Protective effect of hesperidin and naringin against 3-nitropropionic acid induced Huntington's like symptoms in rats: Possible role of nitric oxide

Behavioural Brain Research 206 (2010) 38–46

Contents lists available at ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Protective effect of hesperidin and naringin against 3-nitropropionic acid induced Huntington’s like symptoms in rats: Possible role of nitric oxide Puneet Kumar, Anil Kumar ∗ Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh 160014, India

a r t i c l e

i n f o

Article history: Received 25 June 2009 Received in revised form 17 August 2009 Accepted 24 August 2009 Available online 27 August 2009 Keywords: Flavanoids Huntington’s disease Mitochondria 3-Nitropropionic acid Nitric oxide Oxidative stress

a b s t r a c t 3-Nitropropionic acid (3-NP) is a well known experimental model to study Huntington’s disease (HD) and associated neuropsychiatric problems. Present study has been designed to explore the protective effects of hesperidin, naringin, and their nitric oxide mechanism (if any) against 3-nitropropionic acid induced neurotoxicity in rats. Systemic 3-nitropropionic acid (10 mg/kg) treatment for 14 days in rats significantly induced HD like symptoms in rats as indicated by reduced locomotor activity, body weight, grip strength, oxidative defense and mitochondrial complex enzymes (complex-I, -II, and -IV) activities in striatum. Naringin and hesperidin pretreatment significantly attenuated behavioral alterations, oxidative stress and mitochondrial enzymes complex dysfunction in 3-NP treated group. l-Arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated the protective effect of hesperidin and naringin respectively. Whereas l-NAME (10 mg/kg), a non-selective NOS inhibitor pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect per se. Study highlights the therapeutic potential of hesperidin and naringin against Huntington’s like conditions and further indicates that these drugs might act through nitric oxide mechanism. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Flavonoids are well known for their antioxidant activity, currently being used as dietary supplements for various reasons. Flavonoids are richly available in fruits, vegetables, nuts, seeds, leaves, flowers and barks of plants [1]. Some of the flavonoids have very potent antioxidant capacities, even stronger than that of vitamins C and E [2]. They usually contain aromatic hydroxyl groups in their moiety which is responsible for their antioxidant activity [3]. Researcher’s interest in these bioactive compounds has increased several folds in recent years due to their health benefits/claims particularly against neurodegenerative diseases [4]. Naringin (4 ,5,7-thrihydroxyflavanone-7-rhamnoglucoside) and hesperidin (3 ,5,7-trihydroxy-4 -methoxy-flavanone-7rhamnoglucoside) are members of the flavanone group. Naringin and hesperidin are characterized as potent free radical scavenger [5]. Recent epidemiological and dietary interventional studies both in humans and animals suggest that these flavonoids prevent and delay neurodegeneration, especially in aged-population cognitive dysfunction, mood decline and oxidative pathologies [6]. Antioxi-

∗ Corresponding author. Tel.: +91 172 2534106; fax: +91 172 2541142. E-mail address: [email protected] (A. Kumar). 0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2009.08.028

dants are now being tried as a cocktail to treat these disorders in clinical and preclinical studies [7,8]. Nitric oxide (NO) has been implicated in a variety of pathophysiological conditions including neuroinflammation and neurodegenerative diseases. Flavonoids exert stronger protective activity against peroxynitrite-induced oxidative damage [9]. Flavonoids are well known for their biological activities such as inhibition of nitric oxide synthase and cyclooxygenase expression [10], protection against oxidative stress [11] and modulation of the calcium homeostasis [6]. Several studies documented the potential role of flavonoids and their interaction with nitric oxide pathways in neurodegenerative process including HD [10–12]. These polyphenols act by direct scavenging of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in vitro, including superoxide, peroxyl radicals, singlet oxygen, and peroxynitrite [6,13–16]. 3-Nitropropionic acid (3-NP), a neurotoxin irreversibly inhibits succinate dehydrogenase enzyme (complex-II) in the electron transport chain. 3-NP produces HD like symptoms both in animals and human [12,17,18]. 3-NP intoxication leads to selective striatal lesions which begin in the dorsolateral quadrant of the striatum and later spread to the entire lateral striatum [18]. 3-NP neurotoxin preferentially produces morphological abnormalities and cell death of the striatal medium-sized spiny neurons. Studies reported that 3-NP treatment significantly causes oxidative

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

damage in diverse areas of brain particularly striatum [12,17,18]. Possible source of oxidative damage in 3-NP model is impairment of mitochondrial energy production (inhibition of complex-II), excitotoxicity (NMDA receptor activation), that causes an increase in oxygen flux through mitochondria, leading to elevated ROS production [12,17,18]. Studies have also documented the beneficial effect of antioxidants possibly by free radical scavenging activity (decreases MDA and nitrite concentration) and increased endogenous antioxidant defense (increased levels of superoxide, catalase and glutathione) against 3-NP induced behavioral, biochemical and mitochondrial alterations in rats [8,19,20]. Present study has been designed to explore the effect of hesperidin and naringin and their possible nitric oxide mechanism (if any) against 3-NP induced behavioral, oxidative stress and mitochondrial changes in striatum. 2. Materials and methods 2.1. Animals Male Wistar rats (250–300 g) bred in Central Animal House of the Panjab University, Chandigarh were used. Animals were acclimatized to laboratory conditions prior to experimentation. The animals were kept under standard conditions of light and dark cycle with food and water ad libitum. All the experiments were carried out between 09:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee and carried out in accordance with the Indian National Science Academy Guidelines for the use and care of animals. 2.2. Drugs and treatment schedule The following drugs were used in the present study. 3-NP, l-arginine and lNAME (N(G)-nitro-l-arginine methyl ester) (Sigma Chemicals, St. Louis, MO, USA) were diluted with saline (adjust pH 7.4) and administered intraperitoneally to animals. Hesperidin and naringin were suspended in 0.5% (w/v) sodium carboxymethyl-cellulose (CMC) solution and administered by oral route in a constant volume of 0.5 ml per 100 g of body weight. Animals were randomly divided into 14 groups 10 animals in each. Study was conducted in two phases, effects of hesperidin and naringin were explored in first phase and their interactions with nitric oxide modulators in the second phase. Drug treatment was given for 14 days to all the treatment groups. • Study Phase-1: Group-1 received vehicle or normal saline (i.p.). Group-2 received 3-NP (10 mg/kg, i.p.), Groups 3 and 4 received naringin (100 mg/kg) and hesperidin (100 mg/kg) per se, Groups 5 and 6 received hesperidin (50 and 100 mg/kg) + 3NP (10 mg/kg, i.p.), Groups 7 and 8 received naringin (50 and 100 mg/kg) + 3-NP (10 mg/kg, i.p.). • Study Phase-2: Group-9 received l-arginine (50 mg/kg) + 3-NP (10 mg/kg, i.p.), Group-10 received l-NAME (10 mg/kg) + 3-NP (10 mg/kg, i.p.), Group11 received l-arginine (50 mg/kg) + hesperidin (50 mg/kg) + 3-NP, Group-12 received l-NAME (10 mg/kg) + hesperidin (50 mg/kg) + 3-NP, Group-13 received l-arginine (50 mg/kg) + naringin (50 mg/kg) + 3-NP, Group-14 received l-NAME (10 mg/kg) + naringin (50 mg/kg) + 3-NP. In the present study, l-arginine and l-NAME were administered 1 h prior to hesperidin and naringin treatment whereas hesperidin and naringin were administered 1 h prior to 3-NP treatment. Doses of the drugs were selected on the basis of previous studies [21–23]. 2.3. Measurement of body weight Body weight was recorded on the first and last day of the experiment. Percent change in body weight was calculated as Body weight (1st day − 15th day) × 100 Body weight (1st day)

2.4. Behavioral assessments 2.4.1. Assessment of gross behavioral activity (locomotor activity) The locomotor activity was monitored by using actophotometer (IMCORP, Ambala, India). The motor activity in actophotometer was detected by infrared photo cells installed in the instrument. Animals were placed individually in the activity chamber for 3 min as a habituation period before making actual recording. Each animal was observed over a period of 5 min and expressed as counts per 5 min [12].

39

2.4.2. Rotarod activity Motor incoordination and grip strength was assessed by using rotarod apparatus (Techno, Ambala, India). Animals were exposed to prior training session to acclimatize them on rotarod before starts the actual assessing of drug treatment. Animals were placed on the rotating rod with a diameter of 7 cm (speed 25 rpm). The cut off time was 180 s. Three separate trials after 5 min gap were given to each rat. The average fall of time was recorded and expressed as count per 5 min [21]. 2.5. Dissection and homogenization On day 15, animals were randomized into two groups. First group of animals was used for biochemical and second group for mitochondrial complex enzymes estimation after behavioral assessments. In the biochemical analysis, animals were scarified by decapitation. Striatum was separated from each isolated brain. A 10% (w/v) tissue homogenate were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenate were centrifuged at 10,000 × g at 4 ◦ C for 15 min. Aliquots of supernatants were separated and used for biochemical estimations. 2.6. Measurement of oxidative stress parameters 2.6.1. Measurement of lipid peroxidation The quantitative measurement of lipid peroxidation in striatum was performed according to the method of Wills [24]. The amount of malondialdehyde (MDA), a measure of lipid peroxidation was measured by reaction with thiobarbituric acid at 532 nm using Perkin Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The values were calculated using molar extinction coefficient of chromophore (1.56 × 105 M−1 cm−1 ) and expressed as percentage of vehicle treated group. 2.6.2. Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with Greiss reagent (0.1% N-(1-naphthyl) ethylenediame dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) as described by Green et al. [25]. Equal volumes of supernatant and Greiss reagent were mixed, and incubated for 10 min at room temperature. The absorbance of each sample was determined at 540 nm at Perkin Elmer lambda 20 spectrophotometer. The concentration of nitrite in the supernatants was determined from a sodium nitrite standard curve and expressed as percentage of vehicle treated group. 2.6.3. Catalase estimation Catalase activity was assayed by the method of Luck [26], wherein breakdown of hydrogen peroxides (H2 O2 ) is measured at 240 nm. Briefly, assay mixture consisted of 3 ml of H2 O2 phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), and change in absorbance of each sample was recorded at 240 nm. The results were expressed as micromole H2 O2 decomposed per milligram of protein/min. 2.6.4. Superoxide dismutase activity (SOD) SOD activity was assayed according to the method of Kono [27] wherein reduction of nitrazobluetetrazolium (NBT) was inhibited by the SOD is measured at 560 nm using spectrophotometer. Briefly, reaction was initiated by the addition of the hydroxylamine hydrochloride to the mixture containing NBT and sample. The results were expressed as unit/mg protein. 2.6.5. Protein estimation The protein was measured by biuret method using bovine serum albumin as standard [28]. 2.7. Mitochondrial enzymes estimation 2.7.1. Isolation of rat brain mitochondria Second group of animals were used for mitochondrial isolation as described in the method of Berman and Hastings [29]. The brain regions were homogenized in isolated buffer. Homogenates were centrifuged at 13,000 × g for 5 min at 4 ◦ C. Pellets were resuspended in isolation buffer with ethylene glycol tetra acetic acid (EGTA) and spun again at 13,000 × g at 4 ◦ C for 5 min. The resulting supernatants were transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000 × g at 4 ◦ C for 10 min. Pellets containing pure mitochondria were resuspended in isolation buffer without EGTA. 2.7.2. NADH dehydrogenase activity (Complex-I) Complex-I was measured spectrophotometrically by the method of King and Howard [30]. The method involves catalytic oxidation of NADH to NAD+ with subsequent reduction of cytochrome C. The reaction mixture contained 0.2 M glycyl glycine buffer pH 8.5, 6 mM NADH in 2 mM glycyl glycine buffer and 10.5 mM cytochrome C. The reaction was initiated by the addition of requisite amount of solubilized mitochondrial sample and followed absorbance change at 550 nm for 2 min.

40

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

2.7.3. Succinate dehydrogenase (SDH) activity (Complex-II) SDH was measured spectrophotometrically according to King [31]. The method involves oxidation of succinate by an artificial electron acceptor, potassium ferricyanide. The reaction mixture contained 0.2 M phosphate buffer pH 7.8, 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of mitochondrial sample and change in absorbance was recorded at 420 nm. 2.7.4. Cytochrome oxidase assay (Complex-IV) Cytochrome oxidase activity was assayed in brain mitochondria according to the method of Sottocasa et al. [32]. The assay mixture contained 0.3 mM reduced cytochrome C in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample and change in absorbance was recorded at 550 nm. 2.7.5. Mitochondrial redox activity The MTT assay was based on the reduction of (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-H-tetrazolium bromide) (MTT) by hydrogenase activity in functionally intact mitochondria. The MTT reduction rate was used to assess the activity of the mitochondrial respiratory chain in isolated mitochondria by the method of Liu et al. [33]. Briefly, 100 ␮l mitochondrial samples were incubated with 10 ␮l MTT for 3 h at 37 ◦ C. The blue formazan crystals were solubilized with dimethylsulfoxide and measured by an ELISA reader at 580 nm filter. 2.8. Statistical analysis The data was analyzed by using analysis of variance (ANOVA) followed by Tukey’s test. All the values are expressed as mean ± S.E.M. In all tests, the criterion for statistical significance was P < 0.05.

3. Results 3.1. Effect of hesperidin and naringin on body weight and their interaction with nitric oxide modulator Systemic 3-NP (10 mg/kg) treatment significantly decreased body weight on day 15th as compared to vehicle treated group. Further, hesperidin (100 mg/kg, p.o.) and naringin (100 mg/kg, p.o.) treatment significantly attenuated the loss in body weight as compared to 3-NP treated group (P < 0.05) (Fig. 1). However, lower dose of both hesperidin (50 mg/kg) and naringin (50 mg/kg) did not produce any significant effect on the loss in body weight in 3NP treated group. l-Arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) further

increased loss in body weight which was significant as compared to their effect alone in 3-NP treated group (Fig. 2). However, l-NAME (10 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect per se (Fig. 2). However, hesperidin (100 mg/kg) and naringin (100 mg/kg) per se treatment did not influence significantly o the body weight as compared to vehicle treated group (P < 0.05). 3.2. Effect of hesperidin and naringin on locomotor activity and their interaction with nitric oxide modulators Systemic 3-NP (10 mg/kg) treatment significantly impaired locomotor activity on day 15th as compared to vehicle treated group. Further, hesperidin (100 mg/kg, p.o.) and naringin (50 and 100 mg/kg, p.o.) pretreatment significantly improved locomotor activity as compared to 3-NP treated group (Fig. 3). However, hesperidin (50 mg/kg) did not produce any significant effect on locomotor activity as compared to 3-NP treated group on day 15th. l-Arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg), partially attenuated their protective effects (reduced locomotor activity) which was significant as compared to hesperidin and naringin treatment alone. However, l-NAME (10 mg/kg) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect (improvement in locomotor activity) as compared to their effect per se (P < 0.05) (Fig. 4). However, hesperidin (100 mg/kg) and naringin (100 mg/kg) per se treatment did not influence significantly locomotor activity as compared to vehicle treated group (P < 0.05). 3.3. Effect of hesperidin and naringin on rotarod activity and their interaction with nitric oxide modulators in 3-NP treated rats Systemic 3-NP treatment significantly impaired grip strength performance on day 15th as compared to vehicle treated group. Hesperidin (100 mg/kg, p.o.) and naringin (50 and 100 mg/kg, p.o.) pretreatment significantly improved muscle grip strength (delayed fall of time) as compared to 3-NP treated group (P < 0.05) (Fig. 5).

Fig. 1. Effect of HES and NAR on body weight in 3-nitropropionic acid treated rats. a P < 0.05 versus vehicle treated, b P < 0.05 versus 3-NP, c P < 0.05 versus [HES (50) + 3-NP], e P < 0.05 versus [NAR (50) + 3-NP] treated group (one-way ANOVA followed by Tukey’s test).

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

41

Fig. 2. Effect of HES and NAR and their modification by nitric oxide modulators on body weight in 3-nitropropionic acid treated rats. a P < 0.05 versus vehicle treated, b P < 0.05 versus 3-NP, d P < 0.05 versus [HES (50) + 3-NP], e P < 0.05 versus [l-NAME (10) + 3-NP], f P < 0.05 versus [NAR (50) + 3-NP] treated group (one-way ANOVA followed by Tukey’s test).

However, 50 mg/kg dose of hesperidin did not produce any significant effect on grip strength performance as compared to 3-NP treated group. l-Arginine (50 mg/kg) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated their protective effect (shortened fall of time) as compared to their

effect alone (Fig. 6). Further, l-NAME pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect (delayed fall of time) as compared to their effect alone (P < 0.05) (Fig. 6). However, hesperidin (100 mg/kg) and naringin (100 mg/kg) per se treatment did not alter significantly

Fig. 3. Effect of HES and NAR on locomotor activity in 3-NP treated rats. a P < 0.05 versus vehicle treated, b P < 0.05 versus 3-NP, c P < 0.05 versus [HES (50) + 3-NP], e P < 0.05 versus [NAR (50) + 3-NP] treated group (one-way ANOVA followed by Tukey’s test).

42

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

Fig. 4. Effect of HES and NAR and their modification by nitric oxide modulators on locomotor activity in 3-NP treated rats. a P < 0.05 versus vehicle treated, b P < 0.05 versus 3-NP, d P < 0.05 versus [HES (50) + 3-NP], e P < 0.05 versus [l-NAME (10) + 3-NP], f P < 0.05 versus [NAR (50) + 3-NP] treated group (one-way ANOVA followed by Tukey’s test).

grip strength performance as compared to vehicle treated group (P < 0.05). 3.4. Effect of hesperidin and naringin on brain oxidative damage (lipid peroxidation, nitrite, SOD and catalase) and their interaction with nitric oxide modulators in 3-NP-treated rats Systemic administration of 3-NP for 14 days significantly increased lipid peroxidation, nitrite concentration, depleted SOD and catalase enzyme activity in striatum as compared to vehicle

treated group. However, hesperidin (100 mg/kg) and naringin (50 and 100 mg/kg, p.o.) treatment significantly attenuated lipid peroxidation, nitrite concentration, restored SOD and catalase enzyme activities as compared to 3-NP treated group (P < 0.05) (Table 1). However lower dose of hesperidin (50 mg/kg) did not produce any significant effect on these oxidative stress parameters in 3NP treated group. l-Arginine (50 mg/kg) pretreatment with lower dose of hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated their protective effect (antioxidant like effect) which was significant as compared their effect alone. On the other hand,

Fig. 5. Effect of HES and NAR on rotarod activity in 3-NP treated rats. a P < 0.05 versus vehicle treated, b P < 0.05 versus 3-NP, c P < 0.05 versus [HES (50) + 3-NP], e P < 0.05 versus [NAR (50) + 3-NP] treated group (one-way ANOVA followed by Tukey’s test).

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

43

Fig. 6. Effect of HES and NAR and their modification by nitric oxide modulators on rotarod activity in 3-NP treated rats. a P < 0.05 versus vehicle treated, b P < 0.05 versus 3-NP, d P < 0.05 versus [HES (50) + 3-NP], e P < 0.05 versus [l-NAME (10) + 3-NP], f P < 0.05 versus [NAR (50) + 3-NP] treated group (one-way ANOVA followed by Tukey’s test).

l-NAME (10 mg/kg) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect which was significant as compared to their effect alone. However, hesperidin (100 mg/kg) and naringin (100 mg/kg) per se treatment did not produce any significant effect on these oxidative stress parameters as compared to vehicle treated group (Table 1). 3.5. Effect of hesperidin and naringin on mitochondrial complex enzymes and their interaction with nitric oxide modulators Systemic 3-NP treatment significantly impaired mitochondrial complex enzymes activity as compared to vehicle treated group. Hesperidin (100 mg/kg) and naringin (100 mg/kg, p.o.) treatment

significantly restored mitochondrial complex enzyme (I, II and IV) activities as compared to 3-NP-treated group (P < 0.05) (Table 2). However lower dose of both hesperidin (50 mg/kg) and naringin (50 mg/kg) did not produce any significant effect on mitochondrial complex enzyme activities in 3-NP treated group. Further, l-arginine (50 mg/kg) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated their protective effect on mitochondrial complex enzyme activities as compared to their effect per se. l-NAME (10 mg/kg) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their protective effect as compared to their effect per se. However, hesperidin (100 mg/kg, p.o.) and naringin (100 mg/kg), per se treatment did not produce any significant effect on mito-

Table 1 Effect of HES and NAR and nitric oxide modulator on 3-nitropropionic acid treatment-induced biochemical changes in striatum. Treatment (mg/kg)

MDA (nmol/mg protein) (% of vehicle)

Vehicle 3-NP (10) HES (100) NAR (100) l-A (50) + 3-NP l-NAME (10) + 3-NP HES (50) + 3-NP (10) HES (100) + 3-NP (10) l-A (50) + HES (50) + 3-NP l-NAME (10) + HES (50) + 3-NP NAR (50) + 3-NP (10) NAR (100) + 3-NP (10) l-A (50) + NAR (50) + 3-NP l-NAME (10) + NAR (50) + 3-NP

100 231 109 106 250 225 194 150 238 156 219 144 244 163

± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 9a 11 10 13 8 6b 4b , c 6d 9d , e 10 4b , c 6d 9d , e

Values expressed as % of vehicle treated group. a P < 0.05 versus vehicle. b P < 0.05 versus 3-NP. c P < 0.05 versus [NAR (50) + 3-NP] or [HES (50) + 3-NP]. d P < 0.05 versus [NAR (50) + 3-NP] or [HES (50) + 3-NP]. e P < 0.05 versus [l-NAME (10) + 3-NP].

Nitrite level (␮mol/mg protein) (% of vehicle) 100 183 91 96 196 157 154 130 178 122 161 143 174 117

± ± ± ± ± ± ± ± ± ± ± ± ± ±

7 5a 5 4 6 5 7b 6.5b , c 6 6.5d 5 6b 5 7d , e

SOD (unit/mg protein) (% of vehicle) 100 49 111 105 45 61 62 78 53 82 58 72 50 80

± ± ± ± ± ± ± ± ± ± ± ± ± ±

8 4a 7 4 4.5 3 4.5b 5.5b 4.5 6d 4.5 3b 4 4.5d , e

Catalase ␮M of H2 O2 decomposed/min/mg protein (% of vehicle) 100 44 110 113 41 64 59 85 44 87 56 79 49 85

± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 3.5a 5 3 3.5 2.5 4 4b , c 4 4d 3 3b , c 3 4d , e

44

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

Table 2 Effect of HES and NAR and nitric oxide modulator on 3-nitropropionic acid induced mitochondrial complex enzymes dysfunction in striatum. Treatment (mg/kg)

Complex-I (nmol NADH oxidized/min/mg protein) (% of vehicle)

Vehicle 3-NP (10) HES (100) NAR (100) l-A (50) + 3-NP l-NAME (10) + 3-NP HES (50) + 3-NP (10) HES (100) + 3-NP (10) l-A (50) + HES (50) + 3-NP l-NAME (10) + HES (50) + 3-NP NAR (50) + 3-NP (10) NAR (100) + 3-NP (10) l-A (50) + NAR (50) + 3-NP l-NAME (10) + NAR (50) + 3-NP

100 55 102 106 57 72 77 87 59 99 73 84 61 96

± ± ± ± ± ± ± ± ± ± ± ± ± ±

9.3 6.0a 4.4 4.4 9.8 6.8 4.8b 2.8b , c 8.3d 3.7d , e 8.3 3.9b , c 2.4f 4.8f , e

Complex-II (nmol/min/mg protein) (% of vehicle group) 100 50 104 103 41 54 54 71 45 90 51 74 52 87

± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.3 10.3a 3.1 5.0 9.4 8.2 5.9b 5.5b , c 7.1d 2.9d , e 5.0 6.1b 6.2 3.7f , e

MTT assay (% of vehicle) 100 62 101 101 55 73 73 99 66 95 74 84 70 100

± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.1 4.3a 6.0 2.7 4.9 5.6 1.5b 3.0b 7.8 1.7d 1.8 3.2b 5.8 2.7f , e

Complex-IV (nmol cyto-c oxidized/min/mg protein) (% of vehicle) 100 63 111 110 56 79 73 93 65 104 67 88 56 101

± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.3 5.4a 3.0 5.1 7.0 6.4 6.6 6.1b , c 6.7 4.2d 7.2 4.1b , c 6.4 2.0f , e

Values expressed as % of vehicle treated group. a P < 0.05 versus vehicle. b P < 0.05 versus 3-NP. c P < 0.05 versus [NAR (50) + 3-NP] or [HES (50) + 3-NP]. d P < 0.05 versus [NAR (50) + 3-NP] or [HES (50) + 3-NP]. e P < 0.05 versus [l-NAME (10) + 3-NP]. f P < 0.05 versus [NAR (50) + 3-NP] treated group.

chondrial enzyme complexes as compared to vehicle treated group (Table 2). 3.6. Effect of hesperidin and naringin on MTT ability and its modification by nitric oxide modulators in 3-NP treated rats Systemic 3-NP treatment significantly depleted mitochondrial redox activity as compared to vehicle treated group. Hesperidin (100 mg/kg, p.o.) and naringin (100 mg/kg, p.o.) treatment significantly restored mitochondrial redox activity as compared to 3-NP-treated group (P < 0.05) (Table 2). However, lower dose of both hesperidin (50 mg/kg) and naringin (50 mg/kg) did not produce any significant effect in MTT activity in 3-NP treated group. l-Arginine (nitric oxide precursor) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly attenuated their protective effect (depleted mitochondrial redox activity) as compared to their effect per se. Further, l-NAME (non-selective NOS inhibitor) pretreatment with hesperidin (50 mg/kg) and naringin (50 mg/kg) significantly potentiated their effect (restored mitochondrial redox activity) of hesperidin and naringin which was significant as compared to their effect per se. However, hesperidin (100 mg/kg, p.o.) and naringin (100 mg/kg, p.o.), per se treatment did not cause any significant change in the mitochondrial redox activity as compared to vehicle treated group (Table 2). 4. Discussion Present study shows that, 3-NP significantly caused impairment in locomotor activity, grip strength, body weight, oxidative defense and mitochondrial enzymes complex activities suggesting HD like symptoms in rats. Further hesperidin and naringin drugs pretreatment significantly attenuated these HD like symptoms. There are substantial evidences that a secondary consequence of the gene defect causes an impairment of energy metabolism [18,34]. HD patients often show gradual reduction in body weight despite a normal or increased caloric intake [35]. Supporting to the above observation, 14 days 3-NP treatment caused reduction in body weight which was significantly improved by hesperidin and naringin pretreatment suggesting their therapeutic potential. The main function of the basal ganglia is to control the overall co-ordination of the body movements [36], justifying the analysis

of movement patterns in relation to striatal degeneration [37,38]. As the striatum is the central core area in the basal ganglia that controls co-ordination of motor movement, it is expected that the disturbance in co-ordination of motor movement against 3-NP induced striatal degeneration. This further explains the alteration in locomotor activity as well as grip strength performance in 3NP treated group. Altered locomotion pattern may reflect motor disturbances as observed in HD patients clinically [39]. Further, hesperidin and naringin pretreatment significantly improved locomotor and grip strength performance in 3-NP treated animals, suggesting their therapeutic potential against these behavioral symptoms. However, lower dose of hesperidin did not produce any significant effect on these behavioral parameters (body weight and grip strength) that could be due to its ineffectiveness in low dose. Protective effects of these drugs (hesperidin and naringin) were partially attenuated by l-arginine pretreatment. l-NAME pretreatment with these drugs caused potentiation in the protective effect, suggest the involvement of nitric oxide mechanism in their action. Liu et al. reported naringin and hesperidin block the nitric oxide production from endothelial cells [40]. Flavonoids have been reported to exert a distinct antioxidant action by suppressing the hepatosis-induced increase in the intensity of NO radical generation [41]. Recently, flavonoids have been shown to attenuate several behavioral alterations against ischemic reperfusion and neuroinflammatory induced injury. Further, these studies have suggested the involvement of nitric oxide mechanisms in their protective actions [42,43]. The free radical scavenger and nitric oxide inhibitory property of hesperidin and naringin might be involved in attenuating behavioral alterations against 3-NP treatment. Studies have highlighted that these flavanoids might have therapeutic potential due to their hydrogen donors and free radical scavenging properties [7,44]. Present study investigated 3-NP induced oxidative stress in relation to striatal degeneration. 3-NP administration has also been reported to alter oxidants/antioxidant defense system by unknown mechanisms. These mechanisms have not been fully understood so far. However, several theories and explanations have been proposed such as excitotoxicity, oxidative stress and mitochondrial dysfunction responsible for striatal degeneration. In the present study, 3-NP treatment significantly raised MDA, nitrite concentration and weakened oxidative defense (depleted SOD and

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

catalase). The above observations have also been supported by other researchers from different laboratories [38,45,46]. Further, 3-NP induced oxidative damage was significantly attenuated by hesperidin and naringin pretreatment suggesting their antioxidant like effect. Rajadurai and Prince also reported that flavonoids inhibit alterations in mitochondrial lipid peroxides, oxidative defense enzymes (SOD, catalase, glutathione peroxidase, glutathione-Stransferase and reduced glutathione) in rodents [47]. Kanno et al. reported that naringin increased SOD and catalase activities by upregulating gene expression of SOD and catalase [48]. Further, l-arginine pretreatment with lower dose of hesperidin and naringin caused attenuation of their protective effects. However, l-NAME pretreatment with lower dose of naringin and hesperidin significantly potentiated their protective effect suggesting that these drugs might involve nitric oxide mechanism in producing antioxidant like effect. It seems that hesperidin and naringin showed their protective effect by modulating nitric oxide pathways. However it is still not clear how these drugs modulate nitric oxide pathways to produce their protective effect. Report indicates that naringin suppresses PGE2 and NO concentrations in the aqueous humor [49]. Anti-inflammatory effect of naringin may be due to suppression of PGE2 and NO [49]. Hesperidin is a powerful ONOO-scavenger and promotes cellular defense activity against ONOO-involved diseases [50]. Further, hesperidin has also been reported to cause inhibition on the nitration of bovine serum albumin (BSA) by ONOO [51]. Mitochondria play a key role in maintaining cellular energy balance, cell apoptosis process. Most established function of the cell organelle is to synthesize ATP through oxidative phosphorylation, which is associated with ROS production, including primarily superoxide (O2 •− ) formation by the respiratory chain complexes I and III. Increasing evidences suggest that mitochondrial dysfunction is linked with oxidative damage that plays a crucial role in oxidative neurodegenerative pathologies and therefore mitochondrial scavenging of ROS can be a promising therapeutic approach [51,52]. Supporting to above, 3-NP significantly altered mitochondrial enzyme complex activities that could be due to generation of free radicals in the striatum. These alterations in enzyme mitochondrial complex activities were significantly restored by hesperidin and naringin pretreatment. Rosenstock and its group also reported that 3-NP inhibits complex-II enzyme of the respiratory chain, mitochondrial calcium release, increased ROS, apoptosis [51]. Flavonoids have also been proved to have metal chelating, free radical scavenging properties such as neutralization of superoxide, singlet oxygen and inhibit the hydrogen peroxide induced lipid peroxidation [4,53,54]. It has been demonstrated that flavonoids inhibit the expression of isoforms of inducible nitric oxide synthase, cyclooxygenase and lipooxygenase, which are responsible for the production of nitric oxide, prostanoids and leukotrienes, as well as inflammatory mediators such as cytokines, chemokines or adhesion molecules [55]. Flavonoids significantly claimed to produce beneficial protective effect either acting against oxidative damage or by activation of the antioxidant defense enzyme system [56–58]. Further, l-arginine pretreatment with hesperidin and naringin significantly attenuated their protective effect. However, l-NAME pretreatment with these drugs potentiated their neuroprotective effect on mitochondrial complex enzyme activities suggesting nitric oxide mechanism might also be involved in restoring mitochondrial enzyme complex activities. If the initiating step in the pathological cascade is a depletion of cellular energy stores, then an agent that buffers cellular energy stores could be implicated as effective neuroprotective strategies in the treatment of HD. These observations suggest that hesperidin and naringin treatment might have their protective effect on cellular energy stores by modulating nitric oxide pathways. In the previous studies, the exact mechanisms by which flavonoids exert neuroprotective effects are

45

not fully understood, although evidence from cell studies suggests that flavonoids express a variety of cellular actions that may protect against neuronal injury via the modulation of critical neuronal signalling pathways involved in neuronal death or survival [7,59]. Reports also show that flavonoid decreases NOS-2 activity due to inhibition of NOS-2 mRNA, NOS-2 gene transcription expression and protein expression [60–62]. Flavanoids have been reported to inhibit protein kinase C, phospholipase A2, phospholipase C and phosphodiesterases [63]. Another possibility could be the modulation of NOS-2 induction indirectly by inhibition of the cyclooxygenase and/or lipoxygenase pathways [64]. In conclusion, these results suggest that: (i) 3-nitropropionic acid induces striatal oxidative stress; (ii) hesperidin and naringin protects against 3-nitropropionic acid induced neurotoxicity (iii) protective effect of hesperidin and naringin could be due to their nitric oxide mechanism. Acknowledgments Authors gratefully acknowledged the financial support of University Grants Commission (UGC), New Delhi for carrying out this study. References [1] Middleton Jr E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells. Implications for inflammation, heart disease, and cancer. Pharmacol Rev 2000;52:673–751. [2] Prior RL, Cao G. Analysis of botanicals and dietary supplements for antioxidant capacity: a review. J AOAC Int 2000;83:950–6. [3] Renugadevi J, Prabu SM. Naringenin protects against cadmium-induced oxidative renal dysfunction in rats. Toxicology 2009;256:128–34. [4] Chen YT, Zheng RL, Jia ZJ, Ju Y. Flavonoids as superoxide scavengers and antioxidants. Free Radic Biol Med 1990;9:19–21. [5] Jagetia GC, Reddy TK. Modulation of radiation-induced alteration in the antioxidant status of mice by naringin. Life Sci 2005;77:780–94. [6] Schroeter H, Boyd C, Spencer JP, Williams RJ, Cadenas E, Rice-Evans C. MAPK signaling in neurodegeneration: influences of flavonoids and of nitric oxide. Neurobiol Aging 2002;23:861–80. [7] Cotelle N, Bernier JL, Catteau JP, Pommery J, Wallet JC, Gaydou EM. Antioxidant properties of hydroxy-flavones. Free Radic Biol Med 1996;20:35–43. [8] Kumar P, Kalonia H, Kumar A. Sesamol attenuate 3-nitropropionic acid-induced Huntington-like behavioral, biochemical, and cellular alterations in rats. J Asian Nat Prod Res 2009;11:439–50. [9] Lopez-Lopez G, Moreno L, Cogolludo A, Galisteo M, Ibarra M, Duarte J, et al. Nitric oxide (NO) scavenging and NO protecting effects of quercetin and their biological significance in vascular smooth muscle. Mol Pharmacol 2004;65:851–9. [10] Raso GM, Meli R, Di Carlo G, Pacilio M, Di Carlo R. Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1. Life Sci 2001;68:921–31. [11] Ishige K, Schubert D, Sagara Y. Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms. Free Radic Biol Med 2001;30:433–46. [12] Kumar P, Padi SS, Naidu PS, Kumar A. Cyclooxygenase inhibition attenuates 3-nitropropionic acid-induced neurotoxicity in rats: possible antioxidant mechanisms. Fundam Clin Pharmacol 2007;21:297–306. [13] Nakagawa T, Yokozawa T. Direct scavenging of nitric oxide and superoxide by green tea. Food Chem Toxicol 2002;40:1745–50. [14] Haenen GR, Paquay JB, Korthouwer RE, Bast A. Peroxynitrite scavenging by flavonoids. Biochem Biophys Res Commun 1997;236:591–3. [15] Heijnen CG, Haenen GR, van Acker FA, van der Vijgh WJ, Bast A. Flavonoids as peroxynitrite scavengers: the role of the hydroxyl groups. Toxicol In Vitro 2001;15:3–6. [16] Lorenz P, Roychowdhury S, Engelmann M, Wolf G, Horn TF. Oxyresveratrol and resveratrol are potent antioxidants and free radical scavengers: effect on nitrosative and oxidative stress derived from microglial cells. Nitric Oxide 2003;9:64–76. [17] Patocka J, Bielavsky J, Cabal J, Fusek J. 3-Nitropropionic acid and similar nitrotoxins. Acta Medica (Hradec Kralove) 2000;43:9–13. [18] Alexi T, Hughes PE, Faull RL, Williams CE. 3-Nitropropionic acid’s lethal triplet: cooperative pathways of neurodegeneration. Neuroreport 1998;9:R57–64. [19] Kumar P, Kumar A. Possible neuroprotective effect of Withania somnifera root extract against 3-nitropropionic acid-induced behavioral, biochemical, and mitochondrial dysfunction in an animal model of Huntington’s disease. J Med Food 2009;12:591–600. [20] Kumar P, Kumar A. Effect of lycopene and epigallocatechin-3-gallate against 3nitropropionic acid induced cognitive dysfunction and glutathione depletion in rat: a novel nitric oxide mechanism. Food Chem Toxicol 2009.

46

P. Kumar, A. Kumar / Behavioural Brain Research 206 (2010) 38–46

[21] Kumar P, Kumar A. Neuroprotective effect of cyclosporine and FK506 against 3-nitropropionic acid induced cognitive dysfunction and glutathione redox in rat: possible role of nitric oxide. Neurosci Res 2009;63:302– 14. [22] Akula KK, Dhir A, Kulkarni SK. Nitric oxide signaling pathway in the anticonvulsant effect of adenosine against pentylenetetrazol-induced seizure threshold in mice. Eur J Pharmacol 2008;587:129–34. [23] Kaur G, Tirkey N, Chopra K. Beneficial effect of hesperidin on lipopolysaccharide-induced hepatotoxicity. Toxicology 2006;226:152–60. [24] Wills ED. Mechanisms of lipid peroxide formation in animal tissues. Biochem J 1966;99:667–76. [25] Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131–8. [26] Luck H. Catalase. In: Bergmeyer HU, editor. Methods of enzymatic analysis. New York: Academic Press; 1971. p. 885–93. [27] Kono Y. Generation of superoxide radical during auto-oxidation of hydroxylamine and an assay of superoxide dismutase. Arch Biochem Biophys 1978;186:189–95. [28] Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem 1949;177:751–66. [29] Berman SB, Hastings TG. Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria: implications for Parkinson’s disease. J Neurochem 1999;73:1127–37. [30] King TE, Howard RL. Preparations and properties of soluble NADH dehydrogenases from cardiac muscle. Methods Enzymol 1967;10:275–84. [31] King TE. Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. Methods Enzymol 1967;10:322–31. [32] Sottocasa GL, Kuylenstierna B, Ernster L, Bergstrand A. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol 1967;32:415–38. [33] Liu Y, Peterson DA, Kimura H, Schubert D. Mechanisms of cellular 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolinium bromide (MTT) reduction. J Neurochem 1997;69:581–93. [34] Brouillet E, Guyot MC, Mittoux V, Altairac S, Conde F, Palfi S, et al. Partial inhibition of brain succinate dehydrogenase by 3-nitropropionic acid is sufficient to initiate striatal degeneration in rat. J Neurochem 1998;70:794– 805. [35] Matthews RT, Yang L, Jenkins BG, Ferrante RJ, Rosen BR, Kaddurah-Daouk R, et al. Neuroprotective effects of creatine and cyclocreatine in animal models of Huntington’s disease. J Neurosci 1998;18:156–63. [36] Aldridge JW, Berridge KC, Rosen AR. Basal ganglia neural mechanisms of natural movement sequences. Can J Physiol Pharmacol 2004;82:732–9. [37] Teunissen CE, Steinbusch HW, Angevaren M, Appels M, de Bruijn C, Prickaerts J, et al. Behavioural correlates of striatal glial fibrillary acidic protein in the 3-nitropropionic acid rat model: disturbed walking pattern and spatial orientation. Neuroscience 2001;105:153–67. [38] Kumar P, Kumar A. Possible role of sertraline against 3-nitropropionic acid induced behavioral, oxidative stress and mitochondrial dysfunctions in rat brain. Prog Neuropsychopharmacol Biol Psychiatry 2009;33: 100–8. [39] Hinton SC, Paulsen JS, Hoffmann RG, Reynolds NC, Zimbelman JL, Rao SM. Motor timing variability increases in preclinical Huntington’s disease patients as estimated onset of motor symptoms approaches. J Int Neuropsychol Soc 2007;13:539–43. [40] Liu L, Xu DM, Cheng YY. Distinct effects of naringenin and hesperetin on nitric oxide production from endothelial cells. J Agric Food Chem 2008;56: 824–9. [41] Timoshin AA, Dorkina EG, Paukova EO, Vanin AF. Quercetin and hesperidin decrease the formation of nitric oxide radicals in rat liver and heart under the conditions of hepatosis. Biofizika 2005;50:1145–9. [42] Vafeiadou K, Vauzour D, Lee HY, Rodriguez-Mateos A, Williams RJ, Spencer JP. The citrus flavanone naringenin inhibits inflammatory signaling in glial cells and protects against neuroinflammatory injury. Arch Biochem Biophys 2009;484:100–9.

[43] Gaur V, Aggarwal A, Kumar A. Protective effect of naringin against ischemic reperfusion cerebral injury: possible neurobehavioral, biochemical and cellular alterations in rat brain. Eur J Pharmacol 2009;616:147–54. [44] Haller C, Hizoh I. The cytotoxicity of iodinated radio contrast agents on renal cells in vitro. Invest Radiol 2004;39:149–54. [45] Tunez I, Montilla P, Del Carmen Munoz M, Feijoo M, Salcedo M. Protective effect of melatonin on 3-nitropropionic acid-induced oxidative stress in synaptosomes in an animal model of Huntington’s disease. J Pineal Res 2004;37:252–6. [46] Perez-De La Cruz V, Elinos-Calderon D, Robledo-Arratia Y, Medina-Campos ON, Pedraza-Chaverri J, Ali SF, et al. Targeting oxidative/nitrergic stress ameliorates motor impairment, and attenuates synaptic mitochondrial dysfunction and lipid peroxidation in two models of Huntington’s disease. Behav Brain Res 2009;199:210–7. [47] Rajadurai M, Prince PS. Naringin ameliorates mitochondrial lipid peroxides, antioxidants and lipids in isoproterenol-induced myocardial infarction in Wistar rats. Phytother Res 2009;23:358–62. [48] Kanno S, Shouji A, Asou K, Ishikawa M. Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in P388 cells. J Pharmacol Sci 2003;92:166–70. [49] Shiratori K, Ohgami K, Ilieva I, Jin XH, Yoshida K, Kase S, et al. The effects of naringin and naringenin on endotoxin-induced uveitis in rats. J Ocul Pharmacol Ther 2005;21:298–304. [50] Kim JY, Jung KJ, Choi JS, Chung HY. Hesperetin: a potent antioxidant against peroxynitrite. Free Radic Res 2004;38:761–9. [51] Rosenstock TR, Carvalho AC, Jurkiewicz A, Frussa-Filho R, Smaili SS. Mitochondrial calcium, oxidative stress and apoptosis in a neurodegenerative disease model induced by 3-nitropropionic acid. J Neurochem 2004;88:1220–8. [52] Beal MF, Brouillet E, Jenkins BG, Ferrante RJ, Kowall NW, Miller JM, et al. Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 1993;13:4181–92. [53] Maridonneau-Parini I, Braquet P, Garay RP. Heterogeneous effect of flavonoids on K + loss and lipid peroxidation induced by oxygen-free radicals in human red cells. Pharmacol Res Commun 1986;18:61–72. [54] Pradeep K, Park SH, Ko KC. Hesperidin a flavanoglycone protects against gamma-irradiation induced hepatocellular damage and oxidative stress in Sprague–Dawley rats. Eur J Pharmacol 2008;587:273–80. [55] Tunon MJ, Garcia-Mediavilla MV, Sanchez-Campos S, Gonzalez-Gallego J. Potential of flavonoids as anti-inflammatory agents: modulation of proinflammatory gene expression and signal transduction pathways. Curr Drug Metab 2009;10:256–71. [56] Choi EJ, Ahn WS. Neuroprotective effects of chronic hesperetin administration in mice. Arch Pharm Res 2008;31:1457–62. [57] Gandhi C, Upaganalawar A, Balaraman R. Protection against in vivo focal myocardial ischemia/reperfusion injury-induced arrhythmias and apoptosis by hesperidin. Free Radic Res 2009:1–11. [58] El-Sayed el SM, Abo-Salem OM, Abd-Ellah MF, Abd-Alla GM. Hesperidin, an antioxidant flavonoid, prevents acrylonitrile-induced oxidative stress in rat brain. J Biochem Mol Toxicol 2008;22:268–73. [59] Spencer JP. The interactions of flavonoids within neuronal signaling pathways. Genes Nutr 2007;2:257–73. [60] Olszanecki R, Gebska A, Kozlovski VI, Gryglewski RJ. Flavonoids and nitric oxide synthase. J Physiol Pharmacol 2002;53:571–84. [61] Liang YC, Tsai SH, Tsai DC, Lin-Shiau SY, Lin JK. Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferators-activated receptor-gamma by flavonoids in mouse macrophages. FEBS Lett 2001;496:12–8. [62] Brandi ML. Flavonoids: biochemical effects and therapeutic applications. Bone Miner 1992;19:S3–14. [63] Galati EM, Monforte MT, Kirjavainen S, Forestieri AM, Trovato A, Tripodo MM. Biological effects of hesperidin, a citrus flavonoid (note I): antiinflammatory and analgesic activity. Farmaco 1994;40:709–12. [64] Guerra JA, Molina MF, Abad MJ, Villar AM, Paulina B. Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids isolated from Tanacetum microphyllum. Int Immunopharmacol 2006;6:1723–8.