Cytotoxic Activity of Polyphenol Extracts from Three Oregano Species: Hedeoma patens, Lippia graveolens and Lippia palmeri, and Antiproliferative Potential of Lippia graveolens against Two Types of Breast Cancer Cell Lines (MDA-MB-231 and MCF-7).

Oregano infusions have traditionally been used to treat some diseases related to inflammation and cancer; also, some species have shown antiproliferative activity on cancer cell lines, for example, colon and liver, and this has been attributed to its phytochemical profile, mainly its phenolic compounds. This study aimed to evaluate the cytotoxicity and antiproliferative potential of the polyphenols-rich extracts (PRE) of the oregano species H. patens, L. graveolens, and L. palmeri on breast cancer cell lines. The PRE of the three oregano species were obtained from dried leaves. The extract was characterized by determining antioxidant activity, total phenols content, and identifying the profile of phenolic acids and flavonoids by chromatography UPLC-MS/MS. Furthermore, the cytotoxicity of the extracts was evaluated in vitro on a non-cancer cell line of fibroblast NIH3T3 and the antiproliferative potential on the breast cancer cell lines MDA-MB-231 and MCF-7. L. graveolens showed the highest antioxidant capacity and significantly inhibited the proliferation of MCF-7 and MDA-MB-231 cells at non-cytotoxic concentrations in normal cells, with a similar effect to that cisplatin in MDA-MB-231 cells. Therefore, the polyphenol-rich extract from L. graveolens showed the greatest potential to guide future research on the antiproliferative mechanism of action.

Morphotype divergence and genetic diversity of Hedeoma piperita Benth. in western Mexico.

Hedeoma piperita Benth. is a perennial herb from the Lamiaceae family, which is highly valued for its medicinal and culinary properties by the Purépecha ethnic group in Michoacán, Mexico. The species presents populations of two morphotypes (white and purple corollas) that have not been formally studied. In this work, we aimed to evaluate the morphological and genetic variation between the two morphotypes. We sampled individuals from 15 populations within the Purépecha Plateau in western Mexico to measure 33 quantitative and qualitative morphological variable characters (vegetative and reproductive) and to estimate genetic diversity and structure using six nuclear microsatellite markers. Principal Component Analysis showed a clear separation between populations of the two morphotypes, which differences were statistically significant for all vegetative (n = 11) and reproductive (n = 22) characters. Similarly, Bayesian and multivariate cluster analyses based on the microsatellite data supported the distinction of the two morphotypes, except for one population of the white corolla that was genetically closer to the purple corolla group. Genetic diversity was moderate to low across populations of the two morphotypes, and inbreeding (FIS) was significantly higher in populations of the purple corolla. Our morphological and genetic data support the presence of two divergent morphotypes in H. piperita. This species is of high importance within the Purépecha culture, but unfortunately is declining in the region due to its high extraction rates. Thus, our results are valuable to delineate germplasm zones for future breeding programs and for informing in situ conservation strategies.

Multivariate optimization of a HS-SPME/GC-MS technique for the characterization of volatile compounds present in Hedeoma multiflorum Benth / Optimización Multivariada de una técnica HS-SPME/GC para la caracterización de los compuestos volátiles presentes en Hedeoma multiflorum Benth

The aim of the present work was to optimize the main experimental variables of a procedure using HS-SPME/GC-MS as the analytical methodology to establish the profile of the volatile compounds present in aerial parts of Hedeoma multiflorum Benth. The influence of the type of fiber, equilibrium time, extraction time and extraction temperature on the composition of the volatile compounds was determined using response surface methodology (RSM), and the parameters of the models were corroborated by multiple linear regressions. The results showed that the regression models generated adequately explained the data variation and represented the relationships between the parameters and their responses. The optimal analysis conditions from the contour plots were established (DVB/CAR/PDMS fiber, with a 10 min equilibrium time, 10 min extraction time, and 40°C). Under these conditions, 41 volatile components in the whole plant were determined, which represents more than those reported using hydrodistillation.

El objetivo del presente trabajo fue optimizar las principales variables experimentales de un procedimiento HS-SPME/GC para establecer el perfil de compuestos volátiles presentes en la parte aérea de Hedeoma multiflorum Benth. Se determinó la influencia de las variables tipo de fibra, tiempo de equilibrio, tiempo de extracción y temperatura de extracción sobre la composición de los volátiles, utilizando una met odología de superficie de respuesta (RSM) y los parámetros del modelo se corroboraron por regresión lineal múltiple. Los resultados demostraron que los modelos de regresión generados explican adecuadamente la variación de los datos y representaron significativamente las relaciones reales entre los parámetros y sus respuestas. Las condiciones óptimas de análisis fueron establecidas (DVB/CAR/PDMS, con un tiempo de equilibrio de 10 minutos, un tiempo de extracción de 10 minutos y trabajando a 40°C). Utilizando esta metodología, se determinaron 41 componentes volátiles en planta entera, más que los reportados mediante hidrodestilación.

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.

Protective role of terpenes and polyphenols from three species of Oregano (Lippia graveolens, Lippia palmeri and Hedeoma patens) on the suppression of lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mexican oregano infusions have been traditionally used in México for the treatment of inflammation-related diseases, such as respiratory and digestive disorders, headaches and rheumatism, among others. Nevertheless, there is limited information regarding the phenolic compounds, terpenes and composition as well as biological activity of Mexican oregano. AIM OF THE STUDY: To determine the phenolic and terpene composition and to evaluate the anti-inflammatory potential of three species of Mexican oregano (Lippia graveolens (LG), Lippia palmeri (LP) and Hedeoma patens (HP)) in order to provide a scientific basis for their use. MATERIALS AND METHODS: We obtained methanol and chloroform extracts from dried oregano leaves of each species. We used LC-DAD-ESI-MS/MS and GC-MS to determine the phenolic and terpene profiles of the leaves, respectively. We evaluated anti-inflammatory potential by measuring the effect of Mexican oregano extracts on some pro-inflammatory mediators, such as nitric oxide (NO) and reactive oxygen species (ROS) using lipopolysaccharide(LPS)-stimulated RAW 264.7 macrophage cells and evaluating cyclooxygenase activity (COX-1, COX-2). RESULTS: Nine phenolic compounds (phenolic acids and flavonoids) and 22 terpenes (monoterpenes and sesquiterpenes) were detected in LG, LP and HP. We studied extracts from LG, LP and HP, and fractions from LG and LP in order to know their effect on some pro-inflammatory mediators. The phenolic and terpene extracts from LG, LP and HP exhibited significant inhibitory effect on ROS and NO production and mitochondrial activity in LPS-induced inflammation in RAW 264.7 macrophage cells. Nitric oxide production was also diminished by the terpene LG fraction LGF2 and the LP fractions LPF1, LPF2 and LPF3, confirming that both monoterpenes and sesquiterpenes are active compounds of oregano. Furthermore, the total extracts of LG, LP and HP exhibited non-selective inhibitions against the activity of the cyclooxygenases COX-1 and COX-2. CONCLUSIONS: Our results suggest that Lippia graveolens, Lippia palmeri and Hedeoma patens extracts have the potential to treat inflammatory diseases; their activity is mediated by polyphenols and terpenes. These findings support the claim for their traditional use in the treatment of inflammation-related diseases.

Extraction of volatile oil from aromatic plants with supercritical carbon dioxide: experiments and modeling.

An overview of the studies carried out in our laboratories on supercritical fluid extraction (SFE) of volatile oils from seven aromatic plants: pennyroyal (Mentha pulegium L.), fennel seeds (Foeniculum vulgare Mill.), coriander (Coriandrum sativum L.), savory (Satureja fruticosa Béguinot), winter savory (Satureja montana L.), cotton lavender (Santolina chamaecyparisus) and thyme (Thymus vulgaris), is presented. A flow apparatus with a 1 L extractor and two 0.27 L separators was built to perform studies at temperatures ranging from 298 to 353 K and pressures up to 30.0 MPa. The best compromise between yield and composition compared with hydrodistillation (HD) was achieved selecting the optimum experimental conditions of extraction and fractionation. The major differences between HD and SFE oils is the presence of a small percentage of cuticular waxes and the relative amount of thymoquinone, an oxygenated monoterpene with important biological properties, which is present in the oils from thyme and winter savory. On the other hand, the modeling of our data on supercritical extraction of volatile oil from pennyroyal is discussed using Sovová's models. These models have been applied successfully to the other volatile oil extractions. Furthermore, other experimental studies involving supercritical CO(2) carried out in our laboratories are also mentioned.

Antimicrobial activity of Hedeoma drummondii against opportunistic pathogens.

Hedeoma drummondii is a medicinal plant with diverse properties; however, validations of its medicinal uses are scarce. To evaluate its antimicrobial properties H. drummondii, was tested against opportunistic pathogens of medical importance. Antimicrobial tests were performed by the microdilution method in order to determine the Minimal Inhibitory Concentration (MIC) for each extract. Extracts of this plant showed relevant antimicrobial activity, results revealed that the hexanic extract has stronger activity and broader spectrum compared to acetone and methanol extracts. The activity of hexanic extract may be attributed mainly to the presence of the monoterpenes pulegone and menthol. In conclusion, the hexanic extract possess relevant antibacterial properties which suggests that H. drummondii have bioactive principles; these new data provide scientific support for the use of this plant in traditional medicine, particularly for gastrointestinal diseases.

Antioxidant and cytotoxic properties of an aqueous extract from the Argentinean plant Hedeoma multiflorum.

CONTEXT: Hedeoma multiflorum Benth. (Lamiaceae) is widely used in Argentinean popular medicine for digestive and anti-spasmodic purposes. However, knowledge about its pharmacological properties has been poorly investigated. OBJECTIVE: The antioxidant and cytotoxic properties of an aqueous extract from the plant were investigated for the first time. MATERIAL AND METHODS: Scavenging of stable free radicals of 2,2-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS(+)) and 2,2-diphenyl-1-picryl hydrazyl (DPPH), reducing of ferric (III) iron of ferric reducing ability of plasma (FRAP) reagent, and inhibition of lipid peroxidation (LP) of human plasma and rat brain homogenates were assessed. Cytotoxicity was tested on human polymorphonuclear (PMN) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was assessed by flow cytometric techniques. RESULTS: Extract scavenged ABTS(+) and DPPH (1.78 and 0.78 µmol Trolox equivalent/mg dry extract, respectively) and reduced FRAP reagent (0.66 µmol ascorbic acid equivalent/mg dry extract). LP of human plasma and rat brain was also inhibited in a dose-dependent way (inhibitory concentration 50%=27.0 and 86.0 µg/mL, respectively). Extract is rich in polyphenol compounds (0.96 ± 0.08 µmol equivalent caffeic acid/mg dry matter). Treatment of PMN decreased significantly the cell ability to reduce the MTT salt and increased the hypodiploid nuclei from 4 to 18% quantified using propidium iodide (PI). In the annexin V-Fluorescein isothiocyanate (annexin V-FITC) assay, 26% of treated cells were annexin V-FITC positive and PI negative. Using the 3,3'-dihexyloxacarbocyanine iodide uptake method, the negative fraction of cells was calculated as 29%. DISCUSSION AND CONCLUSION: H. multiflorum extract was found to have a significant antioxidant and pro-apoptotic activities, and a great potential as a source of healthy products.

Antiproliferative effect from the Mexican poleo (Hedeoma drummondii).

Hedeoma drummondii (poleo) has been used by Mexicans to prepare a tea for its pleasant taste. The methanolic extract, polar fractions, and major antioxidant compounds of the Mexican herbal tea obtained from aerial parts of H. drummondii ("poleo") showed antiproliferative effects against the cancer cell lines MCF-7 (human breast) and HeLa (human cervix). Phenolic acids isolated from poleo have significant antiproliferative effects with caffeic acid the most potent. This evidence suggests that herbal poleo tea may have chemopreventive properties.

Free radical scavengers from the Mexican herbal tea "poleo" (Hedeoma drummondii).

The aerial parts of the Lamiaceae Hedeoma drummondii (Benth.) are used in Mexico to prepare a herbal tea and by North American Amerindians as a spice. The methanolic extract of the aerial parts exhibited a strong antioxidant effect measured by the scavenging of the free diphenyl picrylhydrazyl (DPPH) radical. Assay-guided fractionation of the crude methanolic extract allowed the identification of three major active constituents, chlorogenic, caffeic and rosmarinic acid, as well as sideritoflavone derivatives and simple phenolics. The TEAC, FRAP, total phenolic and flavonoid content were determined. The high content of caffeic acid and rosmarinic acid relates to the antioxidant activity of H. drummondii.

Mitigation of pennyroyal oil hepatotoxicity in the mouse.

OBJECTIVES: Pennyroyal oil ingestion has been associated with severe hepatotoxicity and death. The primary constituent, R-(+)-pulegone, is metabolized via hepatic cytochrome P450 to toxic intermediates. The purpose of this study was to assess the ability of the specific cytochrome P450 inhibitors disulfiram and cimetidine to mitigate hepatotoxicity in mice exposed to toxic levels of R-(+)-pulegone. METHODS: 20-g female BALB/c mice were pretreated with either 150 mg/kg of cimetidine intraperitoneal (IP), 100 mg/kg of disulfiram IP, or both. After one hour, mice were administered 300 mg/kg of pulegone IP and were killed 24 hours later. Data were analyzed using ANOVA. Post-hoc t-tests used Bonferroni correction. RESULTS: There was a tendency for lower serum glutamate pyruvate transaminase in the disulfiram and cimetidine groups compared with the R-(+)-pulegone group. The differences were significant for both the cimetidine and the combined disulfram and cimetidine groups compared with the R-(+)-pulegone group. Pretreatment with the combination of disulfiram and cimetidine most effectively mitigated R-(+)-pulegone-induced hepatotoxicity. CONCLUSIONS: Within the limitations of a pretreatment animal model, the combination of cimetidine and disulfiram significantly mitigates the effects of pennyroyal toxicity and does so more effectively than either agent alone. These data suggest that R-(+)-pulegone metabolism through CYP1A2 appears to be more important in the development of a hepatotoxic metabolite than does metabolism via CYP2E1.