J Nat Med DOI 10.1007/s11418-016-0975-3
RAPID COMMUNICATION
Enhanced anti-immobility effects of Sanggenon G isolated from the root bark of Morus alba combined with the a2-antagonist yohimbine in the rat forced swim test Dong Wook Lim1 • Nam-In Baek2 • Yun Tai Kim1,3 • Changho Lee1 In-Ho Kim1 • Daeseok Han1
•
Received: 23 December 2015 / Accepted: 5 February 2016 Ó The Japanese Society of Pharmacognosy and Springer Japan 2016
Abstract In this study, we aimed to determine whether Sanggenon G, an active compound isolated from the root bark of Morus alba, exhibited enhanced anti-immobility activity with the addition of the a2-antagonist yohimbine in rats subjected to forced swim test (FST)-induced depression. Fluoxetine (a selective serotonin reuptake inhibitor) treatment in rats reduced the immobility time, and pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of fluoxetine at 5, 10 and 20 mg/kg. Similarly, Sanggenon G significantly decreased the immobility time, reducing immobility by a maximum of 43.9 % when treated at a dose of 20 mg/kg. Furthermore, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of Sanggenon G at 5 and 10 mg/kg. Our findings suggest that the antidepressant-like effect of Sanggenon G could be facilitated by concomitant use of the a2-antagonist. Further studies are needed to evaluate the potential of Sanggenon G as an alternative therapeutic approach for the treatment of depression. Keywords Sanggenon G Fluoxetine Yohimbine Forced swim test
& Daeseok Han [email protected] 1
Research Group of Innovative Special Food, Korea Food Research Institute, Seongnam 463-746, Korea
2
Graduate School of Biotechnology and Research Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Korea
3
Department of Food Biotechnology, Korea University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, Republic of Korea
Introduction It is well-known that central noradrenergic transmission is regulated by inhibitory a2-adrenoceptors, which are expressed on both somatodendritic areas and axon terminals [1]. Since activation of these receptors induces an inhibition of noradrenaline (NA) release in the brain, it has been proposed that depression is associated with a selective increase in the high-affinity conformation of a2adrenoceptors in the human brain [2]. Moreover, a2-antagonists such as mirtazapine are also able to enhance the increase of NA induced by selective serotonin reuptake inhibitors (SSRIs) or tricyclic antidepressant drugs [3, 4]. Morus alba, one of the best known and most widely distributed trees or shrubs of the family Moraceae, is extensively cultivated in East Asia. The root bark of M. alba (Japanese name ‘Sohakuhi’) has been used in traditional folk medicine for anti-inflammatory, diuretic and antipyretic purposes, and as a sedative [5]. The root bark of M. alba extract has beneficial effects on depression-like behavior in chronic-induced stress in rats [6]. Lee et al. also reported that the root bark of M. alba extract (200 mg/ kg/day for 5 days) had an antidepressant-like effect through decreased hypothalamic–pituitary–adrenocortical (HPA) axis response to the forced swim test (FST), as indicated by an attenuated corticosterone response [7]. Ethyl acetate (EtOAc) soluble fraction of the root bark of M. alba extract (100 mg/kg/day for 7 days) induced a decrease in immobility behavior in the FST [8]. Furthermore, EtOAc soluble fractions (50 and 100 mg/kg/day for 10 days) showed significant anti-stress activity as shown by modifying effects on several chronic restraint stressinduced neurochemical and behavioral parameters [9]. Moreover, the effect of 200 mg/kg M. alba leaf extract in
123
J Nat Med
decreasing the immobility time was comparable to 10 mg/ kg desipramine without an anxiolytic-like effect [10]. Sanggenon G (Fig. 1), an active compound isolated from the root bark of M. alba [11], showed antidepressantlike activity in rats subjected to FST-induced depression through interaction with the serotonergic system for the first time in our previous reports [12]. Considering the inhibitory effects of a2-adrenoceptors on serotonergic function, it was hypothesized that the addition of the a2-antagonist yohimbine to Sanggenon G may results in enhanced antidepressant-like activity in the FST in rats. In the present study, the enhanced effect of Sanggenon G and yohimbine were investigated in response to the rat FST.
Materials and methods Isolation and determination of Sanggenon G Sanggenon G was prepared as described [12]. Briefly, the EtOAc fraction (120 g) from the 80 % methanol (MeOH) extract of the dried root bark M. alba (yield of approximately 17 %) was subjected to SiO2 column chromatography (CC; u 12.5 9 18 cm) and eluted with n-hexane– ethyl acetate (4:1 ? 2:1 ? 1:1, 27 L of each). The eluting solutions were monitored by thin-layer chromatography to produce 41 fractions (MRE-1–MRE-41). Fraction MRE-37 [1.58 g; elution volume/total volume (Ve/Vt), 0.80–0.86] was subjected to ODS CC (u 4 9 10 cm) and eluted with MeOH–H2O (2:1, 4.2 L), yielding 23 fractions (MRE-371–MRE-37-23), including the purified compound Sanggenon G (MRE-37-17; Ve/Vt, 0.28–0.44; 350 mg). The compound was identified as Sanggenon G based on the results of several spectroscopic methods, including NMR, IR, MS, and specific rotation value [(a)20 D -277°, c 0.09, MeOH].
Quantifications of sanggenon G Dried root bark of M. alba powder (100 mg) was extracted with 80 % MeOH (1 mL) in an ultrasonic bath for 1 h at 25 °C. The extract was filtered through a 0.45-lm membrane filter (Woongki Science, Seoul, Korea) and evaporated in a vacuum. A 20-lL aliquot of the extract solution was injected into the HPLC system. Analysis was achieved using a Waters 600 s controller (Milford, MA, USA) with a Waters 2487 UV detector (280 nm). The column was an YMC-Pack ODS-A (Kyoto, Japan, 250 mm 9 4.6 mm, particle size 5 lm). The mobile phase consisted of 0.05 % trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B) eluted at a flow rate of 1.0 mL/min with the following gradient elution—B: 50 % (0.01 min) ? B: 50 % (10 min) ? B: 60 % (25 min) ? B: 100 % (30 min) ? B: 100 % (40 min) ? B: 50 % (45 min). Quantitative analysis was replicated three times. The regression equation and correlation coefficient (r2) were built to be y = 3984.4897x– 3623.4584 and 0.998, respectively. The concentration of Sanggenon G was determined to be 0.446 ± 0.007 mg/g using peak area in the chromatogram and regression equation. The purity of Sanggenon G was determined to be [97 % based on HPLC and 1H-NMR data. Animals Eight-week-old male Sprague–Dawley rats (Samtako Bio Korea, Gyeonggi-do, Korea) weighing 180–210 g were housed with two rats per cage under a controlled temperature (23 ± 1 °C) and a 12-h light/dark cycle (lights on at 07:00 and lights off at 19:00). The rats were allowed at least 1 week for acclimatization before the experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Food Research Institute. Drugs and treatment Yohimbine and fluoxetine (both from Sigma-Aldrich Co., St. Louis, MO, USA) were dissolved in 0.9 % (w/v) saline solution. Sanggenon G was diluted in saline with 1 % Tween 80. Sanggenon G (5, 10, and 20 mg/kg) and fluoxetine (5, 10, and 20 mg/kg) were injected intraperitoneally (i.p., an injection volume of 0.1 mL/100 g body weight) 60 min before the FST. In the combination study, yohimbine (0.5 mg/kg, i.p.) was injected 15 min before fluoxetine or Sanggenon G treatment. FST
Fig. 1 Chemical structure of Sanggenon G isolated from the root bark of M. alba
123
The FST was carried out as previously described [13]. Briefly, in the pretest session, rats were forced to swim for
J Nat Med
15 min in a transparent Plexiglas cylinder (height 50 cm; diameter 20 cm) filled to a depth of 30 cm with water (temperature, 23–25 °C). Twenty-four hours later, the procedure was repeated during a 6-min test session, and the immobility time during the last 4 min was measured by a SMART video tracking system (SMART v3.0; Panlab SL, Barcelona, Spain). Statistical analysis Data analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using Prism 5 (GraphPad Software, Inc., San Diego, CA, USA) for multigroup comparisons. All data are presented as the mean ± standard error (SEM). Differences with p values of \0.05 were considered significant.
Fig. 2 Antidepressant-like effects of treatment with fluoxetine and the synergistic effects of yohimbine on depressive behavior in response to the FST in rats. Fluoxetine (5, 10, and 20 mg/kg) was injected intraperitoneally 60 min before the FST. In the combination study, yohimbine (0.5 mg/kg) was administered 15 min before fluoxetine treatment. Columns show the means ± SEMs (n = 8). *p \ 0.05 and **p \ 0.01 versus the control group. #p \ 0.05 as compared to respective fluoxetine-treated group
Results and discussions In the present study, we examined whether the co-administration of the a2-antagonist yohimbine can enhance the antidepressant-like effects of Sanggenon G treatment in FST-induced depression in rats. The FST is an effective screening tool with good reliability and predictive validity [14]. The state of immobility in the FST is reported to mimic the symptoms of depression in humans and can be reversed by treatment with antidepressant drugs [15]. First, we examined the antidepressant-like effects of the SSRI fluoxetine in the FST. Fluoxetine treatment in rats reduced the immobility time by a maximum of 55.8 % when treated at a dose of 20 mg/kg. Moreover, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of fluoxetine at 5, 10 and 20 mg/kg (Fig. 2). Similarly, Sanggenon G significantly decreased the immobility time by a maximum of 43.9 % when treated at a dose of 20 mg/kg. Furthermore, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of Sanggenon G at 5 and 10 mg/kg (Fig. 3). Previous work by other authors has suggested that the a2-antagonist yohimbine modulates the serotonergic system, and treatment with yohimbine was reported to show beneficial interaction with fluoxetine [16]. Administration of a2-antagonists has been shown to enhance serotonergic neurotransmission via direct inhibition of a2-heteroreceptors located on the serotonergic nerve terminals and indirect stimulation of a1-receptors via inhibition of a2heteroreceptors [17]. Furthermore, Yanpallewar et al. also indicated that combination treatment with an a2-adrenoceptor antagonist and the tricyclic antidepressant, imipramine, elicits neurogenic, neurotrophic and behavioral changes, as compared to imipramine treatment alone [18].
Fig. 3 Antidepressant-like effects of treatment with Sanggenon G and the synergistic effects of yohimbine on depressive behavior in response to the FST in rats. Sanggenon G (5, 10, and 20 mg/kg) was injected intraperitoneally 60 min before the FST. In the combination study, yohimbine (0.5 mg/kg) was administered 15 min before Sanggenon G treatment. Columns show the means ± SEMs (n = 8). **p \ 0.01 versus the control group. #p \ 0.05 as compared to the respective Sanggenon G-treated group
These studies suggest that a2-adrenoceptor antagonism is a putative target for combination antidepressant therapy. In our results, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of Sanggenon G. The results of the present study concluded that the antidepressant-like effect of Sanggenon G could be facilitated by concomitant use of yohimbine. However, further studies are needed to determine the specific mechanisms by which Sanggenon G affects interaction between the serotonin and a2-adrenergic systems. Acknowledgments This study was supported by a grant from the Korea Food Research Institute. Compliance with ethical standards Conflict of interest of interest.
The authors declare that there are no conflicts
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J Nat Med
References 1. Muguruza C, Rodriguez F, Rozas I, Meana JJ, Uriguen L, Callado LF (2013) Antidepressant-like properties of three new alpha2-adrenoceptor antagonists. Neuropharmacology 65:13–19 2. Rodriguez F, Rozas I, Ortega JE, Erdozain AM, Meana JJ, Callado LF (2008) Guanidine and 2-aminoimidazoline aromatic derivatives as alpha2-adrenoceptor antagonists. 2. Exploring alkyl linkers for new antidepressants. J Med Chem 51:3304–3312 3. Rozas I (2009) Improving antidepressant drugs: update on recently patented compounds. Expert Opin Ther Pat 19:827–845 4. Bremner JD (1995) A double-blind comparison of Org 3770, amitriptyline, and placebo in major depression. J Clin Psychiatry 56:519–525 5. Asano N, Yamashita T, Yasuda K, Ikeda K, Kizu H, Kameda Y, Kato A, Nash RJ, Lee HS, Ryu KS (2001) Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.). J Agric Food Chem 49:4208–4213 6. Nade VS, Kawale LA, Naik RA, Yadav AV (2009) Adaptogenic effect of Morus alba on chronic footshock-induced stress in rats. Indian J Pharmacol 41:246–251 7. Lee MS, Park WS, Kim YH, Kwon SH, Jang YJ, Han D, Morita K, Her S (2013) Antidepressant-like effects of Cortex Mori Radicis extract via bidirectional phosphorylation of glucocorticoid receptors in the hippocampus. Behav Brain Res 236:56–61 8. Lim DW, Kim YT, Park JH, Baek NI, Han D (2014) Antidepressant-like effects of the ethyl acetate soluble fraction of the root bark of Morus alba on the immobility behavior of rats in the forced swim test. Molecules 19:7981–7989 9. Nade VS, Yadav AV (2010) Anti-stress effect of ethyl acetate soluble fraction of Morus alba in chronic restraint stress. Pharm Biol 48:1038–1046 10. Sattayasai J, Tiamkao S, Puapairoj P (2008) Biphasic effects of Morus alba leaves green tea extract on mice in chronic forced swimming model. Phytother Res 22:487–492
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11. Jung JW, Ko WM, Park JH, Seo KH, Oh EJ, Lee DY, Lee DS, Kim YC, Lim DW, Han D, Baek NI (2015) Isoprenylated flavonoids from the root bark of Morus alba and their hepatoprotective and neuroprotective activities. Arch Pharm Res 38:2066–2075 12. Lim DW, Jung JW, Park JH, Baek NI, Kim YT, Kim IH, Han D (2015) Antidepressant-like effects of sanggenon G, isolated from the root bark of Morus alba, in rats: involvement of the serotonergic system. Biol Pharm Bull 38:1772–1778 13. Cryan JF, Markou A, Lucki I (2002) Assessing antidepressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 23:238–245 14. Petit-Demouliere B, Chenu F, Bourin M (2005) Forced swimming test in mice: a review of antidepressant activity. Psychopharmacology 177:245–255 15. Renard CE, Dailly E, David DJ, Hascoet M, Bourin M (2003) Monoamine metabolism changes following the mouse forced swimming test but not the tail suspension test. Fundam Clin Pharmacol 17:449–455 16. Sanacora G, Berman RM, Cappiello A, Oren DA, Kugaya A, Liu N, Gueorguieva R, Fasula D, Charney DS (2004) Addition of the alpha2-antagonist yohimbine to fluoxetine: effects on rate of antidepressant response. Neuropsychopharmacology 29:1166–1171 17. Hopwood SE, Stamford JA (2001) Noradrenergic modulation of serotonin release in rat dorsal and median raphe nuclei via alpha(1) and alpha(2A) adrenoceptors. Neuropharmacology 41:433–442 18. Yanpallewar SU, Fernandes K, Marathe SV, Vadodaria KC, Jhaveri D, Rommelfanger K, Ladiwala U, Jha S, Muthig V, Hein L, Bartlett P, Weinshenker D, Vaidya VA (2010) Alpha2adrenoceptor blockade accelerates the neurogenic, neurotrophic, and behavioral effects of chronic antidepressant treatment. J Neurosci 30:1096–1109
RAPID COMMUNICATION
Enhanced anti-immobility effects of Sanggenon G isolated from the root bark of Morus alba combined with the a2-antagonist yohimbine in the rat forced swim test Dong Wook Lim1 • Nam-In Baek2 • Yun Tai Kim1,3 • Changho Lee1 In-Ho Kim1 • Daeseok Han1
•
Received: 23 December 2015 / Accepted: 5 February 2016 Ó The Japanese Society of Pharmacognosy and Springer Japan 2016
Abstract In this study, we aimed to determine whether Sanggenon G, an active compound isolated from the root bark of Morus alba, exhibited enhanced anti-immobility activity with the addition of the a2-antagonist yohimbine in rats subjected to forced swim test (FST)-induced depression. Fluoxetine (a selective serotonin reuptake inhibitor) treatment in rats reduced the immobility time, and pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of fluoxetine at 5, 10 and 20 mg/kg. Similarly, Sanggenon G significantly decreased the immobility time, reducing immobility by a maximum of 43.9 % when treated at a dose of 20 mg/kg. Furthermore, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of Sanggenon G at 5 and 10 mg/kg. Our findings suggest that the antidepressant-like effect of Sanggenon G could be facilitated by concomitant use of the a2-antagonist. Further studies are needed to evaluate the potential of Sanggenon G as an alternative therapeutic approach for the treatment of depression. Keywords Sanggenon G Fluoxetine Yohimbine Forced swim test
& Daeseok Han [email protected] 1
Research Group of Innovative Special Food, Korea Food Research Institute, Seongnam 463-746, Korea
2
Graduate School of Biotechnology and Research Institute of Life Sciences and Resources, Kyung Hee University, Yongin 446-701, Korea
3
Department of Food Biotechnology, Korea University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, Republic of Korea
Introduction It is well-known that central noradrenergic transmission is regulated by inhibitory a2-adrenoceptors, which are expressed on both somatodendritic areas and axon terminals [1]. Since activation of these receptors induces an inhibition of noradrenaline (NA) release in the brain, it has been proposed that depression is associated with a selective increase in the high-affinity conformation of a2adrenoceptors in the human brain [2]. Moreover, a2-antagonists such as mirtazapine are also able to enhance the increase of NA induced by selective serotonin reuptake inhibitors (SSRIs) or tricyclic antidepressant drugs [3, 4]. Morus alba, one of the best known and most widely distributed trees or shrubs of the family Moraceae, is extensively cultivated in East Asia. The root bark of M. alba (Japanese name ‘Sohakuhi’) has been used in traditional folk medicine for anti-inflammatory, diuretic and antipyretic purposes, and as a sedative [5]. The root bark of M. alba extract has beneficial effects on depression-like behavior in chronic-induced stress in rats [6]. Lee et al. also reported that the root bark of M. alba extract (200 mg/ kg/day for 5 days) had an antidepressant-like effect through decreased hypothalamic–pituitary–adrenocortical (HPA) axis response to the forced swim test (FST), as indicated by an attenuated corticosterone response [7]. Ethyl acetate (EtOAc) soluble fraction of the root bark of M. alba extract (100 mg/kg/day for 7 days) induced a decrease in immobility behavior in the FST [8]. Furthermore, EtOAc soluble fractions (50 and 100 mg/kg/day for 10 days) showed significant anti-stress activity as shown by modifying effects on several chronic restraint stressinduced neurochemical and behavioral parameters [9]. Moreover, the effect of 200 mg/kg M. alba leaf extract in
123
J Nat Med
decreasing the immobility time was comparable to 10 mg/ kg desipramine without an anxiolytic-like effect [10]. Sanggenon G (Fig. 1), an active compound isolated from the root bark of M. alba [11], showed antidepressantlike activity in rats subjected to FST-induced depression through interaction with the serotonergic system for the first time in our previous reports [12]. Considering the inhibitory effects of a2-adrenoceptors on serotonergic function, it was hypothesized that the addition of the a2-antagonist yohimbine to Sanggenon G may results in enhanced antidepressant-like activity in the FST in rats. In the present study, the enhanced effect of Sanggenon G and yohimbine were investigated in response to the rat FST.
Materials and methods Isolation and determination of Sanggenon G Sanggenon G was prepared as described [12]. Briefly, the EtOAc fraction (120 g) from the 80 % methanol (MeOH) extract of the dried root bark M. alba (yield of approximately 17 %) was subjected to SiO2 column chromatography (CC; u 12.5 9 18 cm) and eluted with n-hexane– ethyl acetate (4:1 ? 2:1 ? 1:1, 27 L of each). The eluting solutions were monitored by thin-layer chromatography to produce 41 fractions (MRE-1–MRE-41). Fraction MRE-37 [1.58 g; elution volume/total volume (Ve/Vt), 0.80–0.86] was subjected to ODS CC (u 4 9 10 cm) and eluted with MeOH–H2O (2:1, 4.2 L), yielding 23 fractions (MRE-371–MRE-37-23), including the purified compound Sanggenon G (MRE-37-17; Ve/Vt, 0.28–0.44; 350 mg). The compound was identified as Sanggenon G based on the results of several spectroscopic methods, including NMR, IR, MS, and specific rotation value [(a)20 D -277°, c 0.09, MeOH].
Quantifications of sanggenon G Dried root bark of M. alba powder (100 mg) was extracted with 80 % MeOH (1 mL) in an ultrasonic bath for 1 h at 25 °C. The extract was filtered through a 0.45-lm membrane filter (Woongki Science, Seoul, Korea) and evaporated in a vacuum. A 20-lL aliquot of the extract solution was injected into the HPLC system. Analysis was achieved using a Waters 600 s controller (Milford, MA, USA) with a Waters 2487 UV detector (280 nm). The column was an YMC-Pack ODS-A (Kyoto, Japan, 250 mm 9 4.6 mm, particle size 5 lm). The mobile phase consisted of 0.05 % trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B) eluted at a flow rate of 1.0 mL/min with the following gradient elution—B: 50 % (0.01 min) ? B: 50 % (10 min) ? B: 60 % (25 min) ? B: 100 % (30 min) ? B: 100 % (40 min) ? B: 50 % (45 min). Quantitative analysis was replicated three times. The regression equation and correlation coefficient (r2) were built to be y = 3984.4897x– 3623.4584 and 0.998, respectively. The concentration of Sanggenon G was determined to be 0.446 ± 0.007 mg/g using peak area in the chromatogram and regression equation. The purity of Sanggenon G was determined to be [97 % based on HPLC and 1H-NMR data. Animals Eight-week-old male Sprague–Dawley rats (Samtako Bio Korea, Gyeonggi-do, Korea) weighing 180–210 g were housed with two rats per cage under a controlled temperature (23 ± 1 °C) and a 12-h light/dark cycle (lights on at 07:00 and lights off at 19:00). The rats were allowed at least 1 week for acclimatization before the experiments. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Food Research Institute. Drugs and treatment Yohimbine and fluoxetine (both from Sigma-Aldrich Co., St. Louis, MO, USA) were dissolved in 0.9 % (w/v) saline solution. Sanggenon G was diluted in saline with 1 % Tween 80. Sanggenon G (5, 10, and 20 mg/kg) and fluoxetine (5, 10, and 20 mg/kg) were injected intraperitoneally (i.p., an injection volume of 0.1 mL/100 g body weight) 60 min before the FST. In the combination study, yohimbine (0.5 mg/kg, i.p.) was injected 15 min before fluoxetine or Sanggenon G treatment. FST
Fig. 1 Chemical structure of Sanggenon G isolated from the root bark of M. alba
123
The FST was carried out as previously described [13]. Briefly, in the pretest session, rats were forced to swim for
J Nat Med
15 min in a transparent Plexiglas cylinder (height 50 cm; diameter 20 cm) filled to a depth of 30 cm with water (temperature, 23–25 °C). Twenty-four hours later, the procedure was repeated during a 6-min test session, and the immobility time during the last 4 min was measured by a SMART video tracking system (SMART v3.0; Panlab SL, Barcelona, Spain). Statistical analysis Data analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test using Prism 5 (GraphPad Software, Inc., San Diego, CA, USA) for multigroup comparisons. All data are presented as the mean ± standard error (SEM). Differences with p values of \0.05 were considered significant.
Fig. 2 Antidepressant-like effects of treatment with fluoxetine and the synergistic effects of yohimbine on depressive behavior in response to the FST in rats. Fluoxetine (5, 10, and 20 mg/kg) was injected intraperitoneally 60 min before the FST. In the combination study, yohimbine (0.5 mg/kg) was administered 15 min before fluoxetine treatment. Columns show the means ± SEMs (n = 8). *p \ 0.05 and **p \ 0.01 versus the control group. #p \ 0.05 as compared to respective fluoxetine-treated group
Results and discussions In the present study, we examined whether the co-administration of the a2-antagonist yohimbine can enhance the antidepressant-like effects of Sanggenon G treatment in FST-induced depression in rats. The FST is an effective screening tool with good reliability and predictive validity [14]. The state of immobility in the FST is reported to mimic the symptoms of depression in humans and can be reversed by treatment with antidepressant drugs [15]. First, we examined the antidepressant-like effects of the SSRI fluoxetine in the FST. Fluoxetine treatment in rats reduced the immobility time by a maximum of 55.8 % when treated at a dose of 20 mg/kg. Moreover, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of fluoxetine at 5, 10 and 20 mg/kg (Fig. 2). Similarly, Sanggenon G significantly decreased the immobility time by a maximum of 43.9 % when treated at a dose of 20 mg/kg. Furthermore, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of Sanggenon G at 5 and 10 mg/kg (Fig. 3). Previous work by other authors has suggested that the a2-antagonist yohimbine modulates the serotonergic system, and treatment with yohimbine was reported to show beneficial interaction with fluoxetine [16]. Administration of a2-antagonists has been shown to enhance serotonergic neurotransmission via direct inhibition of a2-heteroreceptors located on the serotonergic nerve terminals and indirect stimulation of a1-receptors via inhibition of a2heteroreceptors [17]. Furthermore, Yanpallewar et al. also indicated that combination treatment with an a2-adrenoceptor antagonist and the tricyclic antidepressant, imipramine, elicits neurogenic, neurotrophic and behavioral changes, as compared to imipramine treatment alone [18].
Fig. 3 Antidepressant-like effects of treatment with Sanggenon G and the synergistic effects of yohimbine on depressive behavior in response to the FST in rats. Sanggenon G (5, 10, and 20 mg/kg) was injected intraperitoneally 60 min before the FST. In the combination study, yohimbine (0.5 mg/kg) was administered 15 min before Sanggenon G treatment. Columns show the means ± SEMs (n = 8). **p \ 0.01 versus the control group. #p \ 0.05 as compared to the respective Sanggenon G-treated group
These studies suggest that a2-adrenoceptor antagonism is a putative target for combination antidepressant therapy. In our results, pretreatment with yohimbine significantly enhanced the antidepressant-like behavior of Sanggenon G. The results of the present study concluded that the antidepressant-like effect of Sanggenon G could be facilitated by concomitant use of yohimbine. However, further studies are needed to determine the specific mechanisms by which Sanggenon G affects interaction between the serotonin and a2-adrenergic systems. Acknowledgments This study was supported by a grant from the Korea Food Research Institute. Compliance with ethical standards Conflict of interest of interest.
The authors declare that there are no conflicts
123
J Nat Med
References 1. Muguruza C, Rodriguez F, Rozas I, Meana JJ, Uriguen L, Callado LF (2013) Antidepressant-like properties of three new alpha2-adrenoceptor antagonists. Neuropharmacology 65:13–19 2. Rodriguez F, Rozas I, Ortega JE, Erdozain AM, Meana JJ, Callado LF (2008) Guanidine and 2-aminoimidazoline aromatic derivatives as alpha2-adrenoceptor antagonists. 2. Exploring alkyl linkers for new antidepressants. J Med Chem 51:3304–3312 3. Rozas I (2009) Improving antidepressant drugs: update on recently patented compounds. Expert Opin Ther Pat 19:827–845 4. Bremner JD (1995) A double-blind comparison of Org 3770, amitriptyline, and placebo in major depression. J Clin Psychiatry 56:519–525 5. Asano N, Yamashita T, Yasuda K, Ikeda K, Kizu H, Kameda Y, Kato A, Nash RJ, Lee HS, Ryu KS (2001) Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.). J Agric Food Chem 49:4208–4213 6. Nade VS, Kawale LA, Naik RA, Yadav AV (2009) Adaptogenic effect of Morus alba on chronic footshock-induced stress in rats. Indian J Pharmacol 41:246–251 7. Lee MS, Park WS, Kim YH, Kwon SH, Jang YJ, Han D, Morita K, Her S (2013) Antidepressant-like effects of Cortex Mori Radicis extract via bidirectional phosphorylation of glucocorticoid receptors in the hippocampus. Behav Brain Res 236:56–61 8. Lim DW, Kim YT, Park JH, Baek NI, Han D (2014) Antidepressant-like effects of the ethyl acetate soluble fraction of the root bark of Morus alba on the immobility behavior of rats in the forced swim test. Molecules 19:7981–7989 9. Nade VS, Yadav AV (2010) Anti-stress effect of ethyl acetate soluble fraction of Morus alba in chronic restraint stress. Pharm Biol 48:1038–1046 10. Sattayasai J, Tiamkao S, Puapairoj P (2008) Biphasic effects of Morus alba leaves green tea extract on mice in chronic forced swimming model. Phytother Res 22:487–492
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