Developmental toxicity evaluation of Pimenta pseudocaryophyllus (Gomes) Landrum, (E)-methyl isoeugenol chemotype, in Wistar rats.

BACKGROUND: Pimenta pseudocaryophyllus (Gomes) Landrum (Myrtaceae) has been traditionally used in Brazilian folk medicine. Studies have established the botanical characterization, phytochemistry profile, and pharmacological potential of this species, including antibiotic, anxiolytic, antidepressant, antioxidant, antinociceptive, and anti-inflammatory properties. Despite its widespread use, no previous study has been conducted regarding its toxicological profile, especially during pregnancy. Thus, this study investigated the developmental toxicity of the dry leaf extract of the P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype, in rats. METHODS: First, the dry leaf extract was prepared by a spray-drying technique. Then, pregnant Wistar rats were orally treated with dry extract at doses of 0, 2000, 2500, or 3000 mg/kg from gestational day 6 through 15 (organogenesis period). On gestational day 21, the rats underwent cesarean sections and the reproductive outcomes and biochemistry parameters related to hepatic and renal markers were evaluated. Additionally, the fetuses were examined for external and skeletal variations and malformations. RESULTS: The spray-drying technique preserved the phytocomplex components and showed a satisfactory yield. No relevant differences were seen in the food consumption, reproductive performances, and hepatic and renal biochemical parameters between groups. However, there was a decrease in body weight gain of the dams during the organogenesis period and an increase of minor skeletal variations in the offspring (increased fetal incidences only of delayed ossification of the metacarpals, metatarsals, phalanges, sternebra, and rudimentary ribs) treated with the dry extract. CONCLUSION: The extract of P. pseudocaryophyllus, (E)-methyl isoeugenol chemotype, showed low maternal toxicity and induced minor skeletal variations in the offspring. Birth Defects Research 109:1292-1300, 2017. © 2017 Wiley Periodicals, Inc.

Development and Validation of a HPLC-UV Method for the Evaluation of Ellagic Acid in Liquid Extracts of Eugenia uniflora L. (Myrtaceae) Leaves and Its Ultrasound-Assisted Extraction Optimization.

A simple HPLC-UV method has been developed and validated for the quantification of ellagic acid (EA) in ethanol extracts of Eugenia uniflora L. (Myrtaceae) leaves. The ultrasound-assisted extraction (UAE) optimization was performed using a Box Behnken design (33) combined with response surface methodology to study the effects of the ethanol concentration (%, w/w), extraction time (minutes), and temperature (°C) on the EA concentration. The optimized results showed that the highest extraction yield of EA by UAE was 26.0 µg mL-1 when using 44% (w/w) ethanol as the solvent, 22 minutes as the extraction time, and 59°C as the extraction temperature. The concentration of EA in relation to the predicted value was 93.7% ± 0.4. UAE showed a strong potential for EA extraction.

Phytochemical Analysis and Antimicrobial Activity of Myrcia tomentosa (Aubl.) DC. Leaves.

This work describes the isolation and structural elucidation of compounds from the leaves of Myrcia tomentosa (Aubl.) DC. (goiaba-brava) and evaluates the antimicrobial activity of the crude extract, fractions and isolated compounds against bacteria and fungi. Column chromatography was used to fractionate and purify the extract of the M. tomentosa leaves and the chemical structures of the compounds were determined using spectroscopic techniques. The antibacterial and antifungal activities were assessed using the broth microdilution method. The phytochemical investigation isolated 11 compounds: α-bisabolol, α-bisabolol oxide B, α-cadinol, ß-sitosterol, n-pentacosane, n-tetracosane, quercetin, kaempferol, avicularin, juglanin and guaijaverin. The crude ethanolic extract and its fractions were tested against 15 bacteria and 9 yeasts. The crude extract inhibited the in vitro growth of yeasts at concentration of 4 to 32 µg/mL. The hexane, dichloromethane, ethyl acetate and aqueous fractions inhibited Candida sp. at concentrations of 4 to 256 µg/mL, whereas the Cryptococcus sp. isolates were inhibited only by the hexane and dichloromethane fractions in minimal inhibitory concentrations (MICs) at 16 to 64 µg/mL. The flavonoid quercetin-3-O-α-arabinofuranose (avicularin) was the most active compound, inhibiting Candida species in concentrations of 2 to 32 µg/mL. The MIC values suggest potential activity of this plant species against yeast.

Anxiolytic and antidepressant like effects of natural food flavour (E)-methyl isoeugenol.

(E)-methyl isoeugenol (MIE) is a natural food flavour that constitutes 93.7% of an essential oil from Pimenta pseudocaryophyllus leaf. The leaf extracts of this species are used as a calming agent. As a ubiquitous food additive, the application of MIE for treating mood disorders appears to be globally attractive. Hence, we sought to evaluate general pharmacological activities, anticonvulsant, anxiolytic and antidepressant effects and the possible mechanisms of MIE actions. Administration of MIE was carried out prior to the exposure of a male Swiss mice to general behavioural tests, barbiturate sleep, PTZ-induced convulsion, light dark box (LDB), elevated plus maze (EPM), wire hanging, open field (OF) and forced swimming test (FST). The involvement of monoamine system was studied by mice pretreatment with WAY100635 (antagonist of 5-HT1A), α-methyl-p-tyrosine (AMPT; depletor of catecholamine) or p-chlorophenylalanine (PCPA; depletor of serotonin storage). There was no record of neurotoxic effect or animal's death during the course of general pharmacological tests. MIE at 250 and 500 mg kg(-1) potentiated the hypnotic effect of sodium pentobarbital. However, MIE did not protect against PTZ-induced convulsion. Except for MIE at 500 mg kg(-1), parameters evaluated in the LDB, EPM and OF demonstrated an anxiolytic like property of MIE. This effect was blocked by WAY100635 pretreatment. MIE at 500 mg kg(-1) elicited a reduction in locomotor activity of the mice in the OF. Anti-immobility effect of MIE 250 mg kg(-1) in the FST suggested an antidepressive like property. Unlike AMPT, pretreatment with PCPA reversed the antidepressant like effect of MIE. Our findings demonstrated anxiolytic and antidepressant like properties of (E)-methyl isoeugenol and suggested the participation of serotonergic pathways.

Antidepressive-Like Property of Dichloromethane Fraction of Pimenta pseudocaryophyllus and Relevance of Monoamine Metabolic Enzymes.

Pimenta pseudocaryophyllus popularly referred to as craveiro is considered as a calming agent in different local preparations. The present study attempted to examine antidepressant-like effect of dichloromethane fraction (DF) and role of monoamine oxidase (MAO), tryptophan, and tyrosine hydroxylase. Based on the research focus, tail suspension (TS), forced swimming (FS), and open field (OF) tests were conducted after oral administration of DF (125, 250, or 500 mg/Kg). Ex vivo assay of MAO was also conducted to evaluate inhibitory effect of DF (250 mg/Kg). Administration of DF elicits antidepressant-like response in the TS and FS. However, DF 500 mg/Kg did not alter mice performance in these models. The data obtained in the OF showed a reduction in total crossing and rearing activity; these effects suggest motor interference in TS and FS performance. Mice pretreatment with p-chlorophenylalanine methyl ester (PCPA) (100 mg/kg, i.p.-serotonin biosynthesis inhibitor) for 4 consecutive days or acute administration of α-methyl-p-tyrosine (AMPT) (100 mg/kg, i.p.-catecholamine synthesis inhibitor) blocked anti-immobility effect of DF in the FS. In ex vivo assay of MAO, DF did not inhibit catabolic activity of MAO. Our findings support antidepressant-like activity of DF and suggest an effect that depends on monoamine biosynthesis.

Phytochemical Analysis and Antimicrobial, Antinociceptive, and Anti-Inflammatory Activities of Two Chemotypes of Pimenta pseudocaryophyllus (Myrtaceae).

Preparations from Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) have been widely used in Brazilian folk medicine. This study aims to evaluate the antimicrobial activity of the crude ethanol extracts, fractions, semipurified substances, and essential oils obtained from leaves of two chemotypes of P. pseudocaryophyllus and to perform the antinociceptive and anti-inflammatory screening. The ethanol extracts were purified by column chromatography and main compounds were spectrally characterised (1D and 2D (1)H and (13)C NMR). The essential oils constituents were identified by GC/MS. The broth microdilution method was used for testing the antimicrobial activity. The abdominal contortions induced by acetic acid and the ear oedema induced by croton oil were used for screening of antinociceptive and anti-inflammatory activities, respectively. The phytochemical analysis resulted in the isolation of pentacyclic triterpenes, flavonoids, and phenol acids. The oleanolic acid showed the best profile of antibacterial activity for Gram-positive bacteria (31.2-125 µg mL(-1)), followed by the essential oil of the citral chemotype (62.5-250 µg mL(-1)). Among the semipurified substances, Ppm5, which contained gallic acid, was the most active for Candida spp. (31.2 µg mL(-1)) and Cryptococcus spp. (3.9-15.6 µg mL(-1)). The crude ethanol extract and fractions from citral chemotype showed antinociceptive and anti-inflammatory effects.

Effects of Pimenta pseudocaryophyllus (Gomes) L. R. Landrum, on melanized and non-melanized Cryptococcus neoformans.

In the present study, the in vitro susceptibility and capsular width from both melanized and non-melanized Cryptococcus neoformans cells in the presence of Pimenta pseudocaryophyllus crude extract were determined. The results were compared with those obtained for voriconazole and amphotericin B. Melanization was obtained in minimal medium broth with the addition of L-dopa, and the antifungal susceptibility tests were performed using the broth microdilution method. Capsular width of 30 cells of each one of the isolates in medium with crude extracts of P. pseudocaryophyllus or voriconazole or amphotericin B at a concentration corresponding to 0.5 times the minimal inhibitory concentration (MIC) was measured, and the mean was calculated. The MICs and minimal fungicidal concentrations (MFCs) for plant extract and voriconazole were identical for both melanized and non-melanized C. neoformans isolates, but for amphotericin, the MFCs for melanized cells were up to 8 times higher than for non-melanized cells. The capsular width of C. neoformans cells was smaller (p < 0.001) in the presence crude extract of P. pseudocaryophyllus and of voriconazole regardless melanization. The findings of capsule alterations of C. neoformans verified in this study provide fertile ways for future research into the effects of antifungal agents on the pathogenesis of cryptococcosis.

Involvement of 5-HT1A in the anxiolytic-like effect of dichloromethane fraction of Pimenta pseudocaryophyllus.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal applications of Pimenta pseudocaryophyllus infusion as a diuretic and aphrodisiac agent as well as tranquilizer in the form of tea for the treatment of emotional tension in Brazilian folk medicine has been in practice since time immemorial. Despite its popular therapeutic acceptance and claims, there are scanty scientific reports to corroborate its central biological activities. AIM: To characterize anxiolytic-like effect of the dichloromethane fraction (DF) obtained from ethanolic leaf extract of the Pimenta pseudocaryophyllus and identify mechanisms of action involved while seeking to support its popular use as a soothing agent. MATERIAL AND METHODS: Mice (25-35 g) were treated orally with DF obtained from ethanolic leaf extract of Pimenta pseudocaryophyllus and were submitted to light-dark box (LDB) and elevated plus maze (EPM) tests. Different groups of mice were treated with flumazenil and NAN-190 to identify mechanisms of action involved in the anxiolytic-like effect of DF. RESULTS: Treatment with DF increased number of transitions and time spent in the light compartment of the LDB while the time spent and numbers of entries in the open arm of the LCE were significantly increased. Pre-treatment of the animal with flumazenil (2 mg/kg, i.p.--competitive antagonist of benzodiazepine site of GABA(A) receptor) did not block this effect, thereby excluding participation of benzodiazepine site of the GABA(A) receptor. However, anxiolytic-like effect of DF was reversed by pre-treatment with NAN-190 (0.5 mg/kg, i.p.--an antagonist of the 5-HT(1A) receptor) thereby suggesting involvement of 5-HT(1A) receptor. The thin layer chromatography and high-performance liquid chromatography analysis indicated the predominance of (E)-methyl isoeugenol and oleanolic acid in DF. CONCLUSION: These results support the popular use of Pimenta pseudocaryophyllus as a calming agent and suggest the involvement of 5-HT(1A) receptor.