Honokiol and Magnolol Increase the Number of [3H]Muscimol Binding Sites Three-Fold in Rat Forebrain Membranes In Vitro Using a Filtration Assay,by Allosterically Increasing the Affinities of Low-Affinity Sites

1. The bark of the root and stem of various Magnolia species has been used in Traditional Chinese Medicine to treat a variety of disorders including anxiety and nervous disturbances. The biphenolic compounds honokiol (H) and magnolol (M), the main components of the Chinese medicinal plant Magnolia officinalis, interact with GABA_A receptors in rat brain in vitro. We compared the effects of H and M on [³H]muscimol (MUS) and [³H]flunitrazepam (FNM) binding using EDTA/water dialyzed rat brain membranes in a buffer containing 150 mM NaCl plus 5 mM Tris-HCl, pH 7.5 as well as [³⁵S]t-butylbicyclophosphorothionate (TBPS) in 200 mM KBr plus 5 mM Tris-HCl, pH 7.5. H and M had similar enhancing effects on [³H]MUS as well as on [³H]FNM binding to rat brain membrane preparations, but H was 2.5 to 5.2 times more potent than M. 2. [ ³ H]FNM binding. GABA alone almost doubled [³H]FNM binding with EC₅₀ = 450 nM and 200 nM using forebrain and cerebellar membranes, respectively. In the presence of 5 M H or M the EC₅₀ values for GABA were decreased to 79 and 89 nM, respectively, using forebrain, and 39 and 78 nM, using cerebellar membranes. H and M potently enhanced the potentiating effect of 200 nM GABA on [³H]FNM binding with EC₅₀ values of 0.61 M and 1.6 M using forebrain membranes, with maximal enhancements of 33 and 47%, respectively. Using cerebellar membranes, the corresponding values were 0.25 and 1.1 M, and 22 and 34%. 3. [ ³ H]MUS binding. H and M increased [³H]MUS binding to whole forebrain membranes about 3-fold with EC₅₀ values of 6.0 and 15 M. Using cerebellar membranes, H and M increased [³H]MUS binding ~68% with EC₅₀ values of 2.3 and 12 M, respectively. Scatchard analysis revealed that the enhancements of [³H]MUS binding were due primarily to increases in the number of binding sites (B_max values) with no effect on the high affinity binding constants (K_d values). The enhancing effect of H and M were not additive. 4. [ ³⁵ S]TBPS binding. H and M displaced [³⁵S]TBPS binding from sites on whole rat forebrain membranes with IC₅₀ values of 7.8 and 6.0 M, respectively. Using cerebellar membranes, the corresponding IC₅₀ values were 5.3 and 4.8 M. These inhibitory effects were reversed by the potent GABA_A receptor blocker R5135 (10 nM), suggesting that H and M allosterically increase the affinity of GABA_A receptors for GABA and MUS by binding to sites in GABA_A receptor complexes. 5. Two monophenols, the anesthetic propofol (2,6-diisopropylphenol, P) and the anti-inflammatory diflunisal (2,4-difluoro-4-hydroxy-3-biphenyl carboxylic acid, D) also enhanced [³H]MUS binding, decreased the EC₅₀ values for GABA in enhancing [³H]FNM binding and potentiated the enhancing effect of 200 nM GABA on [³H]FNM binding, although enhancements of [³H]MUS binding for these monophenols were smaller than those for H and M, using forebrain and cerebellar membranes. The enhancing effect of P and D on [³H]MUS binding were almost completely additive. 2,2-biphenol was inactive on [³H]MUS and [³H]FNM binding. These, and other preliminary experiments, suggest that appropriate ortho (C2) and para (C4) substitution increases the GABA-potentiating activity of phenols. 6. The potentiation of GABAergic neurotransmission by H and M is probably involved in their previously reported anxiolytic and central depressant effects.     

和厚朴酚抑制线粒体可溶蛋白诱导小胶质细胞活化的体外实验研究

目的:探讨线粒体可溶蛋白诱导小胶质细胞活化作用及和厚朴酚对小胶质细胞活化的影响。方法:培养BV2小胶质细胞,分空白对照组(control)、线粒体可溶蛋白组(MDP)、和厚朴酚干预组(HNK)。ELISA检测不同浓度线粒体可溶蛋白(MSP)刺激不同时间的细胞上清液中IL-6和TNF-α含量;RT-PCR半定量法测定各组转录因子Klf4的表达情况;倒置相差显微镜观察各组细胞形态变化。结果:1.ELISA:MSP(10μg/ml,100μg/ml)处理7h时,IL-6和TNF-α含量与control组比较显著升高(P0.05);100μg/ml MSP处理1、3、5、7h时,IL-6和TNF-α表达与control组比较明显升高(P0.05),且7h时HNK组含量明显低于MDP组(P0.05)。2.RT-PCR结果显示MDP组KLP4的表达显著高于空白对照组和HNK组(P0.05)。3.倒置相差显微镜观察示空白组胶质细胞形态呈静息状态;MDP组小胶质细胞的胞体变圆或者椭圆,突起消失,呈"阿米巴"状;HNK组活化状态的小胶质细胞明显减少。结论:线粒体可溶蛋白可以激活小胶质细胞,促进白细胞介素IL-6和TNF-α的释放,和厚朴酚能够有效地抑制线粒体可溶蛋白诱导的小胶质细胞活化,其机制可能是通过下调Klf4的表达发挥作用。     

和厚朴酚通过激活SIRT1/FOXO1信号通路抵抗小鼠脓毒症脑损伤

目的:探究和厚朴酚是否通过激活SIRT1/FOXO1信号通路抵抗小鼠脓毒症脑损伤。方法:通过C57BL/6小鼠盲肠结扎穿孔法建立脓毒症脑损伤模型。小鼠随机分为以下6组:假手术(Sham)组;和厚朴酚处理(HKL)组;盲肠结扎穿孔(CLP)组;盲肠结扎穿孔+和厚朴酚处理(CLP+HKL)组;EX527 (SIRT1特异性抑制剂)预处理+盲肠结扎穿孔+和厚朴酚处理(CLP+HKL+EX527)组;EX527预处理+盲肠结扎穿孔(CLP+EX527)组。盲肠结扎穿孔48 h后检测脑组织内水含量、凋亡率及凋亡相关蛋白Bax、Bcl-2和cleaved Caspase-3的表达情况、炎症相关分子IL-1β与TNF-α、SIRT1信号通路相关蛋白表达情况。结果:与CLP组相比,CLP+HKL组脑组织内SIRT1的表达量及活性、Bcl-2表达量明显增加,而脑组织水含量、凋亡率、Bax、cleaved Caspase-3、IL-1β与TNF-α的表达量明显降低(均P 0.05)。EX527可明显抑制HKL的上述脑保护作用(P 0.05)。结论:和厚朴酚主要通过激活SIRT1/FOXO1信号通路,抑制凋亡与炎症,从而缓解脓毒症脑损伤。     

Protective action of honokiol, administered orally, against oxidative stress in brain of mice challenged with NMDA

Neuroprotective effect of honokiol (HK), orally administered, on oxidative damage in the brain of mice challenged with N-methyl-d-aspartic acid (NMDA) was examined. HK (1-100 mg/kg) was administered to Institute of Cancer Research (ICR) male mice through a gavage for 3 days consecutively, and on the third day, NMDA (150 mg/kg) was intraperitoneally (i.p.) administered. Administration of NMDA, causing a lethality of approximately 60%, resulted in a significant decrease of total glutathione (GSH) level and increase of thiobarbituric acid-reactive substances (TBARS) value in brain tissue. Meanwhile, oral administration of HK (> or = 3 mg/kg) for 3 days reduced the lethality (60%) in NMDA-treated group to 10% level, and alleviated the behavioral signs of NMDA neurotoxicity. Moreover, HK pretreatment restored the levels of total GSH and TBARS in the brain tissue to control levels (p<0.01). Additionally, GSH peroxidase activity in cytosolic portion of brain homogenate was also restored significantly (p<0.01), whereas GSH reductase activity was not. Separately, compared to vehicle-treated control, no significant changes in body and brain weight were observed in mice administered with HK. Based on these results, oral intake of HK is suggested to prevent oxidative stress in the brain of mice.     

Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA

The antioxidant properties of magnolol and honokiol were evaluated in the experimental systems of reducing ONOO⁻ and ¹O₂, bleaching β-carotene in linoleic acid (LH) emulsion, and trapping 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS⁺) and 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH), and then were applied to inhibit the oxidation of DNA induced by Cu²⁺/glutathione (GSH) and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). Magnolol and honokiol were active to reduce ONOO⁻ and ¹O₂. Honokiol showed a little higher activity to protect LH and to inhibit Cu²⁺/GSH-induced oxidation of DNA than magnolol. In addition, honokiol exhibited higher activities to trap ABTS⁺ and DPPH than magnolol. In particular, honokiol trapped 2.5 radicals while magnolol only trapped 1.8 radicals in protecting DNA against AAPH-induced oxidation. The obtained results suggested that low antioxidant ability of magnolol may be related to the intramolecular hydrogen bond formed between di-ortho-hydroxyl groups, which hindered the hydrogen atom in hydroxyl group to be abstracted by radicals. Therefore, the antioxidant capacity of magnolol was lower than that of honokiol.     

中药厚朴中群体感应抑制剂的分离与活性评价

目的:从中药厚朴中分离得到群体感应抑制剂,并对其活性进行评价。方法:利用铜绿假单胞菌QSIS-las I对中药厚朴的粗提物进行活性检测及追踪,采用生物自显影薄层色谱、制备薄层色谱和高效液相色谱技术,分离纯化厚朴中的活性化合物,应用核磁共振波谱解析确定其结构。分别利用分光光度法及地衣酚法测定绿脓菌素和鼠李糖脂的产量,并通过半固体培养基检测铜绿假单胞菌的swarming运动,利用RT-PCR检测与QS调控相关基因的m RNA表达的影响。结果:厚朴粗提物具有群体感应抑制活性,其活性化合物结构鉴定为和厚朴酚。亚抑菌浓度下的和厚朴酚能明显抑制毒力因子如鼠李糖脂和绿脓菌素的产量以及swarming运动,且能够有效抑制QS相关基因m RNA水平的表达(P0.05)。结论:和厚朴酚是一种新的群体感应抑制剂,可能发展成为一种治疗细菌感染的潜在新药。     

Effects of Magnolol and Honokiol on the activities of streptococcal glucosyltransferases both in solution and adsorbed on an experimental pellicle

AIMS: To investigate the inhibitory effects of Magnolol and Honokiol on the activity of streptococcal glucosyltransferases (Gtfs). METHODS AND RESULTS: The effect of Magnolol and Honokiol that inhibits the activities of streptococcal GtfB, GtfC, GtfD and GtfS was explored with standard assays. The results showed that both samples can efficiently inhibit the activity of all Gtfs in solution (66.4-96.3%) and adsorbed on the surface of saliva-coated hydroxyapatite (sHA) beads (65.5-92.7%) at concentrations between 1.25 and 5.0 mg ml(-1). Furthermore, Magnolol had a stronger inhibition of four kinds of Gtfs than Honokiol both in solution and adsorbed on the surface of sHA beads at concentrations between 0.04 and 0.63 mg ml(-1) (P < 0.05). CONCLUSIONS: Magnolol had significant effects on the activities of streptococcal Gtfs. SIGNIFICANCE AND IMPACT OF THE STUDY: Magnolol as a natural herb can be developed into a new oral hygiene product to prevent plaque formation.     

Honokiol inhibits in vitro and in vivo growth of oral squamous cell carcinoma through induction of apoptosis,cell cycle arrest and autophagy

Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down‐regulation of Cdk2 and Cdk4 and the up‐regulation of cell cycle suppressors, p21 and p27. In addition, the caspase‐dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3‐MA or bafilomycin, potentiated the honokiol‐mediated anti‐OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5‐FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5‐FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC‐xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.     

Isolation of anti-Saprolegnia lignans from Magnolia officinalis and SAR evaluation of honokiol/magnolol analogs

To control the fish fungal pathogen Saprolegnia, the effects of the petroleum ether extracts of Magnolia officinalis were evaluated by a rapeseed (Brassicanapus) microplate method in vitro. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, honokiol (C₁₈H₁₈O₂) and magnolol (C₁₈H₁₈O₂) were isolated from Magnolia officinalis. Saprolegnia parasitica growth was inhibited significantly when honokiol concentration was >8.0?mg/L, and magnolol concentration was >9.0?mg/L, with EC₅₀ values of 4.38 and 4.92?mg/L, respectively. Six honokiol and magnolol derivatives were designed, synthesized and evaluated for their anti-Saprolegnia activity. According to the results, double bond and hydroxyl played an important role in inhibiting Saprolegnia. Mechanistically, through the scanning electron microscope observation, honokiol and magnolol could cause the Saprolegnia parasitica mycelium tegumental damage including intensive wrinkles and nodular structures. Moreover, compared to traditional drugs kresoxim-methyl (LC₅₀?=?0.66?mg/L) and azoxystrobin (LC₅₀?=?2.71?mg/L), honokiol and magnolol showed a lower detrimental effect on zebrafish, with the LC₅₀ values of 6.00 and 7.28?mg/L at 48?h, respectively. Overall, honokiol and magnolol were promising lead compounds for the development of commercial drugs anti-Saprolegnia.     

Synthesis and anti-neuroinflammatory activity of N-heterocyclic analogs based on natural biphenyl-neolignan honokiol

Novel isoxazole and pyrazole analogs based on natural biphenyl-neolignan honokiol were synthesized and evaluated for their inhibitory activities against nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells. The isoxazole skeleton was constructed via nitrile oxide cycloaddition from oxime 3 and pyrazole was generated by condensation of 4-chromone and alkylhydrazine. Among the analogs, 13b and 14a showed stronger inhibitory activities with IC₅₀ values of 8.9 and 1.2?µM, respectively, than honokiol.