Hydroethanolic Research Articles

Thymus carnosus extracts induce anti-proliferative activity in Caco-2 cells through mechanisms that involve cell cycle arrest and apoptosis

Thymus carnosus is a thyme species rich in bioactive compounds with potential medicinal properties, but the effect of its extract are poorly studied. This work aims to compare the effect of aqueous (decoction) and hydroethanolic (HE) extracts from T. carnosus aerial parts, concerning anti-proliferative activity and underlying mechanisms.Caco-2 and HepG2 cells exposure to extracts induced dose- and time-dependent reduction in cell viability. Decoction showed very low toxicity (IC50 > 500 µg/mL (both cell lines) comparing to HE (IC50 ∼ 30 (Caco-2) and ∼120 (HepG2) µg/mL). Dose-dependent cell cycle arrest was observed in Caco-2 cells exposed to both extracts. At IC50 concentrations, HE extracts only slightly increase Annexin/PI double staining, while decoction extracts significantly increased Annexin/PI fluorescence which results in a dose-dependent large increase of cells in late apoptosis.In conclusion, Thymus carnosus anti-proliferative activity against Caco-2 cells highlights the potential of this plant, and its extracts, as functional food/ingredient with nutraceutical and pharmacological value.

Powders with small microparticle size from Hedera helix and Scrophularia nodosa exhibited high preventive antioxidant activity against H2O2-induced oxidative stress in mouse primary spleen cells.

The purpose of this study was to examine the effects of powder particle size on the cytoprotective and antioxidant activity of Hedera helix (HH) and Scrophularia nodosa (SN), two medicinal plants more commonly known as ivy and figwort, against H2O2-induced oxidative stress in mouse primary spleen cells. Thus, the preventive effects of powders of 3 different granulometric classes (50-100μm, 100-180μm and 180-315μm) and those of the hydroethanolic (HE) extract from HH and SN on oxidative stress were compared by monitoring reactive oxygen species (ROS) formation, malondialdehyde (MDA) production, and the activity of enzymatic antioxidants including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Results showed that pretreatment with the 3 fine powders from both plants generally offered to H2O2-exposed spleen cells, a protection against oxidative stress, highlighted by a significant decrease of both ROS formation and the level of MDA (p<0.001), and a significant increase of GPx activity (p<0.05). The two superfine powders (i.e. 50-100μm and 100-180μm), at 250μg/mL, were more effective in modulating all oxidative stress markers studied than both HE extracts (p<0.01), and the powder with the highest particular size (i.e., 180-315μm) (p<0.01). Compared to untreated cells, our results suggest that pretreatment with powders, in particular the superfine fractions, has relatively restored the levels of antioxidant-related enzymes including GPx, CAT and SOD. In summary, our results suggest differential effects between the 3 different fine powders studied, with the best cytoprotective and antioxidant activities being in favor of the superfine powders.

In vitro Inhibitory Effect of Gymnema sylvestre Extracts and Total Gymnemic Acids Fraction on Select Cytochrome P450 Activities in Rat Liver Microsomes.

Gymnema sylvestre R. Br. is a well-known Indian medicinal herb. Gymnemic acids are pentacyclic triterpenes saponins and active phytoconstituents of Gymnema sylvestre. The study aimed at evaluation of the in vitro rat liver cytochrome P450 (CYP) inhibition potential of extracts and total gymnemic acid (TA)-enriched fractions from G. sylvestre. Standardization of G. sylvestre [ethanolic (EL), hydroethanolic (HE), total acid of ethanolic (TAE), total acid of hydroethanolic (TAHE) and total acid of aqueous (TAAQ) extract] was done with respect to deacyl gymnemic acid (DAGA), using reverse phase-high performance liquid chromatography (RP-HPLC). Total triterpenoid content was determined by vanillin perchloric acid assay. Total triterpene content was found to be the highest in TAAQ (59.86±0.005% w/w) and TAE (49.77±0.009% w/w). TAAQ showed IC50≤50µg/ml for all selected CYP activities. Testosterone 6β-hydroxylation was strongly inhibited by TAE (IC50: 15.48±2.13µg/ml) and was moderately by TAAQ and EL with IC50≥50µg/ml. Flurbiprofen 4'-hydroxylation was subject to strong, weak and moderate inhibition by TAAQ (IC50: 34.67±1.38µg/ml), TAE (IC50:≥50µg/ml) and EL (IC50:>50µg/ml), respectively. Dextromethorphan O-demethylation was inhibited by TAHE and TAAQ. In vitro inhibition studies suggested that TA strongly inhibits activity of selected CYP. This inhibition may possibly be due to triterpenoids and gymnemic acids that have been reported to be present in it. Data also suggest a potential for possible in vivo herb-drug interactions involving G. sylvestre and other medications that are metabolized by the same CYP.

Supplementation with Achyrocline satureioides Inflorescence Extracts to Pregnant and Breastfeeding Rats Induces Tissue-Specific Changes in Enzymatic Activity and Lower Neonatal Survival.

Achyrocline satureioides (AS, family Asteraceae) is a plant widely used in traditional medicine for stomach, digestive, and gastrointestinal disorders during pregnancy. Studies regarding the indiscriminate use of plant infusions during pregnancy are limited. Recent reports have shown that chronic flavonoid supplementation induces toxicity in vivo and raises the mortality rates of healthy subjects. Therefore, we investigated whether supplementation of pregnant and lactating Wistar rats with two AS inflorescence extracts, consisting of an aqueous (AQ) extract similar to a tea (47 mg·kg−1·day) and a hydroethanolic (HA) extract (35 mg·kg−1·day−1) with a higher flavonoid content, could induce redox-related side effects. Total reactive antioxidant potential (TRAP), thiobarbituric reactive species (TBARS), and total reduced thiol (SH) content were evaluated. Superoxide dismutase (SOD) and catalase (CAT) activities were additionally quantified. Our data suggest that both AQ and HA of AS inflorescence extracts may induce symptoms of toxicity in concentrations of (47 mg·kg−1·day) and (35 mg·kg−1·day−1), respectively, in mothers regarding the delivery index and further decrease of neonatal survival. Of note, significant tissue-specific changes in maternal (liver, kidney, heart, and hippocampus) and pups (liver and kidney) biochemical oxidative parameters were observed. Our findings provide evidence that may support the need to control supplementation with the AQ of AS inflorescence extracts during gestation due to potential toxicity in vivo, which might be related, at least in part, to changes in tissue-specific redox homeostasis and enzymatic activity.

Improvement of cytoprotective and antioxidant activity of Rosa canina L. and Salix alba L. by controlled differential sieving process against H2O2-induced oxidative stress in mouse primary splenocytes

In this study, cytoprotective and antioxidant activities of Rosa canina (RC) and Salix alba (SA), medicinal plants, were studied on mouse primary splenocytes by comparing Controlled Differential Sieving process (CDSp), which is a novel green solvent-free process, versus a conventional technique, employing hydroethanolic extraction (HEE). Thus, preventive antioxidant activity of three plant powders of homogeneous particle sizes, 50-100 µm, 100-180 µm and 180-315 µm, dissolved directly in the cellular buffer, were compared to those of hydroethanolic (HE) extract, at 2 concentrations (250 and 500 µg/mL) in H2O2-treated spleen cells. Overall, compared to HE extract, the superfine powders, i. e., fractions < 180 µm, at the lowest concentration, resulted in greater reactive oxygen species (ROS) elimination, increased glutathione peroxidase (GPx) activity and lower malondialdehyde (MDA) production. Better antioxidant and preventive effects in pre-treated cells were found with the superfine powders for SA (i. e., 50-100 µm and 100-180 µm, both p < 0.001), and with the intermediate powder for RC (i. e., 100-180 µm, p < 0.05) versus HE extract. The activity levels of catalase (CAT) and superoxide dismutase (SOD) in pretreated splenocytes exposed to H2O2, albeit reduced, were near to those in unexposed cells, suggesting that pretreatment with the fine powders has relatively restored the normal levels of antioxidant-related enzymes. These findings supported that CDSp improved the biological activities of plants, avoiding the use of organic solvents and thus it could be a good alternative to conventional extraction techniques.

In vitro Evaluation of the Interaction between Amoxicillin and Hydro Ethanolic Extracts of Annickia chlorantha

The discovery of antibiotics has contributed significantly to improving the life expectancy of the global population. Nowadays, the emergence of multi resistant strains of bacteria worldwide is a major concern as it dramatically reduces the choice of effective antibiotics for prevention and treatment of a very common infection in both hospitals and our local communities. The aim of this study was to evaluate the in vitro interaction of amoxicillin and Annickia chlorantha against some Multi resistant bacteria strains. The interaction between Amoxicillin and Annickia chlorantha was studied using the Checkerboard technique. Fractional Inhibitory Concentration (FIC) indices showed that Amoxicillin/Annickia chlorantha combination was synergistic against the clinical stock of Salmonella typhi at concentrations of 5: 15 and 25 mg/mL. The extracts of the stem barks of Annickia chlorantha decreased the minimum inhibitory concentration (MIC) of amoxicillin against Salmonella typhi by 2: 4 and 66 folds respectively. At the same concentrations the combination showed no difference against the reference stocks of Staphylococcus aureus and a dose dependent antagonism against clinical stock of Escherichia coli. The results of this study revealed that the concomitant use of Annickia chlorantha and amoxicillin may potentiate and restore the antibacterial effect of amoxicillin against resistant strains of Salmonella typhi. This implies that the intake of Annickia chlorantha in traditional medicines may affect the effectiveness of a co-administered amoxicillin, which makes this plant a good, environmentally friendly and promising candidate for the development of an improved traditional medicine.

Protection against UV-induced toxicity and lack of mutagenicity of Antarctic Sanionia uncinata.

Antarctica moss Sanionia uncinata (Hedw.) Loeske is exposed in situ to damaging levels of ultraviolet (UV) radiation. This moss has the ability to respond to UV radiation exposure producing secondary metabolites such as flavonoids, and has been recommended as a potential source of photoprotective compounds and antioxidants. The aim of the present paper was to investigate the free-radical scavenging activity and mutagenic and photomutagenic properties of methanolic (ME), hydroethanolic (HE) and ethanolic (EE) extracts of S. uncinata. The phenolic contents were evaluated by high-performance liquid chromatography (HPLC) and spectrophotometry. The findings showed that ME and EE presented the highest phenolic contents and inhibited free radical-scavenging activity against 2,2'-diphenyl-1 picrylhydrazyl (DPPH) and the HPLC analysis indicated several classes of phenolic acids and flavonoids. The sun protection factors (SPF) were determined by an in vitro method and the results showed significant values. The SPF values of BZ-3 at 50μg/mL increased significantly in association with ME, HE and EE. The extracts did not induce mutagenicity in auxotrophic Salmonella typhimurium histidine and photomutagenicity was not detected in the TA102 and TA104 strains after exposure to UV-A at doses of up to 6.5J/cm2 for the TA102 strain and up to 0.24J/cm2 for the TA104 strain. In addition, with the exception of ME, all the extracts induced photoprotective effects in the presence of the TA104 strain at 0.04J/cm2. The present results suggest that S. uncinata extracts did not induce photomutation and showed promise for photoprotection against the photobiological and ROS-inducing effects of the UV-A radiation.

Pre-clinical evidences of Pyrostegia venusta in the treatment of vitiligo

Ethnopharmacological relevanceLeaves of Pyrostegia venusta are popularly used to treat vitiligo; however, none in vivo study showed its ability. Aim of the studyThe overall objective of the present study was to evaluate the antiinflammatory and hyperpigmentant activities of hydroethanolic (HE) extract of leaves from P. venusta in animal models of vitiligo induced by croton oil and monobenzone. Materials and methodsThe antiinflamatory and antioxidative effects of topical and oral administration of HE extract of P. venusta were evaluated in Swiss mice on edema model induced by croton oil, and further the N-acetyl-b-d-glucosaminidase (NAG) activity, cell infiltration, and cytokine and reactive species oxygen (ROS) levels. The involvement on mice pigmentation, cell infiltration and cytokine levels were evaluated on vitiligo model induced by monobenzone in C56BL/6 mice. ResultsHE of P. venusta by gavage (300mg/kg) reduced NAG activity in 32.5±5% on mouse ear edema induced by croton oil. Similarly, cell infiltration was lower (42.3±5.9%) when compared to control group, as well as interleukin-1β (IL-1β), interleukin (IL-6) and tumor necrosis factor-α (TNF-α) levels, in 44.1±9.7%, 71.9±22.2% and to basal levels, respectively. Topical treatment with HE of P. venusta (10%) diminished cell infiltration (67.7±7.1%) and ROS levels (total reduction). P. venusta either by gavage (300mg/Kg) or topically (10%) increased epidermal melanin level (116.5±13% and 100±16.9%, respectively), diminished dermal depigmentation (36.0±6.7% and 38.2±6.2%, respectively), as well as tissue TNF-α levels (68.2±11.6% and 99.2±12.1%, respectively) and cell infiltration (basal levels and 94.3±9.17%, respectively). However, only topical treatment with HE of P. venusta altered melanin specific marker in hair follicles. ConclusionsFor the first time these data show that topical and oral administrations of P. venusta have significant antiinflammatory and hyperpigmentant effects, demonstrating different topical and systemic effects through two animal models. Together these models are capable to mimic several features founded in vitiligo, and the results support the ethnopharmacological use of P. venusta.

Anxiolytic-like, stimulant and neuroprotective effects of Ilex paraguariensis extracts in mice

Yerba-mate (Ilex paraguariensis St. Hil.) is the most used beverage in Latin America with approximately 426 thousand of tons consumed per year. Considering the broad use of this plant, we aimed to investigate the anxiety-like and stimulant activity of both the hydroethanolic (HE) and aqueous (AE) extracts from leaves of I. paraguariensis. Swiss mice were treated with I. paraguariensis HE or AE chronically or acutely, respectively, followed by evaluation in the elevated plus-maze (EPM; anxiety-like paradigm), open field (OF; locomotor activity) or the step-down avoidance task (memory assessment). Following behavioral protocols the brains were collected for evaluation of acetylcholinesterase (AChE) activity ex vivo. Chronic treatment with HE induced an anxiolytic-like effect and increased motor activity besides augmented AChE activity. Additionally, acute treatment with AE prevented the scopolamine-induced memory deficit in the step-down avoidance task. Overall, our results indicate the importance of the I. paraguariensis-induced CNS effects, since it is a widely used nutraceutical. We have reported anxiolytic, stimulant and neuroprotective effects for this plant species. These effects are potentially modulated by the cholinergic system as well as by caffeine.

Evaluation of widely consumed botanicals as immunological adjuvants

Background Many widely used botanical medicines are claimed to be immune enhancers. Clear evidence of augmentation of immune responses in vivo is lacking in most cases. To select botanicals for further study based on immune enhancing activity, we study them here mixed with antigen and injected subcutaneously (s.c.). Globo H and GD3 are cell surface carbohydrates expressed on glycolipids or glycoproteins on the cell surface of many cancers. When conjugated to keyhole limpet hemocyanin (KLH), mixed with an immunological adjuvant and administered s.c. the magnitude of the antibody responses against globo H, GD3 and KLH depend largely on the potency of the adjuvant. We describe here the results obtained using this s.c. immunization model with seven botanicals purported to have immune stimulant effects. Methods Groups of 5–10 mice were immunized with globo H–KLH or GD3–KLH mixed with botanical, saline or positive control immunological adjuvant, s.c. three times at 1 week intervals. Antibody responses were measured 1 and 2 weeks after the 3rd immunization. The following seven botanicals and fractions were tested: (1) H-48 (Honso USA Co.), (2) Coriolus versicolor raw water extract, purified polysaccharide-K (PSK) or purified polysaccharide-peptide (PSP) (Institute of Chinese Medicine (ICM)), (3) Maitake extract (Yukiguni Maitake Co. Ltd. and Tradeworks Group), (4) Echinacea lipophilic, neutral and acidic extracts (Gaia Herbs), (5) Astragalus water, 50% or 95% ethanol extracts (ICM), (6) Turmeric supercritical (SC) or hydro-ethanolic (HE) extracts (New Chapter) or 60% ethanol extract (ICM) and (7) yeast β-glucan (Biotec Pharmacon). Purified saponin extract QS-21 (Antigenics) and semisynthetic saponin GPI-0100 (Advanced BioTherapies) were used as positive control adjuvants. Sera were analyzed by ELISA against synthetic globo H ceramide or GD3 and KLH. Results Consistent significant adjuvant activity was observed after s.c. vaccination with the Coriolus extracts (especially PSK), a 95% ethanol extract of Astragalus and yeast β-glucan, and (to a lesser extent) Maitake. Antibodies against KLH in all cases and against globo H in most cases were induced by these botanicals. Little or no adjuvant activity was demonstrated with H-48 or Echinacea extracts or the Astragalus water extract. Experiments with GD3–KLH as immunogen confirmed the adjuvant activity of the Coriolus, yeast β-glucan and Astragalus extracts. While extraction with ethanol concentrated the active ingredients in Astragalus, it had no impact on Coriolus where the 90% ethanol precipitate and solute were equally active. Conclusions Some, but not all, botanicals purported to be immune stimulants had adjuvant activity in our model. PSK and Astragalus were surprisingly active and are being further fractionated to identify the most active adjuvant components.

Turnera ulmifolia L. (Turneraceae): Preliminary study of its antioxidant activity

Turnera ulmifolia L. is used in Brazilian folk medicine as an anti-inflammatory. Since this activity may be correlated with the presence of antioxidant compounds, a leaf extract was evaluated for its radical scavenging capacity (RSC). The in vitro RSC of a 50% hydroethanolic (HE) extract was evaluated by β-carotene/linoleic acid coupled oxidation system for the inhibition of oxidation and the lipid peroxidation inhibition in rat brain homogenates, using thiobarbituric acid reactive substances (TBARS) and chemiluminescence (CL). Results indicated, through peroxidation suppression, that this extract exhibited greater antioxidative activity (77.4% ± 10%) than α-tocopherol (58.4% ± 3.7%). TBARS and CL inhibition was concentration-dependent and Q 1/2 values were 8.2 and 6.0 μg/mL for TBARS and CL, respectively. For α-tocopherol these values were 7.1 μg/mL (TBARS) and 9.8 μg/mL (CL). Phenolic compounds may be responsible for this antioxidant capacity.