Toxicological effects of aqueous extract of Genipa americana L. leaves on adult zebrafish (Danio rerio): Chemical profile, histopathological effects and lack of genotoxicity.

Genipa americana is a native plant of Brazil with potential applications in folk medicine. Whereas most of the phytochemical and pharmacological studies on this plant have focused on its fruits, the crude extracts of its leaves contain chemical metabolites that may have toxicity to organisms, which have yet to be investigated. This study aimed to determine the main groups of secondary metabolites in the aqueous extract of the leaves of G. americana by phytochemistry and qualitative HPLC, and to evaluate the possible toxicological effects and histopathological changes caused by this extract in zebrafish (Danio rerio) adults, through micronucleus test, nuclear abnormalities and histopathological analyses of gills and liver. While three metabolites of high intensity (phenolic compounds, flavonoids and triterpenes) were found in the phytochemical evaluation, the HPLC showed results compatible with flavonoids and iridoids, all belonging to common classes for this species and the Rubiaceae family. The acute toxicity test did not induce mortality or genotoxicity in zebrafish, but after exposure for 96 h, it was possible to observe injuries to the fish gill tissue, such as lamellar fusion, vasodilation and telangiectasia; in the liver, necrosis was visualized at 40 mg/L, and at higher concentrations (80 and 100 mg/L) induced sinusoidal widening was identified. In conclusion, the results demonstrated the toxic potential of this plant for aquatic species.

Is There Enough Knowledge to Standardize a Cannabis sativa L. Medicinal Oil Preparation with a High Content of Cannabinoids?

Introduction: Cannabis sativa L. medicinal oils are good therapeutic options due to their wide spectrum of pharmacological applications and the easy adjustment of individual doses. The lack of standardization of methodology in the preparation of medicinal oil using the Cannabis crude extract results in elevated variability of cannabinoid concentration in the final product. The elevated variability impairs the understanding of beneficial and adverse effects related to dose-response pharmacological activities. Objective: This study aimed to conduct a review on the current methods of Cannabis oil preparation present in the literature, to demonstrate the most appropriate methodologies to ensure a product with high content of cannabinoids and terpenes. Results: The decarboxylation stage is essential for the conversion of acid cannabinoids into neutral cannabinoids, which are substances with the highest bioavailability. Lower temperatures for longer periods of time instead of high temperatures in less time are highly recommended to ensure that all the acidic cannabinoids have passed through decarboxylation. For the guarantee of a high terpene content, the separate addition of essential oil to the fixed oil prepared from the crude extract should be considered. Ultrasound-assisted extraction is one of the best performing methodologies because it is cheaper than other techniques, such as supercritical fluid extraction, besides that, ultrasound extraction is effective in short extraction times and uses small amounts of solvent when compared with other techniques. Conclusion: Although the literature about the methods of preparation of Cannabis medicinal oil is scarce, it is possible to standardize an optimized, low-cost, and effective Cannabis extractive methodology from the results found in the literature; however, this will depend on new research for methodological validation.

Bauhinia forficata Link Infusions: Chemical and Bioactivity of Volatile and Non-Volatile Fractions.

This study aimed to evaluate Bauhinia forficata infusions prepared using samples available in Rio de Janeiro, Brazil. As such, infusions at 5% (w/v) of different brands and batches commercialized in the city (CS1, CS2, CS3, and CS4) and samples of plant material botanically identified (BS) were evaluated to determine their total phenolic and flavonoid contents (TPC and TFC), antioxidant capacity (ABTS•+, DPPH•, and FRAP assays), phytochemical profile, volatile compounds, and inhibitory effects against the α-amylase enzyme. The results showed that infusions prepared using BS samples had lower TPC, TFC and antioxidant potential than the commercial samples (p < 0.05). The batch averages presented high standard deviations mainly for the commercial samples, corroborating sample heterogeneity. Sample volatile fractions were mainly composed of terpenes (40 compounds identified). In the non-volatile fraction, 20 compounds were identified, with emphasis on the CS3 sample, which comprised most of the compounds, mainly flavonoid derivatives. PCA analysis demonstrated more chemical diversity in non-volatile than volatile compounds. The samples also inhibited the α-amylase enzyme (IC50 value: 0.235−0.801 mg RE/mL). Despite the differences observed in this work, B. forficata is recognized as a source of bioactive compounds that can increase the intake of antioxidant compounds by the population.

Apoptotic effect of ß-pinene on oral squamous cell carcinoma as one of the major compounds from essential oil of medicinal plant Piper rivinoides Kunth.

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck malignancy. Research on essential oils (EOs) has shown important cytotoxic and anti-tumor properties, among others. Piperaceae species are rich in EOs and here we highlight Piper rivinoides Kunth. We investigated the crude EOs from P. rivinoides, their pure major constituents and an enriched fraction with the main EO compounds (EF) as cytotoxic and selective OSCC agents. EOs presented as main compounds (-)-α-pinene, (-)-ß-pinene and limonene. EOs showed an IC50 lower than all isolated compounds, except for (-)-ß-pinene in OSCC cells. The (-)-ß-pinene induced cell death with apoptotic characteristics. Commercial standards showed greater selectivity than EOs, and (-)-ß-pinene was the most selective among them. EF showed higher selectivity compared to crude EOs and carboplatin, turning it into a good candidate as an anticancer fraction. These results are important for the possible development of new treatments for OSCC.

Advanced Chemophenetic Analysis of Essential Oil from Leaves of Piper gaudichaudianum Kunth (Piperaceae) Using a New Reduction-Oxidation Index to Explore Seasonal and Circadian Rhythms.

The aromatic species Piper gaudichaudianum Kunth (Piperaceae) is widely used in Brazil for medicinal and ritualistic applications. In the current study, chemophenetic patterns were realized across season and circadian rhythm based on the chemical profile of essential oils (EOs) from leaves. Hydrodistilled essential oils were analyzed by GC-MS and GC-FID, and a new calculation of metabolite oxidation level, averaged for each individual molecule component of the EO, was used to explore the patterns of metabolism/biosynthesis. This new index used an intermediate calculation, the 'weighted average redox standard' (SRO), to enable a value for mixtures of metabolites to be generated, the 'general mixture redox index' (GMOR). The indices were subjected to a proof-of-concept approach by making comparison to outcomes from multivariate analyses, i.e., PCA and HCA. Chemical analysis demonstrated that the essential oils were dominated by sesquiterpenes, constructed of 15 classes of compound (C-skeletons), and 4 C-skeletons were recognized in the monoterpene group, giving a total of 19. The variation of chemical profiles was distinct at different phenological stages, but stronger chemical variation was evident between day and night as compared to season. Furthermore, due to comprehensive sampling across different regions, nine chemotypes were recognized, including those previously reported. The SRO and GMRO indices demonstrate that phenological variation of chemistry is mainly an outcome of redox fluctuations in terpene biosynthesis, changing from day to night. These indices also corroborate that chemical diversity is increased with oxidative metabolism. Lastly, the current study demonstrates pronounced phenotypic plasticity in P. gaudichaudianum, which makes it a suitable candidate to help further our understanding of chemophenetics and chemical ecology.

Essential Oils from Piper lhotzkyanum Kunth Leaves from Brazilian Atlantic Forest: Chemical Composition and Stability in Different Storage Conditions.

This work aimed to evaluate the impact of different storage conditions and light and temperature exposures on the visual aspect and chemical composition of the essential oil (EO) of Piper lhotzkyanum Kunth, obtained from leaves by hydrodistillation from a region of high altitude. For this purpose, aliquots of the EO were stored for up to 90 days (a) under a refrigerator condition of 5 ± 3°C, (b) under a long-term (LT) condition of 30 ± 2°C and 75 ± 5% relative humidity (RH) and an accelerated condition (AS) of 40 ± 2°C and 75 ± 5% RH, and (c) in a photostability test achieved in amber and colorless glass vials. The changes were monitored on days 0 (control), 60, and 90 for the refrigerator, LT, and AS conditions. All EO chemical analyses were assessed by GC-FID and GC-MS for quantification and identification, respectively. It is reported, for the first time, that the EO of P. lhotzkyanum is rich in the sesquiterpenes ß-elemene and α-zingiberene. No significant changes in the EO was observed, revealing a minimal impact of temperature on the sample at the different storage conditions. However, there was a change in the content of α-zingiberene to bicyclogermacrene after exposure to light. The visual appearance of the samples was altered for all test conditions except the refrigerator condition. These results can potentially contribute to the product development of a bioactive EO from leaves of P. lhotzkyanum, a sesquiterpene rich natural material.

The Influence of Anthocyanidin Profile on Antileishmanial Activity of Arrabidaea chica Morphotypes.

Arrabidaea chica Verlot (crajiru) is a plant used in folk medicine as an astringent, anti-inflammatory, wound healing and to treat fungal and viral diseases such as measles chickenpox and herpes. Arrabidaea chica has several morphotypes recognized but little is known about its chemical variability. In the present study the anthocyanidin profile of A. chica morphotypes collected in two seasons (summer and winter) have been examined and their activity against Leishmania infection compared. High-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD-UV) and by tandem mass spectrometry with electrospray ionization (ESI-MS/MS) were used for anthocyanidin separation and identification. Antileishmanial activity was measured against promastigote forms of Leishmania amazonensis. Multivariate analysis, principal component analysis (PCA) and Pearson's correlation were performed to classify morphotypes accordingly to their anthocyanidin profile. The presence of 6,7,3',4'-tetrahydroxy-5-methoxyflavylium (3'-hydroxy-carajurone) (1), carajurone (2), 6,7,3'-trihydroxy-5,4'-dimethoxy-flavylium (3'-hydroxy-carajurin) (3) and carajurin (4), and three unidentified anthocyanidins were detected. Two different groups were recognized: group I containing 3'-hydroxy-carajurone; and group II with high content of carajurin. Among anthocyanidins identified in the extracts, only carajurin showed significant statistical correlation (p = 0.030) with activity against L. amazonensis. Carajurin could thus be considered as a pharmacological marker for the antileishmanial potential of the species.

Treatment of anal fistula with Baccharis dracunculifolia extract. Experimental study in rats. / Tratamento da fístula anal com extrato de Baccharis dracunculifolia. Estudo experimental em ratos.

OBJECTIVE: to evaluate the efficacy of Baccharis dracunculifolia extract in the treatment of anal fistulas in rats. METHODS: twenty male Wistar rats were submitted to anal fistula and, after 30 days, were divided into three groups: Control Group, with five animals; Carbopol Group, with five animals; and Baccharis dracunculifolia Group, with ten animals. In the Control Group, no treatment was performed. In the Carbopol Group, a daily infusion of Carbopol was performed for 30 days. In the Baccharis dracunculifolia Group, a daily infusion of Carbopol plus Baccharis dracunculifolia extract was performed for 30 days. Specimens were taken for histological analysis after euthanasia. RESULTS: there was no complete closure of the fistulous tract in any of the animals. The mean area of the remaining tract was of 847.2µm2, 565.6µm2 and 372.7µm2, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.001). The mean of the inflammatory process score was of 2.4, 2.4, and 2.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.285), while the mean values of vascular congestion were of 1.6, 1.4, and 1.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.031). CONCLUSION: Baccharis dracunculifolia extract was able to reduce the lumen of the fistulous tracts and the degree of vascular congestion, without, however, reducing the local inflammatory process or totally closing the fistulous tracts.

OBJETIVO: avaliar a eficácia do extrato de Baccharis dracunculifolia no tratamento de fístulas anais em ratos. MÉTODOS: vinte ratos Wistar machos foram submetidos à confecção de fístula anal e, após 30 dias, foram distribuídos em três grupos: Grupo Controle, com cinco animais; Grupo Carbopol, com cinco animais; e Grupo Baccharis dracunculifolia, com dez animais. No Grupo Controle, não se realizou nenhum tratamento. No Grupo Carbopol, realizou-se infusão diária de carbopol, e no Grupo Baccharis dracunculifolia, infusão de extrato de Baccharis dracunculifolia com carbopol, ambos por 30 dias. Foram retirados espécimes para análise histológica após a eutanásia. RESULTADOS: não houve fechamento completo do trajeto fistuloso em nenhum dos animais. A média da área do trajeto resultante foi de 847,2µm2, 565,6µm2 e 372,7µm2, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,001). A média do escore de processo inflamatório foi de 2,4, 2,4 e 2,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,285), enquanto a média de congestão vascular foi de 1,6, 1,4 e 1,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,031). CONCLUSÃO: o extrato de Baccharis dracunculifolia foi capaz de reduzir o lúmen dos trajetos fistulosos e a congestão vascular, sem reduzir, no entanto, o processo inflamatório local ou fechar totalmente os trajetos fistulosos.

Tratamento da fístula anal com extrato de Baccharis dracunculifolia. Estudo experimental em ratos / Treatment of anal fistula with Baccharis dracunculifolia extract. Experimental study in rats

RESUMO Objetivo: avaliar a eficácia do extrato de Baccharis dracunculifolia no tratamento de fístulas anais em ratos. Métodos: vinte ratos Wistar machos foram submetidos à confecção de fístula anal e, após 30 dias, foram distribuídos em três grupos: Grupo Controle, com cinco animais; Grupo Carbopol, com cinco animais; e Grupo Baccharis dracunculifolia, com dez animais. No Grupo Controle, não se realizou nenhum tratamento. No Grupo Carbopol, realizou-se infusão diária de carbopol, e no Grupo Baccharis dracunculifolia, infusão de extrato de Baccharis dracunculifolia com carbopol, ambos por 30 dias. Foram retirados espécimes para análise histológica após a eutanásia. Resultados: não houve fechamento completo do trajeto fistuloso em nenhum dos animais. A média da área do trajeto resultante foi de 847,2µm2, 565,6µm2 e 372,7µm2, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,001). A média do escore de processo inflamatório foi de 2,4, 2,4 e 2,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,285), enquanto a média de congestão vascular foi de 1,6, 1,4 e 1,1, respectivamente, nos Grupos Controle, Carbopol e Baccharis dracunculifolia (p=0,031). Conclusão: o extrato de Baccharis dracunculifolia foi capaz de reduzir o lúmen dos trajetos fistulosos e a congestão vascular, sem reduzir, no entanto, o processo inflamatório local ou fechar totalmente os trajetos fistulosos.

ABSTRACT Objective: to evaluate the efficacy of Baccharis dracunculifolia extract in the treatment of anal fistulas in rats. Methods: twenty male Wistar rats were submitted to anal fistula and, after 30 days, were divided into three groups: Control Group, with five animals; Carbopol Group, with five animals; and Baccharis dracunculifolia Group, with ten animals. In the Control Group, no treatment was performed. In the Carbopol Group, a daily infusion of Carbopol was performed for 30 days. In the Baccharis dracunculifolia Group, a daily infusion of Carbopol plus Baccharis dracunculifolia extract was performed for 30 days. Specimens were taken for histological analysis after euthanasia. Results: there was no complete closure of the fistulous tract in any of the animals. The mean area of the remaining tract was of 847.2µm2, 565.6µm2 and 372.7µm2, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.001). The mean of the inflammatory process score was of 2.4, 2.4, and 2.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.285), while the mean values of vascular congestion were of 1.6, 1.4, and 1.1, in the Control Group, Carbopol Group, and Baccharis dracunculifolia Group, respectively, (p=0.031). Conclusion: Baccharis dracunculifolia extract was able to reduce the lumen of the fistulous tracts and the degree of vascular congestion, without, however, reducing the local inflammatory process or totally closing the fistulous tracts.

The effect of Sebastiania hispida gel on wound model infected by methicillin resistant Staphylococcus aureus.

Medicinal plants are an alternative for the treatment of infected wounds. The objective of this study was to evaluate the efficacy of Sebastiania hispida in an animal model with a wound infected by Staphylococcus aureus. The crude ethanol extract (ExtEtOH) of S. hispida underwent phytochemical analysis, quantification of metabolites and antibacterial activity analysis performed using S. aureus. Wistar rats were used to test healing activity, and the groups evaluated comprised gels of ExtEtOH at the concentrations 0.2 and 2% compared with control groups. Animals were inoculated with the bacteria S. aureus resistant to methicillin. The treatment periods were of 3 and 21 days. Macroscopic and microscopic analysis were conducted and data were submitted to analysis of variance (p < 0.05). Phytochemical and quantification analysis indicated that phenolic compounds and flavonoids are the major constituents, followed by tyterpenes. ExtEtOH 0.2% was the most effective gel against the growth of strains of S. aureus. Histological wound and regression analysis showed that ExtEtOH gels (0.2% and 2%) were similar and effective in promoting wound healing. In the quantification of collagen fibers, the animals from all groups showed a high amount of thick collagen fibers. Thus, ExtEtOH gels based on the shoots of S. hispida can be used for the treatment of infected wounds as a complementary therapy for infected wound closure and further assays are required with other means. The healing effectiveness may be due to the high content of phenolics, flavonoids and triterpenes.

Isolation of a larvicidal compound from Piper solmsianum C.DC. (Piperaceae).

The Aedes aegypti mosquito is one of the major vectors of arboviruses. These diseases have re-emerged and the insecticides used nowadays are toxic to mammals and environment and have only been effective in the short-term. In this context, natural products are an alternative. The genus Piper has many active compounds against arthropods, including neolignans. The present study evaluated the larvicidal potential of the n-hexanic extract of Piper solmsianum and eupomatenoid-6, identified by GC-MS and NMR techniques, from this extract against Ae. aegypti. The crude extract (100 µg/mL) killed 80% and 98.3% of larvae in the first and third day, respectively. Eupomatenoid-6 exhibited LD50 of 19.33 µM and LD90 of 28.68 µM and was then assayed in human fibroblast cells (MRC5), showing an IC50 of 39.30 µM with estimated LD50 of 42.26 mmol/kg. Our results indicate eupomatenoid-6 as a potent insecticide with relatively low toxicity for mammals.

An Overview of Neolignans of the Genus Piper L.: Isolation Methods and Biological Activities.

BACKGROUND: The genus Piper L. has the shikimic acid pathway predominantly expressed, biosynthesizing many cinnamic acid derivatives (CAD). OBJECTIVE: Neolignans comprise an important class of CAD that exhibit a wide range of pharmacological properties such as antibacterial, antitumor, insecticidal, anti-inflammatory, antioxidant, smooth muscle relaxant, neuroprotective, antiprotozoal and against platelet aggregation factor. These substances have been extracted and isolated from Piper species using different technics. RESULTS: The present review aims to summarize extraction and isolation methods and biological activities of the different types of neolignans covering the period from 1968 to January 2016.