Nanoemulsion and nanoencapsulation of a hydroethanolic extract of Nettle (Urtica dioica) and Wormwood (Artemisia absinthium): comparison of antibacterial and anticancer activity.

Introduction: Nanoemulsion and nanoencapsulation are attractive novel methods that can be used for incorporating active plant extracts in food preparations and pharmaceutical formulations. In the current study, we aimed to investigate the anticancer and antibacterial effects of hydroethanolic extracts of Nettle (NE), Wormwood (WE), and the combination of the two plants (CNWE), as well as their nanoemulsion forms (NN, NW, CNNW) and nanoencapsulation forms (CN, CW, and CCNW). Methods: The morphology and structure of the nanoemulsion and nanoencapsulation preparations were assessed utilizing dynamic light scattering (DLS) along with transmission electron microscopy (TEM). The antibacterial activity of the prepared formulations were assessed by determining minimum inhibitory concentration (MIC), zone of inhibition diameter, minimum bactericidal concentration (MBC), along with biofilm growth inhibition against Salmonaella typhimurium and Klebsiella. pneumoniae. The anticancer activity was evaluated via a MTT assay in the colon cancer cell line (HCT116). Results: The nanoemulsion and nanoencapsulation particle size varied between 10 and 50 nm and 60 and 110 nm, respectively. The MIC values were between 11.25 and 95 µg/mL along with MBC values between 11.25 and 190 µg/mL. The highest inhibition of biofilm formation was observed with CCNW against K. pneumoniae (∼78.5%) and S. typhimurium (∼73%). In descending order, the inhibition of biofilm formation was CCNW > CW > CN > CNNW > NN > NW > CNWE > NE > WE against the tested bacteria. The IC50 values for NE, WE, CNWE, NN, NW, CNNW, CN, CW, and CCNW were determined as 250, 170, 560, 380, 312, 370, 250, 420, and 700 µg/mL, respectively. Exposure to a high concentration of NW resulted in a significantly lower HCT116 viability compared to other groups. Taken together, CNNW, and CCNW showed the highest antibacterial and anticancer activitiy. Discussion: Nanoemulsion and nanoencapsulation were effective ways to increase the antibacterial and anticancer activity of the extracts and could be used in the food and pharmaceutical industries.

Rumen fermentation and microbial diversity of sheep fed a high-concentrate diet supplemented with hydroethanolic extract of walnut green husks.

OBJECTIVE: This study aimed to assess the impact of a hydroethanolic extract of walnut green husks (WGH) on rumen fermentation and the diversity of bacteria, methanogenic archaea, and fungi in sheep fed a high-concentrate diet. METHODS: Five healthy small-tailed Han ewes with permanent rumen fistula were selected and housed in individual pens. This study adopted a self-controlled and crossover design with a control period and an experimental period. During the control period, the animals were fed a basal diet (with a ratio of concentrate to roughage of 65:35), while during the treatment period, the animals were fed the basal diet supplemented with 0.5% hydroethanolic extract of WGH. Fermentation parameters, digestive enzyme activities, and microbial diversity in rumen fluid were analyzed. RESULTS: Supplementation of hydroethanolic extract of WGH had no significant effect on feed intake, concentrations of total volatile fatty acids, isovalerate, ammonia nitrogen, and microbial protein (p>0.05). However, the ruminal pH, concentrations of acetate, butyrate and isobutyrate, the ratio of acetate to propionate, protozoa count, and the activities of filter paper cellulase and cellobiase were significantly increased (p<0.05), while concentrations of propionate and valerate were significantly decreased (p<0.05). Moreover, 16S rRNA gene sequencing revealed that the relative abundance of rumen bacteria Christensenellaceae R7 group, Saccharofermentans, and Ruminococcaceae NK4A214 group were significantly increased, while Ruminococcus gauvreauii group, Prevotella 7 were significantly decreased (p<0.05). The relative abundance of the fungus Pseudomonas significantly increased, while Basidiomycota, Fusarium, and Alternaria significantly decreased (p<0.05). However, there was no significant change in the community structure of methanogenic archaea. CONCLUSION: Supplementation of hydroethanolic extract of WGH to a high-concentrate diet improved the ruminal fermentation, altered the structure of ruminal bacterial and fungal communities, and exhibited beneficial effects in alleviating subacute rumen acidosis of sheep.

Deleterious liver-adipose crosstalk in obesity: Hydroethanolic extract of Lampaya medicinalis Phil. (Verbenaceae) counteracts fatty acid-induced fibrotic marker expression in human hepatocytes.

Elevated circulating fatty acids in obesity may induce hepatic steatosis, leading to liver inflammation, fibrosis and nonalcoholic fatty liver disease (NAFLD). On the other hand, impaired communication between hepatocytes and adipose tissue (AT) in obesity influences adipose lipolysis and fibrosis, negatively affecting metabolic function. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in Chilean folk medicine to treat inflammatory diseases. Hydroethanolic extract of lampaya (HEL) contains flavonoids that may explain its anti-inflammatory effect, but it is unknown whether HEL modulates fibrogenic processes in hepatocytes. We studied lipolysis and expression of fibrosis markers after exposure of visceral AT explants from subjects with obesity to HepG2-secreted factors. In addition, we evaluated the effect of HEL on palmitic acid (PA, C16:0) and oleic acid (OA; C18:1)-induced fibrotic marker expression in HepG2 hepatocytes. Results: Exposure to HepG2-secreted factors increased visceral AT lipolysis and expression of CTGF and collagen I. Exposure to OA/PA elevated collagen I, CTGF, fibronectin, α-smooth muscle actin, MMP-2 and MMP-9 expression in HepG2 cells, and these effects were prevented by HEL co-treatment. Conclusion: HEL effect counteracting OA/PA-induced fibrotic marker expression in HepG2 hepatocytes may represent a preventive approach against hepatic fibrosis and deleterious liver-adipose crosstalk in obesity.

Jasminum sambac Cell Extract as Antioxidant Booster against Skin Aging.

Oxidative stress plays a major role in the skin aging process through the reactive oxygen species production and advanced glycation end products (AGEs) formation. Antioxidant ingredients are therefore needed in the skin care market and the use of molecules coming from plant cell cultures provide a unique opportunity. In this paper, the features of an hydroethanolic extract obtained by Jasminum sambac cells (JasHEx) were explored. The antioxidant and anti-AGE properties were investigated by a multidisciplinary approach combining mass spectrometric and bio-informatic in vitro and ex vivo experiments. JasHEx contains phenolic acid derivatives, lignans and triterpenes and it was found to reduce cytosolic reactive oxygen species production in keratinocytes exposed to exogenous stress. It also showed the ability to reduce AGE formation and to increase the collagen type I production in extracellular matrix. Data demonstrated that JasHEx antioxidant properties were related to its free radical scavenging and metal chelating activities and to the activation of the Nrf2/ARE pathway. This can well explain JasHEx anti-inflammatory activity related to the decrease in NO levels in LPS-stimulated macrophages. Thus, JasHEx can be considered a powerful antioxidant booster against oxidative stress-induced skin aging.

Orange thyme: Phytochemical profiling, in vitro bioactivities of extracts and potential health benefits.

Orange thyme (Thymus fragrantissimus) is becoming widely used in food as a condiment and herbal tea, nevertheless its chemical composition and potential bioactivities are largely unknown. Thus the objective of this work is to obtain a detailed phytochemical profile of T. fragrantissimus by exhaustive ethanolic extraction and by aqueous decoction mimicking its consumption. Extracts showed high content in rosmarinic acid, luteolin-O-hexuronide and eriodictyol-O-hexuronide; these were the main phenolic compounds present in orange thyme accounting for 85% of the total phenolic compounds. Orange thyme extracts presented high scavenging activity against nitric oxide and superoxide radicals. Both extracts presented significant inhibitory effect of tyrosinase activity and moderate anti-acetylcholinesterase activity. Both extracts showed a good in vitro anti-inflammatory activity and a weak anti-proliferative/cytotoxic activity against Caco-2 and HepG2 cell lines supporting its safe use. Orange thyme is a very good source of bioactive compounds with potential use in different food and nutraceutical industries.

Wound healing activity of the hydroethanolic extract of the leaves of Maytenus ilicifolia Mart. Ex Reis.

BACKGROUND AND AIM: Maytenus ilicifolia has analgesic, healing, antioxidant and anti-inflammatory properties. This study evaluated effect of the hydroalcoholic extract of M. ilicifolia leaves on skin wound repair. EXPERIMENTAL PROCEDURE: Wounds were induced on mice and treated with the extract. The treatment was performed daily, until day 7 after wound induction. Wound closure was measured and the features of the repaired tissue were investigated, including mast cell quantification, neutrophil and macrophage activities, collagen deposition, angiogenesis, and pro-metalloproteases and metalloproteases 2 and 9 activity (pro-MMPs and MMPs). RESULTS AND CONCLUSION: The M. ilicifolia extract accelerated the closure of wounds. The extract at a concentration of 4% was found to be effective, presenting anti-inflammatory effects and hemoglobin increased, along with increased soluble, total and type III collagens in the wound. In addition, there was an increase in pro-MMP9 and MMP9 activity after day 7th of treatment. The phenolic compounds and tannins present in this plant could be associated with the anti-inflammatory and healing activities observed in this study. Therefore, the ability to modulate essential parameters for accelerated and adequate healing as shown here suggests that the use of standardised extracts of M. ilicifolia and its fractions enriched in polyphenols may represent a therapeutic strategy for the treatment of wounds.

Antifungal Activity of Peruvian Banana Peel (Musa paradisiaca L.) on Candida albicans: An In Vitro Study.

AIM: The purpose of this study was to compare the antifungal activity of three concentrations of a hydroethanolic extract of the Musa × paradisiaca peel against Candida albicans strain ATCC 10231. MATERIALS AND METHODS: The agar diffusion method was used, and the culture medium used was Sabouraud agar. Petri dishes were prepared with concentrations of 10, 30, and 50% of hydroethanolic extract of the M. × paradisiaca peel; nystatin was used as a positive control, and 96% ethanol was used as a negative control. After 24 hours of incubation, each plate was examined, and the diameters (mm) of the growth inhibition halos were measured around each well using a digital vernier caliper. RESULTS: The results showed that the antifungal activity of the extract varied, depending on the concentration, as shown using analysis of variance (ANOVA; p < 0.05). When comparing the different concentrations, it was found by Duncan test that the greatest activity was obtained at 50%. CONCLUSION: It was concluded that the hydroethanolic extract of M. × paradisiaca at 50% exerted a greater antifungal effect on the strain of C. albicans than did the extract at lower concentrations. CLINICAL SIGNIFICANCE: By knowing the antimicrobial effect of M. × paradisiaca, this substance can be effectively used in products aimed to cure candidiasis infection.

Thymus zygis subsp. zygis an Endemic Portuguese Plant: Phytochemical Profiling, Antioxidant, Anti-Proliferative and Anti-Inflammatory Activities.

Thymus zygis subsp. zygis is an endemic Portuguese plant belonging to the Thymus zygis species. Although T. zygis is commonly used as a condiment and as a medicinal herb, a detailed description of the polyphenol composition of hydroethanolic (HE) and aqueous decoction (AD) extracts is not available. In this work, we describe for the first time a detailed phenolic composition of Thymus zygis subsp. zygis HE and AD extracts, together with their antioxidant, anti-proliferative and anti-inflammatory activities. Unlike other Thymus species, T. zygis subsp. zygis extracts contain higher amounts of luteolin-(?)-O-hexoside. However, the major phenolic compound is rosmarinic acid, and high amounts of salvianolic acids K and I were also detected. T. zygis subsp. zygis extracts exhibited significant scavenging activity of ABTS+, hydroxyl (•OH), and nitric oxide (NO) radicals. Regarding the anti-proliferative/cytotoxic effect, tested against Caco-2 and HepG2 cells, the AD extract only slightly reduced cell viability at higher concentrations (IC50 > 600 µg/mL, 48 h exposure), denoting very low toxicity, while the HE extract showed a high anti-proliferative effect, especially at 48 h exposure (IC50 of 85.01 ± 15.10 µg/mL and 82.19 ± 2.46 µg/mL, for Caco-2 and HepG2, respectively). At non-cytotoxic concentrations, both extracts reduced the nitric oxide (NO) release by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (at 50 µg/mL, HE and AD extracts inhibited NO release in ~89% and 48%, respectively). In conclusion, the results highlight the non-toxic effect of aqueous extracts, both resembling the consumption of antioxidants in foodstuff or in functional food. Furthermore, the HE extract of T. zygis subsp. zygis is a source of promising molecules with antioxidant, anti-inflammatory and anticancer activities, highlighting its potential as a source of bioactive ingredients for nutraceutical and pharmaceutical industries.

Hydroethanolic Extract of Lampaya Medicinalis Phil. (Verbenaceae) Decreases Proinflammatory Marker Expression in Palmitic Acid-exposed Macrophages.

BACKGROUND: Obesity is a major health problem associated with increased comorbidities, which are partially triggered by inflammation. Proinflammatory macrophage infiltration in adipose tissue of individuals with obesity increases chronic inflammation. Obesity is associated with elevated plasma levels of saturated fatty acids, such as palmitic acid (PA), which promotes inflammation in vivo and in vitro. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in the folk medicine of Northern Chile to counteract inflammation of rheumatic diseases. Hydroethanolic extract of lampaya (HEL) contains spectrophotometrically defined compounds that may contribute to the observed effect on inflammation. METHODS: We evaluated the phytochemical composition of HEL by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and liquid chromatography-electrospray ionization- tandem mass spectrometry (LC-ESI-MS/MS). We assessed whether the exposure to HEL affects PA-induced expression of proinflammatory factors in THP-1 macrophages. RESULTS: HPLC-DAD and LC-ESI-MS/MS analyses showed the presence of considerable amounts of flavonoids in HEL. The PA-induced phosphorylation of the inflammatory pathway mediators IKK and NF-κB, as well as the elevated expression and secretion of proinflammatory cytokines (IL-6, TNF-α), were reduced in cells pre-exposed to HEL. CONCLUSION: These findings give new insights about the effect of HEL reducing IKK/NF-κB proinflammatory pathway, likely explained by the number of flavonoids contained in the extract. More studies would be needed to define the possible role of Lampaya as a preventive approach in subjects with obesity whose circulating PA might contribute to chronic inflammation.

Hydroethanolic extract from Endopleura uchi (Huber) Cuatrecasas and its marker bergenin: Toxicological and pharmacokinetic studies in silico and in vivo on zebrafish.

Endopleura uchi, is used for the treatment of inflammatory disease and related to the female reproductive tract. The aim of this study was to evaluate the acute toxicity of the Endopleura uchi stem bark hydroethanolic extract (EEu) in zebrafish, emphasizing the histopathological and biochemical parameters, as well as evaluating the in silico pharmacokinetic and toxicological parameters of the phytochemical/pharmacological marker, bergenin, as their metabolites. The animals were orally treated with EEu at a single dose of 75 mg/kg, 500 mg/kg, 1000 mg/kg and 3000 mg/kg. the oral LD50 of the EEu higher to the dose of 3000 mg/kg. Behavioral, biochemical and histopathological changes were dose dependent. In silico pharmacokinetic predictions for bergenin and its metabolites showed moderate absorption in high human intestinal absorption (HIA) and Caco-2 models, reduced plasma protein binding, by low brain tissue binding and no P-glycoprotein (P-Gp) inhibition. Their metabolism is defined by the CYP450 enzyme, in addition to bergenin inhibition of CYP2C9, CYP3A4 and CYP2C19. In the bergenin and its metabolites in silico toxicity test it have been shown to cause carcinogenicity and a greater involvement of the bergenin with the CYP enzymes in the I and II hepatic and renal metabolism's phases was observed. It is possible to suggest that the histopathological damages are involved with the interaction of this major compound and its metabolites at the level of the cellular-biochemical mechanisms which involve the absorption, metabolization and excretion of these possible prodrug and drug.

Beneficial Effects of Algerian Green Alga Ulva lactuca and Its Hydroethanolic Extract on Insulin Resistance and Cholesterol Reverse Transport in High-Fat/Streptozotocin Diabetic Rats.

The aim of this study was to evaluate the impact of the green algae Ulva lactuca and its hydroethanolic extract on insulin resistance and cholesterol reverse transport in type 2 diabetic (T2D) rats. Rats had T2D induced by a high-fat diet (HFD) for 5 weeks followed by intraperitoneal injection of streptozotocin. Diabetic rats were divided into three groups and were fed a HFD in the presence or absence of 1% alga (HFD-Alg) or 1% of its hydroethanolic extract (HFD-Ext), for 4 weeks. The control group consumed 20% casein combined with 5% lipids. Hyperglycemia, insulin resistance, hypercholesterolemia, and hypertriglyceridemia were noted in HFD rats vs control rats. Whole alga and its extract decreased these parameters vs the HFD. Moreover, fecal total cholesterol and triacylglycerols levels were lowered in HFD group vs C group, but were increased with HFD-Alg vs HFD. Compared with the Control, the HFD group had decreased lecithin:cholesterol acyltransferase (LCAT) activity, apolipoprotein A-I (ApoA-I), high-density lipoprotein (HDL3)-phospholipids (PL), and HDL2-cholesteryl ester (CE) levels, but increased HDL3-unesterified cholesterol (UC) levels. Furthermore, compared with the HFD group, the HFD-Alg and HFD-Ext groups had increased LCAT activity, ApoA-I, HDL3-PL, and HDL2-CE levels and decreased HDL3-UC levels. In addition, in the HFD-Ext group, LCAT activity and ApoA-1 levels were decreased vs the HFD-Alg whereas HDL3-UC levels were increased. In conclusion, these results indicate that U. lactuca and its hydroethanolic extract have curative effect on T2D. Therefore, this alga could be considered a functional food supplement for the treatment and prevention of diabetes.

Subacute and subchronic oral toxicity assessments of Acridocarpus smeathmannii (DC.) Guill. & Perr. root in Wistar rats.

Recent adverse herb reactions have stimulated interest documenting the safety profile of medicinal agents. Thus, subacute and subchronic oral toxicity of the hydroethanolic extract of Acridocarpus smeathmannii root (HEASR) in Wistar rats was investigated. In the 28 and 90-day subacute and subchronic toxicity tests, sixty-four rats (n = male: female = 1:1 = 32) were divided into four of eight/group and ninety-six (n = male: female = 1:1 = 48) into twelve/group respectively. Distilled water (10 mL/kg) or HEASR4, HEASR5 and HEASR6 (250, 500 and 1000 mg/kg/day) respectively were administered via oral gavage. Animals were killed humanely 24 h after the last administration. Using standard methods, acute oral toxicity dose of HEAR (2000 mg/kg) was non-lethal in rodents. Subacute administration of HEASR6 increased total bilirubin (p < 0.05) in female rats. HEASR moderately altered both haematological and biochemical indices in rats. HEASR6 administration reduced ovary weight in both studies while follicle stimulating hormone level in male was reduced at all doses used. HEASR modulated lipid peroxidation, sperm quality and elevated cyclooxygenase-2 levels in rats. Histology revealed gastritis and congestions in vital organs. The low-observed adverse effect level for HEASR was below 250 mg/kg for both sexes. Overall, HEASR demonstrated inherent toxicity evidenced by our current findings. The exaggeration of its folklore medicine applications calls for cautions.

Evaluation of anti-HSV-1 activity and toxicity of hydroethanolic extract of Tanacetum parthenium (L.) Sch.Bip. (Asteraceae).

BACKGROUND: Herpes simplex type 1 (HSV-1) is widely distributed throughout the world's population. The virus spreads through direct contact with an infected individual. After primary infection, the virus remains in a latent state, and the recurrence of herpetic lesions is common. Standard treatment is performed with nucleoside analogues, but the selection of resistant strains have occurred, thus requiring the continual search for new antiviral agents. Plant extracts, fractions, and isolated compounds are a good source for studying possible antiviral compounds. HYPOTHESIS: Among plants with antiviral activity, the crude extract of aerial parts of Tanacetum parthenium (L.) Sch.Bip. (Asteraceae) have previously shown to inhibit HSV-1 infection in vitro. METHODS: The present study investigated the chemical composition of a crude hydroethanolic extract (CHE) of T. parthenium, and in vivo safety and therapeutic efficacy against HSV-1 infection. RESULTS: Liquid chromatography-mass spectrometry showed that the CHE was composed of phenolic acids (chlorogenic acids) and sesquiterpene lactones (parthenolide). Acute and subchronic toxicity and genotoxicity tests in vivo showed that oral CHE administration did not result in signs of toxicity, with no genotoxic potential. The CHE was also safe for topical administration, in which no irritation of the epidermis was observed in treated animals. Tests of topical and oral therapeutic efficacy showed that the CHE was effective against HSV-1 infection. Topical administration was the most effective, the results for which were comparable to acyclovir. CONCLUSION: These findings indicate that the CHE from aerial parts of Tanacetum parthenium has in vivo anti-HSV-1 activity and is safe for oral and topical application.

Toxicological evaluation of a standardized hydroethanolic extract from leaves of Plantago australis and its major compound, verbascoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantago australis is a perennial plant widely distributed in Latin America, and its seeds and leaves are used in folk medicine to treat many diseases and conditions. Among its various chemical compounds, verbascoside is one of the most present, and has several pharmacological activities described, but there is not much information about its toxicity. AIMS OF THE STUDY: The aims of this study were to optimize the extraction of verbascoside from P. australis leaves with ultrasound methods, to develop a validated HPLC method to quantify verbascoside, and to evaluate the toxicological safety of the extract and verbascoside using in vitro and in vivo assays. MATERIALS AND METHODS: Dried leaves of P. australis were submitted to different extraction methods (percolation and ultrasound). The optimization of the ultrasound extraction was carried out by complete factorial design (22) and response surface methodology (RSM), followed by HPLC analysis for marker compounds. HPLC analysis was performed to verify the presence of the marker compounds aucubin, baicalein, oleanolic acid, ursolic acid and verbascoside. Mutagenicity was assessed by Salmonella/microsome mutagenicity assay. Cytotoxicity and genotoxicity were evaluated in V79 cells by reduction of tetrazolium salt (MTT) and neutral red uptake (NRU) assays, and alkaline comet assay, respectively. Verbascoside phototoxicity was assessed in 3T3 cells by the NRU phototoxicity assay. Wistar rats were used to perform the acute and sub-chronic toxicity tests. RESULTS: Among the marker compounds, only verbascoside was found in the hydroethanolic extract of P. australis leaves (PAHE); its highest concentration was obtained with the ultrasound-assisted extraction (UAE) method, optimized in 40 min and 25 °C, and the method validation was successfully applied. Neither PAHE nor verbascoside showed mutagenic or genotoxic activities. Cytotoxicity assays demonstrated that both PAHE and verbascoside reduced cell viability only at the highest concentrations, and verbascoside had no phototoxic properties. The in vivo toxicity evaluation of PAHE suggested that the LD50 is higher than 5000 mg/Kg, indicating that this extract is safe for use. In addition, no signs of toxicity were found in subchronic exposure. CONCLUSION: The HPLC method to quantify verbascoside was validated, and the extraction of verbascoside from P. australis leaves through ultrasound method was optimized, yielding an extract with 6% verbascoside. Our results suggest the toxicological safety of PAHE and verbascoside, corroborating the use of P. australis in folk medicine, and also indicate verbascoside as a potential ingredient in topical formulations.

Acridocarpus Smeathmannii (DC.) Guill. & Perr. Root enhanced reproductive behavior and sexual function in male wistar rats: Biochemical and pharmacological mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Novel therapeutic opportunities from medicinal agents continue to arouse scientific interest in recent times. Still, there is a dearth of information as regards experimental evidence generated from medicinal plants that would yield pharmacological agents for the treatment of erectile dysfunction. Acridocarpus Smeathmannii (DC.) Guill. & Perr. Root (ASR) has a long history as an aphrodisiac in African traditional medicine. Thus, this study investigated the reproductive potentials and associated biochemical mechanisms of its hydroethanolic extract (HEASR) in male Wistar rats. Also, the bioactive compounds were identified. MATERIALS AND METHODS: Fifty-four male albino rats (180 ±â€¯20 g) were divided into nine groups of six rats/group. Control, group 1 received normal saline (10 mL/kg). Groups 2-6 rats were administered sildenafil (1.43 mg/kg/day), mesterolone (0.36 mg/kg/day), doxazocin (0.03 mg/kg/day), HEASR1 (50 mg/kg/day) and HEASR2 (200 mg/kg/day) respectively. Others received co-administration of HEASR2 with standard drugs. Treatment lasted for 28 days via oral gavage. RESULTS: An acute oral toxicity of HEASR up to 2 g/kg produced no mortality in mice p.o. while the median lethal dose was estimated to be 810 mg/kg i.p. HEASR2 administration or in combination with sildenafil, mesterolone and doxazocin increased mounting frequencies on day 28 by 77.44%, 122.65%, 148.5% and 93.88% and sperm counts by 38.29%, 55.21%, 42.48%, and 48.98% respectively in treated rats. HEASR2 + sildenafil elevated testosterone and follicle stimulating hormone levels by 36.33% and 24.55% while HEASR2 + doxazocin elevated luteinizing hormone levels by 97.44% in rats. HEASR modulated prostate-specific antigen and malondialdehyde levels respectively. Reduced glutathione, superoxide dismutase, and catalase activities were raised in five selected organs. Serum nitric oxide but not cyclooxygenase-2 or tumor necrosis factor-α levels was moderately improved in rats. CONCLUSION: Overall, the results obtained demonstrated the potential of HEASR as a male reproductive enhancer, thus justify its folklore applications. Further, octadecanoic acid ethyl ester was the most abundant bioactive component present.

Chemical Characterization and Bioactivity of Extracts from Thymus mastichina: A Thymus with a Distinct Salvianolic Acid Composition.

Thymus mastichina, also called mastic thyme or Spanish marjoram, is endemic to the Iberian Peninsula, where it is widely used in folk medicine especially for treating digestive and respiratory systems disorders, and as a condiment to season olives. This work describes for the first time the detailed phenolic composition of exhaustive hydroethanolic extracts and aqueous decoctions of Thymus mastichina. Unlike other species of the Thymus genera, Thymus mastichina extracts contain high amounts of salvianolic acid derivatives, with salvianolic acid A isomer being the main derivative. This isomer was identified in extracts from Thymus mastichina for the first time. Also, an undescribed salvianolic acid derivative in Thymus mastichina was identified and its structure was tentatively described. Extracts from Thymus mastichina showed significant scavenging activity of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical cation, hydroxyl, and nitric oxide radicals. The anti-proliferative effect of both T. mastichina extracts were tested against Caco-2 and HepG2 cells; the hydroethanolic extract showed a high anti-proliferative activity against Caco-2 cells compared to HepG2 cells (at 24 h exposure, the concentration that inhibits 50% of proliferation, IC50, was 71.18 ± 1.05 µg/mL and 264.60 ± 11.78 µg/mL for Caco-2 and HepG2, respectively). Thus, these results make this species a promising candidate for further investigation of its anti-tumoral potential. Therefore, Thymus mastichina can be potentially used as a functional food (used as a decoction or herbal tea) or as a source of bioactive ingredients with antioxidant and anti-proliferative properties.

Wound healing and anti-inflammatory activities induced by a Plantago australis hydroethanolic extract standardized in verbascoside.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantago australis is a popular plant found to be widely spread in Latin America. In folk medicine, the seeds and leaves are used mainly for anti-inflammatory, wound healing, among others. The verbascoside, a phenolic glycoside, is an active chemical component described in this species of plant, which has antioxidant, anti-inflammatory and healing effects. PURPOSE: The aim of the present study was to evaluate whether P. australis hydroethanolic extract (PAHE) standardized in verbascoside could promote wound healing associated with anti-inflammatory action within both in vitro and in vivo models. METHODS: For the wound healing activity, we used a Scratch Test, an assay capable of evaluating the migratory ability of keratinocyte cells (HaCat) in vitro and thereby confirming the activity in rats. For the anti-inflammatory activity, the inflammation was induced with LPS in microglial murine cells (N9). Inflammatory mediators (IL-6, IL-10, IL-12p70, INFγ, MCP-1 and TNFα) were measured and the activity of superoxide dismutase (SOD), catalase (CAT), and mitochondrial membrane potential were evaluated. In addition, using paw edema induced by carrageenan in rats, the anti-inflammatory activity in vivo was analyzed. RESULTS: The PAHE and verbascoside, induced a significant increase in migration of keratinocytes, at all concentrations tested when compared to the negative control. The wound healing activity in vivo showed that the PAHE accelerated the process. The treatments with PAHE and verbascoside induce increases in the antioxidants enzymes, suggesting a possible activation of these enzymes. However, this did not result in an increase in the expression of inflammatory mediators in microglial cells. In LPS activated cells the verbascoside displayed a significant reduction of TNFα, IL-6, IL-12p70, MCP-1 and INFγ, while the PAHE only displayed statistically significant reduction in TNFα. Interestingly, both the compounds could reduce the oxidative parameters in N9 cells activated by LPS. Additionally, pretreatment with PAHE inhibited the paw edema in rats. CONCLUSION: The results suggest that PAHE has wound healing activity, improving cells migration and, as well as was able to reverse the oxidation effect in LPS-activated N9 cells. The wound-healing and anti-inflammatory activities of PAHE were confirmed in vivo. In addition, the presence of verbascoside can be related to PAHE effects, since this compound was capable of increase keratinocytes migration and inhibiting inflammation mediators.

Achillea millefolium L. hydroethanolic extract inhibits growth of human tumor cell lines by interfering with cell cycle and inducing apoptosis.

The cell growth inhibitory activity of the hydroethanolic extract of Achillea millefolium was studied in human tumor cell lines (NCI-H460 and HCT-15) and its mechanism of action was investigated. The GI50 concentration was determined with the sulforhodamine B assay and cell cycle and apoptosis were analyzed by flow cytometry following incubation with PI or Annexin V FITC/PI, respectively. The expression levels of proteins involved in cell cycle and apoptosis were analyzed by Western blot. The extracts were characterized regarding their phenolic composition by LC-DAD-ESI/MS. 3,5-O-Dicaffeoylquinic acid, followed by 5-O-caffeoylquinic acid, were the main phenolic acids, while, luteolin-O-acetylhexoside and apigenin-O-acetylhexoside were the main flavonoids. This extract decreased the growth of the tested cell lines, being more potent in HCT-15 and then in NCI-H460 cells. Two different concentrations of the extract (75 and 100 µg/mL) caused alterations in cell cycle profile and increased apoptosis levels in HCT-15 and NCI-H460 cells. Moreover, the extract caused an increase in p53 and p21 expression in NCI-H460 cells (which have wt p53), and reduced XIAP levels in HCT-15 cells (with mutant p53). This work enhances the importance of A. millefolium as source of bioactive phenolic compounds, particularly of XIAP inhibitors.

Turbo-extraction of glycosides from Stevia rebaudiana using a fractional factorial design

ABSTRACT Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, leaf extract has recently called the attention of food industry as a proposal for natural sweetener. The sweet flavor is attributed to the glycosides, in especial stevioside and rebaudioside A, which are the plant main chemical markers. The aim of the work reported here was to optimize the turbo-extraction of stevia leaves using water, ethanol 70% and 90% (w/w) as green solvents. A 25-2 factorial design was applied to study the linear effects of the drug size, solvent to drug ratio, temperature, time and also the turbolysis speed on the extraction of glycosides. The glycosides exhaustive extraction showed that ethanol 70% gave better results and was used for turbo-extraction. The stevioside and rebaudioside A contents were quantified by a validated method by high performance liquid chromatographic with photodiode array detector. The contents of stevioside and rebaudioside A in fluid extract increased with the drug size, but decreased at high shearing speeds and solvent to drug ratio, while their yields decreased at higher temperature and were not affected by turbo speed. An increase in solvent to drug ratio reduced significantly the glycosides percent in dried extract. Optimal solution for S. rebaudiana leaves turbo-extraction was determined by desirability functions. The optimal extraction condition corresponded to drug size of 780 µm, solvent to drug ratio of 10, extraction time of 18 min; temperature of 23 ºC and turbo speed of 20,000 rpm, resulting in yields of 4.98% and 2.70%, for stevioside and rebaudioside A, respectively. These yields are comparable to the ones recently published for dynamic maceration, but with the advantage of shorter extraction times. This work demonstrates that turbolysis is promising for S. rebaudiana glycosides extraction and stimulate new research on the purification of these extracts, which may become an interesting source of income for developing countries such as India and Brazil.