Evaluation of Lippia scaberrima Sond. and Aspalathus linearis (Burm.f.) R. Dahlgren extracts on human CYP enzymes and gold nanoparticle synthesis: implications for drug metabolism and cytotoxicity.

BACKGROUND: Metabolism is an important component of the kinetic characteristics of herbal constituents, and it often determines the internal dose and concentration of these effective constituents at the target site. The metabolic profile of plant extracts and pure compounds need to be determined for any possible herb-drug metabolic interactions that might occur. METHODS: Various concentrations of the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with fermented and unfermented Aspalathus linearis extract were used to determine the inhibitory potential on placental, microsomal and recombinant human hepatic Cytochrome P450 enzymes. Furthermore, the study investigated the synthesis and characterization of gold nanoparticles from the ethanolic extract of Lippia scaberrima as a lead sample. Confirmation and characterization of the synthesized gold nanoparticles were conducted through various methods. Additionally, the cytotoxic properties of the ethanolic extract of Lippia scaberrima were compared with the gold nanoparticles synthesized from Lippia scaberrima using gum arabic as a capping agent. RESULTS: All the samples showed varying levels of CYP inhibition. The most potent inhibition took place for CYP2C19 and CYP1B1 with 50% inhibitory concentration (IC50) values of less than 0.05 µg/L for the essential oil tested and IC50-values between 0.05 µg/L-1 µg/L for all the other combinations and extracts tested, respectively. For both CYP1A2 and CYP2D6 the IC50-values for the essential oil, the extracts and combinations were found in the range of 1 - 10 µg/L. The majority of the IC50 values found were higher than 10 µg/L and, therefore, were found to have no inhibition against the CYP enzymes tested. CONCLUSION: Therefore, the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with Aspalathus linearis do not possess any clinically significant CYP interaction potential and may be further investigated for their adjuvant potential for use in the tuberculosis treatment regimen. Furthermore, it was shown that the cytotoxic potential of the Lippia scaberrima gold nanoparticles was reduced by twofold when compared to the ethanolic extract of Lippia scaberrima.

Assessment of the in Vitro Antimelanoma Potential of Lippia microphylla Cham (Verbenaceae) Essential Oil.

Essential oils stand out among natural products for their complex composition, frequently described in the literature with a range of biological effects. This study evaluated the cytotoxic activity against several human cancer cell lines of essential oils extracted from the leaves of Lippia microphylla (EO-LM) Cham. (Verbenaceae). The melanoma cell line SK-MEL-28 was the most sensitive to the EO-LM, presenting an IC50 of 33.38±1.16 µg/mL. Afterward, the effects of EO-LM on the cell cycle, induction of apoptosis, and production of reactive oxygen species (ROS) were evaluated. We stated a significant increase in the sub-G1 population, indicating apoptosis, later confirmed by an increase of SK-MEL-28 cells labeled with Annexin V-FITC and by the formation of apoptotic bodies and membrane blebs, observed by confocal microscopy. Additionally, EO-LM reduced the production of ROS, indicating antioxidant activity. Therefore, EO-LM exhibits anti-melanoma activity in vitro, suggesting its potential as an anticancer agent.

Lippia grata essential oil complexed with ß-cyclodextrin ameliorates biochemical and behavioral deficits in an animal model of progressive parkinsonism.

Parkinson's disease (PD) is identified by the loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), and is correlated to aggregates of proteins such as α-synuclein, Lewy's bodies. Although the PD etiology remains poorly understood, evidence suggests a main role of oxidative stress on this process. Lippia grata Schauer, known as "alecrim-do-mato", "alecrim-de-vaqueiro", "alecrim-da-chapada", is a native bush from tropical areas mainly distributed throughout the Central and South America. This plant species is commonly used in traditional medicine for relief of pain and inflammation conditions, and that has proven antioxidant effects. We evaluated the effects of essential oil of the L. grata after its complexed with ß-cyclodextrin (LIP) on PD animal model induced by reserpine (RES). Behavioral assessments were performed across the treatment. Upon completion the treatment, the animals were euthanized, afterwards their brains were isolated and processed for immunohistochemical and oxidative stress analysis. The LIP treatment delayed the onset of the behavior of catalepsy, decreased the number of oral movements and prevented the memory impairment on the novel object recognition task. In addition, the treatment with LIP protected against dopaminergic depletion in the SNpc and dorsal striatum (STRd), and decreased the α-syn immunoreactivity in the SNpc and hippocampus (HIP). Moreover, there was reduction of the oxidative stability index. These findings demonstrated that the LIP treatment has neuroprotective effect in a progressive parkinsonism model, suggesting that LIP could be an important source for novel treatment approaches in PD.

Neuroprotective effects of Lippia javanica (Burm.F.) Spreng. Herbal tea infusion on Lead-induced oxidative brain damage in Wistar rats.

BACKGROUND: Though Lippia javanica (Burm.f.) Spreng antioxidant activity has been demonstrated, its effect in protecting the brain from lead (Pb)-induced oxidative damage is unknown. This study investigated the effect of L. javanica against Pb-induced oxidative stress, inflammation, apoptosis and acetylcholinesterase activity in rat's brain. METHODS: L. javanica herbal tea infusion was prepared, its phytochemical constituent was revealed by liquid chromatography-Mass spectrometer (LC-MS) and was administered simultaneously with Pb. Four groups of male Wistar rats (n = 5/group) were used: control received distilled water; Pb-acetate group received 50 mg Pb/ Kg bodyweight (bw), treatment group received 50 mg Pb/ Kg Pb-acetate + 5 ml/kg bw L. javanica and L. javanica group received 5 ml/Kg bw of L. javanica tea infusion only. After 6 weeks of treatment, oxidative status, acetylcholinesterase activity, inflammation and apoptosis was assessed in brain tissue which was also histologically examined. RESULTS: Mean brain and heart weight was reduced (p < 0.05) while liver and spleen weights were increased (p < 0.05) in Pb exposed animals but were prevented by L. juvanica treatment. Treatment with L. javanica increased (p < 0.05) overall brain antioxidant status (glutathione and superoxide dismutase activities) and reduced lipid peroxidation (p < 0.05) compared to the Pb exposed animals. Pro-inflammatory cytokine tumour necrotic factor-alpha, pro-apoptosis Bax protein and anticholinesterase activity were reduced (p < 0.05) in Pb-L. javanica treated animals compared to the Pb exposed group. Histological examination confirmed neuroprotective effects of L. javanica as evidenced by reduced apoptosis/necrosis and inflammation-induced vacuolization and oedema in the hippocampus. The L. javanica treatment alone had no detrimental effects to the rats. LC-MS analysis revealed L. javanica to be rich in phenolics. CONCLUSIONS: This study demonstrated that L. javanica, rich in phenolics was effective in reducing Pb-induced brain oxidative stress, inflammation, apoptosis, acetylcholinesterase activity and neuronal damage.

Involvement of the PKA pathway and inhibition of voltage gated Ca2+ channels in antihyperalgesic activity of Lippia grata/ß-cyclodextrin.

Neuropathic pain (NP) is a difficult condition to treat because of the modest efficacy of available drugs. New treatments are required. In the study we aimed to investigate the effects of the essential oil from Lippia grata alone or complexed in ß-cyclodextrin (LG or LG-ßCD) on persistent inflammatory and neuropathic pain in a mouse model. We also investigated Ca2+ currents in rat dorsal root ganglion (DRG) neurons. Male Swiss mice were treated with LG or LG/ß-CD (24 mg/kg, i.g.) and their effect was evaluated using an acute inflammatory pleurisy model and nociception triggered by intraplantar injection of an agonist of the TRPs channels. We also tested their effect in chronic pain models: injection of Freund's Complete Adjuvant and partial sciatic nerve ligation (PSNL). In the pleurisy model, LG reduced the number of leukocytes and the levels of TNF-α and IL-1ß. It also inhibited cinnamaldehyde and menthol-induced nociceptive behavior. The pain threshold in mechanical and thermal hyperalgesia was increased and paw edema was decreased in models of inflammatory and neuropathic pain. PSNL increased inflammatory protein contents and LG and LG-ßCD restored the protein contents of TNF-α, NF-κB, and PKA, but not IL-1ß and IL-10. LG inhibited voltage gated Ca2+ channels from DRG neurons. Our results suggested that LG or LG-ßCD produce anti-hyperalgesic effect in chronic pain models through reductions in TNF-α levels and PKA, and inhibited voltage-gated calcium channels and may be innovative therapeutic agents for the management of NP.

Ecophysiological response of Lippia gracilis (Verbanaceae) to duration of salt stress.

The objective of this work was to investigate the effects of salinized water on Lippia gracilis by determining the ion accumulation capacity and morphophysiological changes as a function of salinity and time of exposure to stress. L. gracilis was irrigated with water containing different concentrations of salts and electrical conductivities. The analyses were conducted 25 and 50 days after stress (DAS), in leaf, stem/branches, and roots. Plant height (Hplants), basal stem diameter (Φstem), and mean unitary leaf area (ULA) were determined. After that, the plant material was dried, and the total dry biomass (TDB) (in g plant-1), leaf dry biomass (LDB), stem dry biomass (SDB), and the roots dry biomass (RDB) were determined. Based on these data, the following variables were calculated: total leaf area (TLA) in cm2 plant-1; biomass allocation fraction (BAF) obtained by dividing the biomass of the stem, leaves, and roots, separately, with the total plant biomass; relative growth rate (RGR); net assimilation rate (NAR); leaf area ratio (LAR); specific leaf area (SLA); and sclerophylly index (SI). The concentrations of potassium and sodium ions were quantified by atomic absorption spectrophotometry and the chloride ion concentration was determined by argentometric titration. L. gracilis showed a variable response to salt stress as a function of time, having a considerable capacity to withstand an increasing load of toxic ions, while maintaining a positive growth rate, highlighting the potential use of the species in moderately saline environments.

Prospection of Enzyme Modulators in Aqueous and Ethanolic Extracts of Lippia sidoides Leaves: Genotoxicity, Digestion, Inflammation, and Hemostasis.

The aqueous and ethanolic extracts of Lippia sidoides Cham. were chemically characterized and tested for their action on enzymes involved in processes such as inflammation, blood coagulation, and digestion. Both extracts potentiated the activity of phospholipases A2 present in the venom of Bothrops atrox in 12 % and completely inhibited the hemolysis induced by B. jararacussu and B. moojeni venoms in the proportions between 1 : 0.5 and 1 : 5 (venom/extracts (w/w)). They inhibited the thrombolysis induced by B. moojeni (10 to 25 %), potentiated the thrombolysis induced by the Lachesis muta muta venom (30 to 80 %), prolonged the coagulation time induced by B. moojeni and L. muta muta venoms, and presented antigenotoxic action. Both extracts reduced the activity of α-glycosidases, the aqueous extract inhibited lipases, and the ethanolic extract inhibited α-amylases. The results demonstrate the modulatory action of the extracts on proteases, phospholipases, and digestive enzymes. In addition, the rich phenolic composition of these extracts highlights their potential for nutraceutical use.

Enhanced and green extraction of bioactive compounds from Lippia citriodora by tailor-made natural deep eutectic solvents.

Natural deep eutectic solvents (NADESs) have been postulated as alternative green solvents for the isolation of valuable bioactive compounds from Lippia citriodora. Thus, 11 different NADESs, based on choline chloride (ChCl) as the hydrogen bond acceptor in combination with different hydrogen bond donors (organic acids, polyalcohols, sugars, and urea) were tested. According to the results obtained, ChCl-lactic acid exhibited the highest extraction yield for iridoids, 7.25 mg g-1, phenylpropanoids, 17.23 mg g-1, and flavonoids, 9.02 mg g-1 being significantly greater than phenylpropanoid and flavonoid yields, 15.63 and 5.43 mg g-1 respectively, extracted with methanol as conventional solvent. Subsequently, in order to optimise the most influential microwave assisted extraction (MAE) parameters, a Box-Behnken design paired with a response surface methodology were implemented. Temperature and water content showed a strong effect on the extraction of polyphenol sub-classes, while the effect of irradiation time was less noticeable on extraction yields. Temperature of 63.68 °C, a water content of 32.19% and a microwave irrdiation time of 17.08 min were the optimum conditions provided by the statistical program. The use of NADESs showed potential to facilitate the design and customisation of green tailor-made solvents which have greater extraction capacity than conventional organic solvents.

Growth performance, blood parameters, carcass characteristics and meat quality traits in Potchefstroom Koekoek chickens fed Lippia javanica leaf meal.

Lippia javanica leaves may have nutraceutical bioactivity and thus could be a viable alternative to antibiotic growth promoters in poultry diets. To test this hypothesis, a feeding trial was carried out to determine the effects of dietary inclusion of Lippia javanica leaf meal (LJ) on growth performance, blood parameters, carcass characteristics and meat quality of indigenous Potchefstroom Koekoek (PK) chickens. The leaf meal was used to dilute a commercial chicken grower diet at a rate of 25 or 50 g/kg diet. Two additional experimental diets, a positive control (commercial chicken grower diet with antibiotics, CON+) and a negative control (commercial chicken grower diet without antibiotics, CON-), were also formulated. Feed intake and growth performance were measured over a period of 13 weeks before slaughtering for carcass and meat quality measurements. Blood was collected in week 11 to determine haematological and serum biochemical parameters. Diet had no effect on overall FCE but significantly (P < 0.05) affected bilirubin, alanine aminotransferase (ALT), aspartate transaminase (AST), sodium, potassium, cholesterol and magnesium levels in blood. When included at 50 g/kg, LJ increased carcass weight (P < 0.05). Meat from CON- (6.07) and LJ50 (6.08) chickens had a higher pH than from CON+ (5.9) and LJ25 (5.8) chickens. Breast muscle in CON- chickens had higher (P < 0.05) lightness (L*) value (53.2) compared to chickens fed on other diets. It was observed that meat from LJ25 chickens had the highest for redness value (2.3). It was concluded that L. javanica leaf meal can be included up to 50 g/kg in Potchefstroom koekoek chicken diets without causing negative effects on the birds' feed intake, utilisation and weight gain. Diet-induced changes observed in blood parameters and meat quality fell within the expected normal ranges for indigenous chickens.

Effect of In Vitro Digestion on the Total Antioxidant Capacity and Phenolic Content of 3 Species of Oregano (Hedeoma patens, Lippia graveolens, Lippia palmeri).

Oregano phenolic compounds have been studied for their anti-inflammatory properties. Nonetheless, after ingestion, the gastrointestinal environment can affect their antioxidant stability and thus their bioactive properties. To evaluate the effect of in vitro gastrointestinal (GI) digestion on the phenolic compounds of 3 species of oregano (Hedeoma patens, Lippia graveolens, and Lippia palmeri), the total reducing capacity, total flavonoid content, and antioxidant capacity were evaluated before and after in vitro GI digestion. In addition, the phenolic compounds of the 3 oregano species were identified and quantified by UPLC-PDA before and after in vitro GI digestion. It was shown that the reducing capacity, flavonoid content and antioxidant capacity were affected by the GI digestion process. Moreover, the phenolic compounds identified were apigenin-7-glucoside, scutellarein, luteolin, luteolin-7-glucoside, phloridzin and chlorogenic acid, and their levels were affected by the in vitro GI process. Our results showed that the phenolic compounds from these 3 species of oregano are affected by the in vitro digestion process, and this effect is largely attributable to pH changes. These changes can modify the bioavailability and further anti-inflammatory activity of oregano phenolics, and thus, further research is needed. PRACTICAL APPLICATION: Oregano is a rich source of polyphenols that have shown bioactive properties like anti-inflammatory potential. However, little is known of the gastrointestinal fate of oregano polyphenols which is imperative to fully understand its bioaccessibility. Our results are important to develop new administration strategies which could help protect the antioxidant and anti-inflammatory potential and bioaccessibility of such compounds.

Allelopathic effect of essential oils of medicinal plants in Bidens pilosa L / Efeito Alelopático de oleos essencias de plantas medicinais em Bidens pilosa L

We determined the inhibitory allelopathic effects of the volatile extracts of Cinnamomum zeylanicum Ness, Lippia sidoides Cham. and Cymbopogum nardus L. on seed germination and root growth of seedlings of Bidens pilosa. The experiment was conducted at the Seed Analysis Laboratory of the Department of Plant Science, Federal University of Ceará. For this end, we used oils at the concentrations of 0.01, 0.02, 0.04 and 0.08% (v/v). Five treatments were used for each of the oils arranged in a completely randomized design with four replications of 25 seeds. The seeds were sown in Petri dishes lined with filter paper moistened with distilled water and, aiming at the indirect contact with each oil, two sheets of filter paper were placed on top of the lid, in which three (3) mL of each oil solution were added. Then, the dishes were incubated in a germination chamber at 25°C. The pH did not contribute to alter the results; the volatile extracts of essential oils of C. zeylanicum, L. sidoides and C. nardus inhibited seed germination and root growth of seedlings of B. pilosa, which shows allelopathic potential; and the concentration of 0.08% of oils caused the overall deterioration of the roots and death of seedlings of B. pilosa.

Determinou-se o efeito alelopático inibitório dos extratos voláteis de Cinnamomum zeylanicum Ness, Lippia sidoides Cham. e Cymbopogum nardus L., sobre a germinação de sementes e o crescimento de raiz de plântulas de Bidens pilosa. O experimento foi conduzido no Laboratório de Análises de Sementes do Departamento de Fitotecnia da Universidade Federal do Ceará. Para isso, utilizaram-se óleos essenciais nas concentrações 0,01, 0,02, 0,04 e 0,08%. (v/v). Foram utilizados cinco tratamentos para cada óleo dispostos em delineamento inteiramente casualizado com quatro repetições de 25 sementes. As sementes foram semeadas em placas de Petri forradas com papel de filtro umedecido com água destilada e, visando o contato indireto com cada óleo, foram colocadas duas folhas de papel de filtro na parte superior interna da tampa, onde foi adicionado 3(três) mL da solução de cada óleo. Em seguida, as placas foram incubadas em câmara de germinação a 25ºC. O pH não contribuiu para alterar os resultados; os extratos voláteis de óleos essenciais de C. zeylanicum, L. sidoides and C. nardus inibiram a germinação de sementes e o crescimento da raiz de plântulas de B. pilosa, evidenciando potencial alelopático; a concentração de 0,08% dos óleos causaram a deterioração total das raízes e morte das plântulas de B. pilosa.

Organic fertilization affecting pests and production of Lippia sidoides Cham. (Verbenacea) / Adubação orgânica afetando a ocorrência de pragas e a produção de Óleo essencial em Lippia sidoides Cham. (Verbenacea)

The objective of this paper was to study the effect of four levels of organic fertilization (0, 3, 6 and 9 kg m-2) to produce the essential oil of Lippia sidoides Cham. (Verbenacea) and its pests. A higher number of red mite Tetranychus sp. (Acari: Tetranychiidae) was observed on the abaxial face of the median and basal leaves of L. sidoides plants with eight weeks of age cultivated with 6 kg m-2 of organic fertilization. On the other hand, the smallest number of this mite was noted in control (0 kg m-2). L. sidoides cultivated with 6 kg m-2 of organic fertilization produced a greater quantity of essential oil. The highest number of mealy bug Phenacoccus sp. (Hemiptera: Pseudococcidae) was observed on the apical leaves (both leaf surfaces) of L. sidoides plants with nine and ten weeks of age cultivated with 3 kg m-2 of organic fertilization. No significant effect of the organic fertilization on Aphis gossypii Glover (Hemiptera: Aphididae) was detected. However, a greater number of this aphid and of its predator Scymnus sp. (Coleoptera: Coccinelidae) was observed on the abaxial face of the bottom leaves of L. sidoides plants with eight weeks of age. The dose of organic fertilization recommended for the production of essential oil of L. Sidoides is 6 kg m-2, however, one must pay attention to the fact this dosage favors the attack by Tetranychus sp.. And, when needed, pulverization must always be directed to the lower surface of the leaf.

O objetivo deste trabalho foi avaliar o efeito de quatro níveis de adubação orgânica (0, 3, 6, e 9 kg m-2) na produção de óleo essencial de Lippia sidoides Cham. (Verbenacea) e de suas pragas. Observou-se maior número de ácaro vermelho Tetranychus sp. (Acari: Tetranychiidae) na face inferior das folhas dos terços medianos e basais das plantas de L. sidoides com oito semanas de idade cultivadas com 6 kg m-2 de adubo orgânico. Por outro lado, a menor quantidade deste ácaro foi notada na testemunha (0 kg m-2). L. sidoides cultivada com 6 kg m-2 de adubo orgânico produziu maior quantidade de óleo essencial. Observou-se maior número de cochonilhas Phenacoccus sp. (Hemiptera: Pseudococcidae) nas folhas apicais, sem distinção de face foliar, das plantas de L. sidoides com nove e 10 semanas de idade cultivadas com 3 kg m-2 de adubo orgânico. Não se detectou efeito significativo de adubo orgânico sobre Aphis gossypii Glover (Hemiptera: Aphididae). Contudo, observou-se maior número desse pulgão bem como de seu predador Scymnus sp. (Coleoptera: Coccinelidae) nas faces abaxiais das folhas basais das plantas de L. sidoides com oito semanas de idade. A dose de adubo orgânico recomendada para produção de óleo essencial de L. sidoides é de 6 kg m-2. Contudo, essa dosagem favorece o ataque de Tetranychus sp. e, quando necessário, deve-se dirigir a pulverização sempre para a face inferior da folha, local preferencial de ataque dos artrópodes.

High resolution ultra high pressure liquid chromatography-time-of-flight mass spectrometry dereplication strategy for the metabolite profiling of Brazilian Lippia species.

Plants belonging to the Lippia genus have been widely used in ethnobotany throughout South and Central America and in tropical Africa as foods, medicines, sweeteners and in beverage flavouring. Various taxonomic problems involving some genera from Verbenaceae, including Lippia, have been reported. In this study, the metabolite profiling of fifteen extracts of various organs of six Lippia species was performed and compared using UHPLC-PDA-TOF-MS. Fourteen phenolic compounds that were previously isolated from L. salviaefolia Cham. and L. lupulina Cham. were used as references. The annotation of the remaining LC peaks was based on concomitant online high mass accuracy measurements and subsequent molecular formula assignments following these different steps: (i) elimination of non-coherent putative molecular formulae by heuristic filtering, (ii) verification of the occurrence of remaining molecular formulae in databases, (iii) cross search with reported compounds in the Lippia genus, (iv) match with reported UV spectra, (v) estimation of the chromatographic retention behaviour based on the log P parameter of reference compounds. This strategy is generic and time-saving, avoids isolation/purification procedures, enables an efficient LC peak annotation of most of the studied compounds and is well adapted for plant chemotaxonomic studies. Within this study, the interconversion of four flavanone glucoside isomers was additionally highlighted by analytical HPLC isolation and immediate analysis using fast UHPLC gradients. Dereplication results and hierarchical data analysis demonstrated that L. salviaefolia, L. balansae, L. velutina and L. sidoides displayed significant chemical similarities, while the compositions of L. lasiocalicyna and L. lupulina differed substantially.

Total assignments of 1H and 13C NMR spectra of isocatalpanol and a derivative of tecomaquinone.

Isocatalpanol and tecomaquinone I were obtained from roots of Lippia sidoides, a medicinal plant from northeast Brazil. Reduction of tecomaquinone I with NaBH4 yielded a new derivative. Structural elucidation was done on the basis of spectral data, mainly by high-field NMR and electron ionization mass spectrometry.

[Oregano: properties, composition and biological activity]. / El orégano: propiedades, composición y actividad biológica de sus componentes.

The oregano spice includes various plant species. The most common are the genus Origanum, native of Europe, and the Lippia, native of Mexico. Among the species of Origanum. their most important components are the limonene, gamma-cariofilene, rho-cymenene, canfor, linalol, alpha-pinene, carvacrol and thymol. In the genus Lippia, the same compounds can be found. The oregano composition depends on the specie, climate, altitude, time of recollection and the stage of growth. Some of the properties of this plant's extracts are being currently studied due to the growing interest for substituting synthetic additives commonly found in foods. Oregano has a good antioxidant capacity and also presents antimicrobial activity against pathogenic microorganisms like Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, among others. These are all characteristics of interest for the food industry because they may enhance the safety and stability of foods. There are also some reports regarding the antimutagenic and anticarcinogenic effect of oregano; representing an alternative for the potential treatment and/or prevention of certain chronic ailments, like cancer.