Likewise, plasticity in the temporal organization of neural

Likewise, plasticity in the temporal organization of neural

circuits is proposed to be critical for the context-specific regulation of behavior and physiology (Buzsáki, 2006). Further, dysfunction in the process of neuronal synchronization is implicated in epilepsy and cognitive impairment (Schnitzler and Gross, 2005). Since the principles of neural synchronization apply to systems operating on a range of timescales (Buzsáki and Draguhn, 2004 and Hansel et al., 1995), our study highlights organizational principles that may be relevant for other oscillatory networks (Buzsáki, 2006). All procedures were approved by CP-690550 molecular weight the Institutional Animal Care and Use Committee of the trans-isomer clinical trial Morehouse School of Medicine in accordance with the guidelines of the U.S. National Institutes of Health. Homozygous PERIOD2::luciferase (PER2::LUC) knockin mice (Yoo et al., 2004), backcrossed to a C57Bl/6J background, were

bred and raised under a 24 hr light:dark cycle with 12 hr light and 12 hr darkness (LD12:12, lights on: 0600 EST). Ambient room temperature was maintained at 22°C ± 2°C, and the animals had ab libitum access to water and food (Purina Rodent Chow #5001). For all experiments, adult male PER2::LUC mice (7–9 weeks of age) were transferred to individual wheel-running cages contained within light-tight secondary enclosures. Long-day photoperiods were achieved by an abrupt and symmetrical reduction of the scotophase. Mice were entrained to LD12:12, LD16:8, LD18:6, LD20:4, or LD22:2 for 12 weeks.

LD20:4 entrainment for less than 12 weeks produced SCN reorganization, but with individual differences in whether the pattern was evident Liothyronine Sodium (data not shown). Wheel-running rhythms were monitored and analyzed with the Clocklab data collection and analysis system (Actimetrics). Coronal SCN slices (150 μm) were collected and imaged as described previously (Evans et al., 2011). Unless otherwise stated, mice were sacrificed 2–4 hr before lights-off, since dissections during late subjective day do not reset the phase of the SCN (Davidson et al., 2009). Each SCN slice was cultured on a membrane (Millicell-CM; Millipore) with 1.2 ml of air-buffered medium containing 0.1 mM beetle luciferin (Gold Biotechnologies) and imaged for 5–7 days using a Stanford Photonics XR Mega 10Z cooled intensified charge-coupled device camera. For drug treatments, TTX (2.5 μM, catalog No. 1069; Tocris), the VIP receptor antagonist [4Cl-D-Phe6, Leu17] VIP (20 μM, catalog No. 3054; Tocris), or BIC (200 μM, catalog No. B7686; Sigma) was added to the culture medium and remained for the duration of the recording. For each pharmacological agent, drug dose was selected from published literature (Atkins et al., 2010, Aton et al., 2006 and Yamaguchi et al., 2003), and we independently validated dose efficacy in our preparation (Figures S6A–S6C).

However, it will be critical to

keep in mind that iPS cel

However, it will be critical to

keep in mind that iPS cells will be most powerfully leveraged as tools for biomedical research when they are used alongside existing animal, cell, and molecular models of neural degeneration. If disease-specific iPS cells are to be translated into clinically JQ1 mouse informative models for mechanistic studies and therapeutic drug discovery, several basic requirements must ideally be met. First, it will be important to optimize methods for differentiating stem cells into the particular neural cell type of interest. In the specific case of the spinal motor neurons affected in ALS and SMA and midbrain dopaminergic neurons in PD, methods described in mouse and human embryonic stem cells have translated fairly well into iPS cells, though they are far from perfect. It may further be necessary to identify culture conditions to produce specific subclasses of the desired cell type. For example, in ALS, selective subclasses of motor neurons degenerate whereas other subclasses are preferentially spared (for example motor neurons of the

oculomotor complex in the midbrain controlling eye movements and motor neurons of sacral spinal cord controlling bowel and bladder function). In PD, the A9 nigrostrial dopaminergic selleckchem projection neurons are preferentially affected and are paramount for the motor symptoms that typify this disorder. Second, phenotypic assays relevant to the disease process need to be established and advances in genetic modifications to create isogenic control lines will impart rigorous methods to compare disease versus control phenotypes. Needless to say, iPS cell models alone will not be able to produce clinically important read-outs of memory dysfunction and behavioral changes in AD or frontotemporal dementia, tremor, bradykinesia, and rigidity in PD, or reduced forced vital capacity, swallowing dysfunction,

dysarthria, or limb motor impairment in ALS. However, recapitulation of key molecular, cellular, and anatomical changes involved in disease are well within the scope of disease-related phenotypes in culture. Expected phenotypes based on previously established animal and cellular models and observations from neuropathological studies should serve as a means to establish hypotheses or help validate the specific iPS L-NAME HCl model but the identification of novel mechanisms or cellular phenotypes remains an exciting possibility. Importantly, iPS cell models will allow for the study of human pathophysiology and pharmacologic responses. Lastly, iPS cell-based models may provide a new opportunity to understand selective vulnerability of populations of neurons to discrete degenerative stimuli, a theme common to many neurological disorders. Thus, in coming closer to creating more relevant cellular models of human neurological disease, perhaps what we can create, we can understand. S.

9957, p = 2e−07) To account for the sensory consequences of morp

9957, p = 2e−07). To account for the sensory consequences of morphing on LEC, we assumed that the spatial response of each cell is switched from one map to an independently generated one at some random point during morphing (different assumptions are examined

online in Supplemental Text, Figure S1). The resulting receptive fields are shown for 10 LEC cells in Figure 1B. In Lonafarnib ic50 order to approximate the response dynamics of the EC during environment morphing, we generated the rate maps for both LEC and MEC (10,000 neurons each). To compute the excitatory input to each individual DG neuron, we used a realistic number of inputs (1200 from the MEC and 1500 from LEC; see Experimental Procedures) and summed them. Each synaptic input to the DG was taken from a population of randomly chosen entorhinal neurons, with the synaptic weight randomly assigned according to the synaptic weight distribution derived from the distribution selleck inhibitor of synapse sizes (de Almeida et al., 2009a) as determined by serial EM (Trommald and Hulleberg, 1997). The spatial distribution of firing of 10,000 DG granule cells was computed by applying, at each position, a winner-take-all interaction over the sum of excitation input. This winner-take-all process is governed by the so-called E%-max principle (de Almeida et al., 2009b) derived from the

interaction of excitation with gamma frequency feedback inhibition, a form of inhibition known to exist in this brain region (Bragin et al., 1995, Towers et al., 2002 and Pöschel et al., 2002) that synchronizes the firing of DG cells (reviewed by Bartos et al., 2007). According to this principle, the level of inhibition is set such that cells will fire provided their excitation is within 10% of the cell with maximum excitation. For these cells, their rate is proportional to where they (-)-p-Bromotetramisole Oxalate fall in this 10% range. The value of 10% is computed from d/τm (de Almeida et al., 2009b),

where d = delay of feedback inhibition and τm = membrane time constant, both of which have been experimentally determined. A previous study showed that the interaction of MEC input with this form of inhibition is able to quantitatively account for the size and number of place fields exhibited by active DG cells (de Almeida et al., 2009a). In our simulations, we also take into consideration the LEC. The interaction of the two inputs depends on the ratio (α) of the mean drive of MEC and LEC onto EC. No data is available that would allow us to directly estimate α. However, our results provide for a quantitative estimate of its value (see below). With this simulation framework in place, we investigated whether the cumulative decorrelation of population output from the DG observed during progressive morphing of the arena shape (Leutgeb et al., 2007; population vector [PV] correlation curve, Figure 3A) could be explained by the changes of the LEC spatial response.

Although the risk factors for ACL injuries are

Although the risk factors for ACL injuries are selleck chemical still unclear, many injury prevention programs have been developed for soccer players as well as athletes in other sports. Many studies have been conducted to evaluate the effectiveness of these prevention programs. These training programs can be categorized as balance training, plyometric training, long-duration neuromuscular training, or short-duration warm-up programs. Caraffa et al.63 investigated the effects of balance training on ACL injury rates in male soccer players. The prevention program included 20-min five phases progressive balance training with different balance boards.

The training was performed every day during pre-season and three times a week during the season for a total of three seasons. A total of 10 ACL

injuries occurred to the 300 players in the intervention group, while a total of 70 ACL injuries occurred to the 300 players in the control group. The difference in ACL injury incidence between groups was statistically significant. However how the participants were assigned to the intervention or control group and how the BVD-523 molecular weight proprioceptive training reduced ACL injury incidence were not clear. Söderman et al.64 studied the effects of balance board training on ACL injury rates in female soccer players. A total of 121 players in seven teams were randomized assigned to a training group and 100 players in six teams to a control group. The participants were instructed to perform a 10–15-min balance training on a balance board every day for 30 days and then three times a week for the rest of the season. With a 37% drop-out rate, four ACL injuries occurred among 62 players Ribonucleotide reductase in the intervention group, while one ACL injury occurred among 78 players in the control group during the season. Balance board training could not prevent ACL injury for female soccer players at the given level, which is contradictory to the previous study.63 Pfeiffer et al.65 studied

the effects of a plyometric training program on ACL injuries in high-school female soccer, basketball, and volleyball players. A total of 577 players were included in the training group and 862 players were included in the control group based on their willingness to participate in the training program. The 20-min training program consisted of exercises of jump landing techniques with a focus on a proper alignment of the hip, knee, and ankle. The training was performed twice a week throughout the 9-week season. The difference in the incidence of non-contact ACL injury between training and control groups after training was not statistically different. Heidt et al.66 studied the effects of preseason conditioning on ACL injury rate in high school female soccer players. A total of 300 players were recruited while 42 of them were randomly selected to a conditioning group and rest as control group.

, 2012) One of the best examples

, 2012). One of the best examples BTK inhibitor of neuromodulation by ATP acting at P2X receptors is in magnocellular neurons (MCNs) of the hypothalamus (Figure 6). Endogenous ATP acting at extrasynaptic P2X receptors plays a crucial role in modulating excitatory synaptic transmission in MCNs (Gordon et al., 2005, 2009). Early work suggested that ATP mediates part of the excitatory drive to MCNs following activation of catecholamine containing cells (Day et al., 1993), and electrophysiological studies showed that MCNs expressed functional P2X receptors that mediated membrane potential depolarization and increases in membrane conductance (Hiruma and Bourque, 1995). Later studies found ample evidence

for the presence of P2X receptor mRNAs and proteins that mediated ATP-evoked inward currents and strong elevations in intracellular Ca2+ levels in MCNs. It seems MCNs express a mixture of P2X receptors containing

P2X2, P2X4, and possibly P2X7 subunits (Vavra et al., 2011). It has been shown that P2X receptor signaling mediates the effect of noradrenaline (NA) on AMPA receptor-mediated mEPSCs arriving onto MCNs of the rat paraventricular nucleus (Gordon et al., 2005). Brief applications of NA increased mEPSC amplitudes for ABT-263 clinical trial long periods of time, perhaps permanently, by increasing AMPA receptor insertion into synapses (Figure 6). NA did not act postsynaptically via adrenoceptors in MCNs; instead, NA acted on astrocytes to release ATP. This paper provided

strong evidence that ATP acting via P2X receptors resulted in modulation of glutamatergic synaptic efficacy, and astrocytes were directly implicated as the source of endogenous ATP (Gordon et al., 2005). Bains and colleagues have established found that the ATP signaling mechanism is engaged during afferent activity, thus demonstrating that the effects of endogenous astrocyte derived ATP are likely to be utilized in vivo during physiological action potential firing impinging on MCNs (Gordon et al., 2009). The work on MCNs also provides several interesting points that may be relevant more broadly to the study of brain P2X receptor-mediated signaling. First, the authors failed to find fast ATP synaptic transmission. Second, they found that the sources of endogenous ATP were astrocytes rather than neurons, and that bursts of action potentials were needed to engage astrocyte signaling. Third, activation of P2X receptors resulted in multiplicative scaling of all synapses in MCNs. Multiplicative scaling is a form of homeostatic plasticity, whose underpinnings are only beginning to be explored. Perhaps P2X receptors are also involved in other areas of the brain in this form of plasticity? Fourth, the effect of P2X receptor activation on synaptic efficacy in MCNs took many minutes to develop, even though P2X receptor activation occurs in seconds once ATP is bound (Vavra et al., 2011).

For the various cellular motors to transport mitochondria, they n

For the various cellular motors to transport mitochondria, they need to consume ATP, and the motors need to be attached to

the mitochondria Lenvatinib datasheet via adaptor molecules (Milton/TRAK, Miro, and Syntabulin). Regulation of mitochondrial movement occurs both at the level of motor function, through local alterations of ADP/ATP ratio, and at the level of the attachment of mitochondria to the motors and the tracks they move along, through local changes in [Ca2+]i (Brough et al., 2005; Mironov, 2006). Postsynaptically, increased energy expenditure on glutamate-induced ion fluxes leads to a local rise in [ADP] and a fall of [ATP]. This decreases the energy available to the motor molecules transporting mitochondria, and rebinding of ADP to the motors in particular slows their movement (Mironov, 2007). A similar phenomenon Protease Inhibitor Library solubility dmso occurs in axons

in response to ATP use on pumping out of Na+ at the Ranvier node (Zhang et al., 2010) and so is also expected during energy use on Ca2+ pumping and vesicle trafficking in presynaptic terminals (Figure 5). In addition to this energetic limitation of mitochondrial movement, the rise in [Ca2+]i that occurs presynaptically via voltage-gated Ca2+ channels, and postsynaptically via Ca2+ influx through NMDA receptors (and possibly Ca2+-permeable AMPA/kainate receptors), leads to a parking of mitochondria at the active synapse. Wang and Schwarz (2009) found that a rise in axonal [Ca2+]i in hippocampal neurons leads to mitochondrial stopping, following Ca2+ binding to the adaptor protein Miro, which resulted in kinesin motors detaching from their microtubule tracks (Figure 5, presynaptic side; Ca2+ entry into the mitochondria may be needed for this to occur: Chang Sitaxentan et al., 2011). A similar arrest of mitochondrial movement in dendrites is triggered by Ca2+ entering through postsynaptic

NMDA receptors (Rintoul et al., 2003; MacAskill et al., 2009). In this case the proposed mechanism differed: Ca2+ binding to Miro was suggested to detach Miro from the kinesin motor (Figure 5, postsynaptic side). Calcium may also regulate mitochondrial transport by myosin, since Ca2+ stimulates myosin-actin ATPase activity but also (presumably at higher [Ca2+]i) decreases transport by dissociating calmodulin from myosin (Lu et al., 2006; Taylor, 2007). Speculatively, therefore, a small [Ca2+]i rise may stop microtubule-based transport (MacAskill et al., 2009; Wang and Schwarz, 2009) and promote local actin-based transport, until the mitochondrion encounters a higher [Ca2+]i, which will stop actin-based transport.

They were instructed to learn how to associate three CS shapes wi

They were instructed to learn how to associate three CS shapes with three possible outcomes (40p, 0p, 40/0p), and two colors with either more or less

predictable reward timing. All subjects had learned the associations successfully after the training as shown in a brief questionnaire. However, one subject was excluded because he reported nonexistent changes in color-timing associations after scanning. The scanning session consisted of four experimental blocks learn more of 48 normal and 8 test trials each. The order of trials was randomized and different in each block. Subjects were paid according to the number of successful timing estimates given in test trials. More precisely, the sum of all rewards collected during the experiment (amounting to £30 if no trials were missed) was multiplied by the percentage of test trials in which the time they indicated was within 1 s of the true reward time. On average, subjects earned £15 on the task (min £5, max £26), and were paid an extra £10 for their participation. We carried out t tests and Kolmogorov-Smirnov tests on the timing estimates subjects gave in instrumental test trials. Comparisons were done both between and across groups. We acquired T2∗-weighted EPI images on a 3 T TRIO scanner (Siemens) using a 12-channel head coil. Each of the four blocks consisted of 237 volumes with 43 slices, a 70 ms echo time (TE), resulting in a repetition time (TR) of 3.01 s; the voxel size was 3 ×

3 × 3 mm, flip angle −30°. We used a sequence optimized for orbito-frontal and midbrain regions to minimize signal dropout. We also acquired a high resolution structural find more scan (1 × 1 × 1 mm; 176 partitions, FoV = 256 × 240, TE = 2.48 ms, TR = 7.92 ms, FA = 16°, TI = 910 ms, 50% TI ratio) and a field map (TE1 = 10 ms and TE2 = 12.46 ms, 3 × 3 × 2 mm resolution, 1 mm gap). During scanning peripheral measurements of subject pulse, breathing, Rolziracetam and skin conductance responses were made together with scanner slice synchronization pulses. FMRI analysis was implemented using FMRIB Software

Library (FSL) (Smith et al., 2004). Data were preprocessed using the default options in FSL: Images were motion corrected (Jenkinson et al., 2002) and unwarped using the acquired field maps. Brain matter was segmented from nonbrain (Smith, 2002) before applying Gaussian spatial smoothing with a 5 mm FWHM kernel. Images were high-pass filtered and registered to the high-resolution structural image (7 degrees of freedom) and then the standard MNI152 template using affine registration (12 degrees of freedom) (Jenkinson and Smith, 2001). Further to using a sequence that minimized signal drop-out in midbrain regions, we performed two steps to increase the sensitivity to BOLD responses in the midbrain. These steps were taken because the anatomical location of the VTA makes BOLD signals in the region exquisitely sensitive to both physiological noise and subject motion.

Acute expression of constitutively active CREB in hippocampal neu

Acute expression of constitutively active CREB in hippocampal neurons increased the protein levels of KIF17, NR2B, and NR2A (Figures

8C and 8D). The kif17 and nr2b mRNA levels were also increased, but the nr2a level was not ( Figures 8E and 8F). Inhibition of CREB activity by expression of a dominant-negative form of CREB caused significant decreases in the mRNA and protein levels for NR2B and in the mRNA level for KIF17 ( Figures 8E and 8F). These data suggest that activation of CREB upregulates KIF17 and NR2B by increasing the transcription of each coding gene, but that other mechanisms underlie the increase Sirolimus manufacturer in the level of NR2A. KIF17 has been implicated in the transport of NR2B subunits (Guillaud et al., 2003 and Setou et al., 2000). Consistently, our results revealed a decreased level of motility of NR2B-EGFP clusters (Figures 2G–2J; Movie S1), but normal motility of NR2A-EGFP clusters (Figures 2K–2N; Movie S2) in kif17−/− mouse neurons. These data suggest that KIF17 transports the NR2B subunit, but not the NR2A

subunit. The NR2A subunit is likely to be transported by a different molecular motor. Recent papers have shown data suggesting that Kv4.2 and other neurotransmitter receptors, namely GluR5 and KA2, might also be cargoes for KIF17 (Chu et al., 2006 and Kayadjanian et al., 2007). We studied the expression of Kv4.2, GluR5, and KA2 in the kif17−/− mouse hippocampus.

No significant differences in the levels of these proteins were observed buy GSI-IX between kif17+/+ and kif17−/− mice ( Figure 1B). One possibility is that some molecular motors other than KIF17 support the transport of these proteins, compensating for the loss of function of KIF17 in kif17−/− neurons. Interestingly, significant reductions in the levels of both NR2B and NR2A in kif17−/− mouse synapses were observed (Figures 1G, 2A–2F, and S2B). Further analysis revealed that the level of NR2A in kif17−/− mouse neurons is downregulated at a posttranslational level, but that of NR2B is downregulated at a transcriptional level (Figures 1B–1F and 3A–3C). The NR2A level is decreased in kif17−/− mouse unless neurons due to its accelerated degradation in dendrites ( Figures 3I and 3L). Consistent with this finding, other investigators have reported that NR2A-containing NMDA receptors are easily degraded ( Yashiro and Philpot, 2008), whereas the NR2B subunit is preferentially driven to a recycling pathway ( Lavezzari et al., 2004 and Scott et al., 2004). We also showed that the NR2A degradation is dependent on the ubiquitin-proteasome system ( Figures 3D–3F). Considering these data together with studies reporting KIF17-mediated NR2B transport (Guillaud et al., 2003 and Setou et al., 2000), it is plausible that the accelerated NR2A degradation is caused by the reduction in NR2B transport in kif17−/− mouse neurons.

, 2001) Intra-LC administration of a CRF antagonist during the s

, 2001). Intra-LC administration of a CRF antagonist during the stress prevented the stress-induced excitation and revealed a greater post-stress inhibition that is naloxone-sensitive (Valentino and Wehby, 1988a and Curtis et al., Alectinib 2001). Additionally, LC administration of naloxone alone increased the time taken for LC excitation

to recover to pre-stress levels. This study suggested that opioid inhibition was important in recovery of LC activity from this physiological stressor. Together these findings support a model whereby acute stressors engage both CRF and opioid inputs to the LC (Fig. 2A). CRF is the predominant afferent and shifts LC discharge to a high tonic mode that favors

increased arousal, scanning attention and behavioral flexibility, effects that would be adaptive coping responses to an acute inhibitors threat. At the same time endogenous opioid afferents that have opposing actions are engaged. These function to restrain the CRF excitation and to promote recovery after stressor termination. These CRF/opioid interactions adjust the activity and reactivity of LC neurons so that level of arousal Perifosine and processing of sensory stimuli are optimized to facilitate adaptive behavioral responses to stressors. The protective effects of opioids are apparent in the many studies documenting that morphine administration shortly after a single traumatic event reduces the incidence of PTSD (Bryant et al., 2009 and Holbrook et al., 2010). During acute stress MOR regulation of the LC serves as an adaptive counterbalance that curbs the excitatory effects of CRF and protects against the consequences of a hyperactive

brain norepinephrine system. However, tipping the balance in favor of a MOR influence incurs alternative costs (Fig. 2B). Like the CRF response to stress, the opposing opioid response must be limited. The persistence of an opioid influence can produce enduring modifications in neural circuits that result in opioid tolerance and dependence. Indeed, this may be an underlying basis for the association between stress and substance abuse. A bias toward opioid regulation of the LC was recently demonstrated to occur with repeated tuclazepam social stress, which diminishes CRF function and enhances MOR function in the LC (Chaijale et al., 2013). Unlike acute stressors, repeated social stress decreased LC neuronal discharge rate by 48 h after the last stress and this inhibition was naloxone-sensitive indicating that MOR receptors were occupied. Analysis of CRF1 and MOR protein levels and receptor trafficking in the LC demonstrated that this paradoxical stress-induced inhibition is due to both a loss of CRF-elicited excitation as a result of CRF1 internalization and to increased opioid release and MOR signalling (Chaijale et al., 2013).

14 Butylated hydroxy anisole (BHA) (Himedia, India) was used as s

14 Butylated hydroxy anisole (BHA) (Himedia, India) was used as standard. The extract in methanol was tested at 20–250 μg/ml. DPPH solution was used at 20 μmol/l. DPPH dilution with methanol without extract was control. Percentage of scavenging was calculated as follows, DPPHscavengingactivity(%)=[(Acontrol−Asample)/Acontrol]×100 The data was presented as mean of triplicate. The concentration required for 50% reduction of DPPH radical (IC50) was determined graphically. Lipophilic antioxidants in the extract was measured using β-carotene–linoleic acid system.15

The extract and quercetin in DMSO were tested at 100 μg/ml, 500 μg/ml and 1000 μg/ml. Total reaction volume was 3 ml. The absorbance was recorded at 470 nm at regular time intervals from 0 to1500 min. The control contained 0.2 ml DMSO without extract. The reagent without β-carotene was served as blank. The data is presented as mean of triplicate readings. The antioxidant activity (AA) was expressed as percentage inhibition and calculated using the following equation: AA(%)=[(Degradationrateofcontrol−degradationrateofsample)/Degradationrateofcontrol]×100where

degradation rate = ln (a/b) × 1/t, where ln = natural log, a = initial absorbance (470 nm), b = absorbance (470 nm) after time ‘t’ (in min). A modified thiobarbituric acid Mdm2 inhibitor reactive species (TBARS) assay was used.9 The extract and quercetin were tested at 60 μg/ml, 120 μg/ml, and 600 μg/ml in 250 μl aliquots. The absorbance was measured at 532 nm. The reaction without extract or quercetin served as the control. The test blank contained linoleic acid emulsion without peroxidation treatment. The assay was carried out as described previously with modifications.16 10 μl of extract or quercetin dilutions of 100 μg/ml, 200 μg/ml and 500 μg/ml concentrations incubated for 30 min with 5 μl of calf thymus also DNA (Genei, India. 1 mg/ml) treated with Fenton reagent. Then, the reaction was terminated by adding 30 μl loading buffer (2.5 μg/ml bromophenol blue, 60% sucrose in 1 ml TBE buffer 10 mmol/l and pH 8.0) and 15 μl of which was electrophoresed at 60 eV potential for 30 min in submerged 1% agarose gel. The intact bands without shearing in

the electrophoretogram indicates the DNA protection. HPLC was performed using analytical HPLC system (Agilent Technologies assembled 1100 and 1200 series) equipped with quaternary pump and UV–visible detector. Reversed phase chromatographic analysis was carried out in isocratic conditions using RP-C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles. The separation was carried out in water-acetonitrile-acetic acid (80:20:3, v/v/v) as mobile phase at flow rate of 0.8 ml/min. Quercetin, gallic acid, 4-hydroxy benzoic acid, vanillic acid, epicatechin, ferulic acid, p-coumaric acid, phloroglucinol and chlorogenic acid (Sigma Modulators Aldrich, Germany) were used as reference standards at 300 ppm in methanol. The injection volume was 10 μl. Detection was done at 280 nm and 320 nm.