Major Alkaloid Research Articles

Hydrogen sulfide prevents arecoline-induced neurotoxicity via promoting leptin/leptin receptor signaling pathway.

Arecoline, a major alkaloid of the areca nut, has potential toxicity to the nervous system. Our previous study reveals that the neurotoxicity of arecoline involves ininhibited endogenous hydrogen sulfide (H2 S) generation. Therefore, the present study investigated whether exogenous H2 S protects against arecoline-induced neurotoxicity and further explore the underlying mechanisms focusing on leptin/leptin receptor signaling pathway. The cell viability was measured by CCK-8 kit. The apoptosis were detected by Hoechst 33258 and Annexin V/PI (propidium iodide) staining. The protein expressions were determined by Western blot analysis. Our results demonstrated that NaHS, an exogenous H2 S donor, significantly increases the cell viability, decreases apoptosis ratio, and reduces caspase-3 activity as well as Bax/Bcl-2 ratio in PC12 cells exposed to arecoline, indicating the protection of H2 S against arecoline-induced cytotoxicity and apoptosis. Also, NaHS attenuated arecoline-induced endoplasmic reticulum (ER) stress, as evidenced by the decreases in the expressions of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and Cleaved caspase-12. Meanwhile, NaHS promoted leptin/leptin receptor signaling pathway in arecoline-exposed PC12 cells, as illustrated by upregulations of leptin and leptin receptor expressions. Furthermore, leptin tA, an antagonist of leptin receptor, obviously abolished the inhibitory effects of NaHS on arecoline-induced cytotoxicity, apoptosis, and ER stress in arecoline-exposed PC12 cells. Taken together, these results suggested that H2 S prevents arecoline-induced neurotoxicity via enhancing the leptin/leptin receptor signaling pathway.

Exploration by molecular networking of Strychnos alkaloids reveals the unexpected occurrence of strychnine in seven Strychnos species

IntroductionPlants of the Strychnos genus, which include about 200 species, are used for multiple traditional purposes as hunting poison, for example, and have shown interesting pharmacological properties, especially curarizing and tetanizing, but also against malaria. Many monoterpene indole alkaloids have already been isolated and identified. Among them, there is strychnine, a famous alkaloid that can cause death by asphyxiation. ObjectiveInvestigate alkaloidic molecular diversity from Strychnos genus using molecular networking technique and study the Strychnos genus from a chemotaxonomic point of view. Material and methodsTwenty-eight different species and different plant parts were ground into powder using a grinder. The methanolic extracts were carried out using a pressurized solvent extraction and the alkaloid extract was performed manually with a separating funnel. The extracts were analyzed by HPLC-ESI(+)-Q/TOF. The data were processed using MZmine 2 software and the molecular network was generated on the GNPS platform. The study of the generated molecular network allowed the detection of various alkaloids. Among these is the famous strychnine which has been detected in 7 new Strychnos species not yet described as strychnine producers. This identification was investigated using orthogonal approaches, namely TLC, NMR, HPLC-UV and UHPLC-ESI(+)-Q/TOF analyses. The LOD by HPLC-UV of strychnine was also determined. ResultsFurther analyses allowed to confirm the presence of strychnine in S. densiflora trunk barks but also to show the presence of strychnine with high probability in the trunk barks of S. camptoneura, S. congolana, S. boonei, and S. tchibangensis, and in the leaves of S. usambarensis. About the trunk barks of S. tricalyisoides, the probability of a strychnine content remains low. ConclusionThis work exemplified the efficiency of molecular networking in identifying known metabolites (major and minor alkaloids) involved in the chemotaxonomic study of plants from Strychnos genus.

Biotransformation and pharmacokinetic studies of four alkaloids from Uncaria rhynchophylla in rat plasma by ultra-performance liquid chromatography with tandem mass spectrometry

Uncaria rhynchophylla (Miq.) Miq. ex Havil (U. rhynchophylla), a traditional Chinese medicine that has been officially included in the Chinese Pharmacopeia, is used to treat cardiovascular and central nervous system diseases. The major alkaloids isolated from U. rhynchophylla are two pairs of epimer including rhynchophylline (RIN) and isorhynchophylline (IRN), along with corynoxeine (CN) and isocorynoxeine (ICN). An ultra-performance liquid chromatography with tandem mass spectrometry method (UHPLC–MS/MS), which was highly accurate, stable and sensitive, was established and validated for the simultaneous determination of four alkaloid compounds (RIN, IRN, CN, ICN) in rat plasma samples after oral administration of RIN, IRN, CN, ICN and U. rhynchophylla extract. In this study, the biotransformation and pharmacokinetics of RIN, IRN, CN and ICN were determined for the first time. An ACQUITY UPLC®HSS T3 column (1.8 µm, 2.1 mm × 100 mm) was used to complete the chromatographic separation within 3 min. The isocratic mobile phase was composed of 0.1 % formic acid water (v/v) and acetonitrile, and the flow rate was 0.2 mL/min. The multireaction monitoring mode was adopted, and the tandem mass spectrometry in the positive ion mode was detected by the electrospray ionization source. The method was fully verified and linear at a wide concentration (r > 0.9913), and the linear concentration range was 0.1552–124.5 ng/mL. The intraday and interday precisions of the four analytes were lower than 8.20 % and 13.42 %, respectively. The accuracy range was − 2.64 % and 13.63 %. The extraction recoveries of the analytes exceeded 83.74 %, and the matrix effect range was 98.43–103.7 % in the plasma samples. The four alkaloids can be quickly absorbed into the blood (Tmax, 0.22–3.83 h) and cleared comparatively slowly (T1/2, 7.67–12.13 h). This method has been successfully applied to the biotransformation and pharmacokinetic studies of SD rat plasma after oral administration of U. rhynchophylla extracts. This proved that RIN with IRN and CN with ICN can transform into each other in vivo. The results are of great significance for determining the mechanism of action and guiding the clinical application of U. rhynchophylla extracts.

Preliminary Study of Isolation and Purification Mitragynine from Kratom Leaves

Kratom (Mitragyna speciosa Korth) leaves contain a major alkaloid, mitragynine, which has opioid pain-relieving activity. Kratom and its derivatives are abused as alternatives to other narcotics. Therefore, this preliminary study was aimed to optimize the extraction of mitragynine from kratom investigating the effect of extraction time and ultrasonication on yield and quality of extract (the content of mitragynine). The extraction was carried out sequential extraction at room temperature and treatment of ultrasonication before extraction which was followed the purification process. Ultrasound-assisted extraction increased mitragynine yield to16.88%, compared to extraction without ultrasonication. The extract was purified by column chromatography, eluted with a mixture of n-hexane - ethyl acetate - 25% ammonia with a ratio of 30:15:1 (v/v). Likewise, the highest mitragynine purity obtained was 86.46%.

Correlation patterns prevalence, and co-occurrence of ergot alkaloids in cool-season adapted cereal grains revealed with molecular spectroscopy and LC-MS/MS equipped HPLC system

It is necessary to obtain more recent data on the prevalence and co-occurrence of mycotoxins in feed and food to minimize risks. This study examined the recent presence, co-occurrence, and correlation patterns of six major ergot alkaloids (EAs; i.e. ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) in cool-season adapted barley (n = 57) and wheat (n = 80) submitted by livestock producers and industries for testing ergot alkaloids/mycotoxins by liquid chromatography − tandem mass spectrometry method. Overall, 91% industry-submitted barley samples and 84% industry-submitted wheat samples tested positive for at least one ergot alkaloid and 33% industry-submitted barley and 38% industry-submitted wheat samples were found to be co-contaminated with all six major EAs. The content of total EAs in 9 industry-submitted barley (16%) and 18 industry-submitted wheat (23%) samples exceeded the recommended maximum allowable level for lactating or pregnant animals (250 ppb). All the barley and wheat samples that contained detectable ergosine were found to co-occur with other EAs. Overall, the content of individual EAs was positively correlated with each other and strong correlations (r > 0.8, P < 0.01) were detected between the content of individual EAs and total EAs. These results implied that the industry and producers submitted cool-season adapted barley and wheat samples contaminated with a single EA is likely to contain high levels of other major EAs. The patterns of individual EAs in this study were distinct from previous studies that focus on samples from European countries. Ergocristine was remained as the predominant EAs in the industry-submitted cool-season adapted barley and wheat samples at levels up to 9438.8 and 12416.2 ppb, respectively. While the mean contents of ergosine were the lowest (68.5 and 50.6 ppb for the industry-submitted cool-season adapted barley and wheat samples, respectively). The high prevalence and co-occurrence of EAs indicated that ergot contamination is still posing a significant threat to food and feed industry and more research is expected to reduce the contamination level and explore the toxicological significance of various co-occurrence profiles. A full scale of investigation for all barley and wheat samples is needed to obtain a full picture of mycotoxin contamination.

Standardization of the ethanolic extract of Crinum latifolium leaves by two bioactive markers with antiproliferative activity against TGF-β-promoted prostate stromal cells (WPMY-1)

BackgroundCrinum latifolium L. (Amaryllidaceae) has been used in Southeast Asian traditional medicine to alleviate the symptoms of benign prostatic hyperplasia (BPH). The pathological mechanism of BPH is associated with the induction of prostate stromal cell proliferation through transforming growth factor-beta (TGF-β). Standardization as well as investigation of the potential anti-BPH activity of C. latifolium extract could benefit the further development of BPH-related analyses and provide evidence to support the application of this extract for BPH treatment. This study aimed to standardize and investigate the antiproliferative activity of the ethanolic extract of C. latifolium leaves. The major alkaloids isolated from C. latifolium were also explored for their potential use as bioactive markers.MethodsTwo major alkaloids were isolated from the ethanolic extract of C. latifolium leaves by chromatographic techniques, identified by NMR and MS, and quantified by a validated UHPLC method. Their antiproliferative activity was studied in human prostate stromal cells (WPMY-1) induced by TGF-β. The synergistic effect of combining the two major isolated alkaloids was analyzed by the zero interaction potency (ZIP) model.ResultsTwo alkaloids, lycorine (1) and 6α-hydroxybuphanidrine (2), were isolated from the ethanolic leaf extract of C. latifolium. A UHPLC method for the quantification of (1) and (2) was developed and validated in terms of linearity, precision, and accuracy. The C. latifolium leaf extract contained 0.279 ± 0.003% (1) and 0.232 ± 0.004% (2). The crude extract was more potent than either (1) and (2) alone against TGF-β-treated WPMY-1 cell proliferation. The drug combination study revealed that the greatest synergistic effect of (1) and (2) was achieved at a 1:1 ratio.ConclusionsThe results of this study support the anti-BPH activity of C. latifolium in traditional medicine and suggest that these the two isolated alkaloids may promote the efficacy of the C. latifolium extract. Additionally, major alkaloids (1) and (2) can be used as bioactive markers for the standardization of C. latifolium extracts.

Assessment of Bioactivity-Modulating Pseudo-Ring Formation in Psilocin and Related Tryptamines.

Psilocybin (1) is the major alkaloid found in psychedelic mushrooms and acts as a prodrug to psilocin (2, 4‐hydroxy‐N,N‐dimethyltryptamine), a potent psychedelic that exerts remarkable alteration of human consciousness. In contrast, the positional isomer bufotenin (7, 5‐hydroxy‐N,N‐dimethyltryptamine) differs significantly in its reported pharmacology. A series of experiments was designed to explore chemical differences between 2 and 7 and specifically to test the hypothesis that the C‐4 hydroxy group of 2 significantly influences the observed physical and chemical properties through pseudo‐ring formation via an intramolecular hydrogen bond (IMHB). NMR spectroscopy, accompanied by quantum chemical calculations, was employed to compare hydrogen bond behavior in 4‐ and 5‐hydroxylated tryptamines. The results provide evidence for a pseudo‐ring in 2 and that sidechain/hydroxyl interactions in 4‐hydroxytryptamines influence their oxidation kinetics. We conclude that the propensity to form IMHBs leads to a higher number of uncharged species that easily cross the blood‐brain barrier, compared to 7 and other 5‐hydroxytryptamines, which cannot form IMHBs. Our work helps understand a fundamental aspect of the pharmacology of 2 and should support efforts to introduce it (via the prodrug 1) as an urgently needed therapeutic against major depressive disorder.

Sanguinarine, a major alkaloid from Zanthoxylum nitidum (Roxb.) DC., inhibits urease of Helicobacter pylori and jack bean: Susceptibility and mechanism

Ethnopharmacological relevanceZanthoxylum nitidum (Roxb.) DC. (Z. nitidum) is a traditional Chinese medicine and mainly adopted to treat gastric ulcer, gastritis and stomach cancer. Sanguinarine (SNG), a natural alkaloid isolated from Z. nitidum, possesses significant anti-Helicobacter pylori and gastric protection effects. However, the underlying mechanism is sparsely elucidated. Aim of this studyThe present study aims to explore the inhibition effect, kinetics and potential mechanism of SNG against H. pylori urease (HPU) and jack bean urease (JBU). Materials and methodsThe improved spectrophotometric berthelot method was applied to estimate the inhibitory effect of SNG against HPU and JBU. The Lineweaver-Burk plots were adopted for investigating the inhibitory pattern in enzymatic kinetics. Sulfydryl-containing compounds and competitive active-site Ni2+ binding depressors were used for mechanism research. ResultsSNG remarkably suppressed the activities of HPU and JBU in concentration-and time-dependent mode with IC50 of 0.48 ± 0.14 mM and 0.11 ± 0.02 mM, respectively, in comparison with urease retardant acetohydroxamic acid (0.06 ± 0.01 mM for HPU and 0.03 ± 0.00 mM for JBU, respectively). Kinetic analysis demonstrated that the inhibition of SNG against HPU and JBU were separately characterized by slow-binding, mixed-type and slow-binding, non-competitive type. Addition of sulfydryl-containing reagents (dithiothreitol, glutathione and L-cysteine) and competitive Ni2+ binding restrainers (boric acid and sodium fluoride) significantly abrogated the urease inhibitory effect of SNG, suggesting the significant role of the thiols and Ni2+ for the urease inhibition by SNG. By contrast, interaction with thiol groups possibly contributed to the repression of SNG on JBU. Furthermore, the urease suppression was proved to be partially reversible since the SNG-blocked enzyme could be partly reactivated by glutathione. ConclusionSNG could observably inhibit H. pylori urease targeting the thiols and Ni2+, which indicated that SNG was a new urease suppressant with great promise. The present research also provided scientific evidence for the application of SNG and Z. nitidum treating H. pylori-associated gastrointestinal diseases.

Assessment of the possible health risks associated with the consumption of botanical preparations of Mitragyna speciosa (kratom).

The current report summarises the work performed in the context of the European Food Risk Assessment Fellowship Programme (EU‐FORA), which included the evaluation of health risks associated with the consumption of botanical preparations of Mitragyna speciosa (kratom). Mitragyna speciosa is a tree native to Southeast Asia, where its leaves and preparations of the leaves have been used for centuries, among others, as a stimulant or as a traditional herbal medicine. Preparations of the plant have recently gained increasing popularity in other parts of the world, and are presently also accessible via online platforms, e.g. as food supplements. Kratom has been considered a botanical of possible health concern by the FDA and EFSA, which together with its increasing popularity, makes kratom a subject of international concern. Major alkaloids of the plant, mitragynine and 7‐hydroxymitragynine, are agonists of the μ‐opioid human receptor and are assumed to be mainly responsible for its psychoactive effects. The aim of the present project was to conduct an assessment of potential health risks associated with oral use of kratom‐based preparations. The animal and human data that were evaluated in the course of the current assessment indicate that kratom consumption has the potential to not only lead to adverse neurological effects, including addiction and withdrawal syndrome, but also to elicit distinct organ toxicity with respect to e. g. liver and kidney as target organs. Nevertheless, actual risk characterisation is impeded by considerable uncertainties. Such uncertainties, based on the variability in composition of kratom preparations, insufficient information on dose–response relationships and on limited data on long‐term use effects, currently do not allow the derivation of distinct health based guidance values for kratom/kratom preparations. Further information from well‐designed studies, conducted with kratom preparations that have been clearly defined with respect to their composition, would be required to enable a more refined risk assessment of this botanical.

Plant growth and phytoactive alkaloid synthesis in kratom [Mitragyna speciosa (Korth.)] in response to varying radiance.

Leaves harvested from kratom [Mitragyna speciosa (Korth.)] have a history of use as a traditional ethnobotanical medicine to combat fatigue and improve work productivity in Southeast Asia. In recent years, increased interest in the application and use of kratom has emerged globally, including North America, for its potential application as an alternative source of medicine for pain management and opioid withdrawal syndrome mitigation. Although the chemistry and pharmacology of major kratom alkaloids, mitragynine and 7-hydroxymitragynine, are well documented, foundational information on the impact of plant production environment on growth and kratom alkaloids synthesis is unavailable. To directly address this need, kratom plant growth, leaf chlorophyll content, and alkaloid concentration were evaluated under three lighting conditions: field full sun (FLD-Sun), greenhouse unshaded (GH-Unshaded), and greenhouse shaded (GH-Shaded). Nine kratom alkaloids were quantified using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Greenhouse cultivation generally promoted kratom height and width extension by 93–114% and 53–57%, respectively, compared to FLD-Sun. Similarly, total leaf area and leaf number were increased by 118–160% and 54–80% under such conditions. Average leaf size of plants grown under GH-Shaded was 41 and 69% greater than GH-Unshaded and FLD-Sun, respectively; however, no differences were observed between GH-Unshaded and FLD-Sun treatments. At the termination of the study, total leaf chlorophyll a+b content of FLD-Sun was 17–23% less than those grown in the greenhouse. Total leaf dry mass was maximized when cultivated in the greenhouse and was 89–91% greater than in the field. Leaf content of four alkaloids to include speciociliatine, mitraphylline, corynantheidine, and isocorynantheidine were not significantly impacted by lighting conditions, whereas 7-hydroxymitragynine was below the lower limit of quantification across all treatments. However, mitragynine, paynantheine, and corynoxine concentration per leaf dry mass were increased by 40%, 35%, and 111%, respectively, when cultivated under GH-Shaded compared to FLD-Sun. Additionally, total alkaloid yield per plant was maximized and nearly tripled for several alkaloids when plants were cultivated under such conditions. Furthermore, rapid, non-destructive chlorophyll evaluation correlated well (r2 = 0.68) with extracted chlorophyll concentrations. Given these findings, production efforts where low-light conditions can be implemented are likely to maximize plant biomass and total leaf alkaloid production.

Kratom (Mitragyna speciosa) Validation: Quantitative Analysis of Indole and Oxindole Alkaloids Reveals Chemotypes of Plants and Products.

Many consumers are turning to kratom ( Mitragyna speciosa ) to self-manage pain and opioid addiction. In the United States, an array of capsules, powders, and loose-leaf kratom products are readily available. Additionally, several online sites supply live kratom plants. A prerequisite to establishing quality control and quality assurance standards for the kratom industry, or understanding how alkaloid levels effect clinical outcomes, is the identification and quantitation of major and minor alkaloid constituents within available products and preparations. To this end, an ultra-high performance liquid chromatography-high resolution mass spectrometry method was developed for the analysis of 8 indole alkaloids (7-hydroxymitragynine, ajmalicine, paynantheine, mitragynine, speciogynine, isopaynantheine, speciociliatine, and mitraciliatine) and 6 oxindole alkaloids (isomitraphylline, isospeciofoleine, speciofoline, corynoxine A, corynoxeine, and rhynchophylline) in US-grown kratom plants and commercial products. These commercial products shared a qualitatively similar alkaloid profile, with 12 – 13 detected alkaloids and high levels of the indole alkaloid mitragynine (13.9 ± 1.1 – 270 ± 24 mg/g). The levels of the other major alkaloids (paynantheine, speciociliatine, speciogynine, mitraciliatine, and isopaynantheine) and the minor alkaloids varied in concentration from product to product. The alkaloid profile of US-grown M. speciosa “Rifat” showed high levels of the indole alkaloid speciogynine (7.94 ± 0.83 – 11.55 ± 0.18 mg/g) and quantifiable levels of isomitraphylline (0.943 ± 0.033 – 1.47 ± 0.18 mg/g). Notably, the alkaloid profile of a US-grown M. speciosa seedling was comparable to the commercial products with a high level of mitragynine (15.01 ± 0.20 mg/g). This work suggests that there are several M. speciosa chemotypes.

A Novel Stability Indicating Method for Determination of Major Alkaloid in Black Pepper by RP-HPLC in Different Pharmaceutical Dosage Forms

A Novel Stability Indicating Method for Determination of Major Alkaloid in Black Pepper by RP-HPLC in Different Pharmaceutical Dosage Forms

A Comprehensive Insight into the Phytochemical, Pharmacological Potential, and Traditional Medicinal Uses of Albizia lebbeck (L.) Benth.

Background Albizialebbeck is a deciduous tree having tremendous medicinal utilities, for example, respiratory, skin, gastrointestinal, oral disorders, eye, urinary, genital, anorectal, inflammatory, and neurological disorders, and venereal diseases. Several studies have been undertaken on the medicinal and traditional values of A. lebbeck. Objective The detailed information about its medicinal uses and pharmacological implications is highly scattered and distributed in different data sources. Hence, the study was conducted to supply an inclusive review of its ethnomedicinal uses, phytochemicals, and the available pharmacological attributes supporting its efficiency in traditional medicine. Method Literature surveys were conducted on this medicinal plant via search engines like Google Scholar, PubMed, and Science Direct, and obtained information up to December 2020 has been assessed and analyzed for this study. Results Systematic investigation revealed that A. lebbeck consists of various phytochemicals, including major alkaloids, flavonoids, saponins, and terpenoids. Its crude extract, fraction, and bioactive compounds exhibited potent adulticidal, antiallergic, anticancer, anticonvulsant, antidiabetic, antidiarrheal, anti-inflammatory, antimicrobial, antinociceptive, antioxidant, antiparasitic, antipyretic, antivenom, estrogenic, neuroprotective, nootropic, ovicidal, and wound healing activities. Conclusions This study proposes that A. lebbeck remains a rich source of phytochemicals with various biological activities which possess outstanding therapeutic benefits to humanity across the world. However, studies are required to estimate the potential side effects. Moreover, mechanistic physiognomies of the isolated compounds with known bioactivities are quite limited; thus, forthcoming research needs to focus on the mechanisms of these active phytochemicals to facilitate their potential enrolling for drug discovery.

Piperine Provides Neuroprotection against Kainic Acid-Induced Neurotoxicity via Maintaining NGF Signalling Pathway.

The neuroprotective properties of piperine, the major alkaloid extracted from black pepper, have been under investigation, but its mechanism of action in excitotoxicity is still poorly understood. This study aimed to evaluate the protective effects of piperine with a focus on nerve growth factor (NGF) signalling in a kainic acid (KA) rat model of excitotoxicity. Rats were administered intraperitoneally (i.p.) piperine (10 or 50 mg/kg) before KA injection (15 mg/kg, i.p.). Our results show that KA exposure in rats caused seizure behaviour, intrinsic neuronal hyperactivity, glutamate elevation, hippocampal neuronal damage, and cognitive impairment. These KA-induced alterations could be restored to the normal state by piperine treatment. In addition, piperine decreased the expression of the NGF precursor proNGF and NGF-degrading protease matrix metalloproteinase 9, whereas it increased the expression of proNGF processing enzyme matrix metalloproteinase 7, NGF, and NGF-activated receptor TrkA in the hippocampus of KA-treated rats. Furthermore, KA decreased phosphorylation of the protein kinase B (Akt) and glycogen synthase kinase 3β (GSK3β) in the hippocampus, and piperine reversed these changes. Our data suggest that piperine protects hippocampal neurons against KA-induced excitotoxicity by upregulating the NGF/TrkA/Akt/GSK3β signalling pathways.

New steroidal alkaloids with anti-inflammatory and analgesic effects from Veratrum grandiflorum

Ethnopharmacological relevance“Li-Lu”, the roots and rhizomes of Veratrum grandiflorum (Melianthiaceae), has been historically used as a traditional folk medicine for the treatment of wrist pain, fractures, sores, and inflammation in Yunnan Province, China. However, the anti-inflammatory and analgesic studies of this plant have seldom reported. Aim of the studyTo evaluate the anti-inflammatory and analgesic properties related to the traditional usage of V. grandiflorum both in vitro and in vivo, and further explore the accurate bioactive compounds from the medicinal plant. Materials and methodsPhytochemical investigation was carried out by chromatographic methods and their structures were established based on extensive spectra and comparison with corresponding data in the reported literatures. Anti-inflammatory activities were assessed by the suppression of lipopolysaccharide-activated inflammatory mediators in RAW 264.7 macrophage cells in vitro. Furthermore, anti-inflammatory and analgesic effects were evaluated based on carrageenan-induced paw edema and acetic acid-stimulated writhing in mice. ResultsThe methanol extract (ME) of V. grandiflorum significantly alleviated the paw edema caused by carrageenan and the writhing numbers induced by acetic acid. Subsequent phytochemical investigation led to isolated of 21 steroidal alkaloids, including seven new compounds, veragranines C–I (1–7). Anti-inflammatory test indicated that steroidal alkaloids could decrease the expression of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells at a concentration of 5.0 μg/ml in vitro, comparable to DXM. Moreover, five new steroidal alkaloids (2, 4, 5, 6, and 7) and two major steroidal alkaloids (9 and 13) significantly decreased the numbers of writhing in mice at the doses of 0.5 and/or 1.0 mg/kg (p < 0.01/0.05), roughly comparable to Dolantin™ at 10.0 mg/kg. ConclusionsThe investigation supported the traditional use of V. grandiflorum and provided new steroidal alkaloids as potent analgesic agents.

Bioprospection for new larvicides against Aedes aegypti based on ethnoknowledge from the Amazonian São Sebastião de Marinaú riverside community

Ethnopharmacological relevanceVector-borne diseases represent a huge global burden impacting health systems. Aedes aegypti is the main vector of arboviral diseases including dengue, Zika, chikungunya and urban yellow fever in both tropical and subtropical areas. Ethnopharmacological investigations provide potential avenues for developing new vector control strategies. Aim of the studyThe objective of this study is to document the São Sebastião de Marinaú riverside community's ethnoknowledge of local plants used to control mosquitoes and perform bioguided fractionation to isolate the compounds active against the arboviral disease vector Ae. aegypti. Materials and methodsSemi-structured interviews were conducted with residents of the Marinaú community located in the Caxiuanã National Forest, in the Amazon biome, Pará, Brazil. The plants used to control mosquitoes were subjected to phytochemical studies guided by Ae. aegypti assays. Extracts were obtained from seven species using distinct organic solvents. Active extracts and fractions were separated by chromatographic techniques. Isolated compounds were characterized by NMR, LC/MS and GC/MS. Sample activity against Ae. aegypti larvae and pupae was evaluated after 24, 48 and 72 h exposure. The extracts were also investigated against adult female mosquitoes. The LC50 values were determined by diluting each sample to obtain different concentrations in the respective activity range. ResultsThe Marinaú community uses more than ten plants as a repellent, most of which are trees native to the region. The primary applications of these plants to protect against insect bites were: burning plants (fumigation), application of body oils and bathing in macerated plants. Carapa guianensis is the predominant species used as a repellent. Extracts from Diospyros guianensis fruits, Carapa guianensis seed shells and Aspidosperma nitidum wood demonstrated Ae. aegypti larvicidal activity. The C. guianensis seed shell extract demonstrated a residual larvicidal effect. Plumbagin, stigmasterol, β-sitosterol, betulinic, ursolic and oleanolic acids, and betulin were identified in the D. guianensis extract. The plumbagin, ursolic and oleanolic acids displayed larvicidal activity. Oleanolic, ursolic and betulinic acids, and betulin were considered pupicidal. Aricine, the major alkaloid isolated from A. nitidum wood, also presented larvicidal activity. ConclusionsTen plant species traditionally used by the Marinaú community to afford protection against mosquitoes were reported. C. guianensis, D. guianensis and A. nitidum extracts were considered larvicidal against Ae. aegypti. Four triterpenes stood out as very active compounds against pupae. Aricine, an indole alkaloid, displayed larvicidal activity. Therefore, traditional knowledge of Amazonian plants combined with bioguided fractionation constitutes a strategy for the development of eco-friendly insecticides to control Ae. aegypti, an arbovirus vector.

Genetic and Chemical Profiling of Solenopsis spp. (Hymenoptera: Formicidae) Intercepted in Hawaii

Ants intercepted by the Hawaii Department of Agriculture in 2018 and 2019 identified as Solenopsis spp., and possibly S. invicta, were submitted to the United States Department of Agriculture-Agricultural Research Service-Center for Medical, Agricultural and Veterinary Entomology, Imported Fire Ant and Household Insects Research Unit for further taxonomic assessment using genetic and chemical methods. The 2018 sample consisted of 1 worker ant, and the 2019 sample consisted of 3 worker ants. Thoraces were used for genetic assessment and gasters were used for venom analyses. It was determined readily from genetic and chemical analyses that the intercepted ants were not S. invicta or S. richteri. Mitochondrial COX1 gene sequences were compared with previously published data from Solenopsis species. The closest match for the 2018 sample was S. xyloni, which is native to the shipment's origin (San Diego, California, USA). The 2 major alkaloids found were characteristic of the S. geminata species group. In addition, the alkaloid ratio closely matched that of S. xyloni. Therefore, the alkaloid analysis of the 2018 intercepted ant concurs with the genetic data that the ant was an S. xyloni worker. The COX1 gene sequences of the 2019 interceptions most closely matched S. geminata. The corresponding venom alkaloid profile also eliminated S. invicta and S. richteri as possibilities. However, 1 major alkaloid was unusual (Fig. 2) and normally is found only in trace amounts. We suggest the 2019 specimens are an unusual variant of S. geminata.

Discovery and Biochemical Characterization of N-methyltransferase Genes Involved in Purine Alkaloid Biosynthetic Pathway of Camellia gymnogyna Hung T.Chang (Theaceae) from Dayao Mountain

In the present study, purine alkaloid analysis and transcriptome of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain were performed by high-performance liquid chromatography (HPLC) and RNA-Seq, respectively. The results showed that the major purine alkaloids accumulated in Camellia gymnogyna Hung T. Chang (Theaceae) were theobromine together with a small amount of theacrine and caffeine. Through polymerase chain reaction (PCR), three types of cDNA encoding N-methyltransferases were isolated from the leaves of Camellia gymnogyna Hung T. Chang (Theaceae) and designated GCS1, GCS2, and GCS3. We subsequently expressed GCS1, GCS2, and GCS3 in Escherichia coli and incubated lysates of the bacterial cells with a variety of xanthine substrates in the presence of S-adenosyl-L-methionine as the methyl donor. We found that the recombinant GCS1 proteins catalyzed 1,3,7-trimethyluric acid to produce theacrine, the recombinant GCS3 proteins catalyzed 7-methylxanthine to produce theobromine, while the recombinant GCS2 proteins did not catalyze any xanthine derivatives. Simultaneous analysis of the expressions of GCS1, GCS2, GCS3, and a caffeine synthase gene (TCS1) in Camellia gymnogyna Hung T. Chang (Theaceae) and other tea plants provided a reference for further research on the functions of these genes.

New Insights into the Chemical Composition of Ayahuasca.

Ayahuasca is a psychedelic beverage originally from the Amazon rainforest used in different shamanic settings for medicinal, spiritual, and cultural purposes. It is prepared by boiling in water an admixture of the Amazonian vine Banisteriopsis caapi, which is a source of β-carboline alkaloids, with plants containing N,N-dimethyltryptamine, usually Psychotria viridis. While previous studies have focused on the detection and quantification of the alkaloids present in the drink, less attention has been given to other nonalkaloid components or the composition of the solids suspended in the beverage, which may also affect its psychoactive properties. In this study, we used nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) to study the composition of ayahuasca samples, to determine their alkaloid qualitative and quantitative profiles, as well as other major soluble and nonsoluble components. For the first time, fructose was detected as a major component of the samples, while harmine (a β-carboline previously described as an abundant alkaloid in ayahuasca) was found to be present in the solids suspended in the beverage. In addition, N,N-dimethyltryptamine (DMT), harmine, tetrahydroharmine, harmaline, and harmol were identified as the major alkaloids present in extracts of all samples. Finally, a novel, easy, and fast method using quantitative NMR was developed and validated to simultaneously quantify the content of these alkaloids found in each ayahuasca sample.

The respiratory depressant effects of mitragynine are limited by its conversion to 7-OH mitragynine.

Background and PurposeMitragynine, the major alkaloid in Mitragyna speciosa (kratom), is a partial agonist at the μ opioid receptor. CYP3A‐dependent oxidation of mitragynine yields the metabolite 7‐OH mitragynine, a more efficacious μ receptor agonist. While both mitragynine and 7‐OH mitragynine can induce anti‐nociception in mice, recent evidence suggests that 7‐OH mitragynine formed as a metabolite is sufficient to explain the anti‐nociceptive effects of mitragynine. However, the ability of 7‐OH mitragynine to induce μ receptor‐dependent respiratory depression has not yet been studied.Experimental ApproachRespiration was measured in awake, freely moving, male CD‐1 mice, using whole body plethysmography. Anti‐nociception was measured using the hot plate assay. Morphine, mitragynine, 7‐OH mitragynine and the CYP3A inhibitor ketoconazole were administered orally.Key ResultsThe respiratory depressant effects of mitragynine showed a ceiling effect, whereby doses higher than 10 mg·kg−1 produced the same level of effect. In contrast, 7‐OH mitragynine induced a dose‐dependent effect on mouse respiration. At equi‐depressant doses, both mitragynine and 7‐OH mitragynine induced prolonged anti‐nociception. Inhibition of CYP3A reduced mitragynine‐induced respiratory depression and anti‐nociception without affecting the effects of 7‐OH mitragynine.Conclusions and ImplicationsBoth the anti‐nociceptive effects and the respiratory depressant effects of mitragynine are partly due to its metabolic conversion to 7‐OH mitragynine. The limiting rate of conversion of mitragynine into its active metabolite results in a built‐in ceiling effect of the mitragynine‐induced respiratory depression. These data suggest that such ‘metabolic saturation’ at high doses may underlie the improved safety profile of mitragynine as an opioid analgesic.