Hepatodamianol as hepatoprotective constituent of Turnera diffusa.

It is well known that liver diseases are a major health problem and that there is a lack of hepatoprotective agents. Turnera diffusa (damiana) is a plant with a widespread distribution in México, which has many traditional uses, including the treatment of hepatic illnesses. Based on the bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa, we purified and identified a compound called hepatodamianol (1). This C-glycoside exhibited a four times greater hepatoprotective effect than the widely used hepatoprotective agent silibinin against carbon tetrachloride damage in an in vitro model using HepG2 cells. Hepatodamianol produced no cytotoxic effects and it exhibited a high antioxidant capacity. Therefore, hepatodamianol is a good candidate compound for testing as a hepatoprotective agent in a preclinical trial.

Two Ways to Achieve the Same Goal-Two Validated Quantitative NMR Strategies for a Low-Abundance Natural Product in Standardized Extracts: The Case of Hepatodamianol in Turnera diffusa.

The quantification of low-abundance secondary metabolites in plant extracts is an analytical problem that can be addressed by different analytical platforms, the most common being those based on chromatographic methods coupled to a high-sensitivity detection system. However, in recent years nuclear magnetic resonance (NMR) has become an analytical tool of primary choice for this type of problem because of its reliability, inherent simplicity in sample preparation, reduced analysis time, and low solvent consumption. The versatility of strategies based on quantitative NMR (qNMR), such as internal and external standards and electronic references, among others, and the need to develop validated analytical methods make it essential to compare procedures that must rigorously satisfy the analytical well-established acceptance criteria for method validation. In this work, two qNMR methods were developed for the quantification of hepatodamianol, a bioactive component of T. diffusa. The first method was based on a conventional external standard calibration, and the second one was based on the pulse length-based concentration determination (PULCON) method using the ERETIC2 module as a quantitation tool available in TopSpin software. The results show that both procedures allow the content of the analyte of interest in a complex matrix to be determined in a satisfactory way, under strict analytical criteria. In addition, ERETIC2 offers additional advantages such as a reduction in experimental time, reagent consumption, and waste generated.

Evaluation of antihyperglycemic activity of plants in northeast mexico.

iabetes mellitus is one of the most common non-contagious diseases. In 2017, The International Diabetes Federation reported that around 425 million people suffer from diabetes worldwide. Medications used for the treatment of diabetes lead to unwanted side effects, and thus, new safe drugs are necessary. Some natural plant-based products exhibit anti hyperglycemic activity and low toxicity. The aim of this study was to evaluate the antihyperglycemic activity (using both in vitro and in vivo models) as well as cytotoxicity of the extracts obtained from various plants. Nine extracts from a total of eight plant species were subjected to in vitro α-amylase and α-glucosidase inhibition assays. Subsequently, they were assessed through the ex vivo everted sac assay, and finally, the in vivo antihyperglycemic activity was evaluated. The extracts obtained from Ceanothus coeruleus, Chrysactinia mexicana and Zanthoxylum fagara inhibited the activities of α-amylase and α-glucosidase in the in vitro assays. Ethyl acetate and hydroalcoholic extracts from Jatropha dioica, hydroalcoholic extract from Salvia ballotaeflora and Chrysactinia mexicana, as well as methanolic extract from Ricinus communis and Zanthoxylum fagara significantly reduced the glucose uptake in the ex vivo everted intestinal sac test. All the eight extracts showed antihyperglycemic effect through the in vivo model of the Glucose Tolerance Test, using starch as the carbohydrate source.  The antihyperglycemic effect of the extracts could be mediated through the inhibition of digestive enzymes and/or the absorption of glucose through the intestine. However, the mechanism of action for the hydroalcoholic extract of Salvia texana and the methanolic extract of Turnera diffusa, which showed a strong in vivo antihyperglycemic effect, is unclear.

Peroxisomicine A1, a potential antineoplastic agent, causes micropexophagy in addition to macropexophagy.

Peroxisomicine A1 (PA1) is a potential antineoplastic agent with high and selective toxicity toward peroxisomes of tumor cells. Pexophagy is a selective autophagy process that degrades damaged peroxisomes; this process has been studied mainly in methylotrophic yeasts. There are two main modes of pexophagy in yeast: macropexophagy and micropexophagy. Previous studies showed that peroxisomes damaged by a prolonged exposition to PA1 are eliminated by macropexophagy. In this work, Candida boidinii was grown in methanol-containing media, and PA1 was added to the cultures at 2 µg/mL after they reached the mid-exponential growth phase. Samples were taken at 5, 10, 15, 20, and 25 min after the addition of PA1 and processed for ultrastructural analysis. Typical morphological characteristics of micropexophagy were observed: the direct engulfment of peroxisomes by the vacuolar membrane and the presence of the micropexophagic membrane apparatus (MIPA), which mediates the fusion between the opposing tips of the vacuole to complete sequestration of peroxisomes from the cytosol. In conclusion, here we report that, in addition to macropexophagy, peroxisomes damaged by PA1 can be eliminated by micropexophagy. This information is useful to deepen the knowledge of the mechanism of action of PA1 and of that of pexophagy per se.

Acute Hypoglycemic and Antidiabetic Effect of Teuhetenone A Isolated from Turnera diffusa.

Diabetes mellitus is a chronic degenerative disease that causes long-term complications and represents a serious public health problem. Turnera diffusa (damiana) is a shrub that grows throughout Mexico and is traditionally used for many illnesses including diabetes. Although a large number of plant metabolites are known, there are no reports indicating which of these are responsible for this activity, and this identification was the objective of the present work. Through bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa, teuhetenone A was isolated and identified as the main metabolite responsible for the plant's hypoglycemic activity. Alpha-glucosidase inhibitory activity and cytotoxicity of this metabolite were determined. Hypoglycemic and antidiabetic activities were evaluated in a murine model of diabetes in vivo, by monitoring glucose levels for six hours and comparing them with levels after administering various controls. Teuhetenone A was not cytotoxic at the tested concentrations, and did not show inhibitory activity in the glucosidase test, and the in vivo assays showed a gradual reduction in glucose levels in normoglycemic and diabetic mice. Considering these results, we suggest that teuhetenone A has potential as an antidiabetic compound, which could be further submitted to preclinical assays.

Antioxidant and hepatoprotective activity of Hamelia patens extracts.

Hamelia patens is widely used in the traditional medicine of Mexico and Central America for the treatment of illnesses associated with inflammatory processes. In this study, antioxidant and hepatoprotective activity were assayed on the methanolic crude (ME), hexane (HE), ethyl acetate (AE), and butanol (BE) extracts of H. patens. The total phenolic content (TPC) as mg of gallic acid equivalents per g of dry extract was determined by Folin-Ciocalteu's method (ME=141.58±11.99, HE=33.96±1.13, AE=375.18±13.09, BE=132.08±3.62), and antioxidant activity by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical-scavenging method (EC(50) ME=77.87±5.67, HE=236.64±26.32, AE=45.87±2.24, BE=50.97±0.85µg/mL). Hepatoprotective activity was evaluated through AST activity on HepG2 cells subjected to damage with CCl(4) (ME=62.5±3.41, HE=72.25±2.87, AE=63.50±4.20, BE=43.74±4.03). BE showed the greater hepatoprotective activity and a good antioxidant capacity, while HE did not show hepatoprotective or antioxidant activity. Cytotoxicity was evaluated on Vero cells cultures; none showed significant toxicity.

Activity of Polyphenolic Compounds against Candida glabrata.

Opportunistic mycoses increase the morbidity and mortality of immuno-compromised patients. Five Candida species have been shown to be responsible for 97% of worldwide cases of invasive candidiasis. Resistance of C. glabrata and C. krusei to azoles has been reported, and new, improved antifungal agents are needed. The current study was designed to evaluatethe activity of various polyphenolic compounds against Candida species. Antifungal activity was evaluated following the M27-A3 protocol of the Clinical and Laboratory Standards Institute, and antioxidant activity was determined using the DPPH assay. Myricetin and baicalein inhibited the growth of all species tested. This effect was strongest against C. glabrata, for which the minimum inhibitory concentration (MIC) value was lower than that of fluconazole. The MIC values against C. glabrata for myricitrin, luteolin, quercetin, 3-hydroxyflavone, and fisetin were similar to that of fluconazole. The antioxidant activity of all compounds was confirmed, and polyphenolic compounds with antioxidant activity had the greatest activity against C. glabrata. The structure and position of their hydroxyl groups appear to influence their activity against C. glabrata.

Diterpenes Synthesized from the Natural Serrulatane Leubethanol and Their in Vitro Activities against Mycobacterium tuberculosis.

Seventeen new derivatives of the natural diterpene leubethanol, including some potential pro-drugs, with changes in the functionality of the aliphatic chain or modifications of aromatic ring and the phenolic group, were synthesized and tested in vitro by the MABA technique for their activity against the H37Rv strain of Mycobacterium tuberculosis. Some compounds showed antimycobacterial selectivity indices higher than leubethanol.

Antimicrobial and antioxidant activities and chemical characterization of essential oils of Thymusvulgaris, Rosmarinus officinalis, and Origanum majorana from northeastern México.

There have been no reports of antifungal activity and composition of extracts from Thymus vulgaris, Rosmarinus officinalis or Origanum majorana from northeastern México. Antifungal activity of these oils against Trichophyton rubrum, Trichophyton tonsurans, Trichophyton mentagrophytes, Microsporum gypseum, Microsporum canis and Epidermophyton floccosum was measured by diffusion assay. Additionally, antibacterial and antioxidant activities were evaluated. Antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes was examined by microdilution. Antioxidant activity was assessed by 2,2-difenil-1-picrilhidracil reduction test. The plant oils were characterized by both GC/MS and GC/FID. Oils of T. vulgaris and O. majorana showed growth inhibition activity against dermatophytes, especially T. vulgaris oil, which completely inhibited growth of all tested dermatophytes. The oils also showed bioactivity against bacteria, with minimum inhibitory concentration (MIC) values between 62.5 and 500 µg/mL. The antioxidant activity of the oils was low, with effective concentration (EC50) values <250µg/mL. The major components in the oils were as follows: T. vulgaris, o-cymene, µ-terpinene, thymol and carvacrol; R. officinalis, terpinen-4-ol and 1,8-cineole; O. majorana, terpinen-4-ol and thymol.

Larvicidal and cytotoxic activities of extracts from 11 native plants from northeastern Mexico.

Of all mosquito-borne viral diseases, dengue is spreading most rapidly worldwide. Conventional chemical insecticides (e.g., organophosphates and carbamates) effectively kill mosquitoes at their larval stage, but are toxic to humans. Natural product-based insecticides may be highly specific. Herein, we report the insecticidal activities of 11 native Mexican plants against Aedes aegypti (L). Ether extracts of Ambrosia confertiflora De Candolle, Thymus vulgaris (L.), and Zanthoxylum fagara (L.), and both ether and methanol extracts of Ruta chalepensis L. were significantly larvicidal toward the dengue mosquito after 24 h of exposure. Of them, only the ether extract of A. confertiflora was toxic to Vero cells. In conclusion, the ether extracts of Thymus vulgaris, Z. fagara, and both ether and methanol extracts of Ruta chalepensis L., could be considered as potential bioinsecticides.

Bioactivity of the Genus Turnera: A Review of the Last 10 Years.

Turnera is a genus of plants whose biological activity has been widely studied. The importance of this genus, particularly Turnera diffusa, as a source of treatment for various conditions is evidenced by the large number of new studies that have evaluated its biological activity. Accordingly, the objective of this review was to compile the information published in the last ten years concerning the biological activities reported for Turnera spp. The present work includes 92 publications that evaluate 29 bioactivities and toxicological and genotoxic information on five species of this genus. Among the pharmacological effects reported, the antioxidant, hepatoprotective, neuroprotective, hypoglycemic, and aphrodisiac activities seem more promising. Phytochemicals and standardized plant extracts could offer alternative therapeutic remedies for various diseases. Although several flavonoids, cyanogenic glycosides, monoterpenoids, triterpenoids, and fatty acids have been isolated for Turnera plants, future research should focus on the identification of the main active principles responsible for these pharmacological activities, as well as to perform clinical trials to support the laboratory results.

Synthesis of Leubethanol derivatives and evaluation against Mycobacterium tuberculosis.

Twenty-five derivatives of the natural diterpene leubethanol, including several potential pro-drugs, with changes in the functionality of the aliphatic chain or modifications of the phenolic group, were synthesized and tested in vitro by the MABA technique for their activity against the H37Rv strain of Mycobacterium tuberculosis. Several compounds showed antimycobacterial potencies similar to that of the lead compound and two of them displayed higher selectivity indexes.

Correlation between chromatographic fingerprint and antioxidant activity of Turnera diffusa (Damiana).

In the present contribution, the partial least squares (PLS) method was used to establish a correlation between the antioxidant activity (obtained by DPPH assay) and chromatographic profiles of TURNERA DIFFUSA extracts. Chromatograms were obtained using HPLC-DAD. A model was constructed using 40 samples with 2550 X variables corresponding to the responses obtained at different times; the Y variables consisted of experimental values of antioxidant activity of each extract (measured as EC50). Prior to this analysis, alignment of chromatograms was performed based on consideration of seven high-intensity signals present in all samples. The PLS1 model was validated by cross-validations; its capacity was evaluated using correlation parameters R², root mean square error of calibration (RMSEC), and root mean square error of prediction (RMSEP). The best results were achieved with zero order chromatograms using five-point smoothing (R² = 0.96, RMSEC = 3.31, and RMSEP = 7.86). Under these conditions, the optimal number of components was five. The model was applied to the prediction of antioxidant activity of commercial products; no significant differences were found between the experimental and predicted antioxidant activities for 83 % of them.

Protective effect of four Mexican plants against CCl4-induced damage on the Huh7 human hepatoma cell line.

BACKGROUND: Centaurea americana, Krameria ramosissima, Juglans mollis and Turnera diffusa are used by traditional healers in the northeastern region of Mexico to protect against liver damage. However, the hepatoprotective properties of these plants have not been investigated scientifically. This study reports on the protective effects of these plants using an in vitro assay. MATERIAL AND METHODS: Extracts of plants were tested for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The effects of extracts from these plants on a human hepatoma cell line (Huh7) were evaluated according to cell viability and aspartate aminotransferase and malondialdehyde levels before and after exposure of the cells to carbon tetrachloride (CCl(4)). RESULTS: All extracts reduced DPPH levels by more than 50%. C. americana flower and stem/leaf extracts, the aerial part of T. diffusa, and the nut, leaf and bark of J. mollis extracts were used to assess hepatoprotective activity. The extract of the aerial part of K. ramosissima was toxic. Pretreatment of Huh7 cells with extracts from the flower of C. americana (FCA), the stem/leaf fraction of C. americana (S/LCA), the leaf of J. mollis (LJM) and the bark of J. mollis (BJM) prior to the CCl(4) challenge, protected against CCl(4)-induced liver damage, as evidenced by a significant decrease in the activity of the medium enzyme. The FCA, S/LCA, LJM and BJM extracts showed significant antilipid peroxidant effects in vitro. In conclusion, the hepatoprotective effects of the FCA, S/LCA, LJM and BJM extracts observed in this study may result from their antioxidative properties.

Antimicrobial and antioxidant activities of plants from northeast of Mexico.

Traditional medicine has a key role in health care worldwide. Obtaining scientific information about the efficacy and safety of the plants from our region is one of the goals of our research group. In this report, 17 plants were selected and collected in different localities from northeast Mexico. The dried plants were separated into leaves, flowers, fruit, stems, roots and bark. Each part was extracted with methanol, and 39 crude extracts were prepared. The extracts were tested for their antimicrobial activity using three Gram-negative bacterial strains (Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii), three Gram-positive bacterial strains (Enterococcus faecalis and two Staphylococcus aureus strains), and seven clinically isolated yeasts (Candida albicans, C. krusei, C. tropicalis, C. parapsilosis and C. glabrata); their antioxidant activity was tested using a DPPH free radical assay. No activity against Gram-negative bacteria was observed with any extract up to the maximum concentration tested, 1000 µg ml(-1). We report here for the first time activity of Ceanothus coeruleus against S. aureus (flowers, minimal inhibitory concentration (MIC) 125 µg ml(-1)), C. glabrata (MICs 31.25 µg ml(-1)) and C. parapsilosis (MICs between 31.25 and 125 µg ml(-1)); Chrysanctinia mexicana against C. glabrata (MICs 31.25 µg ml(-1)); Colubrina greggii against E. faecalis (MICs 250 µg ml(-1)) and Cordia boissieri against C. glabrata (MIC 125 µg ml(-1)). Furthermore, this is the first report about antioxidant activity of extracts from Ceanothus coeruleus, Chrysanctinia mexicana, Colubrina greggii and Cyperus alternifolius. Some correlation could exist between antioxidant activity and antiyeast activity against yeasts in the species Ceanothus coeruleus, Schinus molle, Colubrina greggii and Cordia boissieri.

Development and validation of an HPLC-DAD analytical procedure for quality control of damiana (Turnera diffusa), using an antioxidant marker isolated from the plant.

Damiana is a shrub used as a dietary supplement and as an additive in several foods. This report presents the first validated analytical method for QC of products made from damiana. Through bioassay-guided fractionation, the compound showing the best antioxidant activity, determined using 2,2-diphenyl-1-picryl-hydrazyl assay in TLC, was purified and identified as 8-C-beta-[6-deoxy-2-O-(alpha-1-rhamnopyranosyl)-xylo- hexopyranos-3-uloside] (Cl50 5.56 g/mL). This is the only natural source from which this compound has been reported, and has been found in all native damiana samples analyzed. Therefore, it was used as a biomarker to develop an HPLC-diode-array detection analytical procedure using the first-order derivative of the chromatograms. Separation was achieved using an AccQ Tag C18 150 x 3.9 mm (4 microm) column, with a gradient of methanol and 0.1% trifluoroacetic acid in water. The method was shown to be accurate, sensitive, and reproducible, providing a useful quantitative means to apply QC to damiana products.

Screening of north-east Mexico medicinal plants with activities against herpes simplex virus and human cancer cell line.

The plants examined in this study have previous biological activity reports indicating the possibility of found activity against herpes and cancer cell. The aim of this contribution was to carry out a screening of Juglans mollis (Juglandaceae), Persea americana (Lauraceae), Hamelia patens (Rubiaceae), Salvia texana (Lamiaceae), Salvia ballotaeflora (Lamiaceae), Ceanothus coeruleus (Rhamnaceae), Chrysactinia mexicana (Asteraceae) y Clematis drummondii (Ranunculaceae), against HeLa cells, VHS-1 and VHS-2. The method MTT was used to determine the 50% cytotoxic concentration (CC50), in Vero and HeLa cell lines. To determine the 50% inhibitory concentration (IC50) against herpes, the plaque reduction method was used. Results showed that none of the plants exhibited activity against HeLa cells. About antiherpetic activity, J. mollis and S. ballotaeflora extracts present antiherpetic activity in terms of their SI, increasingly interest for further studies on the isolation of compounds with antiherpetic activity and about the mechanisms of action that produce this activity.

Optimization and Validation of a Microscale In vitro Method to Assess α-Glucosidase Inhibition Activity.

BACKGROUND: Microscale in vitro assays are fast, simple, and inexpensive, with reduced reagent quantities, waste, and experimental animal use. However, they have low reproducibility and low correlation with the results of in vivo models, possibly due to differences in precision and accuracy in methodologies between laboratories. OBJECTIVE: The objective was the optimization and validation of an in vitro assay, carried out on microscale, to assess the inhibition of α-glucosidase activity, which is indicative of antihyperglycemic activity. METHODS: The optimization was carried out using a fractional factorial design taking into account the best inhibition percentage and the absorbance of the controls. With the optimized experimental conditions in hand, we carried out method validation. RESULTS: The optimized conditions were as follows: enzyme concentration, 0.55 U/mL; substrate concentration, 111.5 µM; and 17.5 min incubation at 37°C. A linear range between 100 and 310.2 µg/mL of acarbose (r2 0.994) was established. The RSD was <2% and the % error was <3%. The Z factor was >0.96. This method was applied to four plant extracts, one of which was found to be very active. CONCLUSION: The method was found to be accurate, precise, selective, linear, and reliable in evaluating the antihyperglycemic activity of natural extracts in vitro.

Biological Activities of Extracts from Aerial Parts of Salvia pachyphylla Epling Ex Munz.

The antioxidant, antimicrobial, antiproliferative, and enzyme inhibitory properties of five extracts from aerial parts of Salvia pachyphylla Epling ex Munz were examined to assess the prospective of this plant as a source of natural products with therapeutic potential. These properties were analyzed by performing a set of standard assays. The extract obtained with dichloromethane showed the most variety of components, as they yielded promising results in all completed assays. Furthermore, the extract obtained with ethyl acetate exhibited the greatest antioxidant activity, as well as the best xanthine oxidase inhibitory activity. Remarkably, both extracts obtained with n-hexane or dichloromethane revealed significant antimicrobial activity against the Gram-positive bacteria; additionally, they showed greater antiproliferative activity against three representative cell lines of the most common types of cancers in women worldwide, and against a cell line that exemplifies cancers that typically develop drug resistance. Despite that, other extracts were less active, such as the methanolic or aqueous; their results are promising for the isolation and identification of novel bioactive molecules.

Evaluation of thin-layer chromatography methods for quality control of commercial products containing Aesculus hippocastanum, Turnera diffusa, Matricaria recutita, Passiflora incarnata, and Tilia occidentalis.

In Mexico, plant-derived products with health claims are sold as herbal dietary supplements, and there are no rules for their legal quality control. Aesculus hippocastanum, Turnera diffusa, Matricaria recutita, Passiflora incarnata, and Tilia occidentalis are some of the major commercial products obtained from plants used in this region. In this paper, we describe the effectiveness of thin-layer chromatography methods to provide for the quality control of several commercial products containing these plants. Standardized extracts were used. Of the 49 commercial products analyzed, only 32.65% matched the chromatographic characteristic of standardized extracts. A significant number of commercial products did not match their label, indicating a problem resulting from the lack of regulation for these products. The proposed methods are simple, sensitive, and specific and can be used for routine quality control of raw herbals and formulations of the tested plants. The results obtained show the need to develop simple and reliable analytical methods that can be performed in any laboratory for the purpose of quality control of dietary supplements or commercial herbal products sold in Mexico.