Quercetin along with piperine prevents cognitive dysfunction, oxidative stress and neuro-inflammation associated with mouse model of chronic unpredictable stress
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- Rinwa, P. & Kumar, A. Arch. Pharm. Res. (2013). doi:10.1007/s12272-013-0205-4
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Stress occurs in everyday life and persistence of it causes memory loss. Bioflavonoids like quercetin are reported to have poor bioavailability and limited therapeutic potential against stress induced neurological disorders. Therefore, the present study is an attempt to elucidate the therapeutic potency of combination of quercetin with piperine; a bioavailability enhancer against chronic unpredictable stress (CUS)-induced behavioral and biochemical alterations. Laca mice were subjected to a series of stressful events for a period of 28 days. Quercetin (20, 40 and 80 mg/kg, p.o.), piperine (20 mg/kg, p.o.) and their combinations were administered daily 30 min before CUS procedure. Piracetam (100 mg/kg, i.p.) served as a standard control. CUS caused impaired spatial navigation in Morris water maze test and poor retention in elevated plus maze task. Further, there was significant increase in brain oxidative stress markers and neuro-inflammation (TNF-α). This was coupled with marked rise in acetylcholinesterase and serum corticosterone levels. Co-administration of piperine with quercetin significantly elevated their potential to restore these behavioral, biochemical and molecular changes associated with mouse model of CUS. These results suggest that piperine enhances the neuroprotective effects of quercetin against CUS-induced oxidative stress, neuro-inflammation and memory deficits.
KeywordsAcetylcholinesteraseChronic stressMemory deficitsNeuro-inflammationOxidative stress
Stress, initially described by Hans Selye; is any change in environmental conditions that disturb the normal physiological equilibrium and results into a state of threatened homeostasis. Chronic stress exposure is known to be associated with various neurological disorders including memory dysfunction (Radley et al. 2004). Stress causes excess release of corticosterone in the body through dysregulation of hypothalamic–pituitary–adrenocortical (HPA) axis (Kurukulasuriya et al. 2004). Stress-induced increase in corticosterone levels has been found to impair memory retrieval process (Kurukulasuriya et al. 2004). Corticosterone secretion also triggers oxidative stress leading to memory deficits (Sato et al. 2010). The physiological consequences of stress process depend on the intensity and duration of each stressor (Joels 2006). Chronic stress is called as unpredictable when the subjects are unaware of the type and time of stress. Thus, chronic unpredictable stress (CUS) experimental model has been developed to study the development and progress of stress pathology (Willner et al. 1992) and its relation to other neurological disorders. Chronic variable stress is known to cause microglial activation, resulting into neuroinflammation in different brain regions which further causes imbalance in memory functions (Farooq et al. 2012). Generation of reactive oxygen species (ROS) also leads to oxidative stress; a prominent feature in pathogenesis of cognitive impairment (Massaad and Klann 2011).
These days, dietary and medicinal phyto-antioxidants are used in combination in order to increases their effectiveness and reduce the associated side effects. Quercetin is a dietary flavonoid found in a variety of fruits and vegetables such as onions, berries and apples. Quercetin is also said to attenuate stress-induced activation of HPA axis (Kawabata et al. 2010). Previous reports from our laboratory also suggested that quercetin attenuates the behavioral and biochemical alterations produced by immobilization stress (Kumar and Goyal 2008). Further, improvement in learning and memory performance with quercetin in different experimental models has also been studied (Kumar et al. 2008). Quercetin has also been reported to exert free radical scavenging activities (Spencer et al. 2009). Studies have shown that quercetin prevents the ethanol-mediated reduction of intracellular antioxidant defense systems, such as superoxide dismutase, catalase and glutathione reductase (Molina et al. 2003). Therefore, these pieces of evidence suggest the possible beneficial effects of quercetin against behavioral and biochemical changes associated with stress-induced memory/cognitive impairment.
In spite of high pharmacological benefits associated with the use of quercetin, it is not yet approved as a therapeutic agent and this could be because of its poor oral bioavailability. Various strategies have been employed to increase the oral bioavailability of quercetin including use of a bioavailability enhancer. Piperine, a major alkaloid of pepper species, is employed as an adjuvant with quercetin in the given study since it is known to increase the bioavailability of many drugs (Atal et al. 1985). In light of these reports, present study aims to elucidate the beneficial effect of co-administration of piperine with quercetin against CUS-induced oxidative damage, neuro-inflammation and memory deficits in mice.
Materials and methods
Male Laca mice (30–35 g) bred at Central Animal House (CAH) Panjab University, Chandigarh, were used. They were housed under standard (25 ± 2 °C, 60–70 % humidity) laboratory conditions, maintained on a 12 h natural day–night cycle, with free access to standard food and water. Animals were acclimatized to laboratory conditions before the test. The experiments were performed in accordance with the guidelines provided by the Council for the Purpose of Control and Safety of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India and the experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC) of Panjab University (IAEC/282/UIPS/39).
Chronic unpredictable stress procedures
Order of stressors used in the chronic unpredictable stress paradigm
Cold swim (8 °C, 5 min)
Tail pinch (1 min)
Food and water deprivation (24 h)
Swimming at room temperature (24 ± 2 °C, 20 min)
Cold swim (10 °C, 5 min)
Wet bedding (5 h)
Drugs and treatment schedule
Quercetin and piperine were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Piracetam was purchased from CDH, India. All other chemicals used for biochemical estimations were of analytical grade. The animals were randomly divided into nine experimental groups consisting of 8–10 animals each viz Group 1: naïve animals with no stress; Group 2: control animals were exposed to CUS along with an equivalent volume of vehicle; Group 3–5: CUS treated animals received Quercetin (20, 40 and 80 mg/kg, p.o.) respectively; Group 6: CUS treated animals received piperine per se (20 mg/kg; p.o.); Group 7–8: CUS treated animals received quercetin (20 and 40 mg/kg; p.o.) along with piperine (20 mg/kg; p.o.); Group 9: CUS treated animals received standard nootropics, piracetam (100 mg/kg; i.p.). Quercetin and piperine were prepared in peanut oil and administered orally on the basis of body weight (1 ml/100 g). Piracetam was dissolved in normal saline and administered intraperitoneally. Drugs were administered daily 30 min before CUS procedure continuously for 28 days.
Elevated plus maze paradigm
The elevated plus maze (EPM) consisted of two opposite black open arms (16 × 5 cm), crossed with two closed walls of the same dimensions of 12 cm height. The arms were connected with a central square of dimensions 5 × 5 cm. The entire maze was elevated to a height of 25 cm from the floor. Acquisition and retention of memory processes were assessed as previously described (Sharma and Kulkarni 1992). Acquisition of memory was tested on day 20 of CUS procedure. Animal was placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as the initial transfer latency (ITL). Animal was allowed to explore the maze for 20 s after recording the ITL and then returned to the home cage. If the animal could not enter closed arm within 90 s, same was guided to the closed arm and the ITL recording was set as 90 s. Retention of memory was assessed by placing the mouse again in an open arm and the retention latency was noted on day 21 and day 28 of ITL and was termed as the first retention transfer latency (1st RTL) and second retention transfer latency (2nd RTL), respectively.
Morris water-maze test—Morris water-maze apparatus (MWM) is most commonly used model to test memory (Morris 1984). MWM consisted of a large circular pool (150 cm in diameter, 45 cm in height, filled to a depth of 30 cm with water at 28 ± 1 °C). The water was made opaque with a nontoxic white dye. The pool was divided into four equal quadrants using two threads, fixed at a right angle to each other on the rim of the pool. A submerged platform (10 cm²), painted white was placed in the target quadrant 1 cm below the surface to provide an escape area. The position of platform was not altered throughout the training session. The experimenter always stood at the same position, and other visual cues in the room were not disturbed. All trials were completed between 10.00 and 16.00 h.
Retrieval trial—On fifth day (day 28) the platform was removed. Animal was placed in water maze and allowed to explore the maze for 120 s. Mean time spent in all three quadrants, i.e. Q1, Q2 and Q3 were recorded and the time spent in the target quadrant, i.e. Q4 in search of missing platform provided an index of retrieval. Care was taken that relative location of water maze with respect to other objects in the laboratory serving as prominent visual clues was not disturbed during the total duration of study.
Dissection and homogenization
Immediately after the last behavioral test, animals were sacrificed by cervical dislocation for biochemical and molecular analysis. Brain tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10,000×g for 15 min and aliquots of supernatant were separated and used for biochemical estimation.
Measurement of lipid peroxidation
The extent of lipid peroxidation was determined quantitatively by performing the method as described by Wills (1966). The amount of malondialdehyde (MDA) was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer Lambda 20 spectrophotometer (Norwalk, CT, USA). The values were calculated using the molar extinction co-efficient of chromophore (1.56 × 10 M−1 cm−1).
Estimation of nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide was determined by a colorimetric assay with Greiss reagent (0.1 % N-(1-napththyl) ethylene diamine dihydrochloride, 1 % sulphanilamide and 5 % phosphoric acid.) (Green et al. 1982). Equal volumes of the supernatant and the Greiss reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using Perkin Elmer Lambda 20 spectrophotometer (Norwalk, CT, USA). The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve.
Estimation of reduced glutathione
Reduced glutathione in the brain was estimated according to the method of Ellman (1959). Homogenates were precipitated with 1.0 ml of 4 % sulfosalicylic acid by keeping the mixture at 4 °C for 1 h and the samples were immediately centrifuged at 1,200×g for 15 min at 4 °C. The assay mixture contained 0.1 ml of supernatant, 2.7 ml of phosphate buffer of pH 8 and 0.2 ml of 0.01 M dithiobisnitrobenzoic acid (DTNB). The yellow color developed was read immediately at 412 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The results were expressed as nanomoles of reduced glutathione per milligram of protein.
Estimation of superoxide dismutase activity
Superoxide dismutase (SOD) activity was assayed by the method of Kono (1978) where in the reduction of nitrazobluetetrazolium (NBT) was inhibited by the superoxide dismutase and is measured. The assay system consists of EDTA 0.1 mM, sodium carbonate 50 and 96 mM of nitro blue tetrazolium (NBT). In the cuvette, 2 ml of the above mixture, 0.05 ml of hydroxylamine and 0.05 ml of the supernatant was added and auto-oxidation of hydroxylamine was measured for 2 min at 30 s intervals by measuring absorbance at 560 nm using Perkin Elmer Lambda 20 spectrophotometer (Norwalk, CT, USA).
Catalase activity was determined by Luck (1971), wherein the breakdown of hydrogen peroxide (H2O2) is measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H2O2, phosphate buffer and 0.05 ml of supernatant of tissue homogenates (10 %), and the change in absorbance was recorded at 240 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The results were expressed as micromoles of H2O2 decomposed per milligram of protein/min.
The protein content was estimated by biuret method (Gornall et al. 1949) using bovine serum albumin as a standard.
Acetyl cholinesterase (AChE) activity
AChE is a marker of loss of cholinergic neurons in the brain region. The AchE activity was assessed as described by Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of sodium phosphate buffer (pH 8), 0.1 ml of acetylthiocholine iodide and 0.1 ml of DTNB (Ellman reagent). The change in absorbance was measured for 2 min at 30 s intervals at 412 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). Results were expressed as micromoles of acetylthiocholine iodide hydrolyzed per min per mg of protein.
Serum corticosterone estimations
Preparation of serum
Retro-orbital bleeding technique was used to draw blood samples. Blood collected in the test tube was allowed to clot at room temperature. The tubes were then centrifuged at 2,000 rpm for 10 min and the straw colored serum was separated and stored frozen at −20 °C.
For extraction of corticosterone the method of Silber et al. (1958) was modified as described. 0.1–0.2 ml of serum were treated with 0.2 ml of freshly prepared chloroform: methanol mixture (2:1, v/v), followed by 3 ml of chloroform instead of dichloromethane used in the procedure of Silber et al. The samples were vortexed for 30 s and centrifuged at 2,000 rpm for 10 min. The chloroform layer was carefully removed with the help of syringe with a long 16 gauge needle attached to it and was transferred to a fresh tube. The chloroform extract was then treated with 0.1 N NaOH by vortexing rapidly and NaOH layer was rapidly removed. The sample was then treated with 3 ml of 30 N H2SO4 by vortexing vigorously. After phase separation, chloroform layer on top was removed using a syringe as described above and discarded. The tubes containing H2SO4 were kept in dark for 30–60 min and thereafter fluorescence measurements carried out in fluorescence spectrophotometer (make Hitachi, model F-2500) with excitation and emission wavelength set at 472 and 523.2 nm respectively. The standard curve depicting the fluorescence yield versus corticosterone concentration was used for result analysis.
Estimation of brain TNF-α
The quantifications of TNF-α were done with the help and instructions provided by Chemikine and R&D Systems immunoassay kits respectively. All samples were assayed in duplicate and absorbance was read on an ELISA plate reader (iMark™ Microplate absorbance reader, BIO-RAD) and the concentration of each sample was calculated by plotting the absorbance values on standard curve with known concentrations generated by the assay.
All the values were expressed as Mean ± SEM. The behavioral data were analysed by Two-way analysis of variance (ANOVA) followed by Bonferroni’s post test to calculate the statistical significance between various groups. All other test data were analyzed using One way analysis of variance (ANOVA) followed by post hoc Tukey’s test. The criterion for statistical significance was P < 0.05. All statistical procedures were carried out using sigma stat Graph Pad Prism (Graph Pad Software, San Diego, CA).
Effect of quercetin, piperine and their combination on latency time in elevated plus maze (EPM) task
Effects of quercetin, piperine and their interaction on memory performance in Morris water maze test
Effects of quercetin, piperine and their combination on escape latency time in Morris water maze
88.2 ± 3.14
46.3 ± 3.47
34.5 ± 2.42
23.6 ± 2.12
87.6 ± 3.12
78.4 ± 2.53a
73.8 ± 2.22a
68.8 ± 2.30a
84.5 ± 2.17
72.4 ± 3.43
68.3 ± 1.80
60.2 ± 3.41
85.3 ± 3.32
58.5 ± 3.42b
44.1 ± 2.16b,c
33.8 ± 1.16b,c
82.9 ± 3.22
54.6 ± 2.52b
42.6 ± 2.23b
32.5 ± 2.34b
83.6 ± 2.32
74.2 ± 2.34
71.6 ± 1.79
65.2 ± 2.34
84.2 ± 3.31
60.8 ± 3.32
47.4 ± 3.12c,d
34.6 ± 2.23c,d
83.2 ± 3.42
58.3 ± 3.43d
44.4 ± 2.32d
34.6 ± 2.53d
83.4 ± 2.21
50.2 ± 2.24
40.8 ± 3.25
29.8 ± 2.25
Effect of quercetin, piperine and their combination on oxidative biochemical parameters
Effect of quercetin, piperine and their combination on oxidative stress parameters
LPO (mol of MDA/mgpr)
GSH (μmol of GSH/mgpr)
Catalase (μmol of H2O2/min/mgpr)
0.183 ± 0.014
0.084 ± 0.007
294.5 ± 12.42
0.727 ± 0.011
63.48 ± 4.12
0.648 ± 0.012a
0.014 ± 0.005a
736.8 ± 20.22a
0.195 ± 0.032a
11.58 ± 2.30a
0.592 ± 0.017
0.034 ± 0.003
698 ± 16.80
0.227 ± 0.041
16.36 ± 2.12
0.335 ± 0.032b,c
0.055 ± 0.004b
461.1 ± 12.16b,c
0.488 ± 0.016b,c
45.34 ± 3.11b,c
0.319 ± 0.022b
0.060 ± 0.005b
426.6 ± 12.16b
0.503 ± 0.034b
49.79 ± 3.41b
0.629 ± 0.016
0.020 ± 0.003
715.6 ± 15.79
0.221 ± 0.044
17.16 ± 2.48
0.352 ± 0.031c,d
0.058 ± 0.002c,d
471.4 ± 13.12c,d
0.466 ± 0.023c,d
47.17 ± 4.27c,d
0.332 ± 0.042d
0.063 ± 0.003d
446.4 ± 10.22d
0.486 ± 0.053d
50.17 ± 3.11d
0.274 ± 0.021
0.072 ± 0.004
390.8 ± 14.55
0.638 ± 0.025
55.82. ± 4.10
Effect of quercetin, piperine and their combination on brain acetylcholine levels
Effect of quercetin, piperine and their combination on serum corticosterone (CORT) levels
Effect of quercetin, piperine and their combination on tumor necrosis factor (TNF-α) levels
There exist a complex relationship between stressful situations, mind and body’s reaction to stress, and the onset of cognitive disturbances (Bhutani et al. 2009). Chronic administration of variable stresses, a procedure known as “chronic unpredictable stress”, is thought to be a useful animal model to study stress related pathology (Willner et al. 1992). CUS is known to effect different regions of brain which play a critical role in spatial navigation and memory (Churchwell et al. 2010). In the present study, CUS for 28 days significantly impaired cognitive functions, activated HPA axis and caused generation of reactive oxidative stress markers. Further, treatment with quercetin significantly attenuated all these deficits associated with chronic stress. The effect was more pronounced on combination with piperine, which served as a potent bioavailability enhancer in the present study.
CUS is known to severely induce memory deficits in experimental animals (Bhatia et al. 2011). In the study, memory performance in both Morris water maze and elevated plus maze were significant impairment in control group as compared to naïve animals. Quercetin significantly and dose dependently improved cognitive performance in both the mazes indicating its therapeutic efficacy against chronic stress induced memory impairment. These results are also consistent with the previous findings (Bhutada et al. 2010). Cholinergic system of basal forebrain has abundant inputs in hippocampus therefore acetylcholine (ACh) plays an important role in learning and memory process (Anand and Singh 2013). ACh is degraded by the enzyme acetylcholinesterase, which gets modified on stress exposure (Nijholt et al. 2004). In line with above evidences chronic stress in the present study caused a significant increase in the acetylcholinesterase activity, but later was restored by chronic quercetin treatment thereby showing the process of memory retrieval and retention. These results are consistent with the earlier findings (Pachauri et al. 2012).
Glucocorticoids (corticosterone in animals) secreted during stressful events (via HPA activation) are known to influence retention and retrieval of memory (Roozendaal 2002). Similar to the above reports, we found a significant increase in serum corticosterone levels in control animals as compared to the naive group. Studies have documented that quercetin attenuated corticotrophin releasing factor (CRF) induced anxiogenic and depressant-like effects by normalization of HPA axis activity (Bhutada et al. 2010). Similarly, in the present study chronic administration of quercetin significantly reduced increased corticosterone levels associated with HPA axis hyperactivity in CUS animals.
On the other hand, corticosterone administration is also known to promote oxidative stress and consequently causes memory deficits (Sato et al. 2010). Oxygen free radicals can accumulate in brain and have a potent role in neurodegeneration associated with memory loss (Serrano and Klann 2004). Oxidative stress is therefore known to be one of the primary causes for cognitive impairment (Keller et al. 2005). Chronic stress can demolish antioxidant defense system of the brain and promote oxidative stress (Lucca et al. 2009). In our study we found a significant increase in oxidative damage as indicated by increase lipid peroxidation, nitrite concentration, and depletion of reduced glutathione levels, catalase and superoxide dismutase activity, thereby strengthening the hypothesis of oxidative stress induced cognitive deficits. Along with oxidative stress, free radical generation is also known to cause neuroinflammation injury (Moreira et al. 2006). Microglial activation along with release of various inflammatory cytokines (IL-β, TNF-α), reinforce oxidative stress markers and further causes cell death. Similarly, in the present study, CUS resulted in significant increase in TNF-α level, which is one of the prominent markers of neuroinflammation. Quercetin is reported to act as a potent antioxidant due to its multiple effects viz superoxide and hydroxyl radicals scavenging activity, metal chelating property and the ability to inhibit various oxidases (Hanasaki et al. 1994). Quercetin is known to enhance the reduced glutathione levels, increased levels of SOD and catalase in experimental animals (Molina et al. 2003). In line with the above correlates, quercetin in the present study significantly and dose dependently attenuated these oxidative stress markers along with attenuation of enhanced neuroinflammation associated with chronic stress.
In recent years, quercetin has gained much enthusiasm and attention for its research work in the field of neurodegenerative disorders. However, quercetin has a very low oral bioavailability (16.2 %) due to its high metabolic conjugation, and exists mostly as a conjugated form in systemic circulation (Ader et al. 2000). Along with this quercetin is a potent stimulator of the P-glycoprotein (Pgp), which is responsible for the systemic disposition of various lipophilic and amphipathic drugs (Choi et al. 2011). Therefore, all these factors contribute to the low oral bioavailability of quercetin. Piperine, on the other hand is known to increases the oral bioavailability of many drugs through inhibition of Phase II metabolic conjugation (Atal et al. 1985). Earlier, studies have shown that piperine increased the bioavailability of several herbal drugs like curcumin, resveratrol in different in vivo mice models (Moorthi et al. 2012; Johnson et al. 2011) Studies have also reported piperine as a potent inhibitor of systemic Pgp (Singh et al. 2013); thereby inhibiting the deposition of various Pgp associated drugs and increasing their systemic availability. In consistent with the above evidences, we observed a significant potentiation in the effects of quercetin when co-administered with piperine, thereby showing that piperine might have enhanced oral bioavailability of quercetin through inhibition of systemic Pgp and conjugation enzymes. To the best of our knowledge, this is the first in vivo study which states increased bioavailability of quercetin on combination with piperine and thereby potentiating its protective effects. The results of the present study demonstrate that memory restorative potential of quercetin against stress-induced memory/cognitive impairment might be due to its inhibitory effect on the HPA axis, thereby reducing corticosterone levels subsequently reducing ROS and neuro-inflammation.
The current study concludes that quercetin, probably due to its anti-oxidant and anti-inflammatory properties significantly attenuate stress-induced memory/cognitive impairment in mouse model of CUS. Further, the study shows an elevation in the effects of quercetin on combination with piperine, which can be used as a scientific tool to enhance the poor oral bioavailability of quercetin. Besides, these findings represent a valid rationale for co-administration of piperine with quercetin, which might act as a useful and potent adjuvant in the treatment of memory disorders.
Authors gratefully acknowledged the research Grant of Indian Council of Medical Research (ICMR), New Delhi for carrying out this work.