Src Tyrosine Kinase Inhibitory and Antioxidant Activity of Black Chokeberry and Bilberry Fruit Extracts Rich in Chlorogenic Acid.

Edible berries such as the fruits of black chokeberry (Aronia melanocarpa (Michx.) Elliott) and bilberry (Vaccinium myrtillus L.) are considered to be rich in phenolic compounds, which are nowadays attracting great interest due to their promising health benefits. The main objective of our study was to investigate, for the first time, their inhibitory properties on Src tyrosine kinase activity, as this enzyme plays an important role in multiple cellular processes and is activated in both cancer and inflammatory cells. In hydroethanolic fruit extracts, 5.0-5.9% of total polyphenols were determined spectrophotometrically, including high amounts of hydroxycinnamic acid derivatives. HPLC analysis revealed that the black chokeberry and bilberry extracts contained 2.05 mg/g and 2.54 mg/g of chlorogenic acid, respectively. Using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the extracts studied were found to have comparable inhibitory effects on Src tyrosine kinase, with IC50 values of 366 µg/mL and 369 µg/mL, respectively. The results also indicated that chlorogenic acid contributes significantly to the observed effect. In addition, both fruit extracts exhibited antioxidant activity by scavenging DPPH and NO radicals with SC50 values of 153-352 µg/mL. Our study suggested that black chokeberry and bilberry fruits may be beneficial in cancer and other inflammation-related diseases.

Traditional Chinese Medicine Tanreqing Targets Both Cell Division and Virulence in Staphylococcus aureus.

Staphylococcus aureus has been recognized as an important human pathogen and poses a serious health threat worldwide. With the advent of antibiotic resistance, such as the increased number of methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to develop new therapeutical agents. In this study, Chinese traditional medicine Tanreqing (TRQ) has been used as an alternative treating agent against MRSA and we aim to unravel the mode of action of TRQ underlying MRSA inhibition. TRQ treatment affected numerous gene expression as revealed by RNA-seq analysis. Meanwhile, TRQ targeted cell division to inhibit cell growth as shown by illumination microscopy. Besides, we confirmed that TRQ downregulates the expression of virulence factors such as hemolysin and autolysin. Finally, we used a murine model to demonstrate that TRQ efficiently reduces bacterial virulence. Altogether, we have proved TRQ formula to be an effective agent against S. aureus infections.

Comparative Antioxidant, Anti-Acetylcholinesterase and Anti-α-Glucosidase Activities of Mediterranean Salvia Species.

Salvia species have a cosmopolitan distribution and comprise several well-known plants valuable for pharmaceutical and food industries due to their recognized medicinal, food flavouring, and preservative properties. The present study aimed to evaluate and compare the biological activities of seven wild-growing Salvia species from the Mediterranean area (S. fruticosa, S. glutinosa, S. nemorosa, S. officinalis, S. pratensis, S. sclarea, S. verticillata). All studied ethanolic leaf extracts exhibited significant DPPH and NO radical scavenging ability, lipid peroxidation inhibition, and reducing power, as well as moderate iron-chelating properties. Together with S. officinalis and S. fruticosa, S. verticillata showed anti-acetylcholinesterase activity, while S. glutinosa was also found to possess the ability to inhibit α-glucosidase. Total flavonoid (0.37-0.90%), phenolic acid (3.55-12.44%), tannin (1.22-2.60%), and anthocyanin contents (0.03-0.08%) were determined in Salvia leaves. Rosmarinic acid was the predominant hydroxycinnamic acid in all studied sage plants, ranging from 9400 to 38,800 µg/g. The correlation study showed a strong relationship between biological activities and contents of total phenolic acids, total tannins, and rosmarinic acid, indicating their significant contribution to the efficiency of tested Salvia species. Our results highlighted Mediterranean sage plants as rich sources of potent antioxidant, neuroprotective, and hypoglycemic agents which are worthy of further research.

In vitro antibacterial effects of Tanreqing injection combined with vancomycin or linezolid against methicillin-resistant Staphylococcus aureus.

BACKGROUND: Combining conventional drugs and traditional medicine may represent a useful approach to combating antibiotic resistance, which has become a serious threat to global public health. This study aimed to evaluate the potential synergistic interactions between Tanreqing (TRQ) injection, a commercial traditional Chinese medicine formula used for the treatment of upper respiratory tract infection, and selected antibiotics used against methicillin-resistant Staphylococcus aureus (MRSA). METHODS: The minimum inhibitory concentrations (MICs) of TRQ, vancomycin and linezolid against planktonic MRSA strain were determined by the broth microdilution method. The combined effects of TRQ and antibiotics were studied by the checkerboard method and the time-kill curve assay. The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay was employed to determine the inhibitory effect of the test compounds alone and in combination against MRSA embedded in biofilms. RESULTS: MRSA strain was found to be susceptible to TRQ formula with MIC value 4125 µg/ml, while the MIC values for antibiotics, vancomycin and linezolid, were 2.5 µg/ml. The checkerboard analysis revealed that TRQ markedly enhanced activities of the tested antibiotics by reducing their MICs. In the time-kill analysis, TRQ at 1/2 × MIC in combination with vancomycin at 1/2 × MIC, as well as TRQ at 1/8 × MIC in combination with linezolid at 1/2 × MIC decreased the viable colonies by ≥2log10 CFU/ml, resulting in a potent synergistic effect against planktonic MRSA. In contrast to the tested antibiotics, which did not affect mature MRSA biofilms at subinhibitory concentrations, TRQ alone showed strong ability to disrupt preformed biofilms and induce biofilm cell death. The combination of TRQ with vancomycin or linezolid at sub-MIC concentrations resulted in a synergistic antibiofilm effect significantly higher than for each single agent. CONCLUSIONS: This study provides the first in vitro evidence on the synergistic effects of TRQ and vancomycin or linezolid against planktonic and biofilm MRSA, and revealed their optimal combination doses, thereby providing a rational basis for the combination therapies against MRSA.

Antioxidant and Anticholinesterase Potential of Six Thymus Species.

The present study aimed to evaluate antioxidant and acetylcholinesterase (AChE) inhibitory activities of the ethanolic extracts of six selected Thymus species growing in Croatia (T. longicaulis, T. praecox subsp. polytrichus, T. pulegioides, T. serpyllum subsp. serpyllum, T. striatus, and T. vulgaris). Antioxidant effectiveness was assessed using six different assays, in comparison with rosmarinic acid, luteolin, and reference antioxidants. All tested Thymus extracts possessed DPPH (IC50 = 3-6 µg/mL) and nitric oxide (IC50 = 70-177 µg/mL) free radical scavenging activities, strong reducing properties (IC50 = 11-15 µg/mL), ferrous ion chelating activity (IC50 = 126-389 µg/mL), ability to inhibit lipid peroxidation (IC50 = 34-80 µg/mL), and high total antioxidant capacities (238-294 mg AAE/g). AChE inhibitory activity was examined using Ellman's colorimetric method and all tested extracts showed anti-AChE activity in a dose dependent manner. The values of 10-28%, 23-39%, and 64-86% were obtained for tested concentrations of 0.25, 0.5, and 1 mg/mL, respectively. Additionally, the contents of total hydroxycinnamic derivatives, flavonoids, and tannins in dried plant samples were determined spectrophotometrically. Our results highlighted Thymus species as a rich source of natural antioxidants and AChE inhibitors that could be useful in preventing and treating Alzheimer's disease and other neurodegenerative disorders.

Hepatoprotective effects of Micromeria croatica ethanolic extract against CCl4-induced liver injury in mice.

BACKGROUND: Micromeria croatica (Pers.) Schott is an aromatic plant from Lamiaceae family previously found to possess potent in vitro antioxidant activity which is mainly attributed to the high level of polyphenolic substances. The aim of this study was to investigate the hepatoprotective activity and possible underlying mechanisms of Micromeria croatica ethanolic extract (MC) using a model of carbon tetrachloride (CCl4)-induced liver injury in mice. METHODS: Male BALB/cN mice were randomly divided into seven groups: control group received saline, MC group received ethanolic extract of M. croatica in 5% DMSO (100 mg/kg b.w., p.o.), and CCl4 group was administered CCl4 dissolved in corn oil (2 mL/kg, 10% v/v, ip). MC50, MC200 and MC400 groups were treated with MC orally at doses of 50, 200 and 400 mg/kg once daily for 2 consecutive days, respectively, 6 h after CCl4 intoxication. The reference group received silymarin at dose of 400 mg/kg. At the end of experiment, blood and liver samples were collected for biochemical, histopathological, immunohistochemical and Western blot analyses. In addition, major phenolic compounds in MC were quantified by HPLC-DAD. RESULTS: CCl4 intoxication resulted in liver cells damage and oxidative stress and triggered inflammatory response in mice livers. MC treatment decreased ALT activity and prevented liver necrosis. Improved hepatic antioxidant status was evident by increased Cu/Zn SOD activity and decreased 4-hydroxynonenal (4-HNE) formation in the livers. Concomitantly, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were overexpressed. The hepatoprotective activity of MC was accompanied by the increase in nuclear factor-kappaB (NF-κB) activation and tumor necrosis factor-alpha (TNF-α) expression, indicating amelioration of hepatic inflammation. Additionally, MC prevented tumor growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) expression, suggesting the potential for suppression of hepatic fibrogenesis. CONCLUSION: These results of the present study demonstrated that MC possesses in vivo antioxidant and anti-inflammatory activity and exhibits antifibrotic potential, which are comparable to those of standard hepatoprotective compound silymarin.

Acetylcholinesterase inhibitory, antioxidant and phytochemical properties of selected medicinal plants of the Lamiaceae family.

The present study aimed to evaluate acetylcholinesterase (AChE) inhibitory and antioxidant activities of Lamiaceae medicinal plants growing wild in Croatia. Using Ellman's colorimetric assay all tested ethanolic extracts and their hydroxycinnamic acid constituents demonstrated in vitro AChE inhibitory properties in a dose dependent manner. The extracts of Mentha x piperita, M. longifolia, Salvia officinalis, Satureja montana, Teucrium arduini, T. chamaedrys, T. montanum, T. polium and Thymus vulgaris at 1 mg/mL showed strong inhibitory activity against AChE. The antioxidant potential of the investigated Lamiaceae species was assessed by DPPH• scavenging activity and total antioxidant capacity assays, in comparison with hydroxycinnamic acids and trolox. The extracts differed greatly in their total hydroxycinnamic derivatives content, determined spectrophotometrically. Rosmarinic acid was found to be the predominant constituent in most of the investigated medicinal plants (by RP-HPLC) and had a substantial influence on their AChE inhibitory and antioxidant properties, with the exception of Teucrium species. These findings indicate that Lamiaceae species are a rich source of various natural AChE inhibitors and antioxidants that could be useful in the prevention and treatment of Alzheimer's and other related diseases.

Resolution of liver fibrosis by isoquinoline alkaloid berberine in CCl4-intoxicated mice is mediated by suppression of oxidative stress and upregulation of MMP-2 expression.

Liver fibrosis is the result of chronic liver injury, and it represents a widespread medical problem. The aim of this study is to investigate the antifibrotic activity of isoquinoline alkaloid berberine in carbon tetrachloride (CCl4)-induced damage in mice. Hepatic fibrosis was induced by intraperitoneal (i.p.) administration of CCl4 (2 mL/kg, 20% v/v in olive oil) twice a week for 8 weeks. Berberine at the doses of 3 and 9 mg/kg and silymarin at the dose of 50 mg/kg were given i.p. once daily for the next 2 weeks. CCl4 intoxication increased the levels of serum transaminases and induced oxidative stress in the liver. Hepatic fibrosis was evidenced by a massive deposition of collagen, which coincided with increased expression of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1 and the activation of hepatic stellate cells. The high-dose berberine (9 mg/kg) ameliorated oxidative stress, decreased TNF-α and TGF-ß1 expression, increased the levels of matrix metalloproteinase (MMP)-2, and stimulated the elimination of fibrous deposits. Berberine at the dose of 9 mg/kg exhibited stronger therapeutic activity against hepatic fibrosis than silymarin at the dose of 50 mg/kg. In vitro analyses show an important scavenging activity of berberine against oxygen and nitrogen reactive species. The results of this study suggest that berberine could ameliorate liver fibrosis through the suppression of hepatic oxidative stress and fibrogenic potential, concomitantly stimulating the degradation of collagen deposits by MMP-2.

Antioxidant activities and polyphenolic contents of three selected Micromeria species from Croatia.

Antioxidant activities of three selected Micromeria species growing in Croatia (M. croatica, M. juliana and M. thymifolia) were evaluated using five different antioxidant assays, in comparison with plant polyphenolic constituents and reference antioxidants. All studied ethanolic extracts exhibited considerable activity to scavenge DPPH and hydroxyl free radicals, reducing power, iron chelating ability and total antioxidant capacity in the order: M. croatica > M. juliana > M. thymifolia. Total polyphenol (9.69-13.66%), phenolic acid (5.26-6.84%), flavonoid (0.01-0.09%) and tannin (3.07-6.48%) contents in dried plant samples were determined spectrophotometrically. A strong positive correlation between antioxidant activities and contents of phenolic acids and tannins was found, indicating their responsibility for effectiveness of tested plants. Our findings established Micromeria species as a rich source of antioxidant polyphenols, especially the endemic M. croatica.

Evaluation of antioxidant potential of Lavandula x intermedia Emeric ex Loisel. 'Budrovka': a comparative study with L. angustifolia Mill.

A Croatian indigenous cultivar of lavandin, Lavandula x intermedia 'Budrovka' (Lamiaceae) was studied for the phenolic acids, flavonoids, anthocyanins, procyanidins and total tannins, as well as total polyphenols content, in the flower, inflorescence stalk and leaf ethanolic extracts. Antioxidant potentials on these plant part extracts were assessed by the DPPH free radical scavenging activity, iron chelating activity, reducing power, lipid peroxidation inhibition properties and total antioxidant capacity assays. All results were compared with those of Lavandula angustifolia, the only member of the Lavandula genus officially used in modern phytotherapy. Based on the results of our parallel study, we may suggest that Lavandula x intermedia 'Budrovka' is as potent an antioxidant as Lavandula angustifolia and the antioxidant activity of the Lavandula extracts is mainly due to the presence of rosmarinic acid. A good correlation was found between the polyphenol contents and antioxidant activities of the extracts.

In vivo hepatoprotective activity of the aqueous extract of Artemisia absinthium L. against chemically and immunologically induced liver injuries in mice.

AIM OF THE STUDY: This study aimed to evaluate in vivo hepatoprotective activity of the aqueous extract of Artemisia absinthium L. (AEAA), which has been used for the treatment of liver disorders in Traditional Uighur Medicine. MATERIALS AND METHODS: Qualitative and quantitative phytochemical analysis of the AEAA was performed by means of thin layer chromatography and spectrophometric assays. Aqueous extract (50, 100 or 200 mg/kg body weight/day) was administered orally to experimental mice. Liver injury was induced chemically, by a single CCl(4) administration (0.1% in olive oil, 10 ml/kg, i.v.), or immunologically, by injection of endotoxin (LPS, 10 microg, i.v.) in BCG-primed mice. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) in mouse sera, as well as superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) in mouse liver tissues were measured. The biochemical observations were supplemented by histopathological examination. RESULTS: Obtained results demonstrated that the pretreatment with AEAA significantly (P<0.001) and dose-dependently prevented chemically or immunologically induced increase in serum levels of hepatic enzymes. Furthermore, AEAA significantly (P<0.05) reduced the lipid peroxidation in the liver tissue and restored activities of defense antioxidant enzymes SOD and GPx towards normal levels. In the BCG/LPS model, increase of the levels of important pro-inflammatory mediators TNF-alpha and IL-1 was significantly (P<0.01) suppressed by AEAA pretreatment. Histopathology of the liver tissue showed that AEAA attenuated the hepatocellular necrosis and led to reduction of inflammatory cells infiltration. Phytochemical analyses revealed the presence of sesquiterpene lactones, flavonoids, phenolic acids and tannins in the AEAA. CONCLUSIONS: The results of this study strongly indicate the protective effect of AEAA against acute liver injury which may be attributed to its antioxidative and/or immunomodulatory activity, and thereby scientifically support its traditional use.