Rosmarinic Acid Present in Lepechinia floribunda and Lepechinia meyenii as a Potent Inhibitor of the Adenylyl Cyclase gNC1 from Giardia lamblia.

Giardiasis is a parasitosis caused by Giardia lamblia with significant epidemiological and clinical importance due to its high prevalence and pathogenicity. The lack of optimal therapies for treating this parasite makes the development of new effective chemical entities an urgent need. In the search for new inhibitors of the adenylyl cyclase gNC1 obtained from G. lamblia, 14 extracts from Argentinian native plants were screened. Lepechinia floribunda and L. meyenii extracts exhibited the highest gNC1 inhibitory activity, with IC50 values of 9 and 31 µg/mL, respectively. In silico studies showed rosmarinic acid, a hydroxycinnamic acid present in both mentioned species, to be a promising anti-gNC1 compound. This result was confirmed experimentally, with rosmarinic acid showing an IC50 value of 10.1 µM. Theoretical and experimental findings elucidate the molecular-level mechanism of rosmarinic acid, pinpointing the key interactions stabilizing the compound-enzyme complex and the binding site. These results strongly support that rosmarinic acid is a promising scaffold for developing novel compounds with inhibitory activity against gNC1, which could serve as potential therapeutic agents to treat giardiasis.

Phytochemistry, Bioactivity, and Ethnopharmacology of the Genus Lepechinia Willd. (Lamiaceae): A Review.

The genus Lepechinia (Lamiaceae) involves several aromatic shrubs that are distributed only in the American continent, inhabiting mountain areas, mainly in the Andean region of South America. Based on the PRISMA approach, we selected and critically analyzed 48 research articles. From a phytochemical point of view, most of the secondary metabolites reported in Lepechinia spp. are terpenes and terpenoids, with a few exceptions comprising flavonoids and other shikimic acid derivatives. On the one hand, sesquiterpenoids of the guajane, aromadendrane, eudesmane, and cadinane groups are characteristic of essential oils, together with (E)-ß-caryophyllene as the main representative of its chemical family. On the other hand, abietane diterpenoids are the prevalent compounds described in non-volatile fractions. Many biological activities and traditional medical uses have been reported for both pure metabolites and complex mixtures (e.g., essential oils). Regarding ethno-medical uses, the treatment of muscle pain, headache, toothache, diabetes mellitus, uterine tumors, uterine infections, and diarrhea has been reported. Concerning their verified biological activities, insecticidal, antifungal, antioxidant, and anticholinesterase properties have been described. Furthermore, some data concerning anti-herpetic activity have been reported.

A Validated HPLC-UV-ESI-IT-MS Method for the Quantification of Carnosol in Lepechinia mutica, a Medicinal Plant Endemic to Ecuador.

The diphenolic diterpene carnosol was isolated from several species of the family Lamiaceae, including Lepechinia mutica, a medicinal plant endemic to Ecuador. The compound has exhibited high antioxidant, anti-inflammatory, antimicrobial, neuroprotective, and antifungal properties, as well as promising cytotoxicity against prostate, breast, skin, leukemia, and human colon cancer cell lines. In this paper, we developed and validated a simple, accurate, and reliable analytical HPLC-UV-ESI-IT-MS method, carried out on a C18 column, which is potentially suitable to quantify carnosol in plant extracts. The procedure complied with the established ICH validation parameters of analytical range (linearity in the range of 0.19-5.64 µg/g dried leaves; REAVERGE = 4.9%; R2 = 0.99907), analysis repeatability (RSD = 2.8-3.6%), intermediate precision (RSD = 1.9-3.6%), accuracy (estimated as % carnosol recovery in the range of 81 to 108%), and robustness. Finally, the LOD (0.04 µg/mg) and LOQ (0.19 µg/mg) values of carnosol/dried leaves were determined. Using this validated method, the content of carnosol in L. mutica was estimated to be 0.81 ± 0.04 mg/g of dried leaves (0.081%).

Screening and Evaluation of Active Compounds in Polyphenol Mixtures by a Novel AAPH Offline HPLC Method and Its Application.

In this study, we developed a novel offline high-performance liquid chromatography (HPLC) method based on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) radicals for antioxidant screening in 20 polyphenolic compounds and used the Trolox equivalent antioxidant capacity assay to evaluate their antioxidant activity. Compared to the existing offline HPLC methods based on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH), the offline HPLC method based on the AAPH radical is more sensitive. Additionally, we applied this method to Lepechinia meyenii (Walp.) Epling extract and screened out seven antioxidants, caffeic acid, hesperidin, rosmarinic acid, diosmin, methyl rosmarinate, diosmetin, and n-butyl rosmarinate, which are known antioxidants. Therefore, this study provides new insights into the screening of antioxidants in natural extracts.

Controlled Production of Carnosic Acid and Carnosol in Cell Suspensions of Lepechinia meyenii Treated with Different Elicitors and Biosynthetic Precursors.

Lepechinia meyenii is a medicinal plant specialized in the biosynthesis of different types of antioxidants including the diterpenes carnosic (CA) acid and carnosol (CS). Herein we present the results of plant tissue culture approaches performed in this medicinal plant with particular emphasis on the generation and evaluation of a cell suspension system for CA and CS production. The effect of sucrose concentration, temperature, pH, and UV-light exposure was explored. In addition, diverse concentrations of microbial elicitors (salicylic acid, pyocyanin, Glucanex, and chitin), simulators of abiotic elicitors (polyethylene glycol and NaCl), and biosynthetic precursors (mevalonolactone, geranylgeraniol, and miltiradiene/abietatriene) were evaluated on batch cultures for 20 days. Miltiradiene/abietatriene obtainment was achieved through a metabolic engineering approach using a recombinant strain of Saccharomyces cerevisiae. Our results suggested that the maximum accumulation (Accmax ) of CA and CS was mainly conferred to stimuli associated with oxidative stress such as UV-light exposure (Accmax , 6.2 mg L-1 ) polyethylene glycol (Accmax , 6.5 mg L-1 ) NaCl (Accmax , 5.9 mg L-1 ) which simulated drought and saline stress, respectively. Nevertheless the bacterial elicitor pyocyanin was also effective to increase the production of both diterpenes (Accmax , 6.4 mg L-1 ). Outstandingly, the incorporation of upstream biosynthetic precursors such as geranylgeraniol and miltiradiene/abietatriene, generated the best results with Accmax of 8.6 and 16.7 mg L-1 , respectively. Optimized batch cultures containing 100 mg L-1 geranylgeraniol, 50 mg L-1 miltiradiene/abietatriene (95 : 5 %) and 5 g L-1 polyethylene glycol treated with 6 min UV light pulse during 30 days resulted in Accmax of 26.7 mg L-1 for CA and 17.3 mg L-1 for CS on days 18-24. This strategy allowed to increase seven folds the amounts of CA and CS in comparison with batch cultures without elicitation (Accmax , 4.3 mg L-1 ).

Acaricidal Efficacy of Plants from Ecuador, Ambrosia peruviana (Asteraceae) and Lepechinia mutica (Lamiaceae) against Larvae and Engorged Adult Females of the Common Cattle Tick, Rhipicephalus microplus.

Control measures against common cattle tick Rhipicephalus microplus are of the upmost importance because of considerable, deleterious impact on a farm's economy. Due to resistance phenomena to synthetic acaricides being a constraint in affected farms, the search for plant derivatives as acaricides has increased dramatically in recent years. In this work, essential oils obtained from two Ecuadorian plants, Ambrosia peruviana and Lepechinia mutica (EOAp, EOLm), traditionally used as insecticides in indigenous communities, were studied on larvae and engorged females at the parasitic stages of R. microplus. Larvae and females were treated with five (0.0625, 0.125, 0.25, 0.50 and 1%) and six concentrations (0.125, 0.25, 0.50, 1, 2 and 4%), respectively, of each EOsAp/Lm. A 98-99% larval mortality was achieved with 0.5% of both EOsAp/Lm. EOAp inhibited oviposition and egg hatching up to 82% and 80%, respectively, and had an overall efficacy of 93.12%. Efficacy of EOLm was 72.84%, due to the low influence of EOLm on reproductive parameters. By steam distillation and GC-MS analysis, γ-Curcumene was identified as the main constituent (52.02%) in the EOAp and Shyobunol (10.80%) in EOLm. The results suggest that major components of both essential oils should be further studied as promissory acaricides against R. microplus.

Separation and Identification of Antioxidants and Aldose Reductase Inhibitors in Lepechinia meyenii (Walp.) Epling.

We previously reported that Lepechinia meyenii (Walp.) Epling has antioxidant and aldose reductase (AR) inhibitory activities. In this study, L. meyenii was extracted in a 50% MeOH and CH2Cl2/MeOH system. The active extracts of MeOH and 50% MeOH were subjected to fractionation, followed by separation using high-speed counter-current chromatography (HSCCC) and preparative HPLC. Separation and identification revealed the presence of caffeic acid, hesperidin, rosmarinic acid, diosmin, methyl rosmarinate, diosmetin, and butyl rosmarinate. Of these, rosmarinic acid, methyl rosmarinate, and butyl rosmarinate possessed remarkable antioxidant and AR inhibitory activities. The other compounds were less active. In particular, rosmarinic acid is the key contributor to the antioxidant and AR inhibitory activities of L. meyenii; it is rich in the MeOH extract (333.84 mg/g) and 50% MeOH extract (135.41 mg/g) of L. meyenii and is especially abundant in the EtOAc and n-BuOH fractions (373.71-804.07 mg/g) of the MeOH and 50% MeOH extracts. The results clarified the basis of antioxidant and AR inhibitory activity of L. meyenii, adding scientific evidence supporting its traditional use as an anti-diabetic herbal medicine. The HSCCC separation method established in this study can be used for the preparative separation of rosmarinic acid from natural products.

Chemical Composition and Anticholinesterase Activity of the Essential Oil of Leaves and Flowers from the Ecuadorian Plant Lepechinia paniculata (Kunth) Epling.

This work aimed to study the chemical composition, cholinesterase inhibitory activity, and enantiomeric analysis of the essential oil from the aerial parts (leaves and flowers) of the plant Lepechinia paniculata (Kunth) Epling from Ecuador. The essential oil (EO) was obtained through steam distillation. The chemical composition of the oil was evaluated by gas chromatography, coupled to mass spectrometry (GC-MS) and a flame ionization detector (GC-FID). The analyses led to the identification of 69 compounds in total, of which 40 were found in the leaves and 29 were found in the flowers of the plant. The major components found in the oil were 1,8-Cineole, ß-Pinene, δ-3-Carene, α-Pinene, (E)-Caryophyllene, Guaiol, and ß-Phellandrene. Flower essential oil showed interesting selective inhibitory activity against both enzymes AChE (28.2 ± 1.8 2 µg/mL) and BuChE (28.8 ± 1.5 µg/mL). By contrast, the EO of the leaves showed moderate mean inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), with IC50 values of 38.2 ± 2.9 µg/mL and 47.4 ± 2.3 µg/mL, respectively.

Methyl jasmonate enhances ursolic, oleanolic and rosmarinic acid production and sucrose induced biomass accumulation, in hairy roots of Lepechinia caulescens.

BACKGROUND: Ursolic (UA), oleanolic (OA) and rosmarinic (RA) acids are bioactive metabolites found in Lepechinia caulescens that have generated interest for their health benefits, which include antimicrobial, antioxidant, antimutagenic, gastroprotective, antidiabetic, antihypertensive and anti-inflammatory properties, among others. To date, very few attempts have been made to evaluate the potential for simultaneous production of these bioactive compounds, using a biotechnological approach. Hairy root cultures offer a biotechnology approach that can be used to study the factors affecting the biosynthesis and the production of UA, OA and RA. In the current study, we established hairy root cultures of L. caulescens and evaluated the effect of sucrose on biomass accumulation, and the effect of different concentrations and times of exposure of methyl jasmonate (MeJA), on the accumulation of UA, OA and RA. METHODS: Leaves from plants of L. caulescens were inoculated with Agrobacterium rhizogenes strain ATCC 15834. PCR of rolB gene confirmed the transgenic nature of hairy roots. Hairy roots were subcultured in semisolid MSB5 medium, supplemented with 15, 30, 45 or 60 g/L sucrose and after 4 weeks, dry weight was determined. The accumulation of UA, OA and RA of wild plants and hairy roots were determined by HPLC. Finally, the hairy roots were treated with 0, 100, 200 and 300 µM of MeJA and the content of bioactive compounds was analyzed, after 24, 48 and 72 h. RESULTS: High frequency transformation (75%) was achieved, using leaf explants from axenic seedlings, infected with A. rhizogenes. The hairy roots showed an enhanced linear biomass accumulation, in response to the increase in sucrose concentration. The hairy root cultures in MSB5 medium, supplemented with 45 g/L sucrose, were capable to synthesizing UA (0.29 ± 0.00 mg/g DW), OA (0.57 ± 0.00 mg/g DW) and RA (41.66 ± 0.31 mg/g DW), about two, seven and three times more, respectively, than in roots from wild plants. Elicitation time and concentration of MeJA resulted in significant enhancement in the production of UA, OA and RA, with treatments elicited for 24 h, with a concentration of 300 µM of MeJA, exhibiting greatest accumulation. CONCLUSION: This is the first report on development of hairy root cultures of L. caulescens. Future studies should aim towards further improving triterpenes and polyphenolic compound production in hairy roots of L. caulescens, for use in the pharmaceutical and biotechnological industry.

Daily fluctuations in pollination effectiveness explain higher efficiency of native over exotic bees in Lepechinia floribunda (Lamiaceae).

BACKGROUND: Despite Stebbins' principle of the most efficient pollinator being proposed decades ago, the most important pollinators are still mainly identified using the frequency of visits to flowers. This shortcoming results in a gap between the characterization of the flower visitors of a plant species and a reliable estimation of the plant fitness consequences of the mutualistic interaction. The performance of a mutualistic visitor depends on its abundance, behaviour, effectiveness (pollen removal and deposition per unit time) and efficiency (seed set per unit time) conditioned by the temporal matching between pollinator activity and temporal patterns of maturation of the sexual functions of flowers. Although there have been recent attempts to provide a conceptual and methodological framework to characterize pollinators' performance, few have combined all key elements of visitors and plants to provide an accurate estimation of pollinators' performance under natural conditions. METHODS: We complement information on the flower biology and mating system of the sub-shrub Lepechinia floribunda (Lamiaceae) to provide a daily quantitative estimation of performance (effectiveness and efficiency) of the more abundant pollinators, i.e. native bumble-bees (Bombus spp.) and leafcutter bees (Megachile sp.), and the exotic honey-bee (Apis mellifera). KEY RESULTS: Unlike honey-bees or leafcutter bees, native bumble-bees matched the daily pattern of nectar production and stigma receptivity, and showed higher effectiveness and efficiency. Despite the overabundance of honey-bees, visits occurred mainly when stigmas were not receptive, thus reducing the honey-bees' overall performance. CONCLUSIONS: Bumble-bees appear to be the most important pollinators and potential historical mediators of reproductive trait evolution in L. floribunda. Because the production of seeds by bumble-bees involved fewer pollen grains for plants and less investment in floral display than honey-bees, contemporary and expected changes in pollinator abundance may affect future L. floribunda floral evolution. If bumble-bees were to be further displaced by anthropogenic disturbance or by competition with honey-bees, their lower efficiency will select for a larger floral display increasing reproductive costs. This scenario may also impose selection to reduce dichogamy to match honey-bee foraging activity.

Inhibitory effects of compounds isolated from Lepechinia meyenii on tyrosinase.

To contribute enzymatic browning inhibitors to the food industry and also extend knowledge about the phytochemical profile of the anti-tyrosinase plant Lepechinia meyenii, its ethanol extract was subjected to bioguided fractionation. Three hydroxycinnamic acids, p-coumaric acid (1), caffeic acid (2) and rosmarinic acid (3), were isolated as mainly responsible for its activity. Compounds 1, 2 and 3 showed themselves highly effective for inhibiting tyrosinase with IC50 values of 0.30, 1.50 and 4.14 µM, respectively, for monophenolase activity and 0.62, 2.30 and 8.59 µM, respectively for diphenolase activity. This is the first report describing the isolation of the compounds causing the tyrosinase inhibitory activity of L. meyenii extract. The inhibitory kinetics of 1-3 using both L-tyrosine and L-DOPA as substrates was investigated and the results obtained were discussed at molecular level by docking analysis. The resulting compounds 1-3 and a phenolic-enriched fraction of the extract, 2.9-fold more active than the starting material, may be suitable as non-toxic and inexpensive alternatives for the control of deleterious enzymatic darkening.

Phytochemical Study of the Ecuadorian Species Lepechinia mutica (Benth.) Epling and High Antifungal Activity of Carnosol against Pyricularia oryzae.

The plant Lepechinia mutica (Benth.) Epling (family Lamiaceae) is endemic to Ecuador. In the present study, we report some major non-volatile secondary metabolites from the leaves and the chemistry of the essential oil distilled from the flowers. The main identified compounds were carnosol, viridiflorol, ursolic acid, oleanolic acid, chrysothol, and 5-hydroxy-4′,7-dimethoxy flavone. Their structures were determined by X-ray diffraction and NMR and MS techniques. The essential oil showed a chemical composition similar to that distilled from the leaves, but with some qualitative and quantitative differences regarding several minor compounds. The main constituents (>4%) were: δ-3-carene (24.23%), eudesm-7(11)-en-4-ol (13.02%), thujopsan-2-α-ol (11.90%), β-pinene (7.96%), valerianol (5.19%), and co-eluting limonene and β-phellandrene (4.47%). The volatile fraction was also submitted to enantioselective analysis on a β-cyclodextrin column, obtaining the separation and identification of the enantiomers for α-thujene, β-pinene, sabinene, α-phellandrene, limonene and β-phellandrene. Furthermore, the anti-fungal activity of non-volatile secondary metabolites was tested in vitro, with carnosol resulting in being very active against the “blast disease” caused by the fungus Pyricularia oryzae.

Methyl Jasmonate and Salicylic Acid Enhanced the Production of Ursolic and Oleanolic Acid in Callus Cultures of Lepechinia Caulescens.

BACKGROUND: The production of triterpenes from plants for pharmacological purposes varies in concentration, due to genetic and environmental factors. In vitro culture enables the control and increase of these bioactive molecules. OBJECTIVE: To evaluate the effect of plant growth regulators and elicitors in the induction of calli and the production of ursolic acid (UA) and oleanolic acid (OA) in Lepechinia caulescens. MATERIALS AND METHODS: Leaf explants were exposed for the induction of calli at different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). Methyl jasmonate (MJ) and salicylic acid were used as elicitors. High-performance liquid chromatography method was used to quantify UA and OA content in each treatment. RESULTS: Treatment with 3.0 mg/L of 2,4-D and 0.1 mg/L of BAP produced the best results for calli induction and production of UA (1.57 mg/g dry weight [DW]) and OA (1.13 mg/g DW). Both elicitors facilitated the accumulation of triterpenes. CONCLUSION: The combination of auxins and cytokinins showed favorable results for the induction of calli. Variation concerning the accumulation of UA and OA was observed between treatments. MJ increased the production of triterpenes five times after 8 h of exposure, compared to control treatment. There is a greater accumulation of UA (16.58 mg/g DW) and OA (1.94 mg/g DW) in leaves of wild plants. SUMMARY: Callus cultures of Lepechinia caulescens were obtained from leaf explants treated with 2,4-dichlorophenoxyacetic acid and 6-bencylaminopurineResulting cultures were elicited with methyl jasmonate (MJ) and salicylic acid to increase the production of the triterpenes, ursolic acid (UA), and oleanolic acid (OA)The cultures elicited with MJ increased the production of UA and OA five times, as compared to the control. Abbreviations used: 2,4-D: 2,4-dichlorophenoxyacetic acid, BAP: 6-benzylaminopurine, DW: Dry weight, MJ: Methyl jasmonate, OA: Oleanolic acid, PGRs: Plant growth regulators, UA: Ursolic acid, SA: Salicylic acid.

Chemical Composition, Enantiomeric Analysis, AEDA Sensorial Evaluation and Antifungal Activity of the Essential Oil from the Ecuadorian Plant Lepechinia mutica Benth (Lamiaceae).

This study describes the GC-FID, GC/MS, GC-O, and enantioselective GC analysis of the essential oil hydrodistilled from leaves of Lepechinica mutica (Lamiaceae), collected in Ecuador. GC-FID and GC/MS analyses allowed the characterization and quantification of 79 components, representing 97.3% of the total sample. Sesquiterpene hydrocarbons (38.50%) and monoterpene hydrocarbons (30.59%) were found to be the most abundant volatiles, while oxygenated sesquiterpenes (16.20%) and oxygenated monoterpenes (2.10%) were the minor components. In order to better characterize the oil aroma, the most important odorants, from the sensorial point of view, were identified by Aroma Extract Dilution Analysis (AEDA) GC-O. They were α-Pinene, ß-Phellandrene, and Dauca-5,8-diene, exhibiting the characteristic woody, herbaceus, and earthy odors, respectively. Enantioselective GC analysis of L. mutica essential oil revealed the presence of twelve couples and two enantiomerically pure chiral monoterpenoids. Their enantiomeric excesses were from a few percent units to 100%. Moreover, the essential oil exhibited moderate in vitro activity against five fungal strains, being especially effective against M. canis, which is a severe zoophilic dermatophyte causal agent of pet and human infections.

Propagación y descripción morfo-anatómica de salvia chilena (Lepechinia chamaedryoides) / Propagation and morpho-anatomical description of Chilean salvia (Lepechinia chamaedryoides)

The aim of this work was to identify an effective method to improve the rate and percentage of germination and the rooting of seeds and cuttings of Lepechinia chamaedryoides (Balb.) Epling. Morphological changes experienced by the seed during the germination process also were followed by microscopy. This research was conducted during 2009 and 2010 in the Faculty of Agronomy of the University of Concepción. Four germination tests were assayed: a) sulfuric acid scarification and further stratification, b) leaching of inhibitors with different water temperatures plus different concentrations of gibberellic acid, c) thermal scarification plus stratification and d) gibberellic acid application at different concentrations. The highest percentage of germination was obtained with thermal scarification and stratification. Vegetative propagation test consisted of different concentrations of indole butyric acid (IBA) applied to the stem base. Significant differences between treatments were not found. The morphological changes of the seed during the germination process were observed by scanning electron microscope (SEM). These findings allowed identifying characteristics of highly lignified pericarp.

Con el propósito de identificar un método efectivo para obtener un alto porcentaje de germinación y enraizamiento en semillas y estacas de Lepechinia chamaedryoides (Balb.) Epling y observar los cambios morfológicos que experimenta la semilla durante el proceso de germinación, se realizó esta investigación durante el año 2009 y 2010 en la Facultad de Agronomía de la Universidad de Concepción. Se efectuaron cuatro ensayos de germinación: a) escarificación con ácido sulfúrico y estratificación, b) lixiviación de inhibidores con agua a diferentes temperaturas más concentraciones de ácido giberélico, c) escarificación térmica más estratificación y d) aplicación de ácido giberélico, en diferentes concentraciones. El mayor porcentaje de germinación se obtuvo con el tratamiento de escarificación térmica y estratificación. Con respecto a la propagación vegetativa, se realizaron ensayos con diferentes concentraciones de ácido indolbutírico (IBA) aplicado a la base de las estacas, no observándose diferencias significativas entre tratamientos. Los cambios morfológicos de la semilla durante el proceso de germinación se observaron mediante Microscopio Electrónico de Barrido (MEB) lo que permitió identificar características anatómicas y estructurales de un pericarpio altamente lignificado.