Depressive disorders are the most preva- lent form of mental illness worldwide. Major depression is characterized by a change in psychosocial and physical impairment mood as well as lack of interest in the surroundings (Saravi et al., 2016). There are growing re- ports on the incidence of depression in both males and females that imposes a substantial health burden on society (Gu et al., 2014). The post-partum period represents profound physiological and emotional changes in mothers to ensure the well-being and nurtur- ance of the offspring. However, several psy- chiatric disorders can develop in this phase (Perani and Slattery, 2014). Post-partum mood and anxiety disorders affect maternal and infant as well as developing psychiat- ric disorder in later life such as post-partum depression (PPD), post-partum anxiety and post-partum psychosis (Ming and Shinn- Yi, 2016). Several animal methods such as stress-based, high-fat diet-based and pup separation models are used to induce exper- imental PPD (Ming and Shinn-Yi, 2016). There are growing reports of new antide- pressant agents to provide clinically relevant perceptions into the neuropathology that un- derlies the idiopathic disease state of depres- sion (Alimohammadi et al., 2019).
Hesperidin is the major flavonoid isolated from citrus fruits (Li and Schluesener, 2017). The hesperidin molecule is composed of a glycone unit known as hesperetin and a di- saccharide, rutinose (Iranshahi et al., 2015). Hesperidin has several biological effects including antioxidant, anti-inflammatory, antimicrobial, anti-carcinogenic and anti-al- lergic effects and insulin-sensitizing activ- ity (Li and Schluesener, 2017). In addition, hesperidin neuropharmacological properties have been reported for the hesperidin (Ha-
jialyani et al., 2019). It has high potential for radical scavenging and protective effects and can cross blood brain barrier (Khan and Parvez, 2015). Hesperidin promotes neuro- nal survival, differentiation and neuropro- tective capacity of astrocytes (Matias et al., 2017) which have positive effect for stroke, Huntington’s, Alzheimer’s and Parkinson’s disease (Antunes et al., 2014). Several an- tioxidant compounds, such as flavonoids derived from natural products, have demon- strated neuroprotective activity in PPD (An- tunes et al., 2014).
Antioxidant enzymes, such as glutathione peroxidase (GPx), catalase (CAT) and super- oxide dismutase (SOD), also are important mediators in the reduction of oxidative stress (Khan et al. 2012). It is reported that (50 mg/ kg) treatment increased GPx, SOD and CAT activity in mouse model of Parkinson’s dis- ease (Antunes et al., 2014). Hesperidin in the acute (1 mg/kg) and chronic (0.1, 0.3 and 1 mg/kg) levels improved tail suspension test (TST) which improved antidepressant-like effect (Donato et al., 2014). Based on the aforementioned evidence, we sought to in- vestigate effects of the Hesperidin exposure during pregnancy on antidepressant-like ef- fects postpartum in mice.
The NMRI male (n=12) and virgin female mice (n=40, age: 8–10 weeks old and 28–30 gr) were supplied from the Razi Serum and Vaccine Institute (Tehran, Iran). The animals were kept five mice/cage in standard plastic cages at laboratory conditions (temperature of 22 ± 2 °C and 12/h light/dark cycle) with ad libitum access to standard chow pellet (Pars Dam Co, Tehran, Iran) and fresh wa-
ter. The animals acclimatized for 1 week before beginning the study. All experimen- tal procedures were approved by the Animal Ethics Committee of Science and Research Branch of Islamic Azad University, Tehran, Iran (IR.IAU.SRB.REC.1398.117) following the Guidelines for the Care and Use of Lab- oratory Animals in Research. After 1 week of acclimatization, the female mice were caged with fertile male mice. Each morning, the fe- male mice were examined for the presence of sperm or vaginal plug. Presence of the vaginal plug or sperm was defined as onset of preg- nancy. The pregnant mice were randomly as- signed into 4 groups (n = 10 for each group) and provided ad libitum food and water.
Hesperidin (H5254) was purchased from Sigma Aldrich, (Saint Luis, USA) and were dissolved by the sequential addition of di- methyl sulfoxide up to a final concentration of 5%, a water solution of 0.25% Tween 80 up to a final concentration of 20% and saline to complete 100% volume (Donato et al., 2014).
In control group, pregnant mice were
i.p. injected with saline containing 0.05% Tween-80 at gestation day (GD) 5, 8, 11, 14 and 17. In groups 2, 3 and 4, mice were in- jected with 0.1, 0.5 and 1 mg/kg of hesper- idin at GD 5, 8, 11, 14 and 17, respectively. The dosage of the hesperidin was determined based on the previous reports (Antunes et al., 2014; Donato et al., 2014; Khan and Parvez, 2015; Pari et al., 2015) and our pilot study. Then after delivery, antidepressant-like ef- fects of the hesperidin were evaluated using neurobehavioral tests that were done on fe- male mice.
The TST is one of the most common tech- niques for assessing antidepressant-like ac-
tivity in mice (Cryan et al., 2005). The TST was done based on the method stated by Ste- ru et al. (1985). Briefly, the mice were far from nearest objects and were both acousti- cally and visually isolated from observing or interacting each other. Each mouse was then suspended 50 cm above the floor by adhesive tape placed approximately 1 cm from the ex- tremity of the tail, in such a position that it cannot escape or hold on to nearby surfac- es. Immobility time was recorded during a
6 min period. Mice were considered im- mobile only when they had no strong body shaking and movement of the limbs as they hung passively and completely motionless.
The Open field test (OFT) was used to determine possible effects of hesperidin on the locomotor and exploratory activities. The OFT was done using 45×45×30 cm3 poly wood cage. The flour of OFT cage was divided by masking tape markers into 3×3 squares. Each animal was placed individual- ly at the center of the apparatus and observed for 6 min to record the locomotion (number of segments crossed with the four paws) (Donato et al., 2014).
FST was carried out following the protocol as described previously in mice (Castagné et al., 2011). Each mouse was plunged into a glass cylinder (height: 25 cm; diameter: 15 cm) containing 10 cm of water (25 ± 1 °C) for 15 min (pre-test session). Twenty-four hours later, the mouse was placed in the cyl- inder again and left for a 6 min period (test session). The immobility time for mouse was described as when it ceased struggling and remained floating motionless in the water, making only small movements necessary to keep its head above water. The total duration of immobility during the last 4 min of the 6
min testing period was measured.
At the end of the neurobehavioral tests, blood samples were taken from each mouse and serum Malondialdehyde (MDA), SOD, GPx and total antioxidant capacity (TAC) were determined using Zell Bio GmbH (Germany) assay kits (ZB-MDA-48A, ZB-SOD-A48, ZB-
GPX-A48 and ZB-TAC-48A, respectively).
Data was analyzed by one-way analysis of variance (ANOVA) and is presented as the mean ± SEM. For treatments found to have an effect according to the ANOVA, mean
values were compared with Tukey’s test. P≤0.05 was considered to indicate signifi- cant differences between the treatments.
Effect of exposure to different levels of Hes- peridin during pregnancy on immobility time
(S) in TST on postpartum mice is presented in Figure 1. As seen, administration of the differ- ent levels of the hesperidin (0.5 and 1 mg/kg) at GD 5, 8, 11, 14 and 17 significantly decreased immobility time (s) in TST on postpartum mice compared to control group (P≤0.05).
Figure 1. Effect of exposure to different levels of Hesperidin during pregnancy on immo- bility time (sec) in TST on postpartum mice. TST: tail suspension test. There are signifi- cant differences between groups with different superscripts (a, b and c; P ≤ 0.05).
According to the Figure 2, administra- tion of the hesperidin (0.5 and 1 mg/kg) at GD 5, 8, 11, 14 and 17 significantly
decreased immobility time (S) in FST on postpartum mice compared to control group (P≤0.05).
Figure 2. Effect of exposure to different levels of Hesperidin during pregnancy on immo- bility time (sec) in FST on postpartum mice. FST: forced swimming test. There are signifi- cant differences between groups with different superscripts (a, b and c; P ≤ 0.05).
However, pre-partum exposure to the hes- peridin (0.1, 0.5 and 1 mg/kg) had no signif-
icant effect on OFT following delivery com- pared to control group (P>0.05) (Figure 3).
Figure 3. Effect of exposure to different levels of Hesperidin during pregnancy on im- mobility time (S) in OFT on postpartum mice. OFT: open field test.
As seen in Figure 4. administration of the hesperidin (0.5 and 1 mg/kg) during the GD significantly decreased MDA levels on postpartum mice compared to control group
(P≤0.05). Furthermore, administration of the hesperidin (0.5 and 1 mg/kg) significantly in- creased GPx levels on postpartum mice com- pared to control group (P≤0.05) (Figure 5).
Figure 4. Effect of exposure to different levels of Hesperidin during pregnancy on postpartum serum Malondialdehyde (MDA) level n mice. There are significant differences between groups with different superscripts (a and b; P ≤ 0.05).
Figure 5. Effect of exposure to different levels of Hesperidin during pregnancy on postpartum serum glutathione peroxi- dase (GPx) level in mice. There are significant differences between groups with different superscripts (a and b; P ≤ 0.05).
Pre-partum exposure to the hesperidin (0.1,
0.5 and 1 mg/kg) significantly increased SOD levels on postpartum mice compared to control
group (P≤0.05) (Figure 6) but had no signif- icant effect on TAC following delivery com- pared to control group (P>0.05) (Figure 7).
Figure 6. Effect of exposure to different levels of Hesperidin during pregnancy on postpartum serum superoxide dismutase (SOD) level in mice. There are significant differences between groups with different superscripts (a and b; P ≤ 0.05).
Figure 7. Effect of exposure to different levels of Hesperidin during pregnan- cy on postpartum serum total antioxidant capacity (TAC) in mice.
postpartum mice compared to control group.
Depression is a common, chronic, recur- rent illness with severe morbidity. Although a number of research studies have been done on its physiological mechanisms, brain areas underlying this disorder are not yet well un- derstood. Postpartum depression is a severe mood disorder which happens right away after childbirth and is observed by sadness and anx- iety in mothers (O'Hara and McCabe, 2013). According to the results, administration of the different doses of hesperidin (0.5 and 1 mg/ kg) at GD 5, 8, 11, 14 and 17 significantly de- creased immobility time in TST and FST on
Hesperidin (1mg/kg) significantly reduced immobility time in FST in mice (Filho et al., 2013). In a similar study, it was reported hes- peridin (50 mg/kg) improved depressive-like behavior in the TST and memory in the Mor- ris water maze test (Antunes et al., 2014). The antidepressant-like effect of hesperidin has been reported in FST and TST tests (Souza et al., 2013). Moreover, hesperidin suppressed depressive-like behaviors in TST using in- tra-striatal injection of 6-hydroxydopamine in Parkinson's disease (Antunes et al., 2014). Hesperidin (25, 50 and 100 mg/kg) had an- ti-depressant effect in diabetic rats (El-Mara-
sy et al., 2014), our results were in agreement with the reports.
Immobility time in FST resembles a state of despair and mental depression. Stress-in- duced depression like behavioral alterations are routinely determined by TST and FST in rodents. Immobility time in TST and FST reflects the behavioral despair which is sim- ilar to depression in human (Walia and Gil- hotra, 2016). Differences on neurochemical pathways of the FST and TST induced per- formance are reported/have been reported. At face value, these two tests appear very simi- lar but this potency difference appears due to both pharmacokinetic and pharmacodynamic factors (Amin et al., 2015). We also studied effect of the hesperidin on locomotor activity using open field test. AS observed, pre-par- tum exposure to the hesperidin (0.1, 0.5 and 1 mg/kg) had no significant effect on OFT following delivery. It is revealed hesperidin at the levels of 0.1, 0.5 and 1 mg/kg, had no sedative effect, our finding was similar to this report.
Bioavailability is a key step in ensuring the bio efficacy of hesperidin which is affected by physiological conditions. It is selectively me- tabolized by both cytochrome P450 isoforms (CYP1A and CYP1B1) to eriodictyol, indicat- ing that there is Odemethylation of hesperidin in liver. It has higher bio activity compared to the other flavonoids which can be related to the inhibition of phase II metabolism (glucu- ronidation and sulfation of hesperidin). The metabolites of hesperidin are detected in urine but not in feces. In oral administration, more than 40% of the radioactivity of hesperidin
-3-14C was expired as carbon dioxide which indicates further bacterial degradation in the colon than blood circulation (Roohbakhsh et al., 2014). The ability of hesperidin to cross the blood brain barrier makes it an ideal bio-
active substance for treatment of CNS dis- orders (Iranshahi et al., 2015). Hesperidin decreases risk of Parkinson's disease as well as Alzheimer's disease in flavonoid deficient patient (Antuneset al., 2014). The neuropro- tective role of the hesperidin is mediated via anti-inflammatory and antioxidant activities (Menze et al., 2012). Hesperidin (0.01, 0.3 and 1 mg/kg) has antidepressant-like effect in TST and decreased nitrate/nitrite as well as increased hippocampal brain-derived neu- rotrophic factor (BDNF) in the hippocampus of mice (Donato et al., 2014). Based on lit- erature, NO/cGMP pathway has a key role on antidepressant effect of hesperidin (Do- nato et al., 2014). It is reported that nitrate/ nitrite levels decreased in the hippocampus of hesperidin-treated mice. Anti-depressant activity of the hesperidin is inhibited by pre- treatment with L-arginine (processor of nitric oxide). Also, administration of the hesperidin increased the brain-derived neurotrophic fac- tor (BDNF) level in the hippocampus of mice (Donato et al., 2014). Perhaps, antidepres- sant-like activity of the flavonoids mediates via BDNF (Hajialyani et al., 2019). Also, it is reported antidepressant effect of hesperidin is also dependent on nitric oxide (NO)/cGMP pathway (Donato et al., 2014). Hesperidin, acute (1 mg/kg) and chronic (0.1, 0.3 and 1 mg/kg), reduced nitrate/nitrite levels in the hippocampus of mice (Donato et al. 2014). However, the mechanisms and brain areas underlying the pathophysiology of depression are not fully elicited. It is suggested plasma nitrate levels and nitric oxide synthase (NOS) expression increased in the hippocampus of depressed patients. Inhibition of NOS may decrease immobility time in the TST elicited by hesperidin (Donato et al., 2014). Based on the limitation of the study, we were not able to determine interaction of the hesperi-
din with NO pathway.
Based on the findings, pre-partum exposure to the hesperidin (0.1, 0.5 and 1 mg/kg) signifi- cantly increased SOD and GPx levels on post- partum mice compared to control group. It is reported that hesperidin has antioxidant pro- tection against free radicals-induced oxidative damage and photo-damage repair enhancer (Hemanth Kumar et al., 2017). However, Antunes et al. (2014) reported hesperidin (50 mg/kg) treatment attenuated the 6-OHDA-in- duced reduction in GPx, SOD and CAT levels in mouse model of Parkinson’s disease. Also, it is reported hesperidin increased glutathione, SOD, CAT and decreased MDA and nitrite level (Roohbakhsh et al., 2014). Administra- tion of hesperidin (20, 40 and 80 mg/kg) re- versed the levels of serum hepatic CAT, SOD, GPx and glutathione S-transferase (GST) en- zyme levels (Pari et al., 2015) which is sim- ilar to our result. New findings revealed the antioxidant activity of hesperidin mediates by radical scavenging activity and ERK/Nrf2 signaling pathway as well (Elavarasan et al., 2012). Injection of the hesperidin (0.5 and 1 mg/kg) during the GD significantly decreased MDA levels on postpartum mice compared to control group. Hesperidin has protective effect against reactive oxygen species (ROS) production and oxidative stress. Hesperidin enhanced antioxidant enzymes CAT, SOD and GST level (Visnagri et al., 2014). The enzymatic antioxidants CAT, SOD, GPx and GST have crucial role on scavenging ROS. Hesperidin has protective effect against oxi- dative damage due to the ability of enhanced antioxidant activity. There is a correlation between depressive disorders and increased oxidative stress, neuro-inflammation and di- minished anti-oxidant defenses (Black et al., 2014). The positive effect of the antioxidant effects of antidepressants in the treatment of
major depressive disorder is well documented (Da Silva et al., 2014).
Hesperidin–therapy is safe, has a non-ac- cumulative nature with lowest adverse effect, even during the pregnancy period (Hajialyani et al., 2019). Hesperidin administered at dos- es up to 5% for 13 weeks had no mutagenic, toxic, and carcinogenic effects on mice (Garg et al., 2001). In the model of rat colon car- cinogenesis, hesperidin decreased intestinal tumor incidents via antioxidant defense with no toxicity to the liver and colon (Aranga- nathan and Nalini, 2009). Hesperidin is able to decrease streptozotocin–isoproterenol-in- duced myocardial toxicity (Agrawal et al., 2014). Although hesperidin is a safe phyto- chemical, possible interactions of this phy- tochemical should be considered (Hajialyani et al., 2019). In view of our findings, the ob- tained data indicate hesperidin has protective activity against postpartum depression. How- ever, further research is needed to clarify the precise molecular mechanisms involved in the antidepressant effects of the hesperidin.
The authors thank the Faculty of Vet- erinary Medicine, Science and Research Branch, Tehran, Iran for their cooperation. This research was conducted as a part of the DVM thesis of the first author.
The authors declared that there are no conflicts of interest.