Rotenoids and Other Specialized Metabolites from the Roots of Mirabilis multiflora: Opioid and Cannabinoid Receptor Radioligand Binding Affinities.

Mirabilis multiflora is an acclaimed hallucinogen consumed traditionally by the Hopi Indians to induce diagnostic visions. Its root extract afforded a new (3) and four known (2, 5, 6, and 7) 12a-hydroxyrotenoids, a known rotenoid (4), and two known secondary metabolites (1 and 8). The structures of the compounds were elucidated based on spectroscopic and spectrometric data analysis. Electronic circular dichroism data were used to define the (6aS,12aR) absolute configuration of the 12a-hydroxyrotenoids. Compounds 2-7 were screened for their radioligand binding affinities toward the opioid (δ, κ, and µ) and cannabinoid (CB1 and CB2) receptor subtypes. The 6-methoxy-substituted rotenoids 3, 4, and 7 showed the highest receptor binding affinity with moderate selectivity toward the δ-opioid receptor subtype, with negligible binding affinities for CB1 and CB2. Their binding affinities toward the δ-opioid receptor were 64.5% (4), 58.7% (7), and 55.3% (3) at 10 µM, respectively.

Involvement of the Benzodiazepine Site in the Anticonvulsant Activity of Tapinanthus globiferus against Pentylenetetrazole-induced Seizures in Mice.

Tapinanthus globiferus is often referred to as an all-purpose herb for the treatment of stroke and epilepsy. The present study investigates the anticonvulsant effect of methanolic leaf extract, active fractions, and lupeol (isolate) of Tapinanthus globiferus in mice as well as the underlying mechanisms. Following phytochemical studies of T. globiferus, preliminary assays were performed to evaluate MLE-induced toxic effect and behavioral changes. The pentylenetetrazol (70 mg/kg, i. p.)-induced seizure was evaluated in mice that were pretreated orally with vehicle 10 mL/kg, MLE (4, 20, or 100 mg/kg), fractions (F1 to F6), lupeol 10 mg/kg or diazepam (3 mg/kg). Methanolic leaf extract preserved neuron viability as well as the relative organ weight, and hematological and biochemical parameters. The behavioral endpoints, neuromuscular coordination, and sensory response parameters revealed a dose-dependent effect of methanolic leaf extract. This extract, active fractions, lupeol, and diazepam potentiated the hypno-sedative effect of the barbiturate and attenuated PTZ-induced acute seizure. This antiseizure effect was completely reversed by flumazenil 2 mg/kg (benzodiazepine site antagonist). Altogether, the benzodiazepine site-mediated anticonvulsant effects of methanolic leaf extract, active fractions, and lupeol corroborate traditional application of T. globiferus against epilepsy.

LC-MS-Guided Isolation of Insulin-Secretion-Promoting Monoterpenoid Carbazole Alkaloids from Murraya microphylla.

Fifteen new structurally unique monoterpenoid carbazole alkaloids, including two pairs of epimers (1/2 and 3/4), three pairs of enantiomers (6a/6b, 7a/7b, and 8a/8b), and five optically pure analogues (5, 9-12), were obtained from a 95% aqueous EtOH extract of Murraya microphylla by a combination of bioassay- and LC-MS-guided fractionation procedures. Their structures were established based on NMR and HRESIMS data interpretation. The absolute configuration of compound 1 was determined via X-ray crystallographic data analysis and for all compounds by comparison of experimental and calculated ECD data. Compounds 1-5 were assigned as five new thujane-carbazole alkaloids, and compounds 6-12 as 10 new menthene-carbazole alkaloids linked through an ether or carbon-carbon bond. Compounds 1-12 promoted insulin secretion in the HIT-T15 cell line, 1.9-3.1-fold higher than the gliclazide control at 100 µM.

Anti-inflammatory Dimeric 2-(2-Phenylethyl)chromones from the Resinous Wood of Aquilaria sinensis.

Sixteen new 2-(2-phenylethyl)chromone dimers, including four pairs of enantiomers (1a/1b, 3a/3b, 6a/6b, and 8a/8b), along with eight optically pure analogues (2, 4, 5, 7, and 9-12) were isolated from the resinous wood of Aquilaria sinensis. Their structures were determined by extensive spectroscopic analysis (1D and 2D NMR, UV, IR, and HRMS) and experimental and computed ECD data. Compounds 1-10 feature an unusual 3,4-dihydro-2 H-pyran ring linkage connecting two 2-(2-phenylethyl)chromone monomeric units, while compounds 11 and 12 possess an unprecedented 6,7-dihydro-5 H-1,4-dioxepine moiety in their structures. A putative biosynthetic pathway of the representative structures via a diepoxy derivative of a chromone with a nonoxygenated A-ring is also proposed. Compounds 1a/1b, 2, 3a/3b, 5, 7, 8a/8b, and 10-12 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values in the range 7.0-12.0 µM.

ANTIFUNGAL ACTIVITY OF THE ROOT EXTRACTS OF PULSATILLA PATENS AGAINST CANDIDA GLABRATA.

Plants from the genus of Pulsatilla produce a variety of secondary metabolites with biological activity. These species play a special role in herbal medicine and are used in traditional folk medicine to treat many diseases and ailments. Due to their numerous medicinal properties, they are now also widely used as homeopathic preparations. In the present study, the antifungal activity of crude extracts of the root of Pulsatilla patens (L.) Mill. against the yeast Candida glabrata with an IC50 of 9.37 µg/mL is reported.

The hallucinogenic diterpene salvinorin A inhibits leukotriene synthesis in experimental models of inflammation.

Leukotrienes (LTs) are lipid mediators derived from arachidonic acid (AA) involved in a number of autoimmune/inflammatory disorders including asthma, allergic rhinitis and cardiovascular diseases. Salvinorin A (SA), a diterpene isolated from the hallucinogenic plant Salvia divinorum, is a well-established analgesic compound, but its anti-inflammatory properties are under-researched and its effects on LT production is unknown to date. Here, we studied the possible effect of SA on LT production and verified its actions on experimental models of inflammation in which LTs play a prominent role. Peritoneal macrophages (PM) stimulated by calcium ionophore A23187 were chosen as in vitro system to evaluate the effect of SA on LT production. Zymosan-induced peritonitis in mice and carrageenan-induced pleurisy in rats were selected as LT-related models to evaluate the effect of SA on inflammation as well as on LT biosynthesis. SA inhibited, in a concentration-dependent manner, A23187-induced LTB4 biosynthesis in isolated PM. In zymosan-induced peritonitis, SA inhibited cell infiltration, myeloperoxidase activity, vascular permeability and LTC4 production in the peritoneal cavity without decreasing the production of prostaglandin E2. In carrageenan-induced pleurisy in rats, a more sophisticated model of acute inflammation related to LTs, SA significantly inhibited LTB4 production in the inflammatory exudates, along with reducing the phlogistic process in the lung. In conclusion, SA inhibited LT production and it was effective in experimental models of inflammation in which LTs play a pivotal role. SA might be considered as a lead compound for the development of drugs useful in LTs-related diseases.

Nitric Oxide Inhibitory Meroterpenoids from the Fungus Penicillium purpurogenum MHZ 111.

Five new meroterpenoids, purpurogenolides A-E (1-5), and four known metabolites (6-9) were isolated from the solid substrate fermentation cultures of the fungus Penicillium purpurogenum MHz 111. The structures of the new meroterpenoids were elucidated by analysis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of 1 and 5 were determined by single-crystal X-ray crystallographic analysis, and those of 2-4 were elucidated on the basis of experimental and calculated electronic circular dichroism spectra. Compounds 2-4 and 6 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 0.8-30.0 µM.

Nitric Oxide Inhibitory Dimeric Sesquiterpenoids from Artemisia rupestris.

Twelve new dimeric sesquiterpenoids (1-12) were isolated from the dried whole plants of Artemisia rupestris. Their structures were determined using MS and NMR data, and the absolute configurations were elucidated on the basis of experimental and calculated ECD spectra. Compounds 1-9 are presumably formed via biocatalyzed [2+2] or [4+2] cycloaddition reactions. Stereoselectivity of the [4+2] Diels-Alder reaction dictated the formation of endo-products. The dimeric sesquiterpenoids exhibited moderate inhibition on NO production stimulated by lipopolysaccharide in BV-2 microglial cells, with IC50 values in the range 17.0-71.8 µM.

The G protein-biased κ-opioid receptor agonist RB-64 is analgesic with a unique spectrum of activities in vivo.

The hypothesis that functionally selective G protein-coupled receptor (GPCR) agonists may have enhanced therapeutic benefits has revitalized interest for many GPCR targets. In particular, although κ-opioid receptor (KOR) agonists are analgesic with a low risk of dependence and abuse, their use is limited by a propensity to induce sedation, motor incoordination, hallucinations, and dysphoria-like states. Several laboratories have produced a body of work suggesting that G protein-biased KOR agonists might be analgesic with fewer side effects. Although that has been an intriguing hypothesis, suitable KOR-selective and G protein-biased agonists have not been available to test this idea. Here we provide data using a G protein-biased agonist, RB-64 (22-thiocyanatosalvinorin A), which suggests that KOR-mediated G protein signaling induces analgesia and aversion, whereas ß-arrestin-2 signaling may be associated with motor incoordination. Additionally, unlike unbiased KOR agonists, the G protein-biased ligand RB-64 does not induce sedation and does not have anhedonia-like actions, suggesting that a mechanism other than G protein signaling mediates these effects. Our findings provide the first evidence for a highly selective and G protein-biased tool compound for which many, but not all, of the negative side effects of KOR agonists can be minimized by creating G protein-biased KOR agonists.

Anti-inflammatory coumarin and benzocoumarin derivatives from Murraya alata.

Two new rare 8-methylbenzo[h]coumarins, muralatins A and B (1, 2), nine new C-8-substituted coumarins, muralatins C-K (3-11), and 22 known analogues (12-33) were isolated from the leaves of Murraya alata. The absolute configurations of compounds 5, 11, 23, 24, 27, 30, and 33 were assigned via comparison of their specific rotations, by Mosher's method, and by single-crystal X-ray diffraction and electronic circular dichroism (ECD) data of the in situ formed transition metal complexes. A putative biosynthesis pathway to 1 and 2 is proposed, and the chemical synthesis of 1 was accomplished through electrocyclization of 5,7-dimethoxy-8-[(Z)-3-methylbut-1,3-dienyl)]coumarin (12). Compounds 1, 2, 8, 12, and 31 showed inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 values of 6.0-14.5 µM.

Anti-inflammatory Labdane Diterpenoids from Leonurus macranthus.

Twenty new polyoxygenated labdane diterpenoids (1-20) were isolated from the aerial parts of Leonurus macranthus. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of macranthin A (1) and 6-O-deacetylmacranthin A (2) were determined by single-crystal X-ray crystallographic analysis and a modified Mosher's method, respectively. Compounds 1-9, 12, 14, and 19 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 10.0-63.7 µM.

IDENTIFICATION CHALLENGES IN EXAMINATION OF COMMERCIAL PLANT MATERIAL OF PSYCHOTRIA VIRIDIS.

Psychoria viridis (chacruna) is a hallucinogenic plant with psychoactive properties associated with the presence of N,N-dimethyltryptamine (DMT). This species is primarily known as an ingredient of the beverage Ayahuasca, but dry leaves are also smoked by recreational users. The plant is controlled in Poland and France and its proper identification poses many challenges due to the fact that genus Psychotria is relatively large and there are other species that are easily confused with chacruna. The aim of the present work was to develop an effective authentication procedure for the dried and shredded leaves of P. viridis, to be used in comparison of chemical and botanical characteristics of its commercial products. Dried leaves of P. viridis originating from Brazil, Peru and Hawaii were purchased from Internet providers. For DMT identification, thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) methods have been elaborated, validated and applied. In order to clarify the existing differences among samples, chemometric methods have been used. Botanical features and the gas chromatography tandem mass spectrometry (GC-MS) chromatograms have been analyzed using hierarchical cluster analysis (HCA). Our studies revealed significant variety among plant material marketed as P. viridis. Grouping of samples based on their micromorphology features and GC-MS results did not correspond well with the presence of DMT. Based on our results an indisputable identification of dried specimens as P. viridis is very problematic. It is necessary to postulate changes in legislation regarding regulation of P. viridis and replace it with DMT as controlled substance.

Identification of novel functionally selective κ-opioid receptor scaffolds.

The κ-opioid receptor (KOR)-dynorphin system has been implicated in the control of affect, cognition, and motivation, and is thought to be dysregulated in mood and psychotic disorders, as well as in various phases of opioid dependence. KOR agonists exhibit analgesic effects, although the adverse effects produced by some KOR agonists, including sedation, dysphoria, and hallucinations, have limited their clinical use. Interestingly, KOR-mediated dysphoria, assessed in rodents as aversion, has recently been attributed to the activation of the p38 mitogen-activated protein kinase pathway following arrestin recruitment to the activated KOR. Therefore, KOR-selective G protein-biased agonists, which do not recruit arrestin, have been proposed to be more effective analgesics, without the adverse effects triggered by the arrestin pathway. As an initial step toward identifying novel biased KOR agonists, we applied a multifaceted screening strategy utilizing both in silico and parallel screening approaches. We identified several KOR-selective ligand scaffolds with a range of signaling bias in vitro. The arylacetamide-based scaffold includes both G protein- and ß-arrestin-biased ligands, while the endogenous peptides and the diterpene scaffolds are G protein biased. Interestingly, we found scaffold screening to be more successful than library screening in identifying biased ligands. Many of the identified functionally selective ligands are potent selective KOR agonists that are reported to be active in the central nervous system. They therefore represent excellent candidates for in vivo studies aiming at determining the behavioral effects mediated by specific KOR-mediated signaling cascades.

Anti-inflammatory ursane- and oleanane-type triterpenoids from Vitex negundo var. cannabifolia.

Six new polyoxygenated triterpenoids, cannabifolins A-F (1-6), and eight known triterpenoids, 7-14, were isolated from the leaves of Vitex negundo var. cannabifolia. The absolute configuration of cannabifolin A (1) was determined by single-crystal X-ray crystallographic analysis. Compounds 1 and 2 represent a class of rare natural pentacyclic triterpenoids bearing cis-fused C/D rings and are the first examples of 12,19-epoxy ursane- and oleanane-type triterpenoids. Compounds 3, 7, 8, and 14 exhibited inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophages with IC50 values in the range 24.9-40.5 µM.

Anti-inflammatory labdane diterpenoids from Lagopsis supina.

Ten new labdane diterpenoids, lagopsins A-H (1-3, 5, 7-10) and 15-epi-lagopsins C and D (4, 6), together with five known labdane diterpenoids (11-15), were isolated from the whole plants of Lagopsis supina. The absolute configuration of lagopsin A (1) was determined by single-crystal X-ray crystallographic analysis. Compounds 7, 9, 13, and 15 exhibited moderate inhibition of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells with IC50 values in the range 14.9-34.9 µM.

Kappa-opioid receptor-selective dicarboxylic ester-derived salvinorin A ligands.

Salvinorin A, the active ingredient of the hallucinogenic plant Salvia divinorum is the most potent known naturally occurring hallucinogen and is a selective κ-opioid receptor agonist. To better understand the ligand-receptor interactions, a series of dicarboxylic ester-type of salvinorin A derivatives were synthesized and evaluated for their binding affinity at κ-, δ- and µ-opioid receptors. Most of the analogues show high affinity to the κ-opioid receptor. Methyl malonyl derivative 4 shows the highest binding affinity (Ki=2nM), analogues 5, 7, and 14 exhibit significant affinity for the κ-receptor (Ki=21, 36 and 39nM).

In vitro structure-activity relationships of aplysinopsin analogs and their in vivo evaluation in the chick anxiety-depression model.

Aplysinopsins are tryptophan-derived natural products that have been isolated from a variety of marine organisms and have been shown to possess a range of biological activities. In vitro receptor binding assays showed that of the 12 serotonin receptor subtypes, analogues showed a high affinity for the 5-HT2B and 5-HT2C receptor subtypes, with selectivity for 5-HT2B over 5-HT2C. While no conclusions could be drawn about the number and position of N-methylations, bromination at C-4 and C-5 of the indole ring resulted in greater binding affinities, with Ki's as low as 35 nM. This data, combined with previous knowledge of the CNS activity of aplysinopsin analogs, suggested that these compounds may have potential as leads for antidepressant drugs. Compounds 3c, 3u, and 3x were evaluated in the chick anxiety-depression model to assess their in vivo efficacy. Compound 3c showed a modest antidepressant effect at a dose of 30 nM/kg in the animal model.

Triterpenoids and flavonoids from Cecropia schreberiana Miq. (Urticaceae).

Phytochemical investigation of the leaves of Cecropia schreberiana Miq. (Urticaceae) led to the isolation of four triterpenoids (1-4), three flavone C-glycosides (5-7), two flavan-3-ols (8, 9), two flavanolignans (10, 11), and two proanthocyanidins (12, 13). All compounds were isolated from C. schreberiana for the first time. This is the first report demonstrating the presence of arjunolic acid (4), cinchonain Ia (10), and cinchonain Ib (11) in the Urticaceae family. The occurrence of flavanolignans within the family Urticaceae supports the likelihood that such compounds are more common within the class Magnoliopsida than previously thought.

Heteroaromatic salvinorin A analogue (P-3 l) elicits antinociceptive and anxiolytic-like effects.

Previous studies have attributed the prominent analgesic, hallucinogenic, sedative, and anxiolytic properties of Salvia divinorum to Salvinorin A. However, the overall pharmacological profile of this isolate limits its clinical applications. To address these limitations, our study evaluates the C(22)-fused-heteroaromatic analogue of salvinorin A [2-O-salvinorin B benzofuran-2-carboxylate] (P-3l) in mice nociception and anxiety models while assessing possible mechanism of action. In comparison with the control group, orally administered P-3l (1, 3, 10, and 30 mg/kg) attenuates acetic acid-induced abdominal writhing, formalin-induced hind paw licking, the thermal reaction to the hotplate, and/or aversive response in the elevated plus-maze, open field, and light-dark box; and potentiates the effect of morphine and diazepam at sub-effective doses (1.25 and 0.25 mg/kg, respectively) without eliciting significant alterations in relative organ weight, or haematological or biochemical parameters. The in vivo blockade of P-3 l effects by naloxone (non-selective opioid receptor antagonist), naloxonazine (antagonist of specific subtypes mu1 of µ-OR), and nor-binaltorphimine (selective ĸ-OR antagonist) supports initial results from binding assays and the interpretations made possible from computational modeling of the interactions of P-3 l with the opioid receptor subtypes. In addition to the opioidergic mechanism, the blockade of the P-3 l effect by flumazenil suggests benzodiazepine binding site involvement in its biological activities. These results support P-3 l potentially possessing clinical utility and substantiate the need for additional pharmacological characterization.

Salvinorin A reduces mechanical allodynia and spinal neuronal hyperexcitability induced by peripheral formalin injection.

BACKGROUND: Salvinorin A (SA), the main active component of Salvia Divinorum, is a non-nitrogenous kappa opioid receptor (KOR) agonist. It has been shown to reduce acute pain and to exert potent antinflammatory effects. This study assesses the effects and the mode of action of SA on formalin-induced persistent pain in mice. Specifically, the SA effects on long-term behavioural dysfuctions and changes in neuronal activity occurring at spinal level, after single peripheral formalin injection, have been investigated. Moreover, the involvement of microglial and glial cells in formalin-induced chronic pain condition and in SA-mediated effects has been evaluated. RESULTS: Formalin induced a significant decrease of mechanical withdrawal threshold at the injected and contralateral paw as well as an increase in the duration and frequency, and a rapid decrease in the onset of evoked activity of the nociceptive neurons 7 days after formalin injection. SA daily treatment significantly reduced mechanical allodynia in KOR and cannabinoid receptor 1 (CB1R) sensitive manner. SA treatment also normalized the spinal evoked activity. SA significantly reduced the formalin-mediated microglia and astrocytes activation and modulated pro and anti-inflammatory mediators in the spinal cord. CONCLUSION: SA is effective in reducing formalin-induced mechanical allodynia and spinal neuronal hyperactivity. Our findings suggest that SA reduces glial activation and contributes in the establishment of dysfunctions associated with chronic pain with mechanisms involving KOR and CB1R. SA may provide a new lead compound for developing anti-allodynic agents via KOR and CB1R activation.