Ekrem Köksal

Purification and Characterization of Peroxidase from Sweet Gourd (Cucurbita moschata Lam. Poiret)

Peroxidase (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) is an oxidoreductase enzyme found in many fruits and vegetables. This enzyme was purified from sweet gourd (Cucurbita moschata Lam. Poiret) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. Furthermore, optimum pH, optimum temperature, optimum ionic strength, stable pH, and stable temperature conditions were determined as 7.2, 50°C, 0.4 M, 8.0, and 40°C, respectively. The molecular weight (MW) of the enzyme was estimated to be 85 kDa by SDS-PAGE method. The values of Km and Vmax were calculated from the Lineweaver-Burk graph for guaiacol/H2O2 substrate patterns.

Antioxidant activity and polyphenol content of Turkish thyme (Thymus vulgaris) monitored by liquid chromatography and tandem mass spectrometry

ABSTRACT Like tea, the leaves of Turkish thyme (Thymus vulgaris) can be boiled in water to produce an extract. This is widely used as syrup for the treatment of coughs and bronchitis at alternative medicine clinics in many parts of the world. In the current study, we assessed the phenolic content and antioxidant activity of thyme. The antioxidant activities of both ethanol and aqueous extracts of thyme were determined using various in vitro methods. The total phenolic and total flavonoid contents were determined to be a gallic acid equivalent and a quercetin equivalent, respectively. Finally, the quantities of the phenolic compounds were detected using high-performance liquid chromatography and tandem mass spectrometry. The total phenolic compounds in the aqueous extract and ethanol extracts of Turkish thyme were 256.0 μg gallic acid equivalent/mg dried extract and 158.0 μg gallic acid equivalent/mg dried extract, respectively. Conversely, the total flavonoid compounds in both extracts were 44.2 μg and 36.6 μg quercetin equivalent/mg dried extract, respectively. For the first time, we determined phenolic contents and investigated the antioxidant potential of thyme. The results indicate that Turkish thyme is a good dietary source with phenolic properties.

Antioxidant activity and phenolic compounds of ginger (Zingiber officinale Rosc.) determined by HPLC-MS/MS

Oxidative stress related diseases often arise from over production of free radicals and reactive oxygen/nitrogen species. The prevention of these diseases could be possible with the use of natural antioxidant plants that could be promising as therapeutic candidates. Since antioxidant properties of a species could be stem from phenolic compounds, it is, therefore, important to evaluate antioxidant and total/individual phenolic and flavonoid content. For this purpose, we evaluated antioxidant properties of ginger (Zingiber officinale Rosc.) based on three parameters: the antioxidant capacity, total phenolic and flavonoid content as well as identification of phenolic acids of water extract (WEG) and ethanol extract (EEG) of ginger. For antioxidant capacity, we performed FRAP, CUPRAC assay, Fe2+ chelating ability, DPPH and DMPD radical scavenging activities. Also, total phenolic and flavonoid contents in both extracts were also measured via Folin Ciocalteu’s method. For identification of phenolic acids, HPLC-MS/MS method was performed. The results showed that EEG had generally better antioxidant activity than WEG in all assays. HPLC-MS/MS analysis showed that there are at least eight different phenolic acids found in ginger, among which pyrogallol p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were more abundant in both extracts. This study clearly showed that ginger extracts demonstrated effective antioxidant properties and their consumption may reduce or delay the progression of diseases that oxidative stress take place due to lack of antioxidant supplementation.

RP-HPLC/MS/MS Analysis of the Phenolic Compounds, Antioxidant and Antimicrobial Activities of Salvia L. Species

The identification and quantification of the phenolic contents of methanolic extracts of three Salvia L. species namely S. brachyantha (Bordz.) Pobed, S. aethiopis L., and S. microstegia Boiss. and Bal. were evaluated using reverse phase high performance liquid chromatography, UV adsorption, and mass spectrometry (RP-HPLC/MS). In order to determine the antioxidant capacity of these species, cupric ions (Cu2+) reducing assay (CUPRAC) and ferric ions (Fe3+) reducing assay (FRAP) were performed to screen the reducing capacity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was employed for evaluation of the radical scavenging activity for both solvents. In further investigation, the antimicrobial activities of Salvia species were tested using the disc diffusion method against three Gram-positive and four Gram-negative microbial species, as well as three fungi species. The results showed that there is a total of 18 detectable phenols, the most abundant of which was kaempferol in S. microstegia and rosmarinic acids in S. brachyantha and S aethiopis. The other major phenols were found to be apigenin, luteolin, p-coumaric acid, and chlorogenic acid. All species tested showed moderate and lower antioxidant activity than standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ascorbic acid. The ethanolic extracts of Salvia species revealed a wide range of antimicrobial activity. S. brachyantha and S. microstegia showed the highest antimicrobial activities against B. subtilis, whereas S. aethiopis was more effective on Y. lipolytica. None of the extracts showed anti-fungal activity against S. cerevisiae. Thus these species could be valuable due to their bioactive compounds.

Effect of sumac extract on serum oxidative status, RANKL/OPG system and alveolar bone loss in experimental periodontitis in rats

Objectives Sumac (Rhus coriaria L.) is widely used spice which has several properties such as antioxidant, anti-inflammatory and antimicrobial. The purpose of this animal study was to evaluate the effects of sumac extract on levels of receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG) expression, serum oxidative status, and alveolar bone loss in experimental periodontitis. Material and Methods Twenty-four Wistar rats were separated into three groups: non-ligated (NL, n=8), ligature only (LO, n=8), and ligature and treated with sumac extract (S, n=8) (20 mg/kg per day for 11 days). A 4/0 silk suture was placed around the mandibular right first molars subgingivally; after 11 days, the rats were sacrificed, and alveolar bone loss was histometrically measured. The detection of RANKL and OPG were immunohistochemically performed. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Results Alveolar bone loss was significantly greater in the LO group compared to the S and NL groups (p<0.05). The number of inflammatory cell infiltrate (ICI) and osteoclasts in the LO group was significantly higher than that of the NL and S groups (p<0.05). The number of osteoblasts in the LO and S groups was significantly higher than that of the NL group (p<0.05). There were significantly more RANKL-positive cells in the LO group than in the S and NL groups (p<0.05). OPG-positive cells were higher in S group than in LO and NL groups (p<0.05). TOS and OSI levels were significantly reduced in S group compared to LO group (P<0.05) and TAS levels were similar in S and NL group (p>0.05). Conclusions The present study showed that systemic administration of sumac extract may reduce alveolar bone loss by affecting RANKL/OPG balance, TOS and OSI levels in periodontal disease in rats.

The Effectiveness of Crataegus orientalis M Bieber. (Hawthorn) Extract Administration in Preventing Alveolar Bone Loss in Rats with Experimental Periodontitis

The purpose of this animal study was to evaluate the effects of hawthorn (Crataeus orientalis M Bieber.) extract on serum oxidative status and alveolar bone loss in experimental periodontitis. Twenty-seven Wistar rats were assigned to one of the following groups: non- ligated+placebo (saline) (NL, n = 9), ligature only+placebo (saline) (LO, n = 9), and ligature and treated with hawthorn extract in saline (H, n = 9) (100 mg/kg orogastrically, once a day for 11 days). Periodontitis was induced by submerging a 4/0 silk ligature in the sulcus of the mandibular right first molars of rats, and the animals were sacrificed after 11 days. Micro-CT examinations were performed for linear and volumetric parameter assessment of alveolar bone. Periodontal tissues were histopathologically examined to assess the differences among the study groups. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Alveolar bone loss was significantly reduced by hawthorn administration compared to LO group (p<0.05). The number of inflammatory cells and osteoclasts in the LO group was significantly higher than that of the NL and H groups (p< 0.05). The number of osteoblasts in the LO and H groups was significantly higher than that of the NL group (p<0.05). TOS and OSI levels were significantly reduced in H group compared to LO group (P <0.05) and TAS levels were similar in H and NL group (p< 0.05). Hawthorn extract showed inhibitory effect on periodontal inflammation and alveolar bone loss by regulating TAS, TOS and OSI levels in periodontal disease in rats when administered systemically.

Assessment of Antimicrobial and Antioxidant Activities of Nepeta trachonitica: Analysis of Its Phenolic Compounds Using HPLC-MS/MS

Continuing our work on the sources of natural bioactive compounds, we evaluated the antimicrobial and antioxidant activities of Nepeta trachonitica as well as its major phenolic content using the high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) technique. For antioxidant activity, ferric reducing antioxidant power (FRAP) and cupric ion reducing antioxidant capacity (CUPRAC) methods were performed to measure the reducing power and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was employed to evaluate the radical scavenging activity of the sample. For antimicrobial activity, three Gram-positive and four Gram-negative microbial species as well as three fungi species were tested. N. trachonitica appeared to have reasonable antioxidant activity and decent antimicrobial activity as indicated by the inhibition of the organisms’ growth. The most susceptible species were Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 11229 among the organisms tested. Ethanol extract of the plant has the highest effect on Saccharomyces cerevisiae but no effect on Yarrowia lipolytica. The HPLC-MS/MS analysis showed that at least 11 major phenolic compounds of N. trachonitica exist, the major ones being rosmarinic acid, chlorogenic acid and quinic acid. The obtained results suggest that N. trachonitica could be a promising source for food and nutraceutical industries because of its antimicrobial and antioxidant properties and phenolic compounds.

Purification, characterization and inhibition sensitivity of peroxidase from wheat (Triticum aestivum ssp. vulgare)

ABSTRACT The purification and characterization of peroxidase is currently growing interests since peroxidases have implications in various industrial and biochemical applications. In this study, wheat peroxidase was purified using (NH4)2SO4 precipitation, CM-Sephadex cation exchange, and gel filtration chromatographies. Enzyme purity and molecular mass were checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Enzyme kinetics was studied using two substrates: guaiacol and hydrogen peroxide (H2O2). Km and Vmax values were calculated from Lineweaver-Burk graph for each substrate. Wheat peroxidase activity has been enhanced 284-fold. Wheat peroxidase had molecular mass of 38.8 kDa. Km values for guaiacol and H2O2 were found as 2.467 mM and 7.307 mM, respectively. The pH and temperature optima were 5.5 and 40.0°C, respectively. Also, the enzyme was highly inhibited by citric acid and Cetyltrimethylammonium bromide.

Purification, characterization and selected inhibition properties of peroxidase from haricot bean (Phaseolus vulgaris L.)

ABSTRACT In this study, a cationic soluble peroxidase (POD) was homogeneously purified and biochemically characterized. The enzyme purification involved the combination of (NH4)2SO4 precipitation, CM-Sephadex cation exchange, and gel filtration chromatography. The purity of the enzyme was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and one single prominent band was obtained with a molecular weight of approximately 45 kDa. The thermal and pH stability showed that the enzyme was stable at pH 5.0 and at 40°C, respectively. The activity remained stable at pH 4.0 at least for 10 days. Km and Vmax values were calculated from Lineweaver–Burk graph for each substrate. POD exhibited Km values of 0.0154 and 0.065 mM for guaiacol and H2O2, respectively. The enzyme was highly inhibited by sodium azide. This study could enrich the literature regarding the properties of POD from haricot bean that could be applicable to biomedical analysis.

In vitro evaluation of antioxidant and anti-proliferative activities of Gypsophila sphaerocephala (Caryophyllaceae) extracts together with their phenolic profiles

This study was designed to investigate the antioxidant and antiproliferative activities of water and methanol extracts of endemic plant Gypsophila sphaerocephala subsp cappadocica in conjuction with phenolic profiles. Individual phenolics of the extracts were identified and quantified by RP-HPLC analysis. Antioxidant potentials of the extracts were evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging capacity tests, cupric ion reducing antioxidant capacity (CUPRAC) method and Fe2+ chelating assay. Antiproliferative activities of the extracts were tested against MCF-7 (breast adenocarcinoma), HT-29 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma) cell lines. RP-HPLC analysis showed that methanol extract was richer than water extract in terms of phenolic content. In parallel to the phenolic contents, methanol extract showed higher antioxidant activity than water extract by DPPH, CUPRAC and Fe2+ chelating tests while water extract exhibited higher activity by ABTS method. Moreover, methanol extract displayed 1.8-fold, 4.3-fold and 2.6-fold more antiproliferative activity than water extract against MCF-7 cells, HepG2 cells and HT-29 cells, respectively. However, both extracts were found to show moderate antioxidant and antiproliferative activity compared to positive controls. These results suggest that Gypsophila sphaerocephala may be used as a promising source for food and nutraceutical industries due to its considerable antioxidant and antiproliferative potentials together with its rich phenolic content.