interact with the bilayer. The examined compounds included a mixed polyphenol, (-)-epigallocatechin gallate (EGCg); a proanthocyanidin trimer comprising catechin-(4→8)-catechin-(4→8)-catechin (cat?; and a hydrolyzable tannin, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG). These polyphenols were incorporated at different levels into 2H-labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles (MLVs). 31P and 2H solid-state NMR experiments were performed to determine the dynamics of the headgroup region and the hydrophobic acyl chain region of the lipid bilayer upon addition of polyphenols. The chemical shift anisotropy (CSA) width of the 31P NMR spectra decreased upon addition of polyphenols. Addition of PGG induces a dramatic reduction on the CSA width compared with the control lipid bilayer sample, whereas addition of cat? barely reduces the CSA width. The 2H quadupolar splitting of the lipids also decreased upon addition of polyphenols. At the same concentration, PGG substantially reduced the quadrupolar splitting, whereas cat? barely reduced it when compared with the control sample. From a calculation of the order parameters of the acyl chain region of the lipid bilayer, it was concluded that the hydrophobic part of the lipid bilayer was perturbed by PGG, whereas cat? did not cause large perturbations. The data suggest that the polarity of the polyphenols affects the interaction between tannins and membranes. The interactions may relate to the biological activities of polyphenols.     

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.     

Isolation and identification of the first C-17 limonin epimer, epilimonin

Limonoids are a family of highly oxygenated triterpenoid secondary metabolites found in significant quantities in Citrus and reported to possess multiple health promoting properties. This is the first known report of the isolation and characterization of an epimer of limonin. The epimer, named epilimonin, was isolated by fractional crystallization from a mixture consisting mainly of limonin and epilimonin obtained as byproduct from our efforts to isolate limonin glucoside. Side-by-side comparison of the MS, IR, and (1)H and (13)C NMR data of epilimonin and limonin lead to the assignment of C-17 as the site of epimerization. An earlier study on the bioavailability of limonin glucoside in humans had indicated that limonin glucoside was metabolized to give limonin and a second limonin metabolite. Results from analyzing epilimonin by the same chromatographic conditions used for the bioavailability study suggest that the second limonin metabolite was epilimonin.     

Colorimetric method for the estimation of total limonoid aglycones and glucoside contents in citrus juices

A method for estimating the total limonoid aglycone and glucoside concentrations in Citrus samples in terms of limonin and limonin glucoside equivalents is presented. The method consists of extraction followed by colorimetric quantification. The colorimetric quantification was based on the formation of red to orange colored derivatives resulting from the treatment of limonin, limonin glucoside, or a fruit extract with 4-(dimethylamino)benzaldehyde (DMAB) in the presence of perchloric and acetic acids. Absorbance maxima for the limonin and limonin glucoside derivatives were found to be 470 and 503 nm, respectively. The influence of DMAB concentration, reaction time, and solvent composition on color development and sensitivity were investigated and optimal assay conditions established. With a microplate format under these conditions, the limits of detection and quantification were determined to be 0.25 and 0.50 microg/mL for limonin and 0.50 and 1.0 microg/mL for limonin glucoside.     

Differential scanning calorimetry evidence of the enhancement of beta-sitosterol absorption across biological membranes mediated by beta-cyclodextrins

beta-Sitosterol is a plant sterol that has received much attention because of its effectiveness in reducing the absorption of dietary cholesterol, as well as in offering protection from cardiovascular diseases and cancer development. Thus, the knowledge of the interaction of beta-sitosterol with biological membranes can help in understanding its mechanism of action. In the present paper, the differential scanning calorimetry technique has been used to study the interaction of beta-sitosterol with a biomembrane model constituted by dimyristoylphosphatidylcholine multilamellar vesicles. Furthermore, kinetic experiments have been carried out to follow the uptake of beta-sitosterol by biomembranes and the effect of beta-cyclodextrins on such a process. Our results indicate that opportune concentrations of beta-cyclodextrins improve the uptake of beta-sitosterol by phospholipid membranes.     

Effects of divalent cations on nadh‐linked electron transfer in corn root plasma membrane

The effects of calcium (Ca²⁺), cadmium (Cd²⁺), and copper (Cu²⁺) cations on NADH‐linked electron transfer in com root plasma membrane vesicles were investigated. The reduction of both cytochrome c and ferricyanide were slightly stimulated by Ca²⁺ but not significantly affected by Cd²⁺. However, Cd²⁺ induced a redox‐linked increase in light scattering suggesting an increase in the size/volume of the vesicles. The presence of micromolar levels of Cu²⁺ decreased the reduction rates of both cytochrome c and ferricyanide. However, in contrast to ferricyanide reduction, Cu inhibition to the cytochrome c reduction was more effective, and was less sensitive to ionic strength. Copper inhibition changed the Michaelis‐Menten dependence of the ferricyanide reduction but not that of cytochrome c. These results suggest that the reduction of cytochrome c. and ferricyanide must occur at different membrane sites.