In Vitro and In Silico Studies of Neolignans from Magnolia grandiflora L. Seeds against Human Cannabinoids and Opioid Receptors.

Magnolia grandiflora L. (Magnoliaceae) is a plant of considerable medicinal significance; its flowers and seeds have been used in various traditional remedies. Radioligand binding assays of n-hexane seeds extract showed displacement of radioligand for cannabinoid (CB1 and CB2) and opioid δ (delta), κ (kappa), and µ (mu) receptors. Bioactivity-guided fractionation afforded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), which showed higher binding to cannabinoid rather than opioid receptors in radioligand binding assays. Compounds 1-3, together with the dihydro analog of 2 (4), displayed selective affinity towards CB2R (Ki values of 0.29, 1.4, 1.94, and 0.99 µM, respectively), compared to CB1R (Ki 3.85, 17.82, 14.55, and 19.08 µM, respectively). An equal mixture of 2 and 3 (1:1 ratio) showed additive displacement activity towards the tested receptors compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. Due to the unavailability of an NMR or X-ray crystal structure of bound neolignans with the CB1 and CB2 receptors, a docking study was performed to predict ligand-protein interactions at a molecular level and to delineate structure-activity relationships (SAR) of the neolignan analogs with the CB1 and CB2 receptors. The putative binding modes of neolignans 1-3 and previously reported related analogs (4, 4a, 5, 5a, 6, 6a, and 6b) into the active site of the CB1 and CB2 receptors were assessed for the first time via molecular docking and binding free-energy (∆G) calculations. The docking and ∆G results revealed the importance of a hydroxyl moiety in the molecules that forms strong H-bonding with Ser383 and Ser285 within CB1R and CB2R, respectively. The impact of a shift from a hydroxyl to the methoxy group on experimental binding affinity to CB1R versus CB2R was explained through ∆G data and the orientation of the alkyl chain within the CB1R. This comprehensive SAR, influenced by the computational study and the observed in vitro displacement binding affinities, has indicated the potential of magnolia neolignans for developing new CB agonists for potential use as analgesics, anti-inflammatory agents, or anxiolytics.

Pharmacokinetic and Metabolic Profiling of Key Active Components of Dietary Supplement Magnolia officinalis Extract for Prevention against Oral Carcinoma.

Among the three key active components (KACs) of Magnolia officinalis bark extract (ME), 4-O-methylhonokiol and honokiol showed higher antiproliferation activities than magnolol in the oral squamous cancer cell lines (Cal-27, SCC-9, and SCC-4). Oral bioavailabilities of ME-KACs were poor (<0.2%) in C57BL/6 mice primarily due to their extensive first-pass phase II metabolism and poor solubilities. High plasma concentration of glucuronides upon oral administration and faster rate of glucuronidation by intestinal microsomes indicated intestine as one of the major metabolic organs for ME-KACs. Despite the increase in bioavailabilities of ME-KACs (∼8-10-fold) and decrease in AUC0-24 of glucuronides (∼10-fold) upon ME solubility enhancement, systemic exposure of ME-KACs failed to improve meaningfully. In conclusion, we propose a quality-controlled and chemically defined ME mixture, containing an optimized ratio of three KACs, delivered locally in the oral cavity as the most promising strategy for ME use as an oral cancer chemopreventive dietary supplement.

4-O-methylhonokiol protects against diabetic cardiomyopathy in type 2 diabetic mice by activation of AMPK-mediated cardiac lipid metabolism improvement.

Diabetic cardiomyopathy (DCM) is characterized by increased left ventricular mass and wall thickness, decreased systolic function, reduced ejection fraction (EF) and ultimately heart failure. The 4-O-methylhonokiol (MH) has been isolated mainly from the bark of the root and stem of Magnolia species. In this study, we aimed to elucidate whether MH can effectively prevent DCM in type 2 diabetic (T2D) mice and, if so, whether the protective response of MH is associated with its activation of AMPK-mediated inhibition of lipid accumulation and inflammation. A total number of 40 mice were divided into four groups: Ctrl, Ctrl + MH, T2D, T2D + MH. Five mice from each group were sacrificed after 3-month MH treatment. The remaining animals in each group were kept for additional 3 months without further MH treatment. In T2D mice, the typical DCM symptoms were induced as expected, reflected by decreased ejection fraction and lipotoxic effects inducing lipid accumulation, oxidative stress, inflammatory reactions, and final fibrosis. However, these typical DCM changes were significantly prevented by the MH treatment immediately or 3 months after the 3-month MH treatment, suggesting MH-induced cardiac protection from T2D had a memory effect. Mechanistically, MH cardiac protection from DCM may be associated with its lipid metabolism improvement by the activation of AMPK/CPT1-mediated fatty acid oxidation. In addition, the MH treatment of DCM mice significantly improved their insulin resistance levels by activation of GSK-3ß. These results indicate that the treatment of T2D with MH effectively prevents DCM probably via AMPK-dependent improvement of the lipid metabolism.

4-O-methylhonokiol ameliorates type 2 diabetes-induced nephropathy in mice likely by activation of AMPK-mediated fatty acid oxidation and Nrf2-mediated anti-oxidative stress.

Diabetic nephropathy (DN) is one of the most serious long-term complications of type 2 diabetes (T2D). 4-O-methylhonokiol (MH) is one of the biologically active ingredients extracted from the Magnolia stem bark. In this study, we aim to elucidate whether treatment with MH can ameliorate or slow-down progression of DN in a T2D murine model and, if so, whether the protective response of MH correlates with AMPK-associated anti-oxidant and anti-inflammatory effects. To induce T2D, mice were fed normal diet (ND) or high fat diet (HFD) for 3 months to induce insulin resistance, followed by an intraperitoneal injection of STZ to induce hyperglycemia. Both T2D and control mice received gavage containing vehicle or MH once diabetes onset for 3 months. Once completing 3-month MH treatment, five mice from each group were sacrificed as 3 month time-point. The rest mice in each group were sacrificed 3 months later as 6 month time-point. In T2D mice, the typical DN symptoms were induced as expected, reflected by increased proteinuria, renal lipid accumulation and lipotoxic effects inducing oxidative stress, and inflammatory reactions, and final fibrosis. However, these typical DN changes were significantly prevented by MH treatment for 3 months and even at 3 months post-MH withdrawal. Mechanistically, MH renal-protection from DN may be related to lipid metabolic improvement and oxidative stress attenuation along with increases in AMPK/PGC-1α/CPT1B-mediated fatty acid oxidation and Nrf2/SOD2-mediated anti-oxidative stress. Results showed the preventive effect of MH on the renal oxidative stress and inflammation in DN.

RANKL-induced osteoclastogenesis is suppressed by 4-O-methylhonokiol in bone marrow-derived macrophages.

Magnolol, honokiol, and obovatol are well known bioactive constituents of the bark of Magnolia officinalis and have been reported to have beneficial effects in various diseases. We recently isolated a novel active compound, 4-O-methylhonokiol (4-O-MH) from the ethanol extract of M. officinalis, which was previously reported to have pharmacological effects including anti-inflammatory, anti-oxidative, and anti-aging activities. Here, we examined the pharmacological properties of 4-O-MH on osteoblast (bone-forming cells) and osteoclast (bone-resorbing cells) differentiation, and its underlying signaling pathways in primary cultured pre-osteoblasts and bone marrow macrophages. Our results showed that 4-O-MH did not affect cell viability in pre-osteoblasts and did not influence osteoblast differentiation and mineralized nodule formation, as assessed by alkaline phosphatase activity and Alizarin red staining. However, 4-O-MH significantly inhibited TRAP-positive multinuclear osteoclasts and F-actin ring formation during Receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis without cytotoxicity. In addition, 4-O-MH suppressed RANKL-induced critical factors (c-Fos, NF-ATc1, TRAP, and ITB3) for osteoclast differentiation and function. Furthermore, RANKL-mediated signaling, including ERK1/2, AKT, and NF-kB pathways was attenuated by 4-O-MH. Taken together, 4-O-MH has an inhibitory role in RANKL-mediated osteoclastogenesis but not osteoblast differentiation, and our findings also suggest that 4-O-MH is a potential therapeutic agent for bone-destructive diseases such as osteoporosis, alveolar bone resorption, and osteoarthritis.

4-O-Methylhonokiol Protects HaCaT Cells from TGF-ß1-Induced Cell Cycle Arrest by Regulating Canonical and Non-Canonical Pathways of TGF-ß Signaling.

4-O-methylhonokiol, a neolignan compound from Magnolia Officinalis, has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-ß (TGF-ß) signal pathway has an essential role in the regression induction of hair growth, the effect of 4-O-methylhonokiol on the TGF-ß signal pathway has not yet been elucidated. We thus examined the effect of 4-O-methylhonokiol on TGF-ß-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4-O-methylhonokiol, TGF-ß1-induced G1/G0 phase arrest and TGF-ß1-induced p21 expression were decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-ß1-induced canonical pathway. We observed that ERK phosphorylation by TGF-ß1 was significantly attenuated by treatment with 4-O-methylhonokiol. 4-O-methylhonokiol inhibited TGF-ß1-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-ß1-induced noncanonical pathway. These results indicate that 4-O-methylhonokiol could inhibit TGF-ß1-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4-O-methylhonokiol might have protective action on TGF-ß1-induced cell cycle arrest.

4-O-Methylhonokiol Protects HaCaT Cells from TGF-β1-Induced Cell Cycle Arrest by Regulating Canonical and Non-Canonical Pathways of TGF-β Signaling

4-O-methylhonokiol, a neolignan compound from Magnolia Officinalis, has been reported to have various biological activities including hair growth promoting effect. However, although transforming growth factor-β (TGF-β) signal pathway has an essential role in the regression induction of hair growth, the effect of 4-O-methylhonokiol on the TGF-β signal pathway has not yet been elucidated. We thus examined the effect of 4-O-methylhonokiol on TGF-β-induced canonical and noncanonical pathways in HaCaT human keratinocytes. When HaCaT cells were pretreated with 4-O-methylhonokiol, TGF-β1-induced G1/G0 phase arrest and TGF-β1-induced p21 expression were decreased. Moreover, 4-O-methylhonokiol inhibited nuclear translocation of Smad2/3, Smad4 and Sp1 in TGF-β1-induced canonical pathway. We observed that ERK phosphorylation by TGF-β1 was significantly attenuated by treatment with 4-O-methylhonokiol. 4-O-methylhonokiol inhibited TGF-β1-induced reactive oxygen species (ROS) production and reduced the increase of NADPH oxidase 4 (NOX4) mRNA level in TGF-β1-induced noncanonical pathway. These results indicate that 4-O-methylhonokiol could inhibit TGF-β1-induced cell cycle arrest through inhibition of canonical and noncanonical pathways in human keratinocyte HaCaT cell and that 4-O-methylhonokiol might have protective action on TGF-β1-induced cell cycle arrest.

Magnolia bioactive constituent 4-O-methylhonokiol prevents the impairment of cardiac insulin signaling and the cardiac pathogenesis in high-fat diet-induced obese mice.

In obesity, cardiac insulin resistance is a putative cause of cardiac hypertrophy and dysfunction. In our previous study, we observed that Magnolia extract BL153 attenuated high-fat-diet (HFD)-induced cardiac pathogenic changes. In this study, we further investigated the protective effects of the BL153 bioactive constituent, 4-O-methylhonokiol (MH), against HFD-induced cardiac pathogenesis and its possible mechanisms. C57BL/6J mice were fed a normal diet or a HFD with gavage administration of vehicle, BL153, or MH (low or high dose) daily for 24 weeks. Treatment with MH attenuated HFD-induced obesity, as evidenced by body weight gain, and cardiac pathogenesis, as assessed by the heart weight and echocardiography. Mechanistically, MH treatment significantly reduced HFD-induced impairment of cardiac insulin signaling by preferentially augmenting Akt2 signaling. MH also inhibited cardiac expression of the inflammatory factors tumor necrosis factor-α and plasminogen activator inhibitor-1 and increased the phosphorylation of nuclear factor erythroid-derived 2-like 2 (Nrf2) as well as the expression of a Nrf2 downstream target gene heme oxygenase-1. The increased Nrf2 signaling was associated with decreased oxidative stress and damage, as reflected by lowered malondialdehyde and 3-nitrotyrosine levels. Furthermore, MH reduced HFD-induced cardiac lipid accumulation along with lowering expression of cardiac fatty acid translocase/CD36 protein. These results suggest that MH, a bioactive constituent of Magnolia, prevents HFD-induced cardiac pathogenesis by attenuating the impairment of cardiac insulin signaling, perhaps via activation of Nrf2 and Akt2 signaling to attenuate CD36-mediated lipid accumulation and lipotoxicity.

Role of transcription factor Sp1 in the 4-O-methylhonokiol-mediated apoptotic effect on oral squamous cancer cells and xenograft.

Recently, biphenolic components derived from the Magnolia family have been studied for anti-cancer, anti-stress, and anti-inflammatory pharmacological effects. However, the pharmacological mechanism of action of 4-O-methylhonokiol (MH) is not clear in oral cancer. The aim of this study was to investigate the role of MH in apoptosis and its molecular mechanism in oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, as well as tumor xenografts. Here, we demonstrated that MH decreased cell growth and induced apoptosis in HN22 and HSC4 cells through the regulation of specificity protein 1 (Sp1). We employed several experimental techniques such as MTS assay, DAPI staining, PI staining, Annexin-V/7-ADD staining, RT-PCR, western blot analysis, immunocytochemistry, immunohistochemistry, TUNEL assay and in vivo xenograft model analysis. MH inhibited Sp1 protein expression and reduced Sp1 protein levels via both proteasome-dependent protein degradation and inhibition of protein synthesis in HN22 and HSC4 cells; MH did not alter Sp1 mRNA levels. We found that MH directly binds Sp1 by Sepharose 4B pull-down assay and molecular modeling. In addition, treatment with MH or knocking down Sp1 expression suppressed oral cancer cell colony formation. Moreover, MH treatment effectively inhibited tumor growth and Sp1 levels in BALB/c nude mice bearing HN22 cell xenografts. These results indicated that MH inhibited cell growth, colony formation and also induced apoptosis via Sp1 suppression in OSCC cells and xenograft tumors. Thus, MH is a potent anti-cancer drug candidate for oral cancer.

Amelioration of Cognitive Dysfunction in APP/PS1 Double Transgenic Mice by Long-Term Treatment of 4-O-Methylhonokiol.

Alzheimer's disease (AD) is the most common neurodegenerative disease without known ways to cure. A key neuropathologic manifestation of the disease is extracellular deposition of beta-amyloid peptide (Aß). Specific mechanisms underlying the development of the disease have not yet been fully understood. In this study, we investigated effects of 4-O-methylhonokiol on memory dysfunction in APP/PS1 double transgenic mice. 4-O-methylhonokiol (1 mg/kg for 3 month) significantly reduced deficit in learning and memory of the transgenic mice, as determined by the Morris water maze test and step-through passive avoidance test. Our biochemical analysis suggested that 4-O-methylhonokiol ameliorated Aß accumulation in the cortex and hippocampus via reduction in beta-site APP-cleaving enzyme 1 expression. In addition, 4-O-methylhonokiol attenuated lipid peroxidation and elevated glutathione peroxidase activity in the double transgenic mice brains. Thus, suppressive effects of 4-O-methylhonokiol on Aß generation and oxidative stress in the brains of transgenic mice may be responsible for the enhancement in cognitive function. These results suggest that the natural compound has potential to intervene memory deficit and progressive neurodegeneration in AD patients.

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol.

BACKGROUND: Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABAA receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABAA receptors and its GABAA receptor subtype-specificity was established. METHODS: Different recombinant GABAA receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol. RESULTS: 3µM 4-O-methylhonokiol is shown here to potentiate responses of the α1ß2γ2 GABAA receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABAA receptor subtypes to assess GABAA receptor subunits that are responsible for the potentiating effect. CONCLUSION: The much higher potentiation of GABAA receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol. GENERAL SIGNIFICANCE: The results point to the use of 4-O-methylhonokiol as a lead for GABAA receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.

4-O-methylhonokiol inhibits serious embryo anomalies caused by nicotine via modulations of oxidative stress, apoptosis, and inflammation.

BACKGROUND: Since the increasing smoking rate among women has resulted in higher rates of embryonic malformations, it is important to search for an efficient and inexpensive agent that can help reduce the rate of serious fetal anomalies caused by maternal cigarette smoking. In this study, the bioavailability of 4-O-methylhonokiol isolated from Magnolia officinalis was first demonstrated in the mouse embryos exposed to nicotine using a whole embryo culture system. METHODS: Mouse embryos on embryonic day 8.5 were cultured with 1 mM nicotine and/or 4-O-methylhonokiol (1 × 10(-4) or 1 × 10(-3) µM) for 48 hr and were analyzed on the viewpoints of embryo developmental changes, oxidative damages, and apoptotic and inflammatory changes. RESULTS: Embryos exposed to 1 mM nicotine developed not only severe morphological anomalies, increased expressions of tumor necrosis factor-α, interleukin-1ß, and caspase 3 mRNAs; and elevated levels of lipid peroxidation, but also decreased levels of cytoplasmic superoxide dismutase, cytosolic glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, hypoxia inducible factor-1α, and B-cell lymphoma-extra large mRNAs, and reduced superoxide dismutase activity. However, these parameters were significantly improved when embryos exposed to the nicotine were concurrently treated with 4-O-methylhonokiol (1 × 10(-4) or 1 × 10(-3) µM). CONCLUSIONS: These findings indicate that 4-O-methylhonokiol reduces serious embryo anomalies caused by nicotine in mouse embryos via the modulations of oxidative stress, apoptosis, and inflammation, suggesting that 4-O-methylhonokiol may be a preventive and therapeutic agent against the dysmorphology induced by maternal smoking during pregnancy.

Pharmacokinetics and metabolism of 4-O-methylhonokiol in rats.

The purpose of this study was to characterize the pharmacokinetics and metabolism of 4-O-methylhonokiol in rats. The absorption and disposition of 4-O-methylhonokiol were investigated in male Sprague-Dawley rats following a single intravenous (2 mg/kg) or oral (10 mg/kg) dose. Its metabolism was studied in vitro using rat liver microsomes and cytosol. 4-O-Methylhonokiol exhibited a high systemic plasma clearance and a large volume of distribution. The oral dose gave a peak plasma concentration of 24.1±3.3 ng/mL at 2.9±1.9 h and a low estimated bioavailability. 4-O-Methylhonokiol was rapidly metabolized and converted at least in part to honokiol in a concentration-dependent manner by cytochrome P450 in rat liver microsomes, predicting a high systemic clearance consistent with the pharmacokinetic results. It was also shown to be metabolized by glucuronidation and sulfation in rat liver microsomes and cytosol, respectively. 4-O-Methylhonokiol showed a moderate permeability with no apparent vectorial transport across Caco-2 cells, suggesting that intestinal permeation process is not likely to limit its oral absorption. Taken together, these results suggest that the rapid hepatic metabolism of 4-O-methylhonokiol could be the major reason for its high systemic clearance and low oral bioavailability.

Amelioration of Cognitive Dysfunction in APP/PS1 Double Transgenic Mice by Long-Term Treatment of 4-O-Methylhonokiol

Alzheimer's disease (AD) is the most common neurodegenerative disease without known ways to cure. A key neuropathologic manifestation of the disease is extracellular deposition of beta-amyloid peptide (Abeta). Specific mechanisms underlying the development of the disease have not yet been fully understood. In this study, we investigated effects of 4-O-methylhonokiol on memory dysfunction in APP/PS1 double transgenic mice. 4-O-methylhonokiol (1 mg/kg for 3 month) significantly reduced deficit in learning and memory of the transgenic mice, as determined by the Morris water maze test and step-through passive avoidance test. Our biochemical analysis suggested that 4-O-methylhonokiol ameliorated Abeta accumulation in the cortex and hippocampus via reduction in beta-site APP-cleaving enzyme 1 expression. In addition, 4-O-methylhonokiol attenuated lipid peroxidation and elevated glutathione peroxidase activity in the double transgenic mice brains. Thus, suppressive effects of 4-O-methylhonokiol on Abeta generation and oxidative stress in the brains of transgenic mice may be responsible for the enhancement in cognitive function. These results suggest that the natural compound has potential to intervene memory deficit and progressive neurodegeneration in AD patients.

Sensitive determination of 4-O-methylhonokiol in rabbit plasma by high performance liquid chromatography and application to its pharmacokinetic investigation.

A novel high Performance liquid Chromatographie method was developed for the determination of 4-O-methylhonokiol in rabbit plasma and was applied to its pharmacokinetic investigation. Plasma samples were treated by one-fold volume of methanol and acetonitrile to remove the interference proteins. A reverse phase column of SHIM-PACK VP-ODS (150 mm × 4.6 mm, 5.0 Mm) was used to separate 4-O-methylhonokiol in the plasma samples. The detection limit of 4-O-methylhonokiol was 0.2 µg/L and the linear ränge was 0.012 - 1.536 µg/L. The good extraction recoveries were obtained for the spiked samples (84.7%, 89.3% and 87.7% for low, middle and high concentrations of added Standards, respectively). The relative standard deviation of intra-day and inter-day precisions was in the ränge from 0.6% to 13.5%. The pharmacokinetic study of 4-O-methylhonokiol was made and the results from the plasma-concentration curve of 4-O-methylhonokiol showed a two-apartment open model. This work developed a sensitive, stable and rapid HPLC method for the determination of 4-O-methylhonokiol and the developed method has been successfully applied to a pharmacokinetic study of 4-O-methylhonokiol.

Sensitive determination of 4-O-methylhonokiol in rabbit plasma by high performance liquid chromatography and application to its pharmacokinetic investigation / 药物分析学报

A novel high performance liquid chromatographic method was developed for the determination of 4-O- methylhonokiol in rabbit plasma and was applied to its pharmacokinetic investigation. Plasma samples were treated by one-fold volume of methanol and acetonitrile to remove the interference proteins. A reverse phase column of SHIM- PACK VP-ODS （150 mm ×4.6 mm, 5.0 μm） was used to separate 4-O-methylhonokiol in the plasma samples. The detection limit of 4-O-methylhonokiol was 0.2 μg/L and the linear range was 0. 012 - 1. 536 mg/L. The good extraction recoveries were obtained for the spiked samples （84.7%, 89.3% and 87.7% for low, middle and high concentrations of added standards, respectively）. The relative standard deviation of intra-day and inter-day precisions was in the range from 0.6% to 13.5%. The pharmacokinetic study of 4-O-methylhonokiol was made and the results from the plasmaconcentration curve of 4-0-methylhonokiol showed a two-apartment open model. This work developed a sensitive, stable and rapid HPLC method for the determination of 4-O-methylhonokiol and the developed method has been successfully applied to a pharmacokinetic study of 4-O-methylhonokiol.