Biological activity of phenolic lipids.

Phenolic lipids are a very diversified group of compounds derived from mono and dihydroxyphenols, i.e., phenol, catechol, resorcinol, and hydroquinone. Due to their strong amphiphilic character, these compounds can incorporate into erythrocytes and liposomal membranes. In this review, the antioxidant, antigenotoxic, and cytostatic activities of resorcinolic and other phenolic lipids are described. The ability of these compounds to inhibit bacterial, fungal, protozoan and parasite growth seems to depend on their interaction with proteins and/or on their membrane-disturbing properties.

In vitro effects of PNP and PNMC on apoptosis and proliferation in the hen ovarian stroma and prehierarchal follicles.

This study aimed to examine the mRNA expression, activity, and immunolocalisation of apoptosis/proliferation regulating factors following in vitro exposure of the stroma, white (WFs), and yellowish (YFs) follicles of the chicken ovary to 4-nitrophenol (PNP) or 3-methyl-4-nitrophenol (PNMC). PNMC increased the mRNA expression of caspase-3, -8, Apaf-1, and cytochrome c in the ovarian stroma. The activity of caspase-3, -8, and -9 decreased in WFs in both nitrophenol-treated groups. PNP reduced the number of caspase-3-positive cells in the stromal connective tissue (CT) and the theca interna and externa layers of WFs. In the stroma, the proliferating index decreased in the wall of primary follicles in both nitrophenol-treated groups, however, in the CT, the effect of PNMC was opposite. In the theca interna of WFs, PNP diminished the proliferating index. These results suggest that nitrophenols might impact the development of chicken ovarian follicles by affecting cell death and proliferation.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.

Higher cardol homologs (5-alkylresorcinols) in rye seedlings.

The occurrence of alkylresorcinols, polyketide compounds that in the same homologous series as cardol isolated from Anacardium occidentale (cashew) or bilobol from Ginkgo biloba which are derivatives of 1,3-dihydroxy-5-alk(en)ylbenzene, have been demonstrated in developing rye (Secale cereale L.) kernels. The 3-day-old seedlings grown in sterile conditions already contain detectable amounts of phenolic compounds that were identified as alkylresorcinols. This fraction is the mixture of saturated and enoic homologs of various lengths of the aliphatic side chain. The composition of homologs is similar to that determined in mature grains. The relatively high level of alkylresorcinols in mitochondria and plastids (enhanced approximately twice in the absence of light) suggests that their synthetic pathway and/or biological function may be related to these cellular compartments. Resorcinolic lipids, when present in the external medium, are taken up by seedlings in the energy-dependent manner.

Permeability changes of erythrocytes and liposomes by 5-(n-alk(en)yl) resorcinols from rye.

5-(n-Alk(en)yl) resorcinols can induce potassium release from liposomes and erythrocytes. The results suggest that 5-(n-pentyl)resorcinol can induce a specific permeability to protons as well as to potassium and other small molecules. The highest permeability changes were found in the presence of 5-(n-pentadecyl)resorcinol and alkenyl resorcinols. Orcin and resorcin were without effect. The size of permeant as investigated by turbidity measurements indicated that Ca2+ and Mg2+ cannot pass through the alkyl resorcinol-modified membrane but can pass through the alkenyl resorcinol-modified membrane. It was observed that alkenyl resorcinol at a concentration of 15 microM induced not only potassium release but also lysis of erythrocytes.

The effect of 5-(n-alk(en)yl) resorcinols from rye on membrane structure.

The increased membrane permeability for K+, glycerol and erythritol, and membrane lysis induced by alkyl and alkenyl resorcinols, respectively, might be due to the interaction with membrane proteins and the formation of reversed micelles. The 5-(n-alk(en)yl) resorcinols show a very high stability at the air/water interface. The molecular area is 0.28 and 0.37 nm2 (at 30 mN/m) for alkyl and alkenyl resorcinols from rye, respectively. Differential scanning calorimetry experiments show a miscibility of alk(en)yl resorcinols with phosphatidylcholines. Only for alkenyl resorcinols is a small reduction found in the free energy of dipalmitoyl phosphatidylcholine. Electron microscopy studies show protein patching in erythrocyte membranes after the addition of resorcinols. The resorcinol-induced K+ release is not influenced by the presence of proteolytic enzymes, but strongly reduced by bovine serum albumin and glycophorin. 31P-NMR measurements show the occurrence of an isotropic and hexagonal signal in egg phosphatidylcholine in the presence of about 30 mol% alk(en)yl resorcinol.

Dual effect of alkylresorcinols, natural amphiphilic compounds, upon liposomal permeability.

The effect of 5-n-alkylresorcinols, natural amphiphilic compounds, upon properties of phospholipid vesicles depends on their localization asymmetry. A significant increase of the bilayer permeability is observed when the title compounds are present only in the external medium. When these amphiphiles are preincorporated into the bilayer during its formation, the resulting liposomes effectively encapsulate water-soluble solutes which still remain in liposomes after 25 h. Additionally, the size of liposomes made of alkylresorcinol-phosphatidylcholine mixtures after eight cycles of freezing and thawing only (180-200 nm) is severalfold smaller than the size of vesicles prepared in a similar way from phospholipids only and the resulting liposomes are more homogeneous. These liposomes modified with alkylresorcinols are also stable during 40 day storage at both 4 degrees C and 20 degrees C, in contrast to control liposomes that already strongly aggregate after 10 days.

Formation of liposomes by resorcinolic lipids, single-chain phenolic amphiphiles from Anacardium occidentale L.

Resorcinolic lipids isolated from Anacardium occidentale nut oil extract (CNSL), unsaturated congeners of those isolated from bacterial and graminaceous sources, form at alkaline conditions liposomal structures alone as well as in the mixtures with cholesterol, fatty acids or phosphatidylethanolamine. Those vesicular structures show relatively high entrapment of the marker and stability of their size. The retention of the captured solute depends upon the type of resorcinolic lipid and on the temperature, but in general, is lower than control phospholipid liposomes.

The effect of nonadec(en)ylresorcinol on the fluidity of liposome and erythrocyte membranes.

The effect of alk(en)ylresorcinol homologs (5-(n-nonadecyl)- and 5-(n-nonadecenyl)resorcinol) on the mobility of 5-doxyl- and 12-doxylstearate spin probes incorporated into DMPC, DMPC-cholesterol and erythrocyte membranes was studied. It was found that both homologs affect the properties of hydrophobic environment of the membranes: (1) In DMPC vesicles both homologs induce an increase in the order parameter of 5-doxylstearate at temperatures of Tc and above. (2) At higher concentrations of both homologs a decrease in mobility of the 12-doxylstearate was also observed. (3) In the presence of cholesterol in the liposome membrane the influence of alk(en)ylresorcinols on the mobility of spin probes was much greater, depending on the cholesterol content and the position of the probe in the bilayer. (4) In natural membranes (erythrocyte ghosts) both alkyl- and alkenylresorcinols induced a decrease of mobility in the region of 12-doxylstearate as well as in the region closer to the polar head groups of lipids (5-doxylstearate).

Binding of phospholipase A2 to zwitterionic bilayers is promoted by lateral segregation of anionic amphiphiles.

Catalytic action of phospholipase A2 is appreciably influenced by the organization and dynamics of bilayers of glycerophosphocholines (Apitz-Castro et al. (1988) Biochim. Biophys. Acta 688, 341-348). However, such effects of the quality of the interface are not observed with bilayers of glycerophosphoryl methanol and other anionic phospholipids (Jain et al. (1986) Biochim. Biophys. Acta 860, 435-447). Such differences between the catalytic susceptibility of zwitterionic versus anionic bilayers are due to a large difference in the affinity of the enzyme for these interfaces. Binding to phospholipase A2 to zwitterionic interfaces can be promoted in the presence of certain anionic additives. For example in the pre-steady-state phase of hydrolysis, segregation of the nacently produced products of hydrolysis could promote binding of phospholipase A2 to regions of higher anionic charge density in the zwitterionic interface. In this paper we show that the dynamics of segregation of the nacently produced products of hydrolysis in zwitterionic bilayers can be readily followed by monitoring the fluorescence intensity of the cationic dye NK-529 (Yu and Jain (1989) Biochim. Biophys. Acta 980, 15-22). The fluorescence emission characteristics of NK-529 change appreciably due to self-quenching of the bound dye molecules as the fatty acid molecules segregate in the bilayer. The kinetics of segregation of fatty acids during the course of hydrolysis of bilayers of zwitterionic phospholipids by phospholipase A2 exhibits an unequivocal correlation with a variety of phenomena that are observed during the transition from the pre-steady-state phase to the steady-state phase of hydrolysis in the reaction progress curves as a function of temperature and in the presence of lipophilic additives.

Antimutagenic activity of alkylresorcinols from cereal grains.

Alkylresorcinols, natural amphiphilic compounds commonly found in cereal grains, markedly decreased mutagenic activity of four standard mutagens examined in the Ames test. The effect was the strongest in the case of indirect-acting mutagens, benzo[a]pyrene and 2-aminofluorene. In the case of direct-acting mutagens, daunorubicin and methyl methanesulfonate, the diminution of the mutagenic activity by the alkylresorcinols was smaller but still noticeable. In the Sister Chromatid Exchanges test (SCEs) with cultured in vitro human blood-derived lymphocytes, a significant decrease of SCEs frequency induced by benzo[a]pyrene was observed in the presence of alkylresorcinols. These preliminary results seem to be important in the aspect of possible antimutagenic and anticarcinogenic potency of alkylresorcinols found in cereal grains.

Quantum mechanical and experimental oxidation studies of pentadecylresorcinol, olivetol, orcinol and resorcinol.

Resorcinols (pentadecylresorcinol, olivetol, orcinol and resorcinol) exhibit antioxidant properties in liposomal systems. Antioxidant potency depends on the length of the alkyl chain. Pentadecylresorcinol has been demonstrated to be the most active antioxidant, indicating significance of its alkyl chain in a lipid bilayer. Quantum DFT computations demonstrated that hydroxyl group attached to the ring is the first target for the hydrogen abstraction after formation of the radical. However, the carbons of the side chain could also participate in the antioxidant properties of the alkylresorcinols. Formation of the radical at the hydroxyl oxygen initiates changes in the electron density which destabilise the whole system and subsequently leads to oxidation of the ring. The detailed study of lipophilicity and electrostatic properties of resorcinols is discussed.

The effect of 5-(n-alk(en)yl)resorcinols on membranes. I. Characterization of the permeability increase induced by 5-(n-heptadecenyl)resorcinol.

The influence of 5-(n-heptadecenyl)resorcinol, one of the main rye grain resorcinol derivatives, on the erythrocyte membrane permeability for nonelectrolytes differing in molecular size was studied turbidimetrically at various concentrations of the resorcinol derivative studied. The alkenylresorcinol induced increased permeability of the erythrocyte membrane for all the solutes studied (glycerol, m-erythritol, D-glucose, sucrose and polyethylene glycol 1000). At a given concentration of 5-(n-heptadecenyl)resorcinol the highest permeability increases were obtained for the smallest solutes. The membrane lipid to alkenylresorcinol ratio necessary for initiation of the increase of the erythrocyte membrane permeability for the solutes studied varied from 273 to 82 for glycerol and polyethylene glycol 1000, respectively, indicating that this strong membrane perturbing action may be primarily responsible for the biological effect of phenolic lipids.

The effect of 5-(n-alk(en)yl)resorcinols on membranes. II. Dependence of the aliphatic chain length and unsaturation.

The effect of chain length and unsaturation on the haemolytic properties of cereal resorcinolic lipids, (5-n-alk(en)ylresorcinols), was studied using isolated saturated, monoenoic and dienoic homologues. The haemolytic activities of the homologues studied were proportional to the degree of the side chain unsaturation and inversely proportional to the chain length. At temperatures close to physiological of animal organisms the most active were mono- and di-enoic homologues of 5-n-heptadecyl and 5-n-nonadecyl resorcinols. The results might point to the importance of short-chain cereal resorcinolic lipids in animal and human nutrition.

Interaction of alkylresorcinols with proteins.

Alkylresorcinol homologs form stable monomolecular layers at air-water interface. Their interaction with proteins present in the subphase results in an increase of alkylresorcinol molecular packing in the extent dependent upon the protein studied. Strongest effects were observed for proteins with large hydrophobic regions, e.g. glycophorin or serum albumin. Interaction of proteins with alkylresorcinol monolayers is stronger than with phospholipids. A decrease and a shift of intrinsic protein fluorescence upon interaction with the compounds studied support their involvement in alteration of hydrophobic regions. For trypsin, 50% quenching was observed at the alkylresorcinol/trypsin ratio of 0.75. Concomitantly, an apparent inhibition of the enzymatic activity was noted. These results indicate that direct interaction of alkylresorcinols and modulation of enzymatic activities should be recognised as a significant part of the biological effect of these cereal bran components.

Determination of octanol/water partition coefficients for long-chain homologs of orcinol from cereal grains.

Polycratic reversed-phase high-performance liquid chromatography (RPHPLC) was used for estimation of the octanol/water partition coefficient for three highly hydrophobic long chain orcinol homologs. The homologs studied (C 15:0, C 17:0 and C 19:0) showed high preference for hydrophobic phase as evidenced by their high octanol/water partition coefficient (log Po/w) values of 7.02-7.74; 8.71-9.47 and 10.49-11.32 for the 95% prediction interval, respectively. Experimentally estimated values were compared with log P values calculated with the use of several fragmental systems. The experimental values of log Po/w are in best agreement to those calculated with the use of the Klopman system (Klopman, G., Namboodiri, K. & Schochet, M., 1985, J. Comput. Chem. 6, 28-38). The lack of appropriate standard compounds with known log Po/w in the range over 6 markedly affected the accuracy of experimental determinations.

Cereal grain resorcinolic lipids inhibit H2O2-induced peroxidation of biological membranes.

Cereal grain resorcinolic lipids (5-n-alk(en)ylresorcinols) at micromolar concentrations are able to protect the erythrocyte membrane against hydrogen peroxide-induced lipid oxidation. The antioxidative effect is dependent upon chain length of alkylresorcinol molecules. The C15:0 homolog (IC50 of 10 microM) exhibited strongest activity whereas for long chain homologs (C19:0 and C23:0) IC50 values were higher, 32.5 and 59 microM, respectively. The protective effect of alkylresorcinolic antioxidants was also dependent on their incorporation into the membrane, that is governed by their water-membrane partition coefficient. The results obtained show that alkylresorcinols should be recognized as hydrophobic, membrane-localised antioxidants.

Modification of the lipid component modulates nuclear matrix nucleolytic activity.

It was shown that lipid composition of plant nuclear matrix depends on procedure of its isolation. The matrix isolated with the use of lithium diiodosalicylate (LiS) differs in its lipid composition from the preparation isolated with the use of nonionic detergent (Triton X-100). It was also shown that the nucleolytic activity of the matrix is related to its lipid component. Matrix depleted in lipids loses half of its nucleolytic activity which is recovered after supplementation with previously extracted lipids. The extent of recovery of the nucleolytic activity is also dependent on the presence of residual DNA in matrix preparation. The recoveries of nucleolytic activities were higher in matrices not depleted in their DNA content.

Modulation of some nuclear matrix enzymatic activities by free fatty acids.

It was shown that two of main enzymatic activities of plant nucleus and nuclear matrix, namely RNA-polymerasic and DNA-nucleolytic are susceptible to modulation with free fatty acids. The effects observed were dependent to both fatty acid length and degree of unsaturation. In nuclei a stimulation of nuclease activity was observed whereas in matrices short chain fatty acids inhibited the studied activity. The effect of fatty acids on RNA-polymerase was also different in nuclei and matrices. In nuclei all fatty acids studied inhibited polymerasic activity whereas in matrices short chain fatty acids stimulated this activity by up to 80% and the long chain fatty acids inhibited by up over 70%. The overall alteration of studied activities in nuclei and matrices by unsaturated fatty acids was similar. Nucleolytic activity was stronger inhibited and polymerasic activity was stimulated when the effects of linoleic and linolenic acids were studied. The results suggest possible importance of lipid component in nuclear matrix biological function.

Proteolysis of spectrin by trypsin and pronase in the presence of phospholipid suspensions.

The effect of phospholipid suspensions on the proteolysis of isolated spectrin was examined by SDS-polyacrylamide gradient gel electrophoresis. Proteolysis of spectrin in the membranes by trypsin and pronase was also studied. It was found that electrophoretic patterns of spectrin fragments were influenced by the presence of the suspension prepared from phosphatidylethanolamine:phosphatidylserine (60:40) mixture and of phosphatidylcholine. Qualitative changes in the proteolytic patterns obtained after proteolysis of spectrin by pronase in the presence of phosphatidylcholine suspension were observed. The changes in the sensitivity of spectrin towards proteases result probably from changes in the accessibility of some peptide bonds upon the interaction of this extrinsic protein with phospholipids.