Effect of Ginkgo biloba (EGb 761) on differential gene expression.

Supplementation of diets with plant extracts such as ginkgo biloba extract (EGb 761) (definition see editorial) for health and prevention of degenerative diseases is popular. However, it is often difficult to analyse the biological activities of plant extracts due to their complex nature and the possible synergistic and/or antagonistic effects of their components. Genome-wide expression monitoring with high-density oligonucleotide arrays provides one way to examine the molecular targets of plant extracts and may prove a useful tool in evaluating their therapeutic claims. Here, we will briefly describe some of our work on the effect of EGb 761 on differential gene expression in relation to its potential anti-carcinogenic, photoprotective and neuromodulatory properties.

Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.

Effect of selenium and vitamin E deficiency on differential gene expression in rat liver.

To examine the molecular events associated with selenium (Se) and vitamin E (VE) deficiency, we applied cDNA array technology to define the transcriptional response in the liver of Se- and VE-deficient rats. VE deficiency alone did not induce any significant changes in expression profile among the genes evaluated. Se deficiency lead to a down-regulation of Se-dependent cGPx and to an induction of genes, encoding for detoxifying enzymes in liver (cytochrome P450 4B1, UDP-glucuronosyltransferase 1). Combined VE and Se deficiency was characterized by alterations in the expression level of genes encoding for proteins involved in inflammation (multispecific organic anion exporter, SPI-3 serine protease inhibitor) and acute phase response (alpha-1 acid glycoprotein, metallothionein 1). Additionally, a significant down-regulation in the expression level of genes important in the inhibition of apoptosis (defender against cell death 1 protein, Bcl2-L1), cell cycle (G1/S-specific cyclin D1) and antioxidant defense (gamma-glutamylcysteine synthetase catalytic subunit) was demonstrated. The experimental strategy identified several novel Se and VE sensitive genes.

Protein binding of procyanidins: studies using polyacrylamide gel electrophoresis and French maritime pine bark extract.

The application of PAGE to determine the interaction between procyanidins and proteins, as presented here, enables one to directly determine the binding of either a pure of a complex mixture of flavonoids to a particular protein. If the protein of interest is an enzyme, the combination of PAGE with quantitative activity measurements allows identifying whether a change in the enzyme activity is related to the binding. Data presented suggest that PBE and EGb 761 have protein-binding properties, which, in addition to their redox-based effects, could provide a biochemical basis for their action in biological systems.

The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba.

Extracts of Ginkgo biloba leaves are consumed as dietary supplements to counteract chronic, age-related neurological disorders. We have applied high-density oligonucleotide microarrays to define the transcriptional effects in the cortex and hippocampus of mice whose diets were supplemented with the herbal extract. Gene expression analysis focused on the mRNAs that showed a more than 3-fold change in their expression. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1, and microtubule-associated tau were significantly enhanced. Hyperphosphorylated tau is the major constituent of the neurofibrillary tangles in the brains of Alzheimer's disease patients. The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2, calcium and chloride channels, prolactin, and growth hormone (GH), all of which are associated with brain function, were also up-regulated. In the hippocampus, only transthyretin mRNA was upregulated. Transthyretin plays a role in hormone transport in the brain and possibly a neuroprotective role by amyloid-beta sequestration. This study reveals that diets supplemented with Ginkgo biloba extract have notable neuromodulatory effects in vivo and illustrates the utility of genome-wide expression monitoring to investigate the biological actions of complex extracts.

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties.

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha-lipoic acid (alpha-LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha-LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies < or = 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of alpha-LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.

From ancient remedies to modern therapeutics: pine bark uses in skin disorders revisited.

The effect of French maritime pine bark extract (PBE) on the gene expression profile of HaCaT human keratinocytes was studied using high density filter arrays. The expression profile of both control and PBE-treated cells was determined. Interestingly, PBE was shown to downregulate both calgranulin A and B genes which are known to be upregulated in psoriasis and various dermatoses. Thus, PBE could be considered in human dermatoses.

Solar ultraviolet-induced erythema in human skin and nuclear factor-kappa-B-dependent gene expression in keratinocytes are modulated by a French maritime pine bark extract.

The procyanidin-rich French maritime pine bark extract Pycnogenol (PBE) has been investigated for its effect in protecting human skin against solar UV-simulated light-induced erythema. Twenty-one volunteers were given an oral supplementation of Pycnogenol: 1.10 mg/kg body weight (b. wt.)/d for the first 4 weeks and 1.66 mg/kg b. wt./d for the next 4 weeks. The minimal erythema dose (MED) was measured twice before supplementation (baseline MED), once after the first 4 weeks of supplementation, and a last time at the end of the study. The UVR dose necessary to achieve 1 MED was significantly increased during PBE supplementation. Since the activation of the pro-inflammatory and redox-regulated transcription factor NF-kappaB is thought to play a major role in UVR-induced erythema, the effect of PBE was also investigated in the human keratinocyte cell line HaCaT. PBE, added to the cell culture medium, inhibited UVR-induced NF-kappaB-dependent gene expression in a concentration-dependent manner. However, NF-kappaB-DNA-binding activity was not prevented, suggesting that PBE affects the transactivation capacity of NF-kappaB. These data indicate that oral supplementation of PBE reduces erythema in the skin. Inhibition of NF-kappaB-dependent gene expression by PBE possibly contributes to the observed increase in MED.

Inhibition mechanisms of bioflavonoids extracted from the bark of Pinus maritima on the expression of proinflammatory cytokines.

The effect of bioflavonoids extracted from the bark of Pinus maritima, Pycnogenol (PYC), on gene expression of the proinflammatory cytokines interleukin-1beta (IL-1beta) and interleukin-2 (IL-2) were investigated in RAW 264.7 cells and Jurkat E6.1 cells, respectively. PYC exerted strong scavenging activities against reactive oxygen species (ROS) generated by H2O2 in RAW 264.7. In situ ELISA, immunoblot analysis, and competitive RT-PCR demonstrated that pretreatment of LPS-stimulated RAW 264.7 cells with PYC dose-dependently reduced both the production of IL-1beta and its mRNA levels. Furthermore, in the same cells, PYC blocked the activation of nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1), two major transcription factors centrally involved in IL-1beta gene expression. Concordantly, pretreatment of the cells with PYC abolished the LPS-induced IkappaB degradation. We also investigated the effect of PYC on IL-2 gene expression in phorbol 12-myristate 13acetate plus ionomycin (PMA/Io)-stimulated human T-cell line Jurkat E6.1. PYC inhibited the PMA/Io-induced IL-2 mRNA expression. However, as demonstrated in a reporter gene assay system, the mechanism of IL-2 gene transcriptional regulation by PYC was different from the regulation of IL-1beta. PYC inhibited both NF-AT and AP-1 chloramphenicol acetyltransferase (CAT) activities in transiently transfected Jurkat E6.1, but not NF-kappaB CAT activity. We also found that PYC can destabilize PMA/Io-induced IL-2 mRNA by posttranscriptional regulation. All these results suggest that bioflavonids can be useful therapeutic agents in treating many inflammatory, autoimmune, and cardiovascular diseases based on its diverse action mechanisms.

Enzyme inhibition and protein-binding action of the procyanidin-rich french maritime pine bark extract, pycnogenol: effect on xanthine oxidase.

Pycnogenol, an extract from French maritime pine bark (PBE), is a complex mixture of bioflavonoids with reported protective effects against disease. PBE is an effective scavenger of reactive oxygen species, and its main constituents are procyanidins of various chain lengths. To find out the biochemical basis of action of PBE on enzyme activity, involvement of its redox activity and direct binding to the enzyme in its subsequent action on enzyme activity have been investigated. PBE dose-dependently inhibited the activities of xanthine oxidase, xanthine dehydrogenase, horseradish peroxidase, and lipoxygenase, but it did not affect the activities of glucose oxidase, ascorbate oxidase, or elastase. To characterize the mechanism of PBE action, studies were focused on xanthine oxidase and glucose oxidase. Under non-denaturing conditions, PBE changed the electrophoretic mobility of xanthine oxidase but not of glucose oxidase. Gel filtration chromatography confirmed higher molecular weight complexes of xanthine oxidase and xanthine dehydrogenase in the presence of PBE. It was found that hydrophobic bonding might be the dominant mode of interaction between PBE and xanthine oxidase. The importance of the binding in the effect of PBE on enzyme activity was supported by the observation that PBE binds to and inhibits catalase, but not superoxide dismutase. However, no correlation was found between superoxide/hydroxyl radical scavenging activity and the inhibitory effect on xanthine oxidase activity of PBE, various purified flavonoids, or other complex mixtures of bioflavonoids. The results indicate that PBE selectively inhibits xanthine oxidase through binding to the enzyme rather than by the redox activity.

Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and alpha-lipoic acid.

The purpose of these experiments was to examine the effects of dietary antioxidant supplementation with vitamin E (VE) and alpha-lipoic acid (alpha-LA) on biochemical and physiological responses to in vivo myocardial ischemia-reperfusion (I-R) in aged rats. Male Fischer-334 rats (18 mo old) were assigned to either 1) a control diet (CON) or 2) a VE and alpha-LA supplemented diet (ANTIOX). After a 14-wk feeding period, animals in each group underwent an in vivo I-R protocol (25 min of myocardial ischemia and 15 min of reperfusion). During reperfusion, peak arterial pressure was significantly higher (P < 0.05) in ANTIOX animals compared with CON diet animals. I-R resulted in a significant increase (P < 0.05) in myocardial lipid peroxidation in CON diet animals but not in ANTIOX animals. Compared with ANTIOX animals, heart homogenates from CON animals experienced significantly less (P < 0.05) oxidative damage when exposed to five different in vitro radical producing systems. These data indicate that dietary supplementation with VE and alpha-LA protects the aged rat heart from I-R-induced lipid peroxidation by scavenging numerous reactive oxygen species. Importantly, this protection is associated with improved cardiac performance during reperfusion.

Studies of LDL oxidation following alpha-, gamma-, or delta-tocotrienyl acetate supplementation of hypercholesterolemic humans.

In vitro tocotrienols (T3s) have potent vitamin E antioxidant activity, but unlike tocopherols can inhibit cholesterol synthesis by suppressing 3-hydroxy-3-methyl-glutarylCoA (HMG-CoA) reductase. Because hypercholesterolemia is a major risk factor for coronary artery disease and oxidative modification of low-density lipoprotein (LDL) may be involved in atherogenesis, we investigated whether daily supplements of placebo, or alpha-, gamma-, or delta- (alpha-, gamma-, or delta-) tocotrienyl acetates would alter serum cholesterol or LDL oxidative resistance in hypercholesterolemics in a double-blind placebo controlled study. Subjects were randomly assigned to receive placebo (n = 13), alpha- (n = 13), gamma- (n = 12), or delta- (n = 13) tocotrienyl acetate supplements (250 mg/d). All subjects followed a low-fat diet for 4 weeks, then took supplements with dinner for the following 8 weeks while still continuing diet restrictions. Plasma alpha- and gamma-tocopherols were unchanged by supplementation. Plasma T3s were undetectable initially and always in the placebo group. Following supplementation in the respective groups plasma concentrations were: alpha-T3 0.98 +/- 0.80 micromol/l, gamma-T3 0.54 +/- 0.45 micromol/l, and delta-T3 0.09 +/- 0.07 micromol/l. Alpha-T3 increased in vitro LDL oxidative resistance (+22%, p <.001) and decreased its rate of oxidation (p <. 01). Neither serum or LDL cholesterol nor apolipoprotein B were significantly decreased by tocotrienyl acetate supplements. This study demonstrates that: (i) tocotrienyl acetate supplements are hydrolyzed, absorbed, and detectable in human plasma; (ii) tocotrienyl acetate supplements do not lower cholesterol in hypercholesterolemic subjects on low-fat diets; and (iii) alpha-T3 may be potent in decreasing LDL oxidizability.

Ferric nitrilotriacetate induced DNA and protein damage: inhibitory effect of a fermented papaya preparation.

The carcinogen Fe-NTA catalyzes the hydrogen peroxide-derived production of free radicals and possibly acts through a mechanism involving oxidative stress. Fermented papaya preparation (FPP) has been reported as a natural antioxidant able to prevent lipid peroxidation in vitro and in vivo. However, little is known about the antioxidant properties of FPP regarding iron-mediated oxidative damage to DNA and proteins. In the present study FPP protected supercoiled plasmid DNA against Fe-NTA plus H2O2 induced single and double strand breaks. Similar protective effects of FPP were evident when human T-lymphocytes were challenged with Fe-NTA/H2O2 and DNA damage was determined using the Comet assay. Fe-NTA/H2O2 also induced fragmentation of bovine serum albumin (BSA) in vitro and depleted cellular GSH levels in lymphocytes. BSA fragmentation and GSH depletion were dose-dependently counteracted by FPP. EPR spin trapping studies demonstrated that antioxidant properties of FPP are related to both hydroxyl scavenging as well as iron chelating properties.

Effect of bioflavonoids extracted from the bark of Pinus maritima on proinflammatory cytokine interleukin-1 production in lipopolysaccharide-stimulated RAW 264.7.

Currently, bioflavonoids have been known to have strong antioxidant capacities, and a variety of efforts have been made to identify the utilities of bioflavonoids in treating various diseases based on their antioxidant capacities. The effects of bioflavonoids extracted from the bark of Pinus maritima Pycnogenol (PYC) on free radical formation, activation of redox sensitive transcription factors, as well as interleukin-1 beta (IL-1 beta) production were investigated in murine macrophage cell lines. PYC exerted strong scavenging activities against reactive oxygen species generated either by H(2)O(2) or PMA in RAW 264.7 and IC-21 cells, respectively. In situ ELISA, immunoblot analysis, and competitive RT-PCR demonstrated that PYC pretreatment of LPS-stimulated RAW 264.7 cells dose-dependently reduced both the production of IL-1 beta and its mRNA levels. Furthermore, in the same cells, PYC blocked the activation of nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1), two major transcription factors centrally involved in IL-1 beta gene expression. When RAW 264.7 cells were stimulated with LPS, the inhibitor protein I kappa B largely disappeared from cytosolic fractions. However, pretreatment of the cells with PYC abolished the LPS-induced I kappa B degradation. These results suggest that PYC can inhibit the expression of the proinflammatory cytokine IL-1 by regulating redox-sensitive transcription factors. This study may support the possibility that bioflavonoids including PYC can be used as antiinflammatory and immunosuppressive drugs based on their radical scavenging activities.

Antioxidant activity of nasunin, an anthocyanin in eggplant peels.

The free radical scavenging activities and inhibitory effect of lipid peroxidation of a delphinidin derivative in eggplant were investigated. Delphinidin-3-(p-coumaroylrutinoside)-5-glucoside (nasunin), an anthocyanin, was isolated as purple colored crystals from eggplant peels. Using electron spin resonance spectrometry and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals (OH) or superoxide anion radicals (O(2)(-)) generated by the Fenton reaction or the hypoxanthine-xanthine oxidase system were measured as DMPO-OH or DMPO-OOH spin adducts. L-Ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6yl-hydrogen phosphate] potassium salt (EPC-K1) and bovine erythrocyte superoxide dismutase (SOD) were used as standards for OH and O(2)(-) scavengers, respectively. Nasunin showed potent O(2)(-) scavenging (143+/-8 SOD-equivalent U/mg) and OH scavenging (0. 65+/-0.07 EPC-K1-equivalent micromol/mg) activities. Then, by changing the concentration of DMPO to vary the trapping rate of OH, the presence of a competitive reaction between nasunin and OH was studied. The 50% inhibition dose (ID(50)) obtained from the inhibition curve did not change, indicating OH scavenging of nasunin is not due to direct scavenging but inhibition of OH generating system by chelating ferrous ion. Nasunin protection against H(2)O(2)-induced lipid peroxidation in rat brain homogenate was measured at 586 nm using the indicator of malonaldehyde and 4-hydroxyalkenals. Nasunin (<50 microM) protected against lipid peroxidation of brain homogenates. The findings suggest that nasunin is a potent O(2)(-) scavenger and has protective activity against lipid peroxidation.

Thiol homeostasis and supplements in physical exercise.

Thiols are a class of organic sulfur derivatives (mercaptans) characterized by the presence of sulfhydryl residues. In biological systems, thiols have numerous functions, including a central role in coordinating the antioxidant defense network. Physical exercise may induce oxidative stress. In humans, a consistent marker of exercise-induced oxidative stress is blood glutathione oxidation. Physical training programs have specific effects on tissue glutathione metabolism that depend on the work program and the type of tissue. Experimental studies show that glutathione metabolism in several tissues sensitively responds to an exhaustive bout of exercise. Study of glutathione-deficient animals clearly indicates the central importance of having adequate tissue glutathione to protect against exercise-induced oxidative stress. Among the various thiol supplements studied, N-acetyl-L-cysteine and alpha-lipoic acid hold the most promise. These agents may have antioxidant effects at the biochemical level but are also known to influence redox-sensitive cell signaling.

Effect of combined supplementation with vitamin E and alpha-lipoic acid on myocardial performance during in vivo ischaemia-reperfusion.

Reactive oxygen species (ROS) contribute significantly to myocardial ischaemia-reperfusion (I-R) injury. Recently the combination of the antioxidants vitamin E (VE) and alpha-lipoic acid (alpha-LA) has been reported to improve cardiac performance and reduce myocardial lipid peroxidation during in vitro I-R. The purpose of these experiments was to investigate the effects of VE and alpha-LA supplementation on cardiac performance, incidence of dysrhythmias and biochemical alterations during an in vivo myocardial I-R insult. Female Sprague-Dawley rats (4-months old) were assigned to one of the two dietary treatments: (1) control diet (CON) or (2) VE and alpha-LA supplementation (ANTIOXID). The CON diet was prepared to meet AIN-93M standards, which contains 75 IU VE kg-1 diet. The ANTIOXID diet contained 10 000 IU VE kg(-1) diet and 1.65 g alpha-LA kg(-1) diet. After the 14-week feeding period, significant differences (P<0.05) existed in mean myocardial VE levels between dietary groups. Animals in each experimental group were subjected to an in vivo I-R protocol which included 25 min of left anterior coronary artery occlusion followed by 10 min of reperfusion. No group differences (P>0.05) existed in cardiac performance (e.g. peak arterial pressure or ventricular work) or the incidence of ventricular dysrhythmias during the I-R protocol. Following I-R, two markers of lipid peroxidation were lower (P<0.05) in the ANTIOXID animals compared with CON. These data indicate that dietary supplementation of the antioxidants, VE and alpha-LA do not influence cardiac performance or the incidence of dysrhythmias but do decrease lipid peroxidation during in vivo I-R in young adult rats.

Ferulic acid excretion as a marker of consumption of a French maritime pine (Pinus maritima) bark extract.

French maritime pine (Pinus maritima) bark extract (PBE) is a polyphenol-rich food supplement patented under the name of Pycnogenol and known to have strong antioxidant activity and different beneficial effects on human health. Although its biological properties have begun to be extensively studied both in vitro, in laboratory animals and more recently in humans, little is known about its bioavailability. The present study investigated the urinary excretion of free and conjugated ferulic acid, present in quantitatively detectable amounts in PBE, after oral PBE administration to human subjects. Eleven healthy adult subjects (4 women and 7men) consumed either a single dose (200 mg PBE) or two doses of PBE (100 and 200 mg, respectively) within a 48-h interval. Two days before the oral administration of PBE and during the urine sample collection period volunteers adhered to a diet low in polyphenols. Aliquots of all urine production were collected over 24 h. Free and conjugated ferulic acid was assessed in urine by HPLC using diode array detection. A close association between the dietary intake of PBE and the urinary excretion of ferulic acid was detected. Moreover, the results indicate that a considerable proportion of ferulic acid is excreted as glucuronide or sulfate after PBE consumption, varying over the range 2 to 20% between individuals. The kinetics of excretion associated with the administration of 100 mg PBE was quite similar to that obtained after 200 mg PBE. A a biphasic trend was evident in a number of subjects. All subjects studied here displayed a significant, although variable level of excretion of ferulic acid after supplementation with PBE, Thus, the data provide evidence that at least a part of the phenolic components of PBE are absorbed, metabolized, and eliminated by humans.

In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract.

Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 microg/ml (corresponding to 69 microM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 microM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.