Geissoschizine

Deciphering and reprogramming the cyclization regioselectivity in bifurcation of indole alkaloid biosynthesis.[Pubmed:36349266]

Chem Sci. 2022 Sep 28;13(42):12389-12395.

The metabolism of monoterpene indole alkaloids (MIAs) is an outstanding example of how plants shape chemical diversity from a single precursor. Here we report the discovery of novel enzymes from the Alstonia scholaris tree, a cytochrome P450, an NADPH dependent oxidoreductase and a BAHD acyltransferase that together synthesize the indole alkaloid akuammiline with a unique methanoquinolizidine cage structure. The two paralogous cytochrome P450 enzymes rhazimal synthase (AsRHS) and Geissoschizine oxidase (AsGO) catalyse the cyclization of the common precursor Geissoschizine and they direct the MIA metabolism towards to the two structurally distinct and medicinally important MIA classes of akuammilan and strychnos alkaloids, respectively. To understand the pathway divergence, we investigated the catalytic mechanism of the two P450 enzymes by homology modelling and reciprocal mutations. Upon conducting mutant enzyme assays, we identified a single amino acid residue that mediates the space in active sites, switches the enzymatic reaction outcome and impacts the cyclization regioselectivity. Our results represent a significant advance in MIA metabolism, paving the way for discovery of downstream genes in akuammilan alkaloid biosynthesis and facilitating future synthetic biology applications. We anticipate that our work presents, for the first time, insights at the molecular level for plant P450 catalytic activity with a significant key role in the diversification of alkaloid metabolism, and provides the basis for designing new drugs.

Protection against H(2)O(2)-evoked toxicity in HT22 hippocampal neuronal cells by geissoschizine methyl ether via inhibiting ERK pathway.[Pubmed:36304098]

Transl Neurosci. 2022 Oct 10;13(1):369-378.

Oxidative stress is considered as an important mechanism underlying the pathology of neurodegenerative disorders. In this study, we utilized an in vitro model where oxidative stress process was evoked by exogenous hydrogen peroxide (H(2)O(2)) in HT22 murine hippocampal neurons and evaluated the neuroprotective effects of Geissoschizine methyl ether (GME), a naturally occurring alkaloid from the hooks of Uncaria rhynchophylla (Miq.) Jacks. After a 24 h H(2)O(2) (350 muM) insult, a significant decrease in cell survival and a sharp increase in intracellular reactive oxygen species were observed in HT22 cells. Encouragingly, GME (10-200 muM) effectively reversed these abnormal cellular changes induced by H(2)O(2). Moreover, mechanistic studies using Western blot revealed that GME inhibited the increase of phospho-ERK protein expression, but not phospho-p38, caused by H(2)O(2). Molecular docking simulation further revealed a possible binding mode that GME inhibited ERK protein, showing that GME favorably bound to ERK via multiple hydrophobic and hydrogen bond interactions. These findings indicate that GME provide effective neuroprotection via inhibiting ERK pathway and also encourage further ex vivo and in vivo pharmacological investigations of GME in treating oxidative stress-mediated neurological disorders.

Network Pharmacology and Molecular Docking to Explore the Mechanism of Kangxian Decoction for Epilepsy.[Pubmed:36193133]

Evid Based Complement Alternat Med. 2022 Sep 23;2022:3333878.

PURPOSE: Kangxian decoction (KXD) has been used in clinical practice to treat epilepsy. The purpose of this study was to explore the active components of KXD and clarify its antiepileptic mechanism through network pharmacology and molecular docking. METHODS: The components of KXD were collected from the Encyclopedia of Traditional Chinese Medicine (ETCM) database and the literature was searched. Then, active ingredients were screened by SwissADME and potential targets were predicted by the SwissTargetPrediction database. Epilepsy-related differentially expressed genes were downloaded from the Gene Expression Omnibus database. A component-target-pathway network was constructed with Cytoscape. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein‒protein interaction network analysis revealed the potential mechanism and critical targets. Receiver operating characteristic (ROC) curves and box plots in microarray data validated the good diagnostic value and significant differential expression of these critical genes. Molecular docking verified the association between active ingredients and essential target proteins. RESULTS: In our study, we screened the important compounds of KXD for epilepsy, including quercetin, baicalin, kaempferol, yohimbine, Geissoschizine methyl ether, baicalein, etc. KXD may exert its therapeutic effect on epilepsy through the following targets: PTGS2, MMP9, CXCL8, ERBB2, and ARG1, acting on the following pathways: neuroactive ligand-receptor interactions, arachidonic acid metabolism, IL-17, TNF, NF-kappa B, and MAPK signaling pathways. The molecular docking results showed that the active ingredients in KXD exhibited good binding ability to the key targets. CONCLUSION: In this study, we explored the possibility that KXD for epilepsy may act on multiple targets through multiple active ingredients, involving neurotransmitters and neuroinflammatory pathways, providing a theoretical basis for subsequent clinical and experimental studies that will help develop effective new drugs to treat epilepsy.

Properties, Pharmacology, and Pharmacokinetics of Active Indole and Oxindole Alkaloids in Uncaria Hook.[Pubmed:34335255]

Front Pharmacol. 2021 Jul 14;12:688670.

Uncaria Hook (UH) is a dry stem with hook of Ucaria plant and is contained in Traditional Japanese and Chinese medicine such as yokukansan, yokukansankachimpihange, chotosan, Gouteng-Baitouweng, and Tianma-Gouteng Yin. UH contains active indole and oxindole alkaloids and has the therapeutic effects on ailments of the cardiovascular and central nervous systems. The recent advances of analytical technology led to reports of detailed pharmacokinetics of UH alkaloids. These observations of pharmacokinetics are extremely important for understanding the treatment's pharmacological activity, efficacy, and safety. This review describes properties, pharmacology, and the recently accumulated pharmacokinetic findings of UH alkaloids, and discusses challenges and future prospects. UH contains major indole and oxindole alkaloids such as corynoxeine, isocorynoxeine, rhynchophylline, isorhynchophylline, hirsuteine, hirsutine, and Geissoschizine methyl ether (GM). These alkaloids exert neuroprotective effects against Alzheimer's disease, Parkinson's disease, and depression, and the mechanisms of these effects include anti-oxidant, anti-inflammatory, and neuromodulatory activities. Among the UH alkaloids, GM exhibits comparatively potent pharmacological activity (e.g., agonist activity at 5-HT(1A) receptors). UH alkaloids are absorbed into the blood circulation and rapidly eliminated when orally administered. UH alkaloids are predominantly metabolized by Cytochrome P450 (CYP) and converted into various metabolites, including oxidized and demethylated forms. Regarding GM metabolism by CYPs, a gender-dependent difference is observed in rats but not in humans. Several alkaloids are detected in the brain after passing through the blood-brain barrier in rats upon orally administered. GM is uniformly distributed in the brain and binds to various channels and receptors such as the 5-HT receptor. By reviewing the pharmacokinetics of UH alkaloids, challenges were found, such as differences in pharmacokinetics between pure drug and crude drug products administration, food-influenced absorption, metabolite excretion profile, and intestinal tissue metabolism of UH alkaloids. This review will provide readers with a better understanding of the pharmacokinetics of UH alkaloids and their future challenges, and will be helpful for further research on UH alkaloids and crude drug products containing UH.

Putative genes in alkaloid biosynthesis identified in Dendrobium officinale by correlating the contents of major bioactive metabolites with genes expression between Protocorm-like bodies and leaves.[Pubmed:34325653]

BMC Genomics. 2021 Jul 29;22(1):579.

BACKGROUND: Dendrobium officinale, an endangered Chinese herb, possesses extensive therapeutic effects and contains bioactive ingredients such as major polysaccharides, alkaloids, and minimal flavonoids. We first obtained the protocorm-like bodies (PLBs) of this plant through tissue culture in order to determine the distribution of the main secondary metabolites in each organelle and the PLBs. We then analyzed the correlation between gene expression level from comparative transcriptome sequencing and metabolite content in different organs to identify putative genes encoding enzymes involved in the biosynthesis of polysaccharides, alkaloids, and flavonoids. RESULTS: We used seeds as explants for protocorm induction and PLB propagation of D. officinale. The optimal medium formula for PLB propagation was 1/2 MS + alpha-NAA 0.5 mg.L(- 1) + 6-BA 1.0 mg.L(- 1) + 2, 4-D 1.5-2.0 mg.L(- 1) + potato juice 100 g.L(- 1). Stems, PLBs and leaves of D. officinale had the highest content of polysaccharides, alkaloids and flavonoids, respectively. Naringenin was only produced in stem; however, PLBs with high alkaloid content can replace other organs producing alkaloids. The hot water extraction method outperformed the ultrasound-assisted extraction method for extracting polysaccharides from D. officinale. A comparative transcriptome analysis of PLBs and leaves of D. officinale revealed differential expression of genes encoding enzymes involved in polysaccharide, alkaloid and flavonoid biosynthetic pathways. Putative genes encoding enzymes involved in these biosynthetic pathways were identified. Notably, we identified genes encoding the alkaloid biosynthesis enzymes strictosidine beta-D-Glucosidase, Geissoschizine synthase and vinorine synthase in D. officinale. CONCLUSIONS: The identification of candidate genes encoding enzymes involved in metabolite biosynthesis will help to explore and protect this endangered species and facilitate further analysis of the molecular mechanism of secondary metabolite biosynthesis in D. officinale.

Chitosan nanoparticles effectively combat salinity stress by enhancing antioxidant activity and alkaloid biosynthesis in Catharanthus roseus (L.) G. Don.[Pubmed:33714144]

Plant Physiol Biochem. 2021 May;162:291-300.

Chitosan nanoparticles (CSNPs) are non-toxic and biodegradable stimulants of growth and secondary metabolite production, which offer new routes to combat plant stress. Salinity is a common and significant abiotic stress that adversely affects plant growth and development. The possible benefits of CSNPs in salt stress mitigation have not yet been reported in Catharanthus roseus, an important source of anticancer alkaloids. Plants were exposed to 150 mM NaCl as a salt stress treatment, while CSNPs were applied as a foliar spray at 1% concentration. Plant growth was considerably impaired under salt stress conditions; however, CSNPs treatment significantly reversed this effect. Specifically, CSNPs retarded chlorophyll reduction and induced activities of catalase, ascorbate peroxidase, and glutathione reductase. Thus, CSNPs alleviated the oxidative stress, indicated by lower levels of malondialdehyde and H(2)O(2), thereby enabling membrane function retention and enhancing salt tolerance. Higher alkaloid accumulation was observed in salt-stressed plants following CSNP spraying than in controls. Interestingly, the expression levels of mitogen-activated protein kinases (MAPK3), Geissoschizine synthase (GS), and octadecanoid-derivative responsive AP2-domain (ORCA3) genes were significantly elevated in salt-stressed plants sprayed with CSNPs. Overall, CSNP treatment overcame the deleterious effects of salinity in C. roseus by activating the antioxidant defense system, which helps to scavenge reactive oxygen species, and inducing expression of MAPK3, GS, and ORCA3 genes, thus, leading to higher alkaloid accumulation and better protection against salinity stress.

A vital role of chitosan nanoparticles in improvisation the drought stress tolerance in Catharanthus roseus (L.) through biochemical and gene expression modulation.[Pubmed:33610861]

Plant Physiol Biochem. 2021 Apr;161:166-175.

Drought is a main abiotic stress that restricts plant growth and development. The increased global demand of anti-cancer alkaloids extracted from periwinkle (Catharanthus roseus) is mainly related to plant growth and development, which are severely affected by drought. Chitosan nanoparticles (CSNPs) have been used to boost plant growth and defense mechanism, however their impact to alleviate drought stress of C. roseus has not been investigated yet. In this study, control and stressed plants (100 and 50% of field capacity [FC], respectively) were subjected to CSNPs application at 1%. Drought stress considerably reduced plant growth, relative water content (RWC), stomatal conductance and total chlorophyll; however, CSNPs mitigated these effects. They enhanced proline accumulation and the activity of catalase (CAT) and ascorbate peroxidase (APX) with possible mitigation of drought-induced oxidative stress. Therefore, they reduced H(2)O(2) and malondialdehyde (MDA) accumulation, and eventually preserved membrane integrity. Drought stress increased alkaloid accumulation, and further increase was observed with the application of CSNPs. High alkaloid content was associated with induced gene expression of strictosidine synthase (STR), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and Geissoschizine synthase (GS) up to 5.6 folds under drought stress, but more accumulation was noticed with the application of CSNPs. Overall, this study is the first on using CSNPs to mitigate drought stress of C. roseus by inducing the antioxidant potential and gene expression of alkaloid biosynthesis.

Palladium catalyzed reductive Heck coupling and its application in total synthesis of (-)-17-nor-excelsinidine.[Pubmed:35423278]

RSC Adv. 2021 Feb 17;11(13):7570-7574.

Monoterpene indole alkaloids, bearing a highly substituted piperidine ring, are a structurally diverse class of bioactive natural products, found in various parts of the world. Herein, we reported the construction of the key piperidine ring via palladium catalyzed reductive Heck coupling with a good syn selective manner, avoiding the usage of stoichiometric, highly toxic, air sensitive and moisture sensitive Ni(COD)(2). To further showcase the value of this methodology, we realized the total synthesis of the structurally unique zwitterionic monoterpene indole alkaloid (-)-17-nor-excelsinidine in 9 steps, in which the key ammonium-acetate connection (N4-C16) of (-)-17-nor-excelsinidine was constructed via oxidative coupling in excellent yield and high regioselectivity under NBS/pyridine from the enolate of Geissoschizine.

Gender differences in plasma pharmacokinetics and hepatic metabolism of geissoschizine methyl ether from Uncaria hook in rats.[Pubmed:32898626]

J Ethnopharmacol. 2021 Jan 10;264:113354.

ETHNOPHARMACOLOGICAL RELEVANCE: Geissoschizine methyl ether (GM), an indole alkaloid from Uncaria hook, is an active ingredient in the traditional Japanese Kampo medicine yokukansan, which is used to treat neurosis, insomnia, irritability, and night crying in children. AIM OF THE STUDY: Recent our pharmacokinetic studies suggested that there may be gender differences in the plasma concentrations of GM in rats, but not in humans. However, the details of this difference remain unverified. The purpose of this study was to clarify the reasons for the gender differences in rats. MATERIALS AND METHODS: GM plasma pharmacokinetics was compared in male and female rats orally administered yokukansan (4 g/kg). To confirm the involvement of cytochrome P450 (CYP) in GM liver metabolism, GM was incubated with male and female rat liver S9 fraction in the absence or presence of 1-aminobenzotriazole (a nonspecific CYP inhibitor). CYP isoforms involved in GM metabolism were estimated using recombinant rat CYP isoforms and anti-rat CYP antibodies. RESULTS: The maximum GM plasma concentrations were significantly higher in female than in male rats. When GM was incubated with rat liver S9 fractions, GM reduction was more striking in male S9 (69.3%) than that in female S9 (10.0%) and was completely blocked with nonspecific CYP inhibitor 1-aminobenzotriazole. Screening experiments using recombinant rat cytochrome P450 (CYP) isoforms showed that CYP1A1, CYP2C6, CYP2C11, CYP2D1, and CYP3A2 were involved in GM metabolism. Of these CYP isoforms, the use of anti-rat CYP antibodies indicated that male-dependent CYP2C11 and CYP3A2 were predominantly involved in the liver microsomal GM metabolism with gender differences. CONCLUSIONS: These results suggest that the cause of gender differences in plasma GM pharmacokinetics in rats is most likely because of male-dependent CYP2C11 and CYP3A2, and provide also useful information to further evaluate the pharmacological and toxicological effects in future. This study is the first to demonstrate that the gender differences in plasma GM pharmacokinetics in rats are caused by the gender-dependent metabolism of GM.

Different carbon sources and their concentrations change alkaloid production and gene expression in Catharanthus roseus shoots in vitro.[Pubmed:32690131]

Funct Plant Biol. 2020 Dec;48(1):40-53.

To compare the effects of different carbon sources on physiological aspects, especially medicinal alkaloid biosynthesis and related gene expression in Catharantus roseus (L.) G.Don, we employed sucrose and sorbitol with two concentrations (87.64 mM, the equimolar concentration of sucrose in MS basal medium, and 150 mM) on the plant's shoots in vitro in presence of 100 muM methyl jasmonate. The production of plant alkaloids including vincristine, vinblastine, ajmalicine, vindoline and catharantine and their biosynthetic and regulatory gene expression was measured. Both treatments had incremental effects on alkaloid production, upregulated the mitogen-activated protein kinase3 (MAPK3) and a downstream responsive transcription factor, ORCA3, which resulted in elevated transcript contents of the important genes in terpenoid indol alkaloids biosynthetic pathway including peroxidase1 (PRX1), Geissoschizine synthase (GS), strictosidine synthase (STR) and deacetylvindoline acetyltransferase (DAT). Defensive responses such as antioxidant enzymes (catalase, peroxidase and superoxide dismutase) activities and non-enzymatic metabolites (total phenolics, flavonoids and carotenoids) contents increased under both treatments but the effects of sorbitol were stronger. Reduced fresh weight and chlorophylls contents, increased malondialdehyde (MDA) and carotenoid contents were shown after a week under all employed treatments. It seems that replacement of sucrose with sorbitol and also, increased concentrations of both carbon sources via increasing osmotic pressure make stressful conditions for the plant especially in longer times.

A Biolistic-Mediated Virus-Induced Gene Silencing in Apocynaceae to Map Biosynthetic Pathways of Alkaloids.[Pubmed:32557364]

Methods Mol Biol. 2020;2172:93-110.

Monoterpene indole alkaloids (MIAs) are specialized metabolites synthesized in many plants of the Apocynaceae family including Catharanthus roseus and Rauvolfia sp. MIAs are part of the chemical arsenal that plants evolved to face pet and herbivore attacks, and their high biological activities also confer pharmaceutical properties exploited in human pharmacopeia. Developing robust and straightforward tools to elucidate each step of MIA biosynthetic pathways thus constitutes a prerequisite to the understanding of Apocynaceae defense mechanisms and to the exploitation of MIA cytotoxicity through their production by metabolic engineering. While protocols of virus-induced gene silencing (VIGS) based on Agrobacterium-based transformation have emerged, the recalcitrance of Apocynaceae to this type of transformation prompted us to develop an universal procedure of VIGS vector inoculation. Such procedure relies on the delivery of the transforming plasmids through a particle bombardment performed using a biolistic device and offers the possibility to overcome host specificity to silence genes in any plant species. Using silencing of Geissoschizine oxidase as an example, we described the main steps of this biolistic mediated VIGS in C. roseus and R. tetraphylla.

Brain distribution of geissoschizine methyl ether in rats using mass spectrometry imaging analysis.[Pubmed:32350314]

Sci Rep. 2020 Apr 29;10(1):7293.

Geissoschizine methyl ether (GM) is one of the main active ingredients responsible for ameliorating the behavioral and psychological symptoms of dementia (BPSD) in Kampo medicine yokukansan. GM is mainly metabolized into hydroxylated forms (HM-1/2). However, the brain distributions of GM and HM has not been reported in vivo. In this study, therefore, the plasma concentrations and brain distribution of these compounds were examined in vivo using rats injected intravenously with GM. Plasma concentrations were analyzed using liquid chromatography-tandem mass spectrometry analysis and brain distribution using mass spectrometry imaging analysis. Plasma GM and HM-1 concentrations decreased in the 4 h after injection, whereas the concentration of plasma HM-2 increased at 4 h. In the 0.25 h-brain, GM signals were diffusely observed throughout the brain, including the cerebral cortex, hippocampus, striatum, thalamus, amygdala, cerebellum, and cerebral ventricle. HM signals were detected only in the ventricles of the brain at 4 h. These results suggest that plasma GM enters the brain and distributes in the parenchyma of various brain regions involved in BPSD, while plasma HM does not enter the brain parenchyma. This study is also the first to visually demonstrate the brain distribution of GM and its metabolite in vivo.

Lipidomics-based study on the neuroprotective effect of geissoschizine methyl ether against oxidative stress-induced cytotoxicity.[Pubmed:32004630]

J Ethnopharmacol. 2020 May 10;253:112636.

ETHNOPHARMACOLOGICAL RELEVANCE: Lipid homoeostasis is important for neurodevelopment, cell signaling and neurotransmission. Alteration of lipid metabolism has been demonstrated in many neurological disorders and neurodegenerative diseases. Geissoschizine methyl ether (GM) is an active alkaloid ingredient in the traditional Chinese medicine Uncaria hook. It has been shown that GM has strong potency in neuroprotective activity and GM reduces the production of reactive oxygen species by regulating glucose metabolism, which protects neurons against oxidative stress-induced cell death. However, it is unknown whether GM could regulate neuronal lipid metabolism during oxidative challenge. AIM OF THE STUDY: The current study aimed to explore whether GM regulates lipid metabolism in oxidative damaged neurons and to determine the underlying mechanism involved in this neuro-protection. MATERIALS AND METHODS: Using a glutamate-induced oxidative toxicity model in mouse hippocampal neuronal cell line (HT-22 cells), we investigated the effect of GM on glutamate-induced lipid peroxidation, lipotoxicity and mitochondrial dysfunction. In order to clarify the mechanism underlying the neuroprotection by GM, lipid metabolomics was performed to investigate whether GM prevent oxidative stress-induced lipid metabolism disruption. Furthermore, the expression of lipid metabolism-related genes was measured. RESULTS: The results show the protective effect of GM against oxidative stress through blocking glutamate-induced lipid peroxidation and lipotoxicity. Overall, lipidomics analysis revealed that glutamate treatment resulted in different extents of changes in a wide range of lipid classes such as fatty acids (FA), triacylglycerol (TG), sphingomyelin (SM), cardiolipin (CL), lysophosphatidylcholines (LPC). However, GM treatment can significantly reverse glutamate-induced lipids disorder to the homeostasis level. GM prevented the disruption of lipid metabolism by regulating the expression of lipid homeostasis related genes, which contributes to preserve mitochondrial function under oxidative damage. CONCLUSION: These findings clearly demonstrated a novel protective mechanism of GM against glutamate-induced oxidative toxicity in neurons via regulating lipid metabolism. GM may provide an effective approach for the prevention and treatment of oxidative damaged neurons.

Antiepileptic geissoschizine methyl ether is an inhibitor of multiple neuronal channels.[Pubmed:31911638]

Acta Pharmacol Sin. 2020 May;41(5):629-637.

Geissoschizine methyl ether (GM) is an indole alkaloid isolated from Uncaria rhynchophyll (UR) that has been used for the treatment of epilepsy in traditional Chinese medicine. An early study in a glutamate-induced mouse seizure model demonstrated that GM was one of the active ingredients of UR. In this study, electrophysiological technique was used to explore the mechanism underlying the antiepileptic activity of GM. We first showed that GM (1-30 mumol/L) dose-dependently suppressed the spontaneous firing and prolonged the action potential duration in cultured mouse and rat hippocampal neurons. Given the pivotal roles of ion channels in regulating neuronal excitability, we then examined the effects of GM on both voltage-gated and ligand-gated channels in rat hippocampal neurons. We found that GM is an inhibitor of multiple neuronal channels: GM potently inhibited the voltage-gated sodium (Na(V)), calcium (Ca(V)), and delayed rectifier potassium (I(K)) currents, and the ligand-gated nicotinic acetylcholine (nACh) currents with IC(50) values in the range of 1.3-13.3 mumol/L. In contrast, GM had little effect on the voltage-gated transient outward potassium currents (I(A)) and four types of ligand-gated channels (gamma-amino butyric acid (GABA), N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainite (AMPA/KA receptors)). The in vivo antiepileptic activity of GM was validated in two electricity-induced seizure models. In the maximal electroshock (MES)-induced mouse seizure model, oral administration of GM (50-100 mg/kg) dose-dependently suppressed generalized tonic-clonic seizures. In 6-Hz-induced mouse seizure model, oral administration of GM (100 mg/kg) reduced treatment-resistant seizures. Thus, we conclude that GM is a promising antiepileptic candidate that inhibits multiple neuronal channels.