Trichilia catigua and Turnera diffusa phyto-phospholipid nanostructures: Physicochemical characterization and bioactivity in cellular models of induced neuroinflammation and neurotoxicity.

Flavonoid-based therapies supported by nanotechnology are considered valuable strategies to prevent or delay age-related and chronic neurodegenerative disorders. Egg yolk phospholipids were combined with flavonoid-rich extracts obtained from Trichilia catigua A.Juss. (rich in flavan-3-ols and phenylpropanoid derivatives) or Turnera diffusa Willd. ex Schult (dominated by luteolin derivatives) to prepare nanophytosomes. The nanophytosomes showed that size and surface charge of the lipid-based vesicles are dependent of their phenolic composition. In vitro assays with SH-SY5Y cells showed that both formulations protect cells from glutamate-induced toxicity, but not from 6-hydroxydopamine/ascorbic acid. T. diffusa nanophytosomes promote a decrease of nitric oxide produced by BV-2 cells stimulated with interferon-γ. Nanophytosomes dialysed against a mannitol solution, and then lyophilised, allow to obtain freeze-dried products that after re-hydration preserve the essential physicochemical features of the original formulations, and exhibit improved colloidal stability. These results indicate that these flavonoid/phospholipid-based nanophytosomes have suitable features to be considered as tool in the development of therapeutic and food applications.

Chemical Characterization of Flowers and Leaf Extracts Obtained from Turnera subulata and Their Immunomodulatory Effect on LPS-Activated RAW 264.7 Macrophages.

The anti-inflammatory properties of Turnera subulata have been evaluated as an alternative drug approach to treating several inflammatory processes. Accordingly, in this study, aqueous and hydroalcoholic extracts of T. subulata flowers and leaves were analyzed regarding their phytocomposition by ultrafast liquid chromatography coupled to mass spectrometry, and their anti-inflammatory properties were assessed by an in vitro inflammation model, using LPS-stimulated RAW-264.7 macrophages. The phytochemical profile indicated vitexin-2-O-rhamnoside as an important constituent in both extracts, while methoxyisoflavones, some bulky amino acids (e.g., tryptophan, tyrosine, phenylalanine), pheophorbides, and octadecatrienoic, stearidonic, and ferulic acids were detected in hydroalcoholic extracts. The extracts displayed the ability to modulate the in vitro inflammatory response by altering the secretion of proinflammatory (TNF-α, IL-1ß, and IL-6) and anti-inflammatory (IL-10) cytokines and inhibiting the PGE-2 and NO production. Overall, for the first time, putative compounds from T. subulata flowers and leaves were characterized, which can modulate the inflammatory process. Therefore, the data highlight this plant as an option to obtain extracts for phytotherapic formulations to treat and/or prevent chronic diseases.

Evaluation of the testicular protection conferred by damiana (Turnera diffusa Willd.) against amitriptyline-induced testicular toxicity, DNA damage and apoptosis in rats.

Psychiatric drugs, such as antidepressants, are used to treat depression based on their ability to modify chemical imbalances of the key neurotransmitters in the brain, including dopamine, serotonin, and norepinephrine. Amitriptyline, a first-reference tricyclic antidepressant derived from dibenzocycloheptadine, is frequently used, especially in neuropsychiatry, to address general depression, major depressive disorders, and fibromyalgia. Therefore, this study attempted to examine the sexual dysfunction attendant on the use of Amitriptyline by investigating the protective role that can be played by damiana. To this end, this study used damiana (Turnera diffusa Willd.) as adjuvant therapy in male albino rats receiving Amitriptyline. Sixty male albino rats were randomly allocated to six groups, with 10 rats being assigned to each group; the first group was a control, the second was treated with damiana only, the third group was given Amitriptyline, the fourth group received Amitriptyline and damiana simultaneously, the fifth group was given Amitriptyline and post-treated with damiana, and the sixth group was given Amitriptyline and then allowed time for self-healing. The findings of this study suggest that oxidative stress occurs in testicular tissue in rat groups treated with Amitriptyline, as manifested by inappropriate activity of TBARS, SOD, GSH, GR, GST, and GPx. Amitriptyline also repressed reproductive hormonal activity, as confirmed by histopathological lesions, DNA damage, and p53 protein expression. The addition of damiana, however, showed aprotective role in all testicular activity indices.

Thrombin Inhibition: Preliminary Assessment of the Anticoagulant Potential of Turnera subulata (Passifloraceae).

Cardiovascular and thromboembolic disturbances are the main causes of disease-related deaths worldwide. Regardless of the etiological factors involved in thrombus formation, coagulation is mainly activated by thrombin, one of the most important blood clotting molecules. Thus, this study evaluated the Turnera subulata leaf crude extract, its ethyl acetate fraction effect on the coagulation cascade, and its possible side effects. Their phytocomposition indicated polyphenols, mainly flavonol-3-O-glycosylate and a flavone glycoside, without in vitro and in vivo toxicity. Regarding their potential anticoagulants, results displayed partial thromboplastin and prothrombin time activation, and Xa and IIa, and thrombin inhibition by heparin II cofactor, indicating significant anticoagulant activity, suggesting direct and indirect thrombin inhibition as the main mechanism of action. Therefore, T. subulata leaf active compounds exhibit therapeutic potential required to develop phytotherapeutic formulations to assist conventional anticoagulants in clinical treatments.

Selective Inhibition of Human Monoamine Oxidase B by Acacetin 7-Methyl Ether Isolated from Turnera diffusa (Damiana).

The investigation of the constituents that were isolated from Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC50 = 198 nM). Acacetin 7-methyl ether (also known as 5-hydroxy-4', 7-dimethoxyflavone) is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC50 = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC50 for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl (i.e., SelegilineTM or ZelaparTM, a selective MAO-B inhibitor), acacetin 7-methyl ether's selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson's disease.

Anthelmintic activity of plant extracts from Brazilian savanna.

Helminth infections represent a serious problem for the production of small ruminants that is currently aggravated by resistance to anthelmintic products and has induced a search for control alternatives, such as natural products. In this study, extracts of Turnera ulmifolia L. (leaves and roots), Parkia platycephala Benth. (leaves and seeds) and Dimorphandra gardneriana Tul. (leaves and bark), which have been cited in ethnoveterinary studies and selected naturally by goats in the cerrado (Brazilian savanna), were tested in vitro against Haemonchus contortus. Hydroacetonic (ACT) and hydroalcoholic (ETH) extracts were evaluated using an Egg Hatching Assay (EHA), a Larval Exsheathment Inhibition Assay (LEIA) and a Larval Development Assay (LDA). A second set of incubations was performed using polyvinylpolypyrrolidone (PVPP) to determine the influence of polyphenols on the anthelmintic effects of EHA and LEIA. Data from each extract were used to calculate inhibition concentrations (IC50). All tested extracts showed activity against at least one life stage of H. contortus. The use of PVPP revealed that the tannins are not the only extracts of secondary metabolites responsible for the anthelmintic effects. The results showed clear in vitro anthelmintic activities against H. contortus at different stages and indicated the potential use of these species as a promising alternative approach to control helminthic infections of small ruminants.

Phytochemical analysis of hydroethanolic extract of Turnera diffusa Willd and evaluation of its effects on astrocyte cell death / Análise fitoquímica do extrato hidroetanólico de Turnera diffusa Willd e avaliação de seus efeitos na morte de astrócitos

ABSTRACT Objective To evaluate the phytochemical composition of hydroethanolic extracts from powdered aerial parts of Turnera diffusa Willd (Turneraceae; T. diffusa), as well as its toxicity in astrocytes. Methods Chemical analyses of hydroethanolic extract from powdered aerial parts ofT. diffusa were carried out using HPLC-DAD-ESI-MS/MS.In vitro assays using astrocytes culture were performed to evaluate cell death. Results Flavone-C, O-diglycosides, such as, luteolin-8-C-[6-deoxy-2-O-rhamnosyl]-xylo-hexos-3-uloside, apigenin-8-C-[6-deoxy-2-O-rhamnosyl]-xylo-hexos-3-uloside and apigenin-7-O-6”-p-coumaroylglucoside were the main compounds found in this hydroethanolic extract. Concentration time-effect demonstrated the toxicity of this extract at a concentration of 1,000µg/mL in astrocyte culture, after 6 and 24 hours of incubation. Conclusion In phytochemical analyses, important antioxidants (mainly flavonoids) were observed. T. diffusa extracts presented cytotoxic effect in high concentrations, leading to increased cell death in astrocyte culture.

RESUMO Objetivo Avaliar a composição fitoquímica do extrato hidroetanólico das partes aéreas de Turnera diffusa Willd (Turneraceae; T. diffusa) e sua toxicidade em astrócitos. Métodos Análises químicas do extrato hidroetanólico de partes aéreas de T. diffusa foram feitas por HPLC-DAD-ESI-MS/MS. Os ensaiosin vitro utilizaram culturas de astrócitos para avaliar morte celular. Resultados Flavonas-C, O-diglicosídeos, como, luteolina-8-C-[6-deoxi-2-O-raminosil]-xilo-hexos-3-ulosideo, apigenina-8-C-[6-deoxi-2-O-raminosil]-xilo-hexos-3-ulosideo e apigenina-7-O-6”-p-cumaroilglucosídeo foram os principais constituintes encontrados neste extrato hidroetanólico. Uma curva tempo-concentração demonstrou toxicidade desse extrato na concentração de 1.000µg/mL, na cultura de astrócitos após 6 e 24 horas de incubação. Conclusão Nas análises fitoquímicas, importantes antioxidantes, sobretudo flavonoides, foram observados. Extratos de T. diffusa apresentaram efeitos citotóxicos em altas concentrações, ocasionando aumento de morte celular em cultura de astrócitos.

Cytotoxic activity of the methanolic extract of Turnera diffusa Willd on breast cancer cells.

Turnera diffusa Willd, commonly known as Damiana, is employed in traditional medicine as a stimulant, aphrodisiac, and diuretic. Its leaves and stems are used for flavoring and infusion. Damiana is considered to be safe for medicinal use by the FDA. Pharmacological studies have established the hypoglycemic, antiaromatase, prosexual, estrogenic, antibacterial, and antioxidant activity of T. diffusa. The aim of the present study was to evaluate the possible cytotoxic effect of extracts and organic fractions of this plant on five tumor cell lines (SiHa, C-33, Hep G2, MDA-MB-231, and T-47D) and normal human fibroblasts. The results show that the methanolic extract (TdM) displayed greater activity on MDA-MB-231 breast cancer cells (with an IC50 of 30.67 µg/mL) than on the other cancer cell lines. Four organic fractions of this extract exhibited activity on this cancer cell line. In the most active fraction (F4), two active compounds were isolated, arbutin (1) and apigenin (2). This is the first report of a cytotoxic effect by T. diffusa on cancer cells. The IC50 values suggest that the methanolic extract of T. diffusa has potential as an anticancer therapy.

Antioxidant activity and protective effect of Turnera ulmifolia Linn. var. elegans against carbon tetrachloride-induced oxidative damage in rats.

The present study aimed to determine whether the leaves of Turnera ulmifolia Linn. var. elegans extract exert significant antioxidant activity. The antioxidant activity of its hydroethanolic extract (HEETU) was evaluated by assessing (a) its radical scavenging ability in vitro, and (b) its in vivo effect on lipid peroxidation and antioxidant enzyme activities. The in vitro antioxidant assay (DPPH) clearly supported HEETU free radical scavenging potential. Moreover, glutathione content and antioxidant enzyme activities (glutathione peroxidase, superoxide dismutase and catalase) were significantly enhanced in CCl(4)-treated rats due to oral HEETU-treatment (500 mg/kgb.w.) over 7 and 21 days. In addition, an improvement was observed in lipid peroxidation and serum biochemical parameters (aspartate aminotransferase and alanine aminotransferase), indicating a protective effect against CCl(4)-induced liver injuries, confirmed by histopathological studies. The HEETU effect was comparable to the standard drug Legalon® (50 mg/kgb.w.) under the same experimental condition. Quantitative analysis of the HPLC extract revealed the presence of flavonoids, wich mediate the effects of antioxidant and oxidative stress. In conclusion, extract components exhibit antioxidant and hepatoprotective activities in vitro and in vivo.

Gastroprotective activities of Turnera diffusa Willd. ex Schult. revisited: Role of arbutin.

ETHNOPHARMACOLOGICAL RELEVANCE: Turnera diffusa Willd. ex Schult. has been used for the treatment of several human disorders including peptic ulcer. OBJECTIVES OF THE STUDY: The current study is an attempt to evaluate the anti-ulcerogenic activities of arbutin, a major constituent of Turnera diffusa on two ulcer models. The possible involvement of lipid peroxidation, nitric oxide, IL-6, IL-10, TNF-α and mucus barrier mechanism has been investigated. MATERIALS AND METHODS: Effects of arbutin on ulcer index, gastric juice acidity, mucus content and histochemistry, gross and histological gastric lesions, nitric oxide, cytokines levels (IL-6, IL-10 and TNF-α), and thiobarbituric acid reactive substances (TBARS), were evaluated in aspirin or ethanol-induced ulcer in vivo. Acute toxicity of arbutin was also examined in rodent model. MTT assay was used to assess the cytotoxicity of the compound on normal liver cells (WRL-68). RESULTS: Pre-treatment with arbutin or omeprazole protected the gastric mucosa as seen by reduction in ulcer area and mucosal content, reduced or absence of edema, inflammation and leucocytes infiltration on both models. Arbutin significantly (P<0.05) lowered the elevated TBARS level into gasteric homogenate. Arbutin did not produce significant inhibition of NO. This natural compound has modulated the levels of interleukin-6, interleukin-10 and TNF-α. No in vitro or in vivo toxicities for arbutin were observed. CONCLUSION: Thus it can be concluded that Turnera diffusa possesses anti-ulcer activity, which could be attributed to lipid peroxidation inhibitory, immuno modulatory and anti-oxidant mechanisms of arbutin but not to the intervention with nitric oxide inflammation pathway.

Avaliação da atividade anti-Trypanosoma e anti-Leishmania de Mentha arvensis e Turnera ulmifolia / Evaluation of the anti-Trypanosoma and anti-Leishmania activity of Mentha arvensis and Turnera ulmifolia

Tripanosomiasis or “Chagas disease”, caused by Trypanosoma cruzi, affect 10 million people in Latin America. Today, the chemotherapy is the only specific treatment against this disease, being the most used drugs the nifurtimox and benznidazole. Leishmaniasis is a disease caused by parasites of the genus Leishmania, mainly founded in regions with forests, as the Amazonia. Recent reports about the Leishmaniasis indicate a deficit of therapeutical drugs available against this disease and reinforce the necessity of the discovering of new drugs. An interesting approach against these diseases is the use of natural products, as the extracts of plants as Mentha arvensis and Turnera ulmifolia. For the in vitro assays against T. cruzi and Leishmania, was used the clone CL-B5 and promastigote forms, respectively. The cytotoxic assay was performed using fibroblasts. Our results indicated that M. arvensis was active against all strains assayed, inhibiting 65 e 47 percent of the assayed strains (IC50 = 192.3 and 531.9 ug/mL respectively), representing an interesting and alternative source of natural products with anti-kinetoplastida activity.

Doença de Chagas, causada por Trypanosoma cruzi, afeta cerca de 10 milhões de pessoas nas Américas. Atualmente, a quimioterapia é o único tratamento específico disponível para esta doença, onde os medicamentos utilizados são nifurtimox e benzonidazol. Leishmaniose tegumentar Americana no Brasil é causada por uma variedade de espécies de Leishmania e uma grande diversidade destes parasitos pode ser encontrada na Região Amazônica. Revisões recentes na quimioterapia de leishmaniose enfatizam as deficiências dos agentes terapêuticos atualmente disponíveis e mostram a necessidade urgente de novos candidatos. Uma alternativa para substituir esses medicamentos são extratos naturais de Mentha arvensis e Turnera ulmifolia. Foram preparados extratos etanólicos das folhas de M. arvensis e T. ulmifolia. Para os testes in vitro de T. cruzi, foi utilizado o clone CL-B5 e para Leishmania brasiliensis foram utilizadas formas promastigotas. O ensaio de citotoxicidade foi realizado com linhagens de fibroblastos. Nossos resultados indicam que M. arvensis foi eficaz contra as cepas de parasitos testadas apresentando 65 e 47 por cento de inibição em uma concentração de 500 ug/mL (respectivamente, CE50 = 192.3 e 531.9 ug/mL), sendo considerada uma fonte alternativa de produtos naturais com atividade contra T. cruzi e L. brasiliensis.

Composition of three essential oils, and their mammalian cell toxicity and antimycobacterial activity against drug resistant-tuberculosis and nontuberculous mycobacteria strains.

Tuberculosis (TB) is the most ancient epidemic disease in the world and a serious opportunistic disease in HIV/AIDS patients. The increase in multidrug resistant Mycobacterium tuberculosis (MDR-TB, XDR-TB) demands the search for novel antimycobacterial drugs. Essential oils (EOs) have been widely used in medicine and some EOs and their major components have been shown to be active against M. tuberculosis. The aim of this work was to evaluate the antimycobacterial and cell toxicity activities of three EOs derived from Salvia aratocensis, Turnera diffusa and Lippia americana, aromatics plants collected in Colombia. The EOs were isolated by hydrodistillation and analyzed by GC/MS techniques. The EOs were tested against 15 Mycobacterium spp using a colorimetric macrodilution method and against mammalian Vero and THP-1 cells by MTT. The activity was expressed as minimal concentration in microg/mL that inhibits growth, and the concentration that is cytotoxic for 50 or 90% of the cells (CC50 and CC90). The major components were epi-alpha-cadinol (20.1%) and 1,10-di-epi-cubenol (14.2%) for Salvia aratocensis; drima-7,9(11)-diene (22.9%) and viridiflorene (6.6%) for Turnera diffusa; and germacrene D (15.4%) and trans-beta- caryophyllene (11.3%) for Lippia americana. The most active EO was obtained from S. aratocensis, with MIC values below 125 microg mL(-1) for M. tuberculosis Beijing genotype strains, and 200 to 500 microg mL(-1) for nontuberculous mycobacterial strains. The EOs were either partially or non toxic to Vero and THP-1 mammalian cells with CC50 values from 30 to > 100 microg mL(-1), and a CC90 > 100 microg mL(-1). The EOs obtained from the three aromatic Colombian plants are an important source of potential compounds against TB. Future studies using the major EO components are recommended.

Essential oil of Turnera ulmifolia leaves from Cuba.

The chemical composition of the volatile compounds from the leaves of Turnera ulmifolia L. (Turneraceae) from Cuba was studied by GC and GC/MS. Sixty-four volatile compounds were identified, of which the major ones were beta-caryophyllene (21.5%) and (Z)-3-hexen-1-ol (18.4%).

Anti-aromatase activity of the constituents from damiana (Turnera diffusa).

ETHNOPHARMACOLOGICAL RELEVANCE: Damiana (Turnera diffusa Willd. Ex Schult) has traditionally been used as an herbal aphrodisiac. AIM OF THE STUDY: The study was aimed to investigate the anti-aromatse activity and the estrogenic activity of the constituents isolated from Turnera diffusa. MATERIALS AND METHODS: The methanolic extract and 24 compounds isolated from the leaves of Turnera diffusa were evaluated for aromatase activity by using a tritiated-water release assay and for estrogenic activity by using yeast estrogen screen (YES) assay. RESULTS: The methanolic extract demonstrated a dose-dependent inhibitory activity of the aromatase enzyme with the IC(50) value of 63.1 microg/ml. Among the 24 tested compounds, pinocembrin and acacetin showed the most potent inhibition with IC(50) values of 10.8 and 18.7 microM, respectively. Estrogenic activity was also observed in the extract and three compounds including apigenin 7-glucoside, Z-echinacin and pinocembrin with EC(50) values of 10, 20 and 67 microuM, respectively. CONCLUSIONS: The extract of Turnera diffusa and two isolated compounds pinocembrin and acacetin could significantly suppress aromatase activity. Moreover, apigenin 7-glucoside, Z-echinacin and pinocembrin showed estrogenic activity.

Application of low-pressure gas chromatography-ion-trap mass spectrometry to the analysis of the essential oil of Turnera diffusa (Ward.) Urb.

Turnera diffusa Willd. var. afrodisiaca (Ward) Urb. (syn. T. aphrodisiaca) belongs to the family of Turneraceae and is an aromatic plant growing wild in the subtropical regions of America and Africa. It is widely used in the traditional medicine as e.g. anti-cough, diuretic, and aphrodisiac agent. This work presents a 3 min chromatographic analysis using low-pressure (LP) gas chromatography (GC)-ion-trap (IT) mass spectrometry (MS). The combination of a deactivated 0.6 m x 0.10 mm i.d., restrictor with a wide-bore CP-Wax 52 capillary column (10 m x 0.53 mm i.d., 1 microm) reduces the analysis time by a factor of 3-7 in comparison to the use of a conventional narrow bore column. Chromatographic conditions have been optimized to achieve the fastest separation with the highest signal/noise ratio in MS detection. These results allow fast and reliable quality control of the essential oil to be achieved.