Phosphorylation state of pyruvate dehydrogenase and metabolite levels in turkey skeletal muscle in normal and pale, soft, exudative meats.

1. Turkey production has increased dramatically as genetic selection has succeeded in increasing body weight and muscle yield to fulfil increasing consumer demand. However, producing fast-growing, heavily muscled birds is linked to increased heat stress susceptibility and can result in pale, soft, exudative (PSE) meat. Previous studies indicated that pyruvate dehydrogenase kinase 4 (PDK4) is significantly reduced in PSE samples, suggesting this as a candidate gene associated with the development of this problem.2. The objective of this study was to determine whether pre-market thermal challenge results in PSE meat as a result of differential expression of PDK4. Two genetic lines of turkeys were used in this study; the Randombred Control Line 2 (RBC2) and a commercial line. Turkeys were exposed to a pre-market thermal challenge of 12 h at 35°C followed by 12 h at 27°C for 5 d. Birds were slaughtered and processed according to industry standards. Pectoralis major samples were categorised as PSE or normal based on marinade uptake and cook loss indicators. In the first experiment, the relative expression of pyruvate dehydrogenase (PDH) and the phosphorylation state of PDH in normal and PSE turkey meat were analysed by western blotting. In the second experiment, the same samples were used to measure metabolite levels at 5 min post-mortem, comparing the normal to the PSE samples.3. The results of the first experiment showed that PSE samples had significantly lower total PDH (P = 0.029) compared to normal meat. However, there was no significant difference in the degree of phosphorylation of sites 1, 2 or 3. In the second experiment, there were no significant differences in glycogen, lactate, glycolytic potential or ATP when comparing PSE to control samples.4. These results suggested that a reduction in PDK4 expression alone does not explain the development of PSE meat.

Comparison of raw meat quality and protein-gel properties of turkey breast fillets processed by traditional or cold-batter mincing technology.

This study was conducted to evaluate the effects of cold batter mincing on meat quality and protein functionality, using turkey fillets that were chill-boned (CB) or hot-boned (HB) with crust-freeze-air-chilling (HB-»CFAC) at -12°C. For each of four replications, 48 toms (male) were raised and processed at Michigan State University Poultry Farm and Meat Laboratory, respectively. After evisceration, the turkeys were subjected to one of the four treatments: (1) traditional mincing of CB fillets after water immersion chilling (WIC); (2) cold batter mincing of WIC, CB, quarter-sectioned (»), and CB-»CFAC; (3) traditional mincing of HB-»CFAC fillets; and (4) cold batter mincing of HB-»CFAC fillets. Before mincing, the pH and R-values of turkey fillets in HB-»CFAC were higher and lower, respectively, than those in CB fillets. During cold-batter mixing, the initial batter temperatures at -1.5 to -2.1°C reached 1.5°C and 14°C at 6 and 12 min, respectively, and ended at 26 to 31°C at 24 min. During traditional mincing, the initial batter temperatures at 3 to 4°C increased by ∼10°C every 6 min, and ended at 32 to 35°C with higher batter temperatures seen for the 2% salt than the 1% salt batter. Dynamic rheological properties indicated that the cold-batter mincing showed elevated G' compared to the batters of traditional mincing, regardless of mixing time, indicating that the gel-setting temperature was reduced in the cold-batter mincing, potentially due to the different amounts of extracted protein and structural change. After cooking, improved cooking yield and protein functionality were observed in the batter of HB-»CFAC fillets than the batter of CB fillets as well as in the batter of 2% salt than the batter 1% salt (P < 0.05). These results indicated that HB-»CFAC fillets produced superior raw meat quality over the CB fillets, and cold batter mincing of HB-»CFAC fillets significantly improved protein functionality compared with the traditional mincing of CB fillets (P < 0.05).

Influence of temperature and growth selection on turkey pectoralis major muscle satellite cell adipogenic gene expression and lipid accumulation.

P. MAJOR: Immature poults have an inefficient thermoregulatory system, and therefore extreme ambient temperatures can impact their internal body temperature. Satellite cells, the only posthatch myonuclei source, are multipotential stem cells and sensitive to temperature. Selection for faster-growing, high-yielding birds has altered satellite-cell properties. The objective of the current study was to determine how temperature affects adipogenic properties of satellite cells isolated from the pectoralis major ( ) muscle of Randombred Control line ( ) and F line turkeys selected only for increased 16-wk body weight from the RBC2 line. Satellite cells were cultured at 2°C incremental temperatures between 33 and 43°C and compared to cells cultured at the control temperature of 38°C to ascertain temperature effects on lipid accumulation and expression of adipogenic genes: CCAAT/enhancer-binding protein-ß ( ), peroxisome proliferator-activated receptor-γ ( ), and stearoyl-CoA desaturase ( ). During proliferation, the amount of quantifiable lipid in both F and RBC2 satellite cells increased at temperatures above 38°C ( P <  0.01) and decreased at temperatures below 38°C ( P < 0.01). Above 38°C, RBC2 satellite cells had more lipid ( P = 0.02) compared to the F line, whereas there were few differences between lines below 38°C. At 72 h of proliferation, expression of C/EBPß , PPARγ , and SCD decreased ( P ≤  0.02) as temperatures increased from 33 to 43°C in both cell lines. During differentiation expression of C/EBPß increased ( P <  0.01) as temperatures increased from 33 to 43°C in both cell lines. In F line satellite cells, PPARγ expression decreased ( P <  0.01) with increasing temperatures during differentiation, whereas there was no linear trend in RBC2 cells. During differentiation expression of SCD increased as temperatures increased ( P <  0.01) in RBC2 cells, and there was no linear trend within the F line. Results from the current study suggest that environmental temperature can affect p. major satellite cellular fate; however, selection for increased body weight had little impact on these cellular responses.

Temperature effect on proliferation and differentiation of satellite cells from turkeys with different growth rates.

Poultry selected for growth have an inefficient thermoregulatory system and are more sensitive to temperature extremes. Satellite cells are precursors to skeletal muscle and mediate all posthatch muscle growth. Their physiological functions are affected by temperature. The objective of the current study was to determine how temperature affects satellite cells isolated from the pectoralis major (p. major) muscle (breast muscle) of turkeys selected for increased 16 wk body weight (F line) in comparison to a randombred control line (RBC2) from which the F line originated. Pectoralis major muscle satellite cells were thermally challenged by culturing between 33°C and 43°C to analyze the effects of cold and heat on proliferation and differentiation as compared to control temperature of 38°C. Expression levels of myogenic regulatory factors: myogenic differentiation factor 1 (MYOD1) and myogenin (MYOG) were quantified by quantitative polymerase chain reaction (qPCR). At all sampling times, proliferation increased at a linear rate across temperature in both the RBC2 and F lines. Differentiation also increased at a linear rate across temperature from 33 to 41°C at all sampling times in both the F and RBC2 lines. Satellite cells isolated from F line turkeys were more sensitive to both hot and cold temperatures as proliferation and differentiation increased to a greater extent across temperature (33 to 43°C) when compared with the RBC2 line. Expression of MYOD1 and MYOG increased as temperatures increased from 33 to 41°C at all sampling times in both the F and RBC2 lines. These results demonstrate that satellite cell function is sensitive to both cold and hot temperatures and p. major muscle satellite cells from F line turkeys are more sensitive to temperature extremes than RBC2 satellite cells.

Deep transcriptome sequencing reveals differences in global gene expression between normal and pale, soft, and exudative turkey meat.

Previous studies from our laboratory suggested that differential expression of genes between normal and pale, soft, and exudative (PSE) turkey is associated with development of the PSE syndrome. However, a detailed understanding of molecular mechanisms responsible for the development of this meat defect remains unclear. The objective of this study was to extend and complement our previous work by using deep transcriptome RNA sequence analysis to compare the respective transcriptome profiles and identify molecular mechanisms responsible for the etiology of PSE turkey meat. Turkey breasts (n = 43) were previously classified as normal or PSE using marinade uptake as an indicator of quality (high = normal; low = PSE). Total RNA from breast muscle samples with the highest (n = 4) and lowest (n = 4) marinade uptake were isolated and sequenced using the Illumina GA(IIX) platform. The results indicated differential expression of 494 loci (false discovery rate < 0.05). Changes in gene expression were confirmed using quantitative real-time PCR. Pathway analysis of differentially expressed genes suggested abnormalities of calcium homeostasis and signaling pathways regulating actin cytoskeleton structure as well as carbohydrate metabolism and energy production in PSE samples. Dysregulation of postmortem glucose oxidation in PSE turkey was suggested by both dramatic downregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA, the most downregulated gene, and a decrease in the protein product (P = 0.0007) as determined by immunoblot analysis. These results support the hypothesis that differential expression of several genes and their protein products contribute to development of PSE turkey.

Expression profiles for genes in the turkey major histocompatibility complex B-locus.

The major histocompatibility complex (MHC) is a highly polymorphic region of the genome essential to immune responses and animal health. In galliforms, the MHC is divided into 2 genetically unlinked regions (MHC-B and MHC-Y). Many MHC-B genes are involved in adaptive or innate immunity, yet others have nonimmune or unknown functions. The sequenced MHC-B region of the turkey (Meleagris gallopavo) contains 40 genes, the majority of which are predicted transcripts based on comparison with the chicken or quail, without direct evidence for expression. This study was designed to test for the presence of MHC-B gene transcripts in a panel of immune and nonimmune system tissues from domestic turkeys. This analysis provides the first locus-wide examination of MHC-B gene expression in any avian species. Most MHC-B genes were broadly expressed across tissues. Expression of all predicted genes was verified by reverse-transcription PCR, including B-butyrophilin 2 (BTN2), a predicted gene with no previous evidence for expression in any species. Previously undescribed splice variants were also detected and sequenced from 3 genes. Characterization of MHC-B expression patterns helps elucidate unknown gene functions and potential gene coregulation. Determining turkey MHC-B expression profiles increases our overall understanding of the avian MHC and provides a necessary resource for future research on the immunological response of these genes.

Differential gene expression between normal and pale, soft, and exudative turkey meat.

In response to high consumer demand, turkeys have been intensively selected for rapid growth rate and breast muscle mass and conformation. The success in breeding selection has coincided with an increasing incidence of pale, soft, and exudative (PSE) meat defect, especially in response to heat stress. We hypothesized that the underlying mechanism responsible for the development of PSE meat arises from differences in expression of several critical genes. The objective of this study was to determine differential gene expression between normal and PSE turkey meat using a 6K turkey skeletal muscle long oligonucleotide microarray. Breast meat samples were collected from Randombred Control Line 2 turkeys at 22 wk of age, and classified as normal or PSE primarily based on marinade uptake (high = normal, low = PSE). Total RNA was isolated from meat samples with the highest (normal, n = 6) and the lowest (PSE, n = 6) marinade uptake. Microarray data confirmation was conducted using quantitative real-time PCR. Selection of differentially expressed genes for pathway analysis was performed using a combination of fold change (FC) ranking (FC < -1.66, FC >1.66) and false discovery rate (<0.35) as criteria. The calcium signaling pathway was highlighted as the top canonical pathway associated with differential gene expression between normal and PSE turkey. Dramatic downregulation of fast-twitch myosin heavy chain coupled with upregulation of slow-twitch myosin and troponin C suggested a switch of skeletal muscle isoforms, which may alter muscle fiber arrangement and formation of actin-myosin complexes. Changes in expression of genes in the actin cytoskeleton signaling pathway also suggest altered structures of actin filaments that may affect cell motility as well as strength and flexibility of muscle cells. Substantial downregulation of pyruvate dehydrogenase kinase, isozyme 4 was observed in PSE samples, suggesting altered regulation of the aerobic metabolic pathway in the birds that developed PSE meat defect.

Versican, matrix Gla protein, and death-associated protein expression affect muscle satellite cell proliferation and differentiation.

Our previous transcriptional profiling study using a turkey skeletal muscle-specific oligonucleotide microarray revealed over 3,000 genes that were differentially expressed at 3 critical stages of muscle development: 18 d embryonic, 1 d posthatch, and 16 wk of age. The genes versican, matrix Gla protein (MGP), and death-associated protein (DAP) were selected to study for their potential effects on muscle satellite cell proliferation and differentiation, as their functions in other tissues are suggestive of possible key roles in the regulation of myogenesis and they are differentially expressed throughout muscle development in the turkey. Using small interfering RNA to knockdown the expression of these genes during proliferation and differentiation, each of the genes was found to differentially affect proliferation and differentiation. Versican and MGP predominantly affected proliferation with line effects, but later stages of differentiation were affected by the knockdown of versican and MGP. The underexpression of DAP inhibited myotube formation, which is a necessary stage in the development of muscle fibers. Without myotube development, muscle fiber formation will be inhibited or abolished. This is the first report that these genes with no previously documented functions with regard to muscle development play a critical role in muscle cell proliferation and differentiation.

Differential expression of calcium-regulating genes in heat-stressed turkey breast muscle is associated with meat quality.

Aberrant postmortem Ca(2+)-regulation in the early postmortem period is associated with the occurrence of inferior meat quality in turkeys, described as pale, soft, and exudative (PSE). The objective of the current study was to quantify expression of 4 candidate genes responsible for maintaining Ca(2+) homeostasis in turkey skeletal muscle as a function of heat stress: α and ß ryanodine receptors (RYR; Ca(2+)-release channels), the sarco/endoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1), and the sarcoplasmic reticulum, Ca(2+)-storage protein calsequestrin (CASQ1). Two genetic lines of turkeys were used: a growth-selected commercial line and a randombred control line. Market-age birds were subjected to one of 5 heat stress treatments: no heat, 1 d, 3 d, 5 d, or 7 d of heat followed by 7 d of ambient temperature. Breast muscle samples were harvested and classified as normal or PSE using the meat quality parameters percentage of marinade uptake and percentage of cook loss. These parameters differed significantly by line, heat stress treatment, and meat quality status. Expression of candidate genes was measured using TaqMan quantitative PCR. Heat treatment was associated with significantly enhanced expression of αRYR, ßRYR, and CASQ1 in normal muscle from both lines. Conversely, mRNA abundance of these genes was reduced in PSE muscle from both lines and recovered or increased by 7 d + 7 d of rest. Genetic line differences were observed at several time points. Expression of SERCA1 in both normal and PSE samples from both lines was unchanged or trended downward with heat stress. Taken together, genetic line and heat-stress treatment affected the expression of important Ca(2+)-regulating genes in association with meat quality status. The data suggest that birds whose meat leads to PSE may fail to respond to heat stress appropriately due to a delay in the upregulation of the important calcium-regulating genes: αRYR, ßRYR, and CASQ1.

Differential expression of genes characterizing myofibre phenotype.

Skeletal muscle is composed of metabolically heterogeneous myofibres that exhibit high plasticity at both the morphological and transcriptional levels. The objective of this study was to employ microarray analysis to elucidate the differential gene expression between the tonic-'red' anterior latissimus dorsi (ALD) muscle, the phasic-'white' posterior latissimus dorsi (PLD) and 'mixed'-phenotype biceps femoris (BF) in 1-week-and 19-week-old male turkeys. A total of 170 differentially expressed genes were identified in the muscle samples analysed (P < 0.05). Gene GO analysis software was utilized to identify top gene networks and metabolic pathways involving differentially expressed genes. Quantitative real-time PCR for selected genes (BAT2D, CLU, EGFR and LEPROT) was utilized to validate the microarray data. The largest differences were observed between ALD and PLD muscles, in which 32 genes were over-expressed and 82 genes were under-expressed in ALD1-PLD1 comparison, and 70 genes were over-expressed and 70 under-expressed in ALD19-PLD19 comparison. The largest number of genes over-expressed in ALD muscles, as compared to other muscles, code for extracellular matrix proteins such as dystroglycan and collagen. The gene analysis revealed that phenotypically 'red' BF muscle has high expression of glycolytic genes usually associated with the 'white' muscle phenotype. Muscle-specific differences were observed in expression levels of genes coding for proteins involved in mRNA processing and translation regulation, proteosomal degradation, apoptosis and insulin resistance. The current findings may have large implications in muscle-type-related disorders and improvement of muscle quality in agricultural species.

Characterization of a 6K oligonucleotide turkey skeletal muscle microarray.

Consumer demand for lean, inexpensive meat products has driven the domestic turkey (Meleagris gallopavo) industry to unprecedented production; however, this has coincided with an increase in growth-induced myopathies and meat quality defects. With the aim of developing a new tool for the study of turkey growth and development at the muscle transcriptome level, a 6K oligonucleotide microarray was constructed, the Turkey Skeletal Muscle Long Oligo (TSKMLO) microarray. Skeletal muscle samples were collected at three critical stages in muscle development: 18-day embryo (hyperplasia), 1-day post-hatch (hypertrophy), and 16-week (market age) from two genetic lines of turkeys: RBC2, a line maintained without selection pressure, and F, a line selected from the RBC2 line for increased 16-week body weight. Oligonucleotides were designed from sequences obtained from skeletal muscle cDNA libraries from the three developmental stages. Several unique controls, including mismatch and distance controls and scrambled sequences, were designed for 30 genes. Quality control hybridizations were completed, confirming the validity and repeatability of the array. Control features were evaluated across two larger experiments comparing developmental stage within genetic line or genetic line within each developmental stage, totaling 70 arrays. Mismatch and scrambled sequences appeared to be useful controls of specific hybridization for most genes. In addition, quantitative real-time RT-PCR confirmed microarray results. This creation and assessment of the TSKMLO array provides a valuable community resource for the study of gene expression changes related to turkey muscle growth and development.

Pale, soft, exudative turkey--The role of ryanodine receptor variation in meat quality.

The poultry industry has made significant advances in growth rate, feed efficiency, and breast muscle yield through intensive breeding of turkeys. However, a meat quality problem known as pale, soft, exudative (PSE) meat presents the industry with a major challenge during periods of stress, such as the onset of a prolonged heat wave. The biochemical characteristics of PSE turkey are strikingly similar to those of PSE pork. Abnormally rapid postmortem metabolism, stimulated in part by high concentrations of calcium ions, may be one of the underlying factors associated with the incidence of PSE turkey. This presentation summarizes our studies on the avian ryanodine receptors and suggests that heat stress may alter the expression pattern of splice variants of ryanodine receptors, which, in turn, could affect postmortem calcium homeostasis.

Characterization of expressed sequence tags from turkey skeletal muscle.

This study was designed to identify important muscle gene homologues in the turkey. Three skeletal muscle cDNA libraries representing distinct muscle developmental stages were constructed. A total of 20,042 clones were sequenced resulting in 13,023 finished high-quality sequences (trimmed, quality scored and masked) for analysis. Sequence clustering produced 1113 contigs and 4144 singletons (5257 putative transcripts). Sequences were compared by blastn to the chicken whole-genome sequence and to the Ensembl and NCBI databases to identify homologous sequences. These surveys indicated that most of the important muscle genes are included in the sequence collection. Examination of contigs identified 1288 single nucleotide polymorphisms and in 320 of those the minor allele was observed to be present in more than one sequence. This resource provides sequence variants for numerous genes in the turkey, as demonstrated by the SNP haplotypes that were constructed for 10 genes. Sequences obtained in this study provide the basis for constructing a skeletal muscle-focused microarray, a tool that will facilitate the analysis of genes expressed during turkey muscle development, as well as the expression of genes underlying the genetic basis of muscle characteristics associated with meat quality.

Characterization of a cardiac complementary deoxyribonucleic acid library from the Turkey (Meleagris gallopavo).

Although the domestic turkey (Meleagris gallopavo) is a valuable agricultural commodity, genetic studies on this species lag behind those of other agricultural species. In this study, we examined expressed sequence tags (EST) from a turkey cardiac cDNA library constructed from 4 birds representing 2 developmental stages. A collection of 3,937 EST sequences were sequenced and analyzed for gene annotation and sequence variation. Clustering of sequences resulted in 353 contigs and 874 singletons (1,227 putative transcripts). All EST sequences were compared by BLASTN to the chicken whole genome sequence and to Ensembl and National Center for Biotechnology Information databases. The majority of significant matches correspond to genes found in the chicken. Sequence polymorphisms were identified in 310 contigs, 64 where the minor allele was observed to be present in more than 1 sequence. This study gives species-specific insight into the cardiac transcriptome of turkeys and provides resources for future studies of cardiac function.

Comparison of iron-catalyzed DNA and lipid oxidation.

Lipid and DNA oxidation catalyzed by iron(II) were compared in HEPES and phosphate buffers. Lipid peroxidation was examined in a sensitive liposome system constructed with a fluorescent probe that allowed us to examine the effects of both low and high iron concentrations. With liposomes made from synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine or from rat liver microsomal lipid, lipid peroxidation increased with iron concentration up to the range of 10--20 microM iron(II), but then rates decreased with further increases in iron concentration. This may be due to the limited amount of lipid peroxides available in liposomes for oxidation of iron(II) to generate equimolar iron(III), which is thought to be important for the initation of lipid peroxidation. Addition of hydrogen peroxide to incubations with 1--10 microM iron(II) decreased rates of lipid peroxidation, whereas addition of hydrogen peroxide to incubations with higher iron concentrations increased rates of lipid peroxidation. Thus, in this liposome system, sufficient peroxide from either within the lipid or from exogenous sources must be present to generate equimolar iron(II) and iron(III). With iron-catalyzed DNA oxidation, hydrogen peroxide always stimulated product formation. Phosphate buffer, which chelates iron but still allows for generation of hydroxyl radicals, inhibited lipid peroxidation but not DNA oxidation. HEPES buffer, which scavenges hydroxyl radicals, inhibited DNA oxidation, whereas lipid peroxidation was unaffected since presumably iron(II) and iron(III) were still available for reaction with liposomes in HEPES buffer.

Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin.

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) in the presence of nanomolar Ca(2+) concentrations. However, the role of CaM activation in the mechanisms that control Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscle and in the heart remains unclear. In media that contained 100 nM Ca(2+), the rate of (45)Ca(2+) release from porcine skeletal muscle SR vesicles was increased approximately threefold in the presence of CaM (1 microM). In contrast, cardiac SR vesicle (45)Ca(2+) release was unaffected by CaM, suggesting that CaM activated the skeletal RyR1 but not the cardiac RyR2 channel isoform. The activation of RyR1 by CaM was associated with an approximately sixfold increase in the Ca(2+) sensitivity of [(3)H]ryanodine binding to skeletal muscle SR, whereas the Ca(2+) sensitivity of cardiac SR [(3)H]ryanodine binding was similar in the absence and presence of CaM. Cross-linking experiments identified both RyR1 and RyR2 as predominant CaM binding proteins in skeletal and cardiac SR, respectively, and [(35)S]CaM binding determinations further indicated comparable CaM binding to the two isoforms in the presence of micromolar Ca(2+). In nanomolar Ca(2+), however, the affinity and stoichiometry of RyR2 [(35)S]CaM binding was reduced compared with that of RyR1. Together, our results indicate that CaM activates RyR1 by increasing the Ca(2+) sensitivity of the channel, and further suggest differences in CaM's functional interactions with the RyR1 and RyR2 isoforms that may potentially contribute to differences in the Ca(2+) dependence of channel activation in skeletal and cardiac muscle.

Regulation of RYR1 activity by Ca(2+) and calmodulin.

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.

Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn.

Anti-oxidant bioassay-directed extraction of the fresh leaves and stems of Ocimum sanctum and purification of the extract yielded the following compounds; cirsilineol [1], cirsimaritin [2], isothymusin [3], isothymonin [4], apigenin [5], rosmarinic acid [6], and appreciable quantities of eugenol. The structures of compounds 1-6 were established using spectroscopic methods. Compounds 1 and 5 were isolated previously from O. sanctum whereas compounds 2 and 3 are here identified for the first time from O. sanctum. Eugenol, a major component of the volatile oil, and compounds 1, 3, 4, and 6 demonstrated good antioxidant activity at 10-microM concentrations. Anti-inflammatory activity or cyclooxygenase inhibitory activity of these compounds were observed. Eugenol demonstrated 97% cyclooxygenase-1 inhibitory activity when assayed at 1000-microM concentrations. Compounds 1, 2, and 4-6 displayed 37, 50, 37, 65, and 58% cyclooxygenase-1 inhibitory activity, respectively, when assayed at 1000-microM concentrations. Eugenol and compounds 1, 2, 5, and 6 demonstrated cyclooxygenase-2 inhibitory activity at slightly higher levels when assayed at 1000-microM concentrations. The activities of compounds 1-6 were comparable to ibuprofen, naproxen, and aspirin at 10-, 10-, and 1000-microM concentrations, respectively. These results support traditional uses of O. sanctum and identify the compounds responsible.

Cyclooxygenase active bioflavonoids from Balaton tart cherry and their structure activity relationships.

Several flavonoids and isoflavonoids isolated from Balaton tart cherry were assayed for prostaglandin H endoperoxide synthase (PGHS-1) enzyme or cyclooxygenase isoform-1 (COX-1) activity. Genistein showed the highest COX-1 inhibitory activity among the isoflavonoids studied, with an IC50 value of 80 microM. Kaempferol gave the highest COX-1 inhibitory activity among the flavonoids tested, with an IC50 value of 180 microM. The structure-activity relationships of flavonoids and isoflavonoids revealed that hydroxyl groups at C4', C5 and C7 in isoflavonoids were essential for appreciable COX-1 inhibitory activity. Also, the C2-C3 double bond in flavonoids is important for COX-1 inhibitory activity. However, a hydroxyl group at the position decreased COX-1 inhibitory activity by flavonoids.

Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids.

The polyphenolic structures of flavonoids and isoflavonoids confer them with the ability to scavenge free radicals and to chelate transition metals, a basis for their potent antioxidant abilities. Another possible contributory mechanism toward their antioxidant activities is their ability to stabilize membranes by decreasing membrane fluidity. In this study, the effects of representative flavonoids, isoflavonoids, and their metabolites on membrane fluidity and their preferential localization in the membrane were investigated using large unilamellar vesicles (LUVs) as the membrane models. These results were compared with those of cholesterol and alpha-tocopherol. Changes in fluorescence anisotropy values for a series of n-(9-anthroyloxy) fatty acid probes (n = 6, 12, 16) upon addition of the test compounds were used to monitor alterations in membrane fluidity at graded depths in lipid bilayer. The results of the study suggest that the flavonoids and isoflavonoids, similar to cholesterol and alpha-tocopherol, partition into the hydrophobic core of the membrane and cause a dramatic decrease in lipid fluidity in this region of the membrane. Localization of flavonoids and isoflavonoids into the membrane interiors and their resulting restrictions on fluidity of membrane components could sterically hinder diffusion of free radicals and thereby decrease the kinetics of free radical reactions.