Chinese Journal of Natural Medicines 2015, 13(3): 0183−0191
Chinese Journal of Natural Medicines
Flavonoid-rich fraction of the Monodora tenuifolia seed extract attenuates behavioural alterations and oxidative damage in forced-swim stressed rats EKEANYANWU Raphael Chukwuma1*, NJOKU Obioma Uzoma2 1
Department of Biochemistry, Faculty of Medicine, Imo State University Owerri, Imo State, Nigeria;
2
Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria Nsukka, Enugu State, Nigeria
Available online 20 Mar. 2015
[ABSTRACT] The antidepressant effects of the flavonoid-rich fraction of Monodora tenuifolia seed extract were examined by assessing the extent of attenuation of behavioural alterations and oxidative damage in the rats that were stressed by forced swim test. Compared with the model control group, the altered behavioural parameters were attenuated significantly (P < 0.05) in the group treated with the flavonoid-rich fraction (100 and 200 mg·kg−1), comparable to the group treated with the standard drug, fluoxetine (10 mg·kg−1). The flavonoid-rich fraction and fluoxetine improved significantly (P < 0.05) the activities of the antioxidant enzymes such as superoxide dismutase and catalase as well as other biochemical parameters such as reduced glutathione, protein, and nitrite in the brain of the stressed rats. These results suggested that the flavonoid-rich fraction of Monodora tenuifolia seed extract exerted the antidepressant-like effects which could be useful in the management of stress induced disease. [KEY WORDS] Monodora tenuifolia; Antidepressant; Forced swim test; Depression; Oxidative damage
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2015)03-0183-09
Introduction Recent findings have shown that the Monodora tenuifolia seed extract exerts several biological effects such as antimicrobial [1], antidiarrhoeal [2] and antioxidant activities [3]. In traditional medicine practice, the plant is widely used to relieve toothache, dysentery, diarrhoea, dermatitis, and head ache, and used as vermifuge [2]. The reported antioxidant potential of the plant [3] makes it important in the management of stress induced conditions such as depression. Depression is a common disorder associated with high rates of chronicity, relapse, and recurrence, resulting in psy-
[Received on] 01-Mar.-2014 [Research funding] This work was supported by the Tertiary Education Trust Fund, Nigeria with grant number 2012. [*Corresponding author] E-mail: [email protected], Tel: +2348032744572 (EKEANYANWU Raphael Chukwuma) All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
chosocial and physical impairments [4]. The current therapy for depression is only effective in a certain part of the patient population and associated with several side effects [5-7]. Therefore, there is an urgent need for the discovery of alternative therapy for the treatment of depression. Herbal therapies may be an effective alternative in the treatment of depression; and, indeed, the search for biologically active compounds from medicinal plants for psychiatric illnesses, including depression, has progressed significantly in the past decade [4]. Although Monodora tenuifolia has antidepressant potential, there is no sufficient evidence for its effects in animal models of depression. Guarrera et al.[8] have found that a flavonoid-rich diet is beneficial, through modulating the expression of certain disease-associated genes.. Thus, this study aimed at examining the antidepressant-like action of the flavonoid-rich fraction of the aqueous-alcoholic extract from the seed of Monodora tenuifolia using different models that are predictive of antidepressant activity [7] and investigating the level of oxidative damage in the brain of the stressed rats after treatment with the fraction and a known antidepressant drug.
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Materials and Methods Collection and preparation of plant materials Fresh seeds of Monodora tenuifolia were collected in University of Nigeria Nsukka at Enugu State and authenticated by a taxonomist, Mr A. Ozioko of the Department of Botany through comparison with a voucher specimen present in the herbarium. The fresh seeds were shade dried for 48 h, reduced in size to a coarse texture using a manual blender (Corona, Landers Colombia), and packed in airtight containers. Extraction and determination of total flavonoid contents of the extract fractions The aqueous-alcoholic extract and fractions of the ground Monodora tenuifolia seeds were obtained by solvent-solvent extraction as previously reported by Ekeanyanwu and Njoku [9]. Briefly, 5.5 kg of the ground Monodora tenuifolia seeds was macerated twice with 10 L of 70 % ethanol for 72 h at room temperature. The combined macerate was passed through Whatman No.4 filter paper and mixed thoroughly with 1.8 L of chloroform to partition the aqueous-alcoholic extract. Two distinct layers were obtained: the upper aqueous layer and the lower chloroform layer. The two layers were drawn out separately and the chloroform fraction (CLF) was dried in vacuo, and weighed. The main (aqueous portion) extract (2.75 L) was extracted three times with an equal volume of ethyl acetate for 48 h at room temperature. The ethyl acetate fractions (EAF) were combined, dried in vacuo, and weighed. The remaining aqueous extracts were stored away. 11.6 g of the dried EAF was then suspended in 200 mL of absolute methanol and allowed to settle. The dissolved part was separated from the residue by filtration through a Whatman No.4 filter paper and then concentrated in vacuo, weighed, and called methanol fraction (MEF). The total flavonoid content of the different fractions was determined qualitatively on a TLC plate and quantitatively by a spectrophotometer method. The total flavonoid content of the fractional extracts was determined according to the method of Chang et al. [10] using quercetin as standard. Animals Wistar Albino rats of both sexes weighing 30–50 g bred in the animal house at the Zoological garden, University of Nigeria, Nsukka, were used in the present study. The animals were housed under standard laboratory conditions of temperature and humidity, maintained on 12 h light and dark cycle, with free access to food and water. Each group was consisted of minimum of six rats. The procedures in this study were performed in accordance with the National Institute of Health Care Guide for the Care and Use of Laboratory Animals and approved by the Ethics committee of the National Institute of Council (US). All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiment.
Experimental design Forced swim test and measurement of immobility period This test was consisted of two parts, an initial training period of 15 min and an actual test for 5 min at 24 h later. The rats were individually forced to swim inside a vertical borosilicate glass jar (25 cm × 12 cm × 25 cm) containing water to a height of 15 cm at 25 ± 2 °C for a 5-min session every day for 7 days. When they were placed in the glass jar for the first time the rats were initially highly active, vigorously swimming in circles, and trying to climb the wall or diving to the bottom. After 2–3 min, their activity began to subside and was interspersed with phases of immobility or floating of increasing length. After 5–6 min, immobility reached a plateau where the rat remained immobile for approximately 80% of the time. After 15 min in the water, the rats were removed, wiped with dry cloth and allowed to dry before being returned to their home cages. The glass jar was emptied and washed thoroughly after testing for each rat. The rats were again placed in the jar 24 h later after initial administration of the drug and extracts and their activity was recorded within 5 min. The duration of immobility was measured using a stop watch. An animal was judged to be immobile whenever it remained floating passively in the water in a slightly hunched but upright position, its nose just above the surface, with no additional activity other than that necessary to keep its head above water [11]. There were four groups in this present study: Group I: Normal control (Unstressed) treated by gavage (p.o.) with vehicle (2 % W/V of DMSO): Group II: Experimental control (Stressed) treated with vehicle as Group I; Groups III and IV: Experimental groups treated with flavonoid-rich fraction (MTF) of Monodora tenuifolia seed extract (100 and 200 mg·kg−1 p.o., respectively) at 60 min before exposure to stress. Group V: Positive control group treated with Fluoxetine hydrochloride (FLU) (10 mg·kg−1, p.o.) at 30 min before exposure to stress. Behavioural assessment Various behavioural parameters were assessed in the rats on Day 8 after forced swim stress test. All the animals were tested for behavioural alterations such as locomotion, anxiety, memory, muscle contraction, and pain. The locomotor activity was recorded using a digital actophotometer (INCO, Ambala, India) for a period of 5 min [12]. The mirror chamber test was used as a measure of anxiety [11]. The elevated plus maze test developed by Kulkarni [13] was used as a test for noting cognitive behaviour. The evaluation of muscle coordination (muscle strength and grip) was carried out using the horizontal rotating rod (rota rod) test, which determines an animal’s ability to support its own body weight by holding onto the rotating rod [14]. The tail immersion assay was used to assess hyperalgesia in rats after PTZ (pentylene tetrazole) kindling by administering with PTZ (80 mg·kg−1, i.p.).[15]
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Biochemical tests for oxidative damage in stressed rats Dissection and homogenisation of brain tissues On Day 8, after behavioural analyses, the rats were immediately sacrificed by decapitation. The whole brains were removed and 10 % (W/V) tissue homogenate was prepared in 0.1 mol·L−1 phosphate buffer (pH 7.4). The homogenates were centrifuged for 20 min at 10 000 g and the supernatants were used for analyses of nitrite, protein, lipid peroxidation and reduced glutathione levels. The post nuclear fractions for catalase assay were obtained by centrifugation of the homogenates at 1 000 r·min−1 for 20 min at 4 °C and that for the superoxide dismutase assay were obtained at 12 000 g for 60 min at 4 °C. Biochemical Assays The content of malondialdehyde, a biomarker of lipid peroxidation, was assayed in form of thiobarbituric acid-reactive substances by the method of Wills [16]. Reduced glutathione in brain was analyzed according to the method described by Ellman [17]. 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 (1 : 1 solution of 1 % Sulfanilamide in 5 % phosphoric acid and 0.1% napthylamine diamine dihydrochloric acid in water) as reported by Green et al. [18]. The protein content was measured according to the method of Lowry et al. [19] using bovine serum albumin as standard. The catalase activity was assessed by the method of Luck [20], wherein the breakdown of H2O2 was measured at 240 nm. The assay of Superoxide dismutase was based on the inhibition of the formation of NADH-phenazine methosulphate-nitroblue tetrazolum formazon [21]; the colour formed at the end of the reaction was extracted into butanol and measured at 560 nm. Statistical analysis The data are presented as mean ± SD. The values of various treated groups were compared with control by independent sample t-test, two-way ANOVA and Spearman correlation; P < 0.05 was considered statistically significant.
Results Body weights At the beginning of the experiment, there were no significant differences in body weight among the five groups (P > 0.05), with the values (mean ± SD) being 40.17 ± 1.9, 41.88 ± 1.6, 43.77 ± 0.76, 45.19 ± 0.21, and 43.67 ± 0.3 for groups 1–5 respectively. The body weights at the end of the treatments were also not significantly different among the groups (P > 0.05); the values were 44.14 ± 0.9, 45.69 ± 1.2, 49.09 ± 1.1, 48.07 ± 0.8, and 48.88 ± 1.4 for groups 1–5, respectively. Therefore, the results suggested an absence of systemic toxicity in the stressed rats with or without treatment with the flavonoid-rich fraction or fluoxetine. Effect of the flavonoid-rich fraction on Immobility time After treatment with the flavonoid-rich fraction for seven consecutive days, there was a significant reduction
in immobility time in the forced swim test, in a dose-dependent manner, compared with the experimental control (stressed) rats (P < 0.05, Fig. 1). The reduction in immobility after the treatment with the flavonoid-rich fraction demonstrated its antidepressant-like potentials, since decreased swimming performance would increase the immobility time in the forced swim test. Fluoxetine was used as the standard drug for antidepressant activity on the forced swim test. As presented in Fig. 1, fluoxetine caused a significant reduction in immobility time.
Fig. 1 Effect of the flavonoid-rich fraction on the immobility time Flavonoid-rich in the stressed rats. Fluoxetine was used as a positive control
Effects of the flavonoid-rich fraction on muscle coordination, locomotor activity, hyperalgesia and anxiety The rats subjected to seven days of forced swim stress exhibited significant decreases in muscle coordination (as indicated by decreased fall off time; P < 0.05, Fig. 2), locomotor activity (as indicated by decreased ambulatory movements; P < 0.05, Fig. 3) and hyperalgesia (as indicated by decreased tail flicking time; P < 0.05, Fig. 4). There were also significant changes in anxiety-like behaviours (increased latency to enter in mirror chamber, decreased number of entries and time spent in the mirror chamber), compared to the unstressedrats (all P < 0.05; Fig. 5). Administration of the flavonoid-rich fraction and fluoxetine significantly improved the muscle coordination (as indicated by increased fall off time, Fig. 2), locomotor activity (as indicated by increased ambulatory movement, Fig. 3) and tail flick time (Fig. 4), in a dose-dependent manner. The treatment alsoexerted anti-anxiety like effect (decreased time latency to enter in mirror chamber, increased number of entries and duration in mirror chamber) in stressed rat (Fig. 5). The standard drug, fluoxetine, was more effective than the flavonoid-rich fraction (P < 0.05, Figs. 2–5).
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Fig. 2 Effect of the flavonoid-rich fraction on Muscle coordination (fall off time) Fig. 5 Effect of the flavonoid-rich fraction on Anxiety
Fig. 3 Effect of the flavonoid-rich fraction on locomotor activity (No. of counts/min)
Fig. 4 Effect of the flavonoid-rich fraction on hyperalgesia (tail flicking time)
Effect of the flavonoid-rich fraction on memory The elevated plus maze data (Fig. 6) revealed that forced swim test for 7 consecutive days induced a significant reduction in open arm entries and time spent in open arm (P < 0.05), which was effectively reversed by the treatment with the flavonoid-rich fraction (P < 0.05) and by the standard drug, fluoxetine (P < 0.05).
Fig. 6
Effect of the flavonoid-rich fraction on Memory
Effects of the flavonoid-rich fraction on forced swim stressinduced biochemical alterations in brain The forced swim stress significantly increased malondialdehyde and nitrite levels and decreased reduced glutathione level, superoxide dismutase activity, catalase activity and protein levels, compared to the normal control group of (unstressed) rats (P < 0.05, Fig. 7) Administration of the flavonoid-rich fraction significantly reduced the oxidative damage (decreased malondialdehyde and nitrite levels and increased reduced glutathione level, superoxide dismutase activity, catalase activity and protein) as compared to the experimental control group of (stressed) rats (P < 0.05) (Fig. 7). Fluoxetine which served as the standard drug reduced oxidative damage in stressed rats much more effectively than the flavonoid-rich fraction (Fig. 7).
Discussion The total flavonoid contents (quercetin equivalent) of chloroform fraction, ethyl acetate fraction, and methanol fraction were found to be 12.1 ± 0.01, 12.5 ± 0.04, and 9.2 ± 0.01 mg·g−1, respectively. The ethyl acetate fractionhad the highest content of total flavonoid and was subsequently
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Fig. 7 Effect of the flavonoid-rich fraction on the malondialdehyde level and lipid peroxidation status (A), catalase activity (B), reduced glutathione activity (C), superoxide dismutase activity (D), protein level (E), nitrite level (F)
used as the flavonoid-rich fraction in animal studies. Alcohol extract of Monodora tenuifolia seed has been reported to possess potent antioxidant activity [12]. We hypothesized it could be effective in the management of diseases caused by fatigue and oxidative stress. To test the hypothesis, we evaluated the effects of the flavonoid-rich fraction on behavioural alterations and oxidative stress parameters in stressed rats. The main findings of the present study can be summarized as follows: (a) the forced swim stress procedure caused depressive-like behaviours in vehicle treated rats, as observed in various assays such as the locomotor activity test, mirror chamber test, elevated plus maze test, and tail immersion test; (b) significant reduction in depressive-like behaviours was evident in the stressed rats treated with flavonoid-rich fraction and fluoxetine (positive control); (c) forced swim test procedures induced oxidative damage by increasing lipid peroxidation and decreasing antioxidant defences in the vehicle treated rats; and (d) the flavon-
oid-rich fraction and fluoxetine treatments exerted protective effects against forced swim stress induced oxidative stress.in the rat brain. Stress is envisaged as the balance between environmental, physical and/or psychological stimuli capable of altering physiological homeostasis and the body’s ability to cope up with such stressful conditions makes a crucial determinant of health and disease [22]. Any kind of stress influences brain functions and may lead to several neurodegenerative diseases [23]. Depression is a complex disorder and the mechanism underlying its pathogenesis is unknown. Clinical and preclinical evidences indicate that stressful life events and chronic stresses are risk factors for developing depression [24]. The forced swim test is the most commonly employed behavioural model of misery. Although this behavioural model does not mimic the human state of major depression, it is used to screen antidepressants molecules. It induces unavoidable misery that is similar to human depression [25].
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Animal models are widely used for preclinical screening for antidepressants [26-27]. The animals treated with antidepressants struggle more, even in desperate situation, and spend less time with immobility [28]. A significant number of studies have reported that rats exposed to stress exhibit depressive-like behaviours, evidenced by increased immobility period in behavioural despair tests, particularly in the forced swim test [29]. Therefore, the present data (Fig. 1) were in agreement with previous findings that showed consistent depressive-like behaviour induced by repeated and unpredictable stress in rats. The treatment with the flavonoid-rich fraction and fluoxetine significantly increased the time spent on the revolving rod by rats (Fig. 2). It also reduced hyperalgesia, thus showing antihyperalgesic effect. Anxiety is a psychological, physiological and behavioural state induced in animals and humans by stress-like conditions. It is characterised by fear and annoyance and is neurochemically linked to the serotonin level [30]. Anxiety was reduced in stressed rats treated with the flavonoid-rich fraction (Fig. 5). The elevated plus maze test is used to evaluate psychomotor performance and emotional aspects of rodents [31] . The model is based on natural dislike of rodents for open spaces (afraid if possibility of falling off). Rodents tend to avoid the open areas, especially when they are brightly lit, favouring darker and more enclosed spaces. Out elevated plus maze data (Fig. 6) showed that the forced swim test for seven consecutive days induced a significant reduction in open arm entries and the time spent in open arm (P < 0.05). Treatment with the flavonoid-rich fraction for seven days reversed the forced swim stress-induced changes in both open arm entries and time spent in open arms significantly (P < 0.05). Standard drug, fluoxetine also increased the open arm entries and time spent in open arm (P < 0.05). This effect is observed with drugs that promote glucocorticoid production and release in the adrenal cortex [32]. Decreased muscle relaxant activity, increased locomotor activity and tail flick time in rats suggested that the flavonoid-rich fraction could have antidepressant-like effects in the stressed rats. It was reported that certain flavonoids could interact with the GABA (γ-aminobutyric acid), benzodiazepine receptor complex in brain [30]. GABA is the most important inhibitory neurotransmitter in the human central nervous system. GABA is involved in epilepsy, sedation and anxiolysis, and works through binding to GABAA (γ-aminobutyric acid type A) receptors. GABAA receptors are heteromeric GABA-gated chloride channels. The transmembrane ion channel is opened by a stimulus generated by GABA, which allows influx of chloride ions. This results in a decrease in the depolarizing effects of an excitatory input, thereby depressing excitability [33]. Many flavonoids are found to be ligands for GABAA receptors in the central nervous system, which leads to the hypothesis that they act as
benzodiazepine-like molecules. This is supported by their behavioural effects in animal models of anxiety, sedation and convulsion [34]. Oxidative stress has been associated in the pathophysiology of many neurological disorders. It is well known that oxidative stress plays a role in the origin and effects of anxiety and stress [35] . The associations between stresses and diseases in which reactive oxygen species are involved have been established [36]. Stress also causes the formation of reactive oxygen species and oxidants and induces oxidative damage to lipids, resulting in alterations in membrane functions, protein damage, and reduction in intracellular antioxidant defence in different areas of the brain. The reactive oxygen species like hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide, produced during normal cellular metabolic functions, produce oxidative damage in brain [37]. The brain is more vulnerable to oxidative stress because of its elevated consumption of oxygen and the consequent generation of large amounts of reactive oxygen species. Since oxygen radicals form abundantly during stress, changes in antioxidant system are expected [38]. In the present study, we investigated the influence of forced swim stress on oxidative stress-related parameters in rat brain such as markers of oxidative damage to lipids and antioxidant capacity. A previous study has shown that lipid peroxidation is increased in rat brain after 21 days of exposure to different stressors [29] . A potential relationship between increased lipid peroxidation in stress-based depression models has also been reported [39] . The molecular mechanism mediating stress-induced lipid peroxidation and the relationship between oxidative stress and depressive-like behaviour are not completely understood. Our results showed that flavonoid-rich fraction prevented forced swim stress-induced lipid peroxidation, suggesting a potential relationship between oxidative stress and depressive-like behaviour. Conversely, the treatment with fluoxetine restored the lipid peroxidation in the brain of stressed rat. It cannot be concluded whether the antioxidant potentials of the flavonoid-rich fraction which seemed to be responsible for reversing forced swim stress induced brain lipid peroxidation, are enough to elicit the observed antidepressant-like effects observed in our study. The primary antioxidant defence system, which involves synchronized effects of superoxide dismutase, catalase and glutathione, has consistently been studied in depression [39]. The present study found a decrease in catalase activity and reduced glutathione level in stressed rats, indicating an alteration antioxidant brain defence in relation to the forced swim stress induced depressive-like behaviour. A decreased activity of catalase is associated with large amount of H2O2 available to react with transition metals and to generate the radical hydroxyl (the most
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harmful radical), resulting in increased lipid peroxidation and, as consequence, neuronal damage [39]. In addition, since glutathione represents a main cellular non-protein antioxidant and redox regulator in protecting oxygen species [39], the observed reduction in glutathione levels further reinforces the belief the forces swim stress model induces increased oxidative damage in rat brain. Administration of the flavonoid-rich fraction and fluoxetine, which prevented forced swim stress induced depressant-like behaviour and lipid peroxidation, also prevented forced swim stress-induced decrease in catalase activity. The results were relevant because catalase is an important protein that might mediate the depressant-like effects and lipid peroxidation induced in the forced swim stress model, as well as the beneficial effects and lipid peroxidation induced after the treatment with flavonoid-rich fraction and fluoxetine. The results were particularly important, when taking into account the recent finding pointing to catalase as an important enzyme whose levels are changed in the course of depression in humans [40]. Our findings on oxidative stress-related parameters were also in agreement with basic and clinical study results that depression is accompanied by significantly lower antioxidant defence against lipid peroxidation [39]. In vitro and in vivo studies indicate that flavonoids exert antioxidant effects. Human studies with large doses of flavonoid aglycones have produced disappointing results, but emerging evidence suggests that chronic ingestion of flavonoid-rich fruit or vegetables extract attenuates oxidative stress [41]. In the present study, the increased levels of lipid peroxidation and nitrite in forced swim stressed groups indicated that the stress caused significant oxidative damage and depleted superoxide dismutase, catalase, reduced glutathione and protein (Fig. 7). Increased oxidative damage and weak antioxidant defense events have been implicated in major depression [42] and enzymic superoxide dismutase, catalase, and non-enzymic reduced glutathione play important roles during the process by scavenging reactive oxygen species or preventing their formation [43].
The active principle(s) responsible for the antidepressant-like effects of the flavonoid-rich fraction is/are not known, but much of the fraction’s action could be related to the presence of certain flavonoids. The present study provided evidence supporting that the flavonoid-rich fraction of Monodora tenuifolia seed extract shares some pharmacological effects with established antidepressant, at least at nonclinical level. Further studies are needed to unravel the exact mechanism of action of the antidepressant effect of the flavonoid rich fraction of Monodora tenuifolia seed extract in rodents and to elucidate the structure of the bioactive compound(s) present in the seed extract.
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Cite this article as: EKEANYANWU Raphael Chukwuma, NJOKU Obioma Uzoma. Flavonoid-rich fraction of the Monodora tenuifolia seed extract attenuates behavioural alterations and oxidative damage in forced-swim stressed rats [J]. Chinese Journal of Natural Medicines, 2015, 13(3): 183-191
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Chinese Journal of Natural Medicines
Flavonoid-rich fraction of the Monodora tenuifolia seed extract attenuates behavioural alterations and oxidative damage in forced-swim stressed rats EKEANYANWU Raphael Chukwuma1*, NJOKU Obioma Uzoma2 1
Department of Biochemistry, Faculty of Medicine, Imo State University Owerri, Imo State, Nigeria;
2
Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria Nsukka, Enugu State, Nigeria
Available online 20 Mar. 2015
[ABSTRACT] The antidepressant effects of the flavonoid-rich fraction of Monodora tenuifolia seed extract were examined by assessing the extent of attenuation of behavioural alterations and oxidative damage in the rats that were stressed by forced swim test. Compared with the model control group, the altered behavioural parameters were attenuated significantly (P < 0.05) in the group treated with the flavonoid-rich fraction (100 and 200 mg·kg−1), comparable to the group treated with the standard drug, fluoxetine (10 mg·kg−1). The flavonoid-rich fraction and fluoxetine improved significantly (P < 0.05) the activities of the antioxidant enzymes such as superoxide dismutase and catalase as well as other biochemical parameters such as reduced glutathione, protein, and nitrite in the brain of the stressed rats. These results suggested that the flavonoid-rich fraction of Monodora tenuifolia seed extract exerted the antidepressant-like effects which could be useful in the management of stress induced disease. [KEY WORDS] Monodora tenuifolia; Antidepressant; Forced swim test; Depression; Oxidative damage
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2015)03-0183-09
Introduction Recent findings have shown that the Monodora tenuifolia seed extract exerts several biological effects such as antimicrobial [1], antidiarrhoeal [2] and antioxidant activities [3]. In traditional medicine practice, the plant is widely used to relieve toothache, dysentery, diarrhoea, dermatitis, and head ache, and used as vermifuge [2]. The reported antioxidant potential of the plant [3] makes it important in the management of stress induced conditions such as depression. Depression is a common disorder associated with high rates of chronicity, relapse, and recurrence, resulting in psy-
[Received on] 01-Mar.-2014 [Research funding] This work was supported by the Tertiary Education Trust Fund, Nigeria with grant number 2012. [*Corresponding author] E-mail: [email protected], Tel: +2348032744572 (EKEANYANWU Raphael Chukwuma) All the authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
chosocial and physical impairments [4]. The current therapy for depression is only effective in a certain part of the patient population and associated with several side effects [5-7]. Therefore, there is an urgent need for the discovery of alternative therapy for the treatment of depression. Herbal therapies may be an effective alternative in the treatment of depression; and, indeed, the search for biologically active compounds from medicinal plants for psychiatric illnesses, including depression, has progressed significantly in the past decade [4]. Although Monodora tenuifolia has antidepressant potential, there is no sufficient evidence for its effects in animal models of depression. Guarrera et al.[8] have found that a flavonoid-rich diet is beneficial, through modulating the expression of certain disease-associated genes.. Thus, this study aimed at examining the antidepressant-like action of the flavonoid-rich fraction of the aqueous-alcoholic extract from the seed of Monodora tenuifolia using different models that are predictive of antidepressant activity [7] and investigating the level of oxidative damage in the brain of the stressed rats after treatment with the fraction and a known antidepressant drug.
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Materials and Methods Collection and preparation of plant materials Fresh seeds of Monodora tenuifolia were collected in University of Nigeria Nsukka at Enugu State and authenticated by a taxonomist, Mr A. Ozioko of the Department of Botany through comparison with a voucher specimen present in the herbarium. The fresh seeds were shade dried for 48 h, reduced in size to a coarse texture using a manual blender (Corona, Landers Colombia), and packed in airtight containers. Extraction and determination of total flavonoid contents of the extract fractions The aqueous-alcoholic extract and fractions of the ground Monodora tenuifolia seeds were obtained by solvent-solvent extraction as previously reported by Ekeanyanwu and Njoku [9]. Briefly, 5.5 kg of the ground Monodora tenuifolia seeds was macerated twice with 10 L of 70 % ethanol for 72 h at room temperature. The combined macerate was passed through Whatman No.4 filter paper and mixed thoroughly with 1.8 L of chloroform to partition the aqueous-alcoholic extract. Two distinct layers were obtained: the upper aqueous layer and the lower chloroform layer. The two layers were drawn out separately and the chloroform fraction (CLF) was dried in vacuo, and weighed. The main (aqueous portion) extract (2.75 L) was extracted three times with an equal volume of ethyl acetate for 48 h at room temperature. The ethyl acetate fractions (EAF) were combined, dried in vacuo, and weighed. The remaining aqueous extracts were stored away. 11.6 g of the dried EAF was then suspended in 200 mL of absolute methanol and allowed to settle. The dissolved part was separated from the residue by filtration through a Whatman No.4 filter paper and then concentrated in vacuo, weighed, and called methanol fraction (MEF). The total flavonoid content of the different fractions was determined qualitatively on a TLC plate and quantitatively by a spectrophotometer method. The total flavonoid content of the fractional extracts was determined according to the method of Chang et al. [10] using quercetin as standard. Animals Wistar Albino rats of both sexes weighing 30–50 g bred in the animal house at the Zoological garden, University of Nigeria, Nsukka, were used in the present study. The animals were housed under standard laboratory conditions of temperature and humidity, maintained on 12 h light and dark cycle, with free access to food and water. Each group was consisted of minimum of six rats. The procedures in this study were performed in accordance with the National Institute of Health Care Guide for the Care and Use of Laboratory Animals and approved by the Ethics committee of the National Institute of Council (US). All efforts were made to minimize animals suffering and to reduce the number of animals used in the experiment.
Experimental design Forced swim test and measurement of immobility period This test was consisted of two parts, an initial training period of 15 min and an actual test for 5 min at 24 h later. The rats were individually forced to swim inside a vertical borosilicate glass jar (25 cm × 12 cm × 25 cm) containing water to a height of 15 cm at 25 ± 2 °C for a 5-min session every day for 7 days. When they were placed in the glass jar for the first time the rats were initially highly active, vigorously swimming in circles, and trying to climb the wall or diving to the bottom. After 2–3 min, their activity began to subside and was interspersed with phases of immobility or floating of increasing length. After 5–6 min, immobility reached a plateau where the rat remained immobile for approximately 80% of the time. After 15 min in the water, the rats were removed, wiped with dry cloth and allowed to dry before being returned to their home cages. The glass jar was emptied and washed thoroughly after testing for each rat. The rats were again placed in the jar 24 h later after initial administration of the drug and extracts and their activity was recorded within 5 min. The duration of immobility was measured using a stop watch. An animal was judged to be immobile whenever it remained floating passively in the water in a slightly hunched but upright position, its nose just above the surface, with no additional activity other than that necessary to keep its head above water [11]. There were four groups in this present study: Group I: Normal control (Unstressed) treated by gavage (p.o.) with vehicle (2 % W/V of DMSO): Group II: Experimental control (Stressed) treated with vehicle as Group I; Groups III and IV: Experimental groups treated with flavonoid-rich fraction (MTF) of Monodora tenuifolia seed extract (100 and 200 mg·kg−1 p.o., respectively) at 60 min before exposure to stress. Group V: Positive control group treated with Fluoxetine hydrochloride (FLU) (10 mg·kg−1, p.o.) at 30 min before exposure to stress. Behavioural assessment Various behavioural parameters were assessed in the rats on Day 8 after forced swim stress test. All the animals were tested for behavioural alterations such as locomotion, anxiety, memory, muscle contraction, and pain. The locomotor activity was recorded using a digital actophotometer (INCO, Ambala, India) for a period of 5 min [12]. The mirror chamber test was used as a measure of anxiety [11]. The elevated plus maze test developed by Kulkarni [13] was used as a test for noting cognitive behaviour. The evaluation of muscle coordination (muscle strength and grip) was carried out using the horizontal rotating rod (rota rod) test, which determines an animal’s ability to support its own body weight by holding onto the rotating rod [14]. The tail immersion assay was used to assess hyperalgesia in rats after PTZ (pentylene tetrazole) kindling by administering with PTZ (80 mg·kg−1, i.p.).[15]
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Biochemical tests for oxidative damage in stressed rats Dissection and homogenisation of brain tissues On Day 8, after behavioural analyses, the rats were immediately sacrificed by decapitation. The whole brains were removed and 10 % (W/V) tissue homogenate was prepared in 0.1 mol·L−1 phosphate buffer (pH 7.4). The homogenates were centrifuged for 20 min at 10 000 g and the supernatants were used for analyses of nitrite, protein, lipid peroxidation and reduced glutathione levels. The post nuclear fractions for catalase assay were obtained by centrifugation of the homogenates at 1 000 r·min−1 for 20 min at 4 °C and that for the superoxide dismutase assay were obtained at 12 000 g for 60 min at 4 °C. Biochemical Assays The content of malondialdehyde, a biomarker of lipid peroxidation, was assayed in form of thiobarbituric acid-reactive substances by the method of Wills [16]. Reduced glutathione in brain was analyzed according to the method described by Ellman [17]. 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 (1 : 1 solution of 1 % Sulfanilamide in 5 % phosphoric acid and 0.1% napthylamine diamine dihydrochloric acid in water) as reported by Green et al. [18]. The protein content was measured according to the method of Lowry et al. [19] using bovine serum albumin as standard. The catalase activity was assessed by the method of Luck [20], wherein the breakdown of H2O2 was measured at 240 nm. The assay of Superoxide dismutase was based on the inhibition of the formation of NADH-phenazine methosulphate-nitroblue tetrazolum formazon [21]; the colour formed at the end of the reaction was extracted into butanol and measured at 560 nm. Statistical analysis The data are presented as mean ± SD. The values of various treated groups were compared with control by independent sample t-test, two-way ANOVA and Spearman correlation; P < 0.05 was considered statistically significant.
Results Body weights At the beginning of the experiment, there were no significant differences in body weight among the five groups (P > 0.05), with the values (mean ± SD) being 40.17 ± 1.9, 41.88 ± 1.6, 43.77 ± 0.76, 45.19 ± 0.21, and 43.67 ± 0.3 for groups 1–5 respectively. The body weights at the end of the treatments were also not significantly different among the groups (P > 0.05); the values were 44.14 ± 0.9, 45.69 ± 1.2, 49.09 ± 1.1, 48.07 ± 0.8, and 48.88 ± 1.4 for groups 1–5, respectively. Therefore, the results suggested an absence of systemic toxicity in the stressed rats with or without treatment with the flavonoid-rich fraction or fluoxetine. Effect of the flavonoid-rich fraction on Immobility time After treatment with the flavonoid-rich fraction for seven consecutive days, there was a significant reduction
in immobility time in the forced swim test, in a dose-dependent manner, compared with the experimental control (stressed) rats (P < 0.05, Fig. 1). The reduction in immobility after the treatment with the flavonoid-rich fraction demonstrated its antidepressant-like potentials, since decreased swimming performance would increase the immobility time in the forced swim test. Fluoxetine was used as the standard drug for antidepressant activity on the forced swim test. As presented in Fig. 1, fluoxetine caused a significant reduction in immobility time.
Fig. 1 Effect of the flavonoid-rich fraction on the immobility time Flavonoid-rich in the stressed rats. Fluoxetine was used as a positive control
Effects of the flavonoid-rich fraction on muscle coordination, locomotor activity, hyperalgesia and anxiety The rats subjected to seven days of forced swim stress exhibited significant decreases in muscle coordination (as indicated by decreased fall off time; P < 0.05, Fig. 2), locomotor activity (as indicated by decreased ambulatory movements; P < 0.05, Fig. 3) and hyperalgesia (as indicated by decreased tail flicking time; P < 0.05, Fig. 4). There were also significant changes in anxiety-like behaviours (increased latency to enter in mirror chamber, decreased number of entries and time spent in the mirror chamber), compared to the unstressedrats (all P < 0.05; Fig. 5). Administration of the flavonoid-rich fraction and fluoxetine significantly improved the muscle coordination (as indicated by increased fall off time, Fig. 2), locomotor activity (as indicated by increased ambulatory movement, Fig. 3) and tail flick time (Fig. 4), in a dose-dependent manner. The treatment alsoexerted anti-anxiety like effect (decreased time latency to enter in mirror chamber, increased number of entries and duration in mirror chamber) in stressed rat (Fig. 5). The standard drug, fluoxetine, was more effective than the flavonoid-rich fraction (P < 0.05, Figs. 2–5).
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Fig. 2 Effect of the flavonoid-rich fraction on Muscle coordination (fall off time) Fig. 5 Effect of the flavonoid-rich fraction on Anxiety
Fig. 3 Effect of the flavonoid-rich fraction on locomotor activity (No. of counts/min)
Fig. 4 Effect of the flavonoid-rich fraction on hyperalgesia (tail flicking time)
Effect of the flavonoid-rich fraction on memory The elevated plus maze data (Fig. 6) revealed that forced swim test for 7 consecutive days induced a significant reduction in open arm entries and time spent in open arm (P < 0.05), which was effectively reversed by the treatment with the flavonoid-rich fraction (P < 0.05) and by the standard drug, fluoxetine (P < 0.05).
Fig. 6
Effect of the flavonoid-rich fraction on Memory
Effects of the flavonoid-rich fraction on forced swim stressinduced biochemical alterations in brain The forced swim stress significantly increased malondialdehyde and nitrite levels and decreased reduced glutathione level, superoxide dismutase activity, catalase activity and protein levels, compared to the normal control group of (unstressed) rats (P < 0.05, Fig. 7) Administration of the flavonoid-rich fraction significantly reduced the oxidative damage (decreased malondialdehyde and nitrite levels and increased reduced glutathione level, superoxide dismutase activity, catalase activity and protein) as compared to the experimental control group of (stressed) rats (P < 0.05) (Fig. 7). Fluoxetine which served as the standard drug reduced oxidative damage in stressed rats much more effectively than the flavonoid-rich fraction (Fig. 7).
Discussion The total flavonoid contents (quercetin equivalent) of chloroform fraction, ethyl acetate fraction, and methanol fraction were found to be 12.1 ± 0.01, 12.5 ± 0.04, and 9.2 ± 0.01 mg·g−1, respectively. The ethyl acetate fractionhad the highest content of total flavonoid and was subsequently
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Fig. 7 Effect of the flavonoid-rich fraction on the malondialdehyde level and lipid peroxidation status (A), catalase activity (B), reduced glutathione activity (C), superoxide dismutase activity (D), protein level (E), nitrite level (F)
used as the flavonoid-rich fraction in animal studies. Alcohol extract of Monodora tenuifolia seed has been reported to possess potent antioxidant activity [12]. We hypothesized it could be effective in the management of diseases caused by fatigue and oxidative stress. To test the hypothesis, we evaluated the effects of the flavonoid-rich fraction on behavioural alterations and oxidative stress parameters in stressed rats. The main findings of the present study can be summarized as follows: (a) the forced swim stress procedure caused depressive-like behaviours in vehicle treated rats, as observed in various assays such as the locomotor activity test, mirror chamber test, elevated plus maze test, and tail immersion test; (b) significant reduction in depressive-like behaviours was evident in the stressed rats treated with flavonoid-rich fraction and fluoxetine (positive control); (c) forced swim test procedures induced oxidative damage by increasing lipid peroxidation and decreasing antioxidant defences in the vehicle treated rats; and (d) the flavon-
oid-rich fraction and fluoxetine treatments exerted protective effects against forced swim stress induced oxidative stress.in the rat brain. Stress is envisaged as the balance between environmental, physical and/or psychological stimuli capable of altering physiological homeostasis and the body’s ability to cope up with such stressful conditions makes a crucial determinant of health and disease [22]. Any kind of stress influences brain functions and may lead to several neurodegenerative diseases [23]. Depression is a complex disorder and the mechanism underlying its pathogenesis is unknown. Clinical and preclinical evidences indicate that stressful life events and chronic stresses are risk factors for developing depression [24]. The forced swim test is the most commonly employed behavioural model of misery. Although this behavioural model does not mimic the human state of major depression, it is used to screen antidepressants molecules. It induces unavoidable misery that is similar to human depression [25].
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Animal models are widely used for preclinical screening for antidepressants [26-27]. The animals treated with antidepressants struggle more, even in desperate situation, and spend less time with immobility [28]. A significant number of studies have reported that rats exposed to stress exhibit depressive-like behaviours, evidenced by increased immobility period in behavioural despair tests, particularly in the forced swim test [29]. Therefore, the present data (Fig. 1) were in agreement with previous findings that showed consistent depressive-like behaviour induced by repeated and unpredictable stress in rats. The treatment with the flavonoid-rich fraction and fluoxetine significantly increased the time spent on the revolving rod by rats (Fig. 2). It also reduced hyperalgesia, thus showing antihyperalgesic effect. Anxiety is a psychological, physiological and behavioural state induced in animals and humans by stress-like conditions. It is characterised by fear and annoyance and is neurochemically linked to the serotonin level [30]. Anxiety was reduced in stressed rats treated with the flavonoid-rich fraction (Fig. 5). The elevated plus maze test is used to evaluate psychomotor performance and emotional aspects of rodents [31] . The model is based on natural dislike of rodents for open spaces (afraid if possibility of falling off). Rodents tend to avoid the open areas, especially when they are brightly lit, favouring darker and more enclosed spaces. Out elevated plus maze data (Fig. 6) showed that the forced swim test for seven consecutive days induced a significant reduction in open arm entries and the time spent in open arm (P < 0.05). Treatment with the flavonoid-rich fraction for seven days reversed the forced swim stress-induced changes in both open arm entries and time spent in open arms significantly (P < 0.05). Standard drug, fluoxetine also increased the open arm entries and time spent in open arm (P < 0.05). This effect is observed with drugs that promote glucocorticoid production and release in the adrenal cortex [32]. Decreased muscle relaxant activity, increased locomotor activity and tail flick time in rats suggested that the flavonoid-rich fraction could have antidepressant-like effects in the stressed rats. It was reported that certain flavonoids could interact with the GABA (γ-aminobutyric acid), benzodiazepine receptor complex in brain [30]. GABA is the most important inhibitory neurotransmitter in the human central nervous system. GABA is involved in epilepsy, sedation and anxiolysis, and works through binding to GABAA (γ-aminobutyric acid type A) receptors. GABAA receptors are heteromeric GABA-gated chloride channels. The transmembrane ion channel is opened by a stimulus generated by GABA, which allows influx of chloride ions. This results in a decrease in the depolarizing effects of an excitatory input, thereby depressing excitability [33]. Many flavonoids are found to be ligands for GABAA receptors in the central nervous system, which leads to the hypothesis that they act as
benzodiazepine-like molecules. This is supported by their behavioural effects in animal models of anxiety, sedation and convulsion [34]. Oxidative stress has been associated in the pathophysiology of many neurological disorders. It is well known that oxidative stress plays a role in the origin and effects of anxiety and stress [35] . The associations between stresses and diseases in which reactive oxygen species are involved have been established [36]. Stress also causes the formation of reactive oxygen species and oxidants and induces oxidative damage to lipids, resulting in alterations in membrane functions, protein damage, and reduction in intracellular antioxidant defence in different areas of the brain. The reactive oxygen species like hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide, produced during normal cellular metabolic functions, produce oxidative damage in brain [37]. The brain is more vulnerable to oxidative stress because of its elevated consumption of oxygen and the consequent generation of large amounts of reactive oxygen species. Since oxygen radicals form abundantly during stress, changes in antioxidant system are expected [38]. In the present study, we investigated the influence of forced swim stress on oxidative stress-related parameters in rat brain such as markers of oxidative damage to lipids and antioxidant capacity. A previous study has shown that lipid peroxidation is increased in rat brain after 21 days of exposure to different stressors [29] . A potential relationship between increased lipid peroxidation in stress-based depression models has also been reported [39] . The molecular mechanism mediating stress-induced lipid peroxidation and the relationship between oxidative stress and depressive-like behaviour are not completely understood. Our results showed that flavonoid-rich fraction prevented forced swim stress-induced lipid peroxidation, suggesting a potential relationship between oxidative stress and depressive-like behaviour. Conversely, the treatment with fluoxetine restored the lipid peroxidation in the brain of stressed rat. It cannot be concluded whether the antioxidant potentials of the flavonoid-rich fraction which seemed to be responsible for reversing forced swim stress induced brain lipid peroxidation, are enough to elicit the observed antidepressant-like effects observed in our study. The primary antioxidant defence system, which involves synchronized effects of superoxide dismutase, catalase and glutathione, has consistently been studied in depression [39]. The present study found a decrease in catalase activity and reduced glutathione level in stressed rats, indicating an alteration antioxidant brain defence in relation to the forced swim stress induced depressive-like behaviour. A decreased activity of catalase is associated with large amount of H2O2 available to react with transition metals and to generate the radical hydroxyl (the most
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harmful radical), resulting in increased lipid peroxidation and, as consequence, neuronal damage [39]. In addition, since glutathione represents a main cellular non-protein antioxidant and redox regulator in protecting oxygen species [39], the observed reduction in glutathione levels further reinforces the belief the forces swim stress model induces increased oxidative damage in rat brain. Administration of the flavonoid-rich fraction and fluoxetine, which prevented forced swim stress induced depressant-like behaviour and lipid peroxidation, also prevented forced swim stress-induced decrease in catalase activity. The results were relevant because catalase is an important protein that might mediate the depressant-like effects and lipid peroxidation induced in the forced swim stress model, as well as the beneficial effects and lipid peroxidation induced after the treatment with flavonoid-rich fraction and fluoxetine. The results were particularly important, when taking into account the recent finding pointing to catalase as an important enzyme whose levels are changed in the course of depression in humans [40]. Our findings on oxidative stress-related parameters were also in agreement with basic and clinical study results that depression is accompanied by significantly lower antioxidant defence against lipid peroxidation [39]. In vitro and in vivo studies indicate that flavonoids exert antioxidant effects. Human studies with large doses of flavonoid aglycones have produced disappointing results, but emerging evidence suggests that chronic ingestion of flavonoid-rich fruit or vegetables extract attenuates oxidative stress [41]. In the present study, the increased levels of lipid peroxidation and nitrite in forced swim stressed groups indicated that the stress caused significant oxidative damage and depleted superoxide dismutase, catalase, reduced glutathione and protein (Fig. 7). Increased oxidative damage and weak antioxidant defense events have been implicated in major depression [42] and enzymic superoxide dismutase, catalase, and non-enzymic reduced glutathione play important roles during the process by scavenging reactive oxygen species or preventing their formation [43].
The active principle(s) responsible for the antidepressant-like effects of the flavonoid-rich fraction is/are not known, but much of the fraction’s action could be related to the presence of certain flavonoids. The present study provided evidence supporting that the flavonoid-rich fraction of Monodora tenuifolia seed extract shares some pharmacological effects with established antidepressant, at least at nonclinical level. Further studies are needed to unravel the exact mechanism of action of the antidepressant effect of the flavonoid rich fraction of Monodora tenuifolia seed extract in rodents and to elucidate the structure of the bioactive compound(s) present in the seed extract.
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Cite this article as: EKEANYANWU Raphael Chukwuma, NJOKU Obioma Uzoma. Flavonoid-rich fraction of the Monodora tenuifolia seed extract attenuates behavioural alterations and oxidative damage in forced-swim stressed rats [J]. Chinese Journal of Natural Medicines, 2015, 13(3): 183-191
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