https://orcid.org/0000-0002-1812-9844
Abstract
The exposure of neurons to an excessive excitatory stimulation induces the alteration of the normal neuronal function. Mood disorders are among the first signs of alterations in the central nervous system function. Magnolia officinalis bark extract has been extensively used in the traditional medicine systems of several countries, showing several pharmacological activities. Honokiol, the main constituent of M. officinalis, is a GABA modulator and a CB1 agonist, which is deeply investigated for its role in modulating mood disorders.
Thus, we evaluated the possible neuroprotective effect of a standardized M. officinalis bark extract (MOE), enriched in honokiol, and its effect on animal mood behavioural tests and in an in vitro model of excitotoxicity.
MOE showed neuroprotective effect using SH-SY5Y cells, by normalizing brain-derived neurotrophic factor release. Then, we tested the effect of MOE in different behavioural tests evaluating anxiety and depression and we observed a selective anxiolytic-like effect. Finally, we confirmed the involvement of CB1 in the final effect of MOE by the co-administration of the CB1 antagonist, AM251.
These results suggest that MOE could be considered an effective and safe anxiolytic candidate with neuroprotective activity.
Introduction
The exposure of the central nervous system (CNS) to an excessive excitatory stimulation or to a toxic substance induces the alteration of the normal neuronal function, which may lead to a permanent damage causing the complete degeneration of the synaptic function.[1] Anxiety, depression and cognitive impairments are among the first signs to recognize alterations in the CNS function.[2] Mood disorders are some of the most important global problems in term of cost and public health. Indeed, according to WHO, depression is one of the most diffuse disability worldwide and 1 in 13 people globally suffers from anxiety.[3] The research of novel drug candidates for the management of mood disorders represents an important target, in particular because the available conventional medicines are often ineffective and are characterized by several undesired effects, limiting their prolonged use.[4] Nowadays, natural products are considered promising therapeutic tools for the management of these conditions, and several studies have been conducted to confirm their efficacy and safety.[5]
Recently, lot of efforts have been put in the investigation of the involvement of the endocannabinoid system in the pathophysiology of mood disorders.[6,7] In particular, cannabinoid receptor (CB) 1 activation was demonstrated to affect the glutamatergic synaptic transmission, reducing the hyperexcitability state of neuronal cells,[8] and the increased activity of CB1 receptor was related to the induction of anxiolytic effects.[9] The use of Cannabis sativa L. in the management of anxiety is still controversial, indeed frequent cannabis users have been reported to be constantly subject to anxiety.[10]
Magnolia officinalis Rehder & E.H. Wilson (M. officinalis) is an herb used in Chines and Japanese traditional medicine systems, for the treatment of gastrointestinal complaints, microbial infections, stress and anxiety.[11,12] The part of the plant to be of medicinal interest is the bark, which mainly contains lignans, and, in particular, honokiol and its isomer magnolol. This two phenolic compound enhanced the GABAA receptors, inducing a tonic GABAergic neurotrasmission[13] and possessed neuroprotective effects against oxidative stress.[14–16] The chemical structure of honokiol is similar to that of some cannabinoid receptors ligands and this led to the finding that honokiol act as an agonist at CB1. Another characteristic lignan present in M. officinalis bark is magnolol, a honokiol isomer, which was reported to be a partial agonist at both CB1 and CB2.[17]
In the attempt to investigate herbal phytocomplexes with potential anxiolytic activity through the modulation of the endocannabinoid system, but without the psychotropic effects induced by C. sativa, thus we focused on M. officinalis. Specifically, the aim of this work was to investigate the possible neuroprotective effect of a standardized M. officinalis bark extract, enriched in honokiol, and to evaluate its possible use for the management of mood disorders by determining its anxiolytic-like activity in behavioural experiments. Finally, we evaluated the involvement of the endocannabinoid system in the effects produced by the extract.
Material and Methods
Chemicals and drug administration
Magnolia officinalis Rehder & E.H. Wilson bark extract (MOE, extraction solvent: ethanol 96% v/v, standardized to contain 40% honokiol, Naturex Inc., South Hackensack, NJ, USA) and L-theanine (TEA, Giellepi, Spa, Milan Italy) were dissolved in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Aldrich, Milan, Italy) to reach the final concentration of 1 μg/ml according to cell viability experiments. The excitotoxic stimulation of SH-SY5Y cells was induced using monosodium glutamate (GLU). A stock solution (800 mM) was prepared in sterile DMEM and then added to cell culture.
AM251 (Tocris, Bristol, UK), a known CB1 antagonist, was dissolved in dimethyl sulfoxide/Tween 80/0.9% saline (1:1:18) and diluted in DMEM to the final concentration of 5 µM. For behavioural tests, MOE was dissolved in saline (0.9% NaCl) and administered in a volume of 10 ml/kg by gavage (p.o.) 45 min before the experimental procedure at the dose of 30 mg/kg, except for the dose–response curve where doses of MOE ranging from 1 to 30 mg/kg were tested. Diazepam (DIAZ, 1 mg/kg i.p.), amitriptyline (AMI, 10 mg/kg i.p.) (Sigma Aldrich), dissolved in saline, and AM251 (1 mg/kg i.p) were administered 30 min before tests.[18]
Quantification of honokiol by HPLC-DAD
A 1 mg/ml solution was obtained by dissolving MOE in ethanol 96%. Ten microlitres were injected into a HPLC-DAD system, consisting of a Shimadzu Prominence LC 2030 3D instrument, equipped with a Bondpak C18 column (10 µm, 125 Å, 3.9 mm, Waters Corporation). The mobile phase consisted of ddH2O + 0.1% v/v formic acid (A) and acetonitrile + 0.1% v/v formic acid (B). The following method was applied: A 35% at 0 min for 3 min, then from 35% to 10% at 10 min, A 10% for 2 min and from 10% to 35% at 14 min, then A 35% for 1 min. Flow rate was set to 1.2 ml/min and column temperature to 28°C. Chromatograms were recorded at 292 nm. A calibration curve was set up using concentrations of honokiol (Sigma-Aldrich) ranging from 0.008 to 0.500 mg/ml, with R2 > 0.99.
Cell culture and cell viability
SH-SY5Y (human neuroblastoma cells, RRID:CVCL_0019), were a kind gift of Prof. Lorenzo Corsi (University of Modena and Reggio Emilia, Italy), were cultured in DMEM and F12 Ham’s nutrients mixture (Sigma-Aldrich), both containing 10% heat-inactivated foetal bovine serum (FBS; Sigma-Aldrich), 10 mg/ml L-glutamine (Sigma-Aldrich), and 100U/ml penicillin-streptomycin solution (Sigma-Aldrich), in a humidified atmosphere with 5% CO2 at 37°C.[18]
For cell viability test, a 4-h pre-treatment with MOE and TEA, followed by stimulation with GLU 80 mM for 24 h, were administered to SH-SY5Y. Before the administration of MOE and TEA, SH-SY5Y were pre-treated with AM251. Cell viability was performed using a Cell Counting Kit (CCK-8, Sigma-Aldrich). Briefly, SH-SY5Y were seeded (5 × 105) in 96-well plates and allowed to grow until 70–80% confluence. A MP96 microplate reader spectrophotometer (Safas, Monte Carlo, Principality of Monaco) was used for measuring the absorbance at 450 nm. Three independent experiments (N = 3; n = 6) were performed, and cell viability was calculated by normalizing the values to the control’s mean.
Non-competitive sandwich ELISA assay for BDNF
SH-SY5Y (1.5 × 105) were seeded in 48-well plates and cultured until confluence. After a 4-h pre-treatment with MOE or TEA, cells were stimulated with GLU 80 mM for 24 h. The level of brain-derived neurotrophic factor (BDNF) released in the culture medium was measured using the human BDNF ELISA kit (Abcam, Milan, Italy) according to the manufacturer’s instructions.[18] The samples were analysed in three independent experiments (N = 3; n = 6).
Animals
CD1 male mice (4–6 weeks of age) weighting approximately 22–24 g (Envigo, Varese, Italy) were housed in the Ce.S.A.L. (Centro Stabulazione Animali da Laboratorio, University of Florence) vivarium and used one day after their arrival. Mice were housed in standard cages, kept at 23 ± 1°C with a 12-h light/dark cycle, light on at 7 a.m., and fed with standard laboratory diet and tap water ad libitum. Twenty-four hours before the behavioural test, the animals were acclimatized by placing the cages in the experimental room. All tests were conducted during the light phase. The experimental protocol was approved by the Institution’s Animal Care and Research Ethics Committee (University of Florence, Italy), under license from the Italian Department of Health (54/2014-B). Mice were treated in accordance with the relevant European Union (Directive 2010/63/EU, the council of 22 September 2010 on the protection of animals used for scientific purposes) and international regulations (Guide for the Care and Use of Laboratory Animals, US National Research Council, 2011). All studies involving animals are reported in accordance with the ARRIVE guidelines.[19] The experimental protocol was designed to minimize the number of animals used and their suffering. The G power software was used to perform a power analysis to choose the number of animals per experiment.[20]
Investigation of anxiolytic-like activity
Marbles test
Mice were individually placed in a cage (27 × 16 × 14 cm) and allowed to move freely for 5 min. After this adaptation time, 20 glass marbles (1 cm diameter) were positioned in the cage. The number of buried marbles (at least two-thirds) registered in 30 min was used as a measure of anxiety behaviour in mice.[21] The experiments were conducted blindly.
Light dark box (LDB)
The LDB apparatus consisted of a box (50 × 20.5 × 19 cm), divided in one dark (black) and one illuminated by a bulb lamp (60-W; light) compartments. The two compartments were separated by an insert with a small door (10 cm × 4 cm) at floor level that allowed mice to freely move across compartments.[21] After each test, 70% ethanol was used to remove the olfactory cues and allowed to dry before the next subject was tested. Two different parameters were detected: the time (s) spent in the light chamber and the number of transitions from a chamber to the other one.
Novelty suppressed feeding test (NSFT)
Mice were deprived of food overnight (for a maximum of 12 h). The day after, a single pellet of food was placed in the centre of the container, and animals were individually placed in a corner. The latency to reach the pellet in the centre of the cage and the amount of pellet eaten (mg) was recorded in 5 min.[18] The experiments were conducted blindly.
Evaluation of antidepressant activity
Tail suspension test (TST)
Each animal was suspended (50 cm above the ground) for a total of 6 min. A depressant-like behaviour was observed in the last 4 min (mice are passive and motionless), and the time of immobility was registered.[21]
Evaluation of locomotor side effects
Rotarod test
Rotorod was used to assess the locomotor side effects induced by the treatments as previously described.[22] The motor function was evaluated by counting the number of falls in 30 s.
Hole-board test
The hole-board test is a frequently-used test which allow to assess if a drug can alter the spontaneous mobility and exploratory activity of the animals.[22] Mice were individually evaluated for 5 min.
Statistical analysis
Behavioural test: results are reported as mean ± standard error mean (SEM), using eight mice per group. The statistical analysis was performed using one-way and two-way analysis of variance, followed by Tukey and Šidák post hoc test, respectively. In vitro experiments: results are reported as the mean ± SEM of three independent experiments. A P value lower than 0.05 was considered significant. All the statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA).
Results
Quantification of honokiol by HPLC-DAD
The HPLC-DAD chromatogram of MOE is reported in Figure 1. By comparison with the reference analytical standard, honokiol (peak 1) was identified as the main constituent of MOE, with RT = 2.8 min. The amount of honokiol in MOE was found to be 46.22% w/w, which is consistent with the value declared by the vendor. A second important peak (peak 2) at RT = 3.0 min was also observed and was tentatively assigned to magnolol, by comparison of the UV spectrum with the available literature.[23] The UV spectra of peak 1 and 2 are reported as supplementary material in Supplementary Figure S1.
(a) HPLC-DAD chromatogram of MOE, recorded at 292 nm. Peak 1 (RT = 2.8 min) was identified as honokiol by comparison with reference analytical standard; peak 2 (RT = 3.0 min) was tentatively assigned to magnolol by comparison of the UV spectrum. (b) HPLC-DAD chromatogram of the purified honokiol.
Neuroprotective effect of MOE in SH-SY5Y cell
To establish the maximum non-toxic concentration of MOE, increasing concentrations of MOE were added to the SH-SY5Y cell culture. As cell viability started to decrease at 10 µg/ml, for further experiments 1 µg/ml was chosen as the maximum concentration that did not affect cell viability (Figure 2a). GLU-stimulated neuronal cell viability was strongly reduced in the in vitro model of excitotoxicity.[18] The pretreatment with MOE prevented this neurotoxic effect, showing an efficacy similar to TEA 1 µg/ml (used as reference GLU receptor antagonist) (Figure 2b). The dysregulation of BDNF in the CNS has been considered to have a strong correlation with the onset of mood disorders. The GLU-induced BDNF release in SH-SY5Y cells was completely counteracted by the pretreatment with MOE and TEA, with similar potency.
(a) Cell viability test of MOE on SH-SY5Y cells. (b) Neuroprotective effect induced by MOE in GLU-stimulated SH-SY5Y cells compared to TEA. (c) MOE reduced the release of BDNF in GLU-stimulated SH-SY5Y cells, similarly to TEA. ***P < 0.001 vs. non-treated cells (dashed line), °°°P < 0.001, °°P < 0.01 vs. CTRL.
MOE reduced the anxiety-like symptoms without showing antidepressant-like effects
In mice, anxiety-like behaviour can be observed by registering the number of buried marbles in the marble’s test. Indeed a high number of hidden marbles correspond to an increased state of anxiety of the animals.[24] As showed in the dose–response curve, mice treated with MOE 1 and 10 mg/kg buried the same number of marbles, compared to the VEH group, used as the anxiety-like behaviour control. 20 mg/kg MOE started to show an effect, reducing the number of hidden marbles. This effect became significant at the dose of 30 mg/kg, with a trend similar to that obtained for diazepam (DIAZ) (Figure 3a). To confirm this activity, we performed the LDB test, in which 30 mg/kg MOE increased the time spent in the light chamber, compared to the VEH group, with an efficacy comparable to DIAZ, confirming also in this test a potential anxiolytic-like activity (Figure 3b). Indeed, this parameter represents an index of the mice mood, with less time spent in the dark chamber corresponding to a more evident sedative effect of the test substance.[25] The number of transitions across the compartments was used as a second behavioural parameter in the LDB test to investigate the anxiolytic activity of MOE. Consistently with data on the time spent in the light, 30 mg/kg MOE increased the number of transitions across the two chambers with a similar efficacy to the reference drug (Figure 3c). In the NFST, MOE reduced the latency to feed (Figure 3d) similarly to DIAZ without altering the food consumption compared to the VEH group (Figure 3e). We did not observe any effect in the TST, in which MOE did not affect the immobility time compared to VEH group. On the contrary, AMI, a well-known antidepressant drug used as reference compound (ref), significantly reduced the immobility time (Figure 3f).
Effects of MOE reduced anxiety behaviours with a trend similar to diazepam (DIAZ): (a, b) Marbles test (MT), (b, c) Light Dark box (LDB), (d, e) Novelty Suppressed Feeding Test (NSFT). (f) Effects of MOE on depression behaviours on tail suspension test (TST), compared to amitriptyline (AMI). ***P < 0.001, **P < 0.01, *P < 0.05 vs. VEH. §§§P < 0.001; §P < 0.05.
MOE did not alter the motor coordination and exploratory activity
MOE did not alter the motor coordination of animals. Figure 4a shows that the number of falls in the rotarod test following MOE administration decreased during the experiment. Moreover, in the hole-board test, the exploratory activity and the spontaneous mobility of MOE-treated animals was not altered, in comparison to the VEH group (Figure 4b). These results exclude the possibility of MOE-induced excessive sedation or cognitive impairment.
(a) Lack of impairment of motor coordination observed with rotarod test, 2 h time course after p.o. (b) Spontaneous mobility and exploratory activity measured by the hole board test, 45 min after p.o.
The endocannabinoid system mediates the anxiolytic-like effect of MOE
The anxiolytic-like effect of MOE was completely prevented by AM251, a CB1-antagonist. Indeed, the co-administration of AM251 with MOE reduced the time spent in the light chamber (Figure 5a) and the number of transitions (Figure 5b) across the two chambers in the LDB. The role of CB1 in the MOE anxiolytic-like effect was also confirmed by the reduction of the neuroprotective effect of MOE in GLU-stimulated-SHSY5Y cells observed in the presence of AM251. This effect was not observed with TEA, highlighting a different mechanism of action between the two compounds.
MOE lost the neuroprotective effect in the presence of AM251 in (a, b) the light dark box test (LDB) and (c) on SH-SY5Y cell viability. ***P < 0.001 vs. CTRL VEH, °°°P < 0.001 vs. CTRL AM251, §§§P < 0.001 vs. MOE VEH.
Discussion
Glutamatergic excitotoxicity is one of the major responsible for the insurgence of neuronal damage, leading to the development of several disorders, including mood disorders (i.e. anxiety, stress, depression), and neurodegenerative diseases.[2] The first part of the present study demonstrated that, when exposed to an excessive glutamatergic stimulation, the neuronal cells SH-SY5Y human neuroblastoma cells lost their normally activity. TEA, a green tea amino acid, is a structural analog of GLU, and acts as an antagonist at GLU receptors, reducing the glutamatergic transmission.[26] Consistently, in our work, TEA completely prevented the impairment of neuronal cell viability induced by GLU. Confirming our results, the work of Di and colleagues supports the neuroprotective effect of TEA against GLU-induced excitotoxicity, through the modulation of NMDA receptor in the APP transgenic SH-SY5Y cells.[27]
Similarly, MOE counteracted the toxic effect produced by GLU in neuronal cells. This activity could be related to the presence of honokiol in the extract. Honokiol, the most abundant constituent of the extract, was reported to induce beneficial effects on neurotoxicity,[14,28,29] based on the fact that honokiol inhibited the GLU-induced intracellular ROS generation, thus, reducing neuronal cells damage.
The alteration of BDNF levels is strongly related to the loss of normal neuronal functions.[30] In our experimental model, the hyper-activation of the GLU receptors induced the release of BDNF, which could be considered as a mechanism of defence against the excitotoxic stimuli. After MOE and TEA pre-treatment, the GLU-induced excitotoxicity was attenuated, leading to a reduction of BDNF release.
These results suggest that M. officinalis extract could be considered a good candidate for the management of mood disorders, being these diseases closely connected with excitotoxicity states. The absence of a safe and effective therapy supports the need to find some novel therapies beside conventional medicine. Herbal medicine is frequently used in complementary medicine, and is generally considered them safer than conventional medicine.[31]
M. officinalis bark is a traditional botanical drug used for the treatment of clinical depression and anxiety-related disorders. To the best of our knowledge, this is the first work in which the effect of a chemically characterized M. officinalis bark extract is tested in experimental models of mood disorders. Ham and colleagues described the anxiolytic effect of an ethanolic extract of Magnolia obovata Thunb. Leaves, by evaluating its activity on GABA transmission.[32] Similarly, we observed a reduced anxiety behaviour in mice using a M. officinalis bark extract. Surprisingly, we did not observe effects on depressant-like symptoms. This is in contrast with other published works. Indeed, 20 mg/kg honokiol administered p.o. was reported to reduce the symptoms associated to depression in mice.[33] However, these discrepancies might be related to the different doses used in these research protocols. The concentration used by Pitta and colleagues is about twice than that used in this work. Consistent with this hypothesis, another work described the effect of honokiol in depression, but only after a repeated oral administration for 11 days.[25]
These results suggested that MOE is a selective and safe anxiolytic agent, not inducing locomotor impairment or excessive sedative effects different to the currently available drugs.[5,34]
Honokiol has been studied for its affinity at CB1 and CB2 receptors, being a structural analogue of THC. Rempel and colleagues described that honokiol extracted from M. officinalis bark is a CB1 agonist and a CB2 antagonist.[35] In the last decades, the endocannabinoid system attracted the scientific interest for its possible involvement in the neurobiology of anxiety. CB1 receptor agonists may act both as anxiolytic or anxiogenic agent, depending on the route of administration, dose and the areas of the brain involved.[36] This peculiar effect on anxiety could be considered a consequence of the balance between GABA and glutamate transmission.[37] In the last part of this work, we tested our honokiol-enriched M. officinalis bark extract in the light dark box test in co-treatment with AM251, a well-known CB1 antagonist, confirming the involvement of the endocannabinoid system in the final effect of MOE. To deeply investigate the role of CB1, we confirmed this result in vitro on neuronal cells, where we observed a significant reduction of the neuroprotective activity of MOE after GLU stimulation, when AM251 was co-administered.
Conclusions
The oral administration of MOE showed a selective anxiolytic-like activity and a neuroprotective role in SH-SY5Y cells by reducing the excitotoxicity induced by GLU, through the modulation of CB1 receptors. These results suggest that MOE could be considered a novel and safe selective anxiolytic candidate with neuroprotective activity.
Author Contributions
V.B.: Investigation, methodology, formal analysis and writing original draft. P.G.: Investigation, writing original draft. F.M., E.M. and M.B.: writing review and editing. N.G.: Conceptualization, methodology, writing original draft, writing review and editing.
Funding
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Conflict of Interest
None declared.
References
