Repellency effect of Pilocarpus spicatus A. St.-Hil essential oil and nanoemulsion against Rhipicephalus microplus larvae.

Rhipicephalus microplus, the cattle tick, is a major cause of economic losses in bovine production. Due to the widespread acaricidal resistance to commercially available products, as well as their toxicity and environmental impact, alternative control methods are required. Nanoformulations produced from plant extracts as bioactive substances are very promising as innovative acaricidal agents. Thus, the aim of this study was to evaluate the in vitro repellent activity of Pilocarpus spicatus essential oil and its nanoemulsion against R. microplus, using larval repellent test (RT). The essential oil was extracted by hydrodistillation, using a Clevenger-type apparatus. The nanoemulsion was prepared with 5% essential oil, 5% tween 80, and 90% water, using the phase inversion method (50 mg/mL). Limonene was the major component (46.8%) of the essential oil, as determined by gas chromatography-mass spectrometry (GC/MS) and confirmed by flame ionization detection (GC/FID). According to the RT results, the essential oil had a repellent activity greater than 69%, from concentrations of 3.12 mg/mL (69.81 ± 10%) to 50 mg/mL (98.10 ± 0.6%), whereas the nanoemulsion at 50 mg/mL presented repellent activities of 97.14 ± 1.37% and 97.89 ± 0.52% 6 and 10 h after treatment, respectively. These values regarding to total repellency were very close to those calculated for mortality corrected by Abbott's formula. The phase inversion method preserved the chemical and physical characteristics of the essential oil since both reached an equal repellent effect at the same concentration. Therefore, P. spicatus essential oil and nanoemulsion had excellent repellent activities against R. microplus larvae, demonstrating its potential for future use as an alternative for tick control.

Phytochemical and antimicrobial study of Pilocarpus pennatifolius Lemaire.

The investigation of the crude extract of leaves and bark of Pilocarpus pennatifolius Lemaire allowed isolated of a not yet described coumarin, together with three known coumarins (bergapten, xanthotoxin and dimethyl allyl xanthyletin), and a not yet described imidazole alkaloid. All structures were established by means of spectral analysis, including extensive 2D NMR studies. In addition, the alkaloid had its absolute stereochemistry determined by X-ray diffraction. Meanwhile, extracts and pure compounds were tested against various strains of bacteria and fungi, showing promising antimicrobial activities. We highlight the activities of crude bark methanol extract (CBME), of the leaf basic acetate fraction (LBAcF), and of compound 2 against the Gram negative bacteria Shigella flexneri (MICs = 7.8, 7.8 and 3.12 µg·mL-1, respectively), of compound 5 against the Gram positive Enterococcus fecalis (MIC = 1.56 µg·mL-1), and against two Gram negative bacteria Salmonella enteritidis (MIC = 1.56 µg·mL-1), and Pseudomonas aeruginosa (MIC = 6.25 µg·ml-1). On the other hand, CBME and compounds 3-5 showed excellent activity against the fungus Candida krusei with MICs of 15.6, 1.56, and 3.12 µg·mL-1 respectively, as actives or better than the antifungal standard fluconazole (MIC = 3.12 µg·mL-1).

Computational quantum chemistry, molecular docking, and ADMET predictions of imidazole alkaloids of Pilocarpus microphyllus with schistosomicidal properties.

Schistosomiasis affects million people and its control is widely dependent on a single drug, praziquantel. Computational chemistry has led to the development of new tools that predict molecular properties related to pharmacological potential. We conducted a theoretical study of the imizadole alkaloids of Pilocarpus microphyllus (Rutaceae) with schistosomicidal properties. The molecules of epiisopiloturine, epiisopilosine, isopilosine, pilosine, and macaubine were evaluated using theory models (B3lyp/SDD, B3lyp/6-31+G(d,p), B3lyp/6-311++G(d,p)). Absorption, distribution, metabolization, excretion, and toxicity (ADMET) predictions were used to determine the pharmacokinetic and pharmacodynamic properties of the alkaloids. After optimization, the molecules were submitted to molecular docking calculations with the purine nucleoside phosphorylase, thioredoxin glutathione reductase, methylthioadenosine phosphorylase, arginase, uridine phosphorylase, Cathepsin B1 and histone deacetylase 8 enzymes, which are possible targets of Schistosoma mansoni. The results showed that B3lyp/6-311++G(d,p) was the optimal model to describe the properties studied. Thermodynamic analysis showed that epiisopiloturine and epiisopilosine were the most stable isomers; however, the epiisopilosine ligand achieved a superior interaction with the enzymes studied in the molecular docking experiments, which corroborated the results of previous experimental studies on schistosomiasis.

Amanita muscaria (fly agaric): from a shamanistic hallucinogen to the search for acetylcholine.

The mushroom Amanita muscaria (fly agaric) is widely distributed throughout continental Europe and the UK. Its common name suggests that it had been used to kill flies, until superseded by arsenic. The bioactive compounds occurring in the mushroom remained a mystery for long periods of time, but eventually four hallucinogens were isolated from the fungus: muscarine, muscimol, muscazone and ibotenic acid. The shamans of Eastern Siberia used the mushroom as an inebriant and a hallucinogen. In 1912, Henry Dale suggested that muscarine (or a closely related substance) was the transmitter at the parasympathetic nerve endings, where it would produce lacrimation, salivation, sweating, bronchoconstriction and increased intestinal motility. He and Otto Loewi eventually isolated the transmitter and showed that it was not muscarine but acetylcholine. The receptor is now known variously as cholinergic or muscarinic. From this basic knowledge, drugs such as pilocarpine (cholinergic) and ipratropium (anticholinergic) have been shown to be of value in glaucoma and diseases of the lungs, respectively.

Seasonal change in main alkaloids of jaborandi (Pilocarpus microphyllus Stapf ex Wardleworth), an economically important species from the Brazilian flora.

Pilocarpus microphyllus Stapf ex Wardleworth (jaborandi, Rutaceae) is one of the most important Brazilian medicinal species owing to its content of pilocarpine (PIL), an alkaloid used for treating glaucoma and xerostomia. This species contains another alkaloid, epiisopiloturine (EPI), which has demonstrated effectiveness against schistosomiasis. The aim of this work was to assess seasonal changes of PIL and EPI in three populations of cultivated P. microphyllus from northeastern Brazil over one year, including the dry and rainy seasons. Alkaloid profiles were correlated to phenotypic and genetic patterns in the morphological and molecular characterizations. PIL was the primary alkaloid and its levels differed among populations in all months except September. The S01 population (green line) showed an especially high PIL content compared to populations S02 and S03 (traditional line), which had similar alkaloid contents. PIL content gradually decreased in the three populations in the rainy season.EPI content was significantly different between the green line (S01) and the traditional line (S02 and S03).S01 had a significantly lower EPI content in all months, demonstrating that it was not the best source for EPI extraction. Inter simple sequence repeat (ISSR) markers and morphological analyses clearly separated S01 from S02 and S03, in agreement with the alkaloid results. This study shows the first correlation between the chemical, morphological, and molecular markers of P. microphyllus and highlights the potential benefits of a multidisciplinary research approach aimed at supporting both industry and conservation of natural resources.

Anthelmintic, Antibacterial and Cytotoxicity Activity of Imidazole Alkaloids from Pilocarpus microphyllus Leaves.

Pilocarpus microphyllus Stapf ex Wardlew (Rutaceae), popularly known as jaborandi, is a plant native to the northern and northeastern macroregions of Brazil. Several alkaloids from this species have been isolated. There are few reports of antibacterial and anthelmintic activities for these compounds. In this work, we report the antibacterial and anthelmintic activity of five alkaloids found in P. microphyllus leaves, namely, pilosine, epiisopilosine, isopilosine, epiisopiloturine and macaubine. Of these, only anthelmintic activity of one of the compounds has been previously reported. Nuclear magnetic resonance, HPLC and mass spectrometry were combined and used to identify and confirm the structure of the five compounds. As regards the anthelmintic activity, the alkaloids were studied using in vitro assays to evaluate survival time and damaged teguments for Schistosoma mansoni adult worms. We found epiisopilosine to have anthelmintic activity at very low concentrations (3.125 µg mL-1 ); at this concentration, it prevented mating, oviposition, reducing motor activity and altered the tegument of these worms. In contrast, none of the alkaloids showed antibacterial activity. Additionally, alkaloids displayed no cytotoxic effect on vero cells. The potent anthelmintic activity of epiisopilosine indicates the potential of this natural compound as an antiparasitic agent. Copyright © 2017 John Wiley & Sons, Ltd.

In vitro effects of Pilocarpus microphyllus extracts and pilocarpine hydrochloride on Rhipicephalus (Boophilus) microplus.

The aim of this study was to assess the activity of aqueous (AE) and ethanolic extracts (EE) and pilocarpine hydrochloride, which were extracted and isolated from Pilocarpus microphyllus (Jaborandi), respectively, on Rhipicephalus (Boophilus) microplus. High performance liquid chromatography (HPLC) was performed to quantify these compounds. Larval packet and adult immersion tests were conducted with different concentrations. Five AE and EE concentrations, ranging from 6.2 to 100.0 mg mL-1, and six concentrations of pilocarpine hydrochloride, ranging from 0.7 to 24.0 mg mL-1, were tested. The lethal concentration (LC50) of each extract for larvae and engorged females was calculated through Probit analysis. The concentration of pilocarpine hydrochloride obtained from the EE and the AE was 1.3 and 0.3% (m/m), respectively. Pilocarpine hydrochloride presented the highest acaricidal activity on larvae (LC50 2.6 mg mL-1) and engorged females (LC50 11.8 mg mL-1) of R.(B.) microplus, followed by the EE which presented LC50 of 56.4 and 15.9 mg mL-1, for larvae and engorged females, respectively. Such results indicate that pilocarpine hydrochloride has acaricidal activity, and may be the primary compound responsible for this activity by P. microphyllus EE.

In vitro effects of Pilocarpus microphyllus extracts and pilocarpine hydrochloride on Rhipicephalus (Boophilus) microplus / Efeitos in vitro do extrato de Pilocarpus microphyllus e do cloridrato de pilocarpina sobre Rhipicephalus (Boophilus) microplus

Abstract The aim of this study was to assess the activity of aqueous (AE) and ethanolic extracts (EE) and pilocarpine hydrochloride, which were extracted and isolated from Pilocarpus microphyllus (Jaborandi), respectively, on Rhipicephalus (Boophilus) microplus. High performance liquid chromatography (HPLC) was performed to quantify these compounds. Larval packet and adult immersion tests were conducted with different concentrations. Five AE and EE concentrations, ranging from 6.2 to 100.0 mg mL–1, and six concentrations of pilocarpine hydrochloride, ranging from 0.7 to 24.0 mg mL–1, were tested. The lethal concentration (LC50) of each extract for larvae and engorged females was calculated through Probit analysis. The concentration of pilocarpine hydrochloride obtained from the EE and the AE was 1.3 and 0.3% (m/m), respectively. Pilocarpine hydrochloride presented the highest acaricidal activity on larvae (LC50 2.6 mg mL–1) and engorged females (LC50 11.8 mg mL–1) of R.(B.) microplus, followed by the EE which presented LC50 of 56.4 and 15.9 mg mL–1, for larvae and engorged females, respectively. Such results indicate that pilocarpine hydrochloride has acaricidal activity, and may be the primary compound responsible for this activity by P. microphyllus EE.

Resumo O objetivo desse estudo foi avaliar a atividade dos extratos aquoso (AE) e etanólico (EE) e do cloridrato de pilocarpina, que foram, respectivamente, extraídos e isolado de Pilocarpus microphyllus (Jaborandi), sobre Rhipicephalus (Boophilus) microplus. Cromatografia líquida de alta eficiência foi realizada para quantificação dos compostos. Testes de pacote de larvas e de imersão de adultos foram realizados com diferentes concentrações. Cinco concentrações do AE e EE variando de 6,2 a 100,0 mg mL–1 e seis concentrações do cloridrato de pilocarpina variando de 0,7 a 24,0 mg mL–1 foram testadas. A concentração letal (CL50) de cada extrato para larvas e fêmeas ingurgitadas foi estimada por meio da análise Probit. A concentração de cloridrato de pilocarpina obtida do EE e AE foi de 1,3 e 0,3% (m/m), respectivamente. O cloridrato de pilocarpina apresentou a maior atividade carrapaticida sobre larvas (CL50 2,6 mg mL–1) e fêmeas ingurgitadas (CL50 11,8 mg mL–1) de R. (B.) microplus, seguido do EE que apresentou CL50 de 56,4 e 15,9 mg mL–1, para larvas e fêmeas ingurgitadas, respectivamente. Tais resultados indicam que o cloridrato de pilocarpina apresenta atividade carrapaticida e pode ser o principal responsável pela atividade acaricida do EE de P. microphyllus.

Unraveling the biosynthesis of pilocarpine in Pilocarpus microphyllus.

Pilocarpine is found exclusively in species of Pilocarpus and the presence of other imidazole alkaloids has been reported in several species of the genus. Pilocarpine has several important pharmaceutical applications. Although several imidazole alkaloids related to pilocarpine have been reported in the previous years, little is still known about its biosynthetic route. At most, histidine has been reported as the precursor of pilocarpine. Based on our own previous reports and in an experiment where pilocarpine and related alkaloids (pilosine, trachyllophiline and anhydropilosine) were supplied to P. microphyllus leaves and the alkaloid profile analyzed by UPLC-MS, we suggest a biosynthesis pathway for pilocarpine. Further experiments using labeled precursors associated with transcriptome data may allow us to understand the whole biosynthesis pathway and its genetic control.

Estudo fitoquímico e avaliação in vitro da atividade anti-Trypanosoma cruzi cepa Y de Pilocarpus spicatus St. Hil. (Rutaceae) / Phytochemical study and in vitro evaluation of the anti-Trypanosoma cruzi Y strain activity of Pilocarpus spicatus A. St. Hil.

A investigação química da espécie Pilocarpus spicatus, popularmente conhecida como jaborandi e usada na medicina tradicional para doenças como estomatite, febre, bronquite e psoríase, teve por objetivo o isolamento e/ou identificação de substâncias ativas e a avaliação da atividade antiparasitária dos extratos frente às formas epimastigotas de Trypanosoma cruzi. O estudo resultou na identificação de nove substâncias, tais como: tridecanona, 2-heptadecanona, espatulenol, aromadendreno, β-cariofileno, ácido 3α-hidroxitirucala-7,24-dien-21-óico, (+)-isoangenomalina, episesamina e sesamina. As estr uturas dos compostos foram elucidadas por análises espectroscópicas e comparação com dados da literatura. Os extratos hexânico e metanólico de folhas e raízes foram testados in vitro contra o Trypanosoma cruzi cepa Y e apresentaram atividade tripanomicida.

The chemical investigation of the species Pilocarpus spicatus - popularly known as jaborandi and used in traditional medicine for diseases, such as stomatitis, fever, bronchitis and psoriasis - aimed to isolate and / or identify the active substances and evaluate the antiparasitic activity of the extracts against the Trypanosoma cruzi epimastigote forms. The study resulted in the identification of nine substances, such as tridecanone, 2-heptadecanone, spathulenol, aromadendrene, β-caryophyllene, 3α-hydroxytirucalla-7,24-dien-21-oic acid, (+)-isoangenomaline, episesamin and sesamin. The structures were elucidated by spectroscopic analysis and comparison with literature data. The hexane and methanol extracts from leaves and roots were tested in vitro against Trypanosoma cruzi Y strain and showed trypanocidal activity.

Industrial Scale Isolation, Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis.

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), (1)H and (13)C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.

HPLC-ESI-MS/MS of imidazole alkaloids in Pilocarpus microphyllus.

Pilocarpine, an important imidazole alkaloid, is extracted from the leaves of Pilocarpus microphyllus (Rutaceae), known in Brazil as jaborandi and used mainly for the treatment of glaucoma. Jaborandi leaves also contain other imidazole alkaloids, whose pharmacological and physiological properties are unknown, and whose biosynthetic pathways are under investigation. In the present study, a HPLC method coupled with ESI-MS(n) was developed for their qualitative and quantitative analysis. This method permits the chromatographic separation of the imidazole alkaloids found in extracts of jaborandi, as well as the MS/MS analysis of the individual compounds. Thus two samples: leaves of P. microphyllus and a paste that is left over after the industrial extraction of pilocarpine; were compared. The paste was found to contain significant amounts of pilocarpine and other imidazole alkaloids, but had a slightly different alkaloid profile than the leaf extract. The method is suitable for the routine analysis of samples containing these alkaloids, as well as for the separation and identification of known and novel alkaloids from this family, and may be applied to further studies of the biosynthetic pathway of pilocarpine in P. microphyllus.