Hypoglycemic Effects of Extracts Obtained from Endemic Betonica bulgarica Degen and Neic.

The increasing prevalence of diabetes mellitus, together with the limited access of many patients to conventional antidiabetic drugs and the side effects resulting from their use, are the reason for the ever-increasing need for new agents. One of the most important strategies used in the therapy of this disease is to reduce the postprandial blood glucose level by inhibiting the carbohydrate-degrading enzymes α-amylase and α-glucosidase. The purpose of the present study was to provide in vitro evidence for the potential hypoglycemic effect of leaf and inflorescence aqueous extracts of Bulgarian endemic species Betonica bulgarica Degen and Neic. Total phenolic and flavonoid contents and antioxidant activities were determined by spectrophotometric methods. Qualitative and quantitative determinations of principal phenolic acids and flavonoids were performed using HPLC with a dual absorbance detector. The plant extracts were able to retard the enzymatic breakdown of starch to glucose with 50% inhibiting concentrations of 1.86 mg/mL and 1.54 mg/mL respectively for leaf and flower extract. Some of the plant constituents are proven inhibitors of α-amylase and/or α-glucosidase, but their adsorption on starch seems to be one additional mechanism for the inhibition of glucose release. Combination index analysis carried out with binary mixtures of acarbose and plant extracts showed a tendency toward synergism with an increase in concentrations and level of inhibition.

Chromatographic Profile and Redox-Modulating Capacity of Methanol Extract from Seeds of Ginkgo biloba L. Originating from Plovdiv Region in Bulgaria.

Oxidative stress underlies the pathogenesis of many diseases, which determines the interest in natural substances with antioxidant properties. Ginkgo biloba L. leaves are well known and widely used in the pharmaceutical industry, but the therapeutic properties of the seeds are less studied. This study aimed to identify the chromatographic profile and to evaluate the antioxidant properties of methanol extract from seeds of G. biloba (GBSE). In the GBSE, flavonoids and terpenes were found as terpenes predominated. The GBSE antioxidant capacity determined by 2,2 azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and 1-diphenyl-2-picrylhydrazyl (DPPH) methods were equal to 1.34% and 0.58% of the activity of reference substance Trolox, respectively. The results of the ferric reducing antioxidant power method showed that the effect of concentration 1 mg/mL (w/v) GBSE was equal to 7.418 mM FeSO4 used as a standard. The cupric reducing antioxidant capacity activity of the GBSE was found to be 215.39 µmol Trolox/g GBSE and is presented as Trolox equivalent. The metal chelation effect of 1 mg/mL (w/v) GBSE was equal to that obtained for 0.018 mM EDTA. In conclusion, GBSE showed a good ability to neutralize ABTS and DPPH radicals and could have a beneficial effect in pathological conditions with oxidative stress etiology.

Phytochemical Constituents and Pharmacological Potential of Tamus communis Rhizomes.

Tamus communis L. is a plant distributed in a number of geographical areas whose rhizome has been used for centuries as an anti-inflammatory and analgesic remedy. This review aims to summarize the current knowledge of the chemical composition and biological activity of the extracts or individual compounds of the rhizome. The data for the principal secondary metabolites are systematized: sterols, steroidal saponins, phenanthrenes, dihydrophenanthrenes, etc. Results of biological tests for anti-inflammatory action, cytotoxicity, anticholinesterase effect, and xanthine oxidase inhibition are presented. Some open questions about the therapeutic properties of the plant are also addressed.

Selective Anticancer Properties, Proapoptotic and Antibacterial Potential of Three Asplenium Species.

The ferns Asplenium ceterach L., Asplenium scolopendrium L. and Asplenium trichomanes L. have wide application in traditional medicine worldwide. However, the scientific research on their anticancer and antibacterial properties is insufficient. The present article aims to provide more information on this topic. Extracts derived from the aerial parts of A. ceterach, A. scolopendrium and A. trichomanes were examined using a panel of in vitro assays with different bacterial and mammalian cells. The cytotoxicity and anticancer activity of the samples were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays with three human (A549, FL, HeLa) and three murine (3T3, TIB-71, LS48) cell lines. Inhibitory effects on the growth of Gram-positive (Bacillus cereus) and Gram-negative (Pseudomonas aeruginosa) bacteria were determined by the agar diffusion assay. Apoptosis-inducing properties of the extracts were analyzed by flow cytometry. Superoxide dismutase (SOD) activity in extract-treated cells was investigated by ELISA. The obtained results demonstrate selective anticancer activity of all three Asplenium species. The extract from A. ceterach displayed the strongest inhibitory properties against human cervical cancer cells and bacterial cells. It induced a lower level of cytotoxicity against mouse cell lines, indicating a species-specific effect. The extract from A. trichomanes demonstrated better anticancer and antibacterial properties than the sample from A. scolopendrium. Further experiments linked the mechanism of action of A. ceterach extract with oxidative stress-inducing potential and strong proapoptotic potential against the cervical cancer cell line HeLa. A. trichomanes and A. scolopendrium extracts appeared to be potent inducers of necrotic cell death.

An experimental study on phytochemical composition and memory enhancing effect of Ginkgo biloba seed extract.

INTRODUCTION: The Ginkgo biloba L. tree is considered as one of the oldest species on Earth. It is known as a "living fossil" dating back approximately 200 million years. Both the leaves and seeds of this tree have been used for millennia in traditional Chinese medicine. AIM: To study the phytochemical profile of Gingko biloba seed extract (GBSE) and its memory enhancing effects. MATERIALS AND METHODS: Liquid chromatography with mass detection (LC-MS) was performed for phytochemical analyses of the extracts. For the in vivo experiments, male Wistar rats were divided randomly into 5 groups (n=8): saline; piracetam;  GBSE 50; 100, and 200 mg/kg b.w. Y-maze, T-maze, step-down passive avoidance and novel object recognition test (NORT) were performed. The observed parameters were: percentage of spontaneous alternations (% SA), working memory index, latency of reaction and recognition index, respectively. Statistical analysis was done using SPSS 19. RESULTS: LC-MS analysis showed the presence of the flavonoids quercetin, kaempferol and isorhamnetin (as aglycones), the ginkgolides A, B, C, J, and bilobalide. In Y-maze task, the groups treated with 50 and 100 mg/kg of GBSE significantly increased the % SA during the memory test compared to saline (p<0.05). In T-maze test, the three experimental groups with GBSE significantly increased the working memory index in comparison with that of the control group (p<0.05). In step-down test, the animals receiving 100 mg/kg b.w. GBSE, notably increased the latency during both retention tests (p<0.05 and p<0.01, respectively). In NORT, only the animals with the middle dose of GBSE ameliorated the recognition index when compared to saline (p<0.05). CONCLUSIONS: GBSE enhances spatial working memory, recognition memory, and short- and long-term recall in naïve rats due to the synergic effects of detected flavonoids and terpene lactones on brain functions. The brain structures involved are probably the hippocampus and prefrontal cortex.

Receptor-mediated biological effects of extracts obtained from three Asplenium species.

This study reports the effects of aqueous extracts obtained from three fern species of Bulgarian origin: Asplenium ceterach L., Asplenium scolopendrium L., and Asplenium trichomanes L. on the contractility and bioelectrogenesis of rat gastric smooth muscle tissues. In the concentration range 0.015-0.150 mg/mL the three extracts contracted smooth muscle tissues in a concentration-dependent manner. The contractions caused by A. ceterach L. and A. scolopendrium L. extracts (0.150 mg/mL) were reduced by ketanserin (5 × 10-7 and 5 × 10-6 mol/L), an antagonist of serotonin 5-HT2 receptor. The contraction evoked by A. trichomanes L. (0.150 mg/mL) was significantly reduced by 1 × 10-6 mol/L atropine, an antagonist of muscarinic receptors, and turned into relaxation against the background of 3 × 10-7 mol/L galantamine. After combined pretreatment with galantamine and l-arginine (5 × 10-4 mol/L), this relaxation become more pronounced. The study demonstrates that constituents of A. ceterach L. and A. scolopendrium L. extracts act as agonists of 5-HT2 receptors and cause contraction by activating serotonergic signaling system. A. trichomanes L.-induced reaction is an additive result of two opposite-in-character effects. The dominant contraction is initiated by inhibition of acetylcholinesterase activity. The relaxation develops with pre-inhibited acetylcholinesterase, it is significantly potentiated by l-arginine, and therefore associated with nitrergic signaling pathway.

Cytotoxic effect of Ginkgo biloba kernel extract on HCT116 and A2058 cancer cell lines.

While the pharmacology of Ginkgo biloba leaf extract has been studied extensively, little is known about the pharmacological potential of Ginkgo biloba seeds, although they contain similar active ingredients that are responsible for the therapeutic effects of the leaf extract. In this study we used 70%-methanol Ginkgo biloba kernel extract, quantified its bioactive constituents and tested their cytotoxic effect on two cancer cell lines, A2058 and HCT116, and the non-tumor cell line McCoy-Plovdiv. We studied the biological effect of the extract by real-time analysis in the xCELLigence system, WST-1 assay and LIVE/DEAD viability assay. We show that the extract significantly perturbed the viability of cancer cells in a concentration- and time-dependent manner. In contrast, non-cancerous McCoy-Plovdiv cells sustained their proliferation potential even at high concentrations of the extract. Therefore, we propose that the active constituents of the Ginkgo biloba endosperm extract may interact additively or synergistically to protect against cancer.

Naturally present metal ions in plants could interfere with common antioxidant assays.

Most of the commonly applied assays used to assess antioxidant properties of plant extracts exploit the ability of some biologically active metabolites to participate in oxidation-reduction reactions with metal ions. On the other hand, most plants contain different chelated metal ions whose levels depend on the geographic origin, soil, and environmental pollutions. In this study the levels of redox-active metal ions in three plant sources were measured and extracts of these botanicals were treated with ChelexⓇ - an ion exchanger that is noteworthy for its ability to bind transition metal ions. The original and chelated extracts were subjected to three antioxidant assays based on single electron transfer. The results obtained showed statistically significant differences between the original and Chelex-treated extracts suggesting that the naturally present metal ions could interfere with the results of the three most commonly applied antioxidant methods.•The proposed pre-analytical procedure is simple and does not require special instrumental equipment.•Preliminary depletion of redox active metal ions, namely iron and copper ions could improve reproducibility of the analytical methods.•The method allows a more reliable comparison of antioxidant properties of particular botanical species from different geographic regions.

Biochemical Changes in Experimental Rat Model of Abdominal Compartment Syndrome.

BACKGROUND: Increased intra-abdominal pressure (IAP) causes tissue ischemia, subsequent hypoxia, and impairment of normal tissue metabolism. Elevation of IAP above 20 mmHg leads to progression of abdominal compartment syndrome (ACS) that is associated with organ dysfunction or failure not previously manifested. AIM: To evaluate the eff ects of diff erent grades and time of exposure to IAP on biochemical parameters and oxidative stress in organs aff ected by ischemia using previously developed rat model. RESULTS: Three experimental groups exposed to diff erent IAP and time frames were tested for liver, kidney, and pancreas injury by measuring the activities of tissue specifi c enzymes in blood serum. Elevated activities of aspartate aminotransferase, pancreatic amylase, lipase, and higher concentrations of D-lactate, urea, and creatinine were found in some of the experimental groups compared to a control group of animals not subjected to increased IAP. Increased levels of biomarkers of oxidative stress as well as decrease in concentration of the major cellular antioxidant glutathione indicated the presence of oxidative injury as a result of elevated IAP. CONCLUSIONS: The developed rat model is appropriate to study the mechanism and manifestation of tissue injury during diff erent grades of elevated IAP but also to test approaches aimed to attenuate the detrimental eff ects of ACS. This study also underlines the necessity of using not a single but a set of biochemical parameters in order to assess the severity of tissue injury during elevated IAP and progression to ACS.

HPLC Quantification of astaxanthin and canthaxanthin in Salmonidae eggs.

Astaxanthin and canthaxanthin are naturally occurring antioxidants referred to as xanthophylls. They are used as food additives in fish farms to improve the organoleptic qualities of salmonid products and to prevent reproductive diseases. This study reports the development and single-laboratory validation of a rapid method for quantification of astaxanthin and canthaxanthin in eggs of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis М.). An advantage of the proposed method is the perfect combination of selective extraction of the xanthophylls and analysis of the extract by high-performance liquid chromatography and photodiode array detection. The method validation was carried out in terms of linearity, accuracy, precision, recovery and limits of detection and quantification. The method was applied for simultaneous quantification of the two xanthophylls in eggs of rainbow trout and brook trout after their selective extraction. The results show that astaxanthin accumulations in salmonid fish eggs are larger than those of canthaxanthin. As the levels of these two xanthophylls affect fish fertility, this method can be used to improve the nutritional quality and to minimize the occurrence of the M74 syndrome in fish populations.

Effect of Thiocyanate on Iodine Status of Pregnant Women.

The aim of this study was to assess the thyroid status of pregnant women on the basis of biochemical indicators and to evaluate the potential risk of developing iodine deficiency as a result of tobacco smoke exposure by assessing the association between urinary thiocyanate levels and the manifestation of iodine deficiency. The study included 219 pregnant women from the town of Plovdiv and Plovdiv District in Southern Bulgaria. The levels of urinary iodine, thyroid-stimulating hormone (TSH), free thyroxine (FT4), and urinary thiocyanate as indicators of tobacco smoke exposure were measured. Most of the pregnant women (60.1 %) were found to have iodine deficiency, 10.6 % of them had TSH values greater than 4 mIU/L, and 16.4 % had FT4 below 9 pmol/L. There were negative correlations between urinary iodine levels and thiocyanate/creatinine ratio (R = -0.148, р = 0.034) and between thiocyanate/creatinine ratio and FT4 (R = -0.379, p < 0.0001); thiocyanate/creatinine ratio and serum TSH were positively correlated (R = 0.169, p = 0.019). Logistic regression analysis showed that pregnant women in whom the thiocyanate/creatinine ratio was greater than the median value of 3.57 mg/g had a 3.882-fold higher risk of developing iodine deficiency (urinary iodine <150 µg/L) than the pregnant women with lower thiocyanate levels (OR = 3.882, 95 % CI 1.402-10.751, p = 0.009). Higher levels of urinary thiocyanate were found in women exposed to tobacco smoke, and quantification of these ions in urine provided a fast non-invasive method to monitor thiocyanate load. Due to the competitive inhibition of iodine intake by thiocyanates, their levels should be carefully monitored, especially in cases of severe iodine deficiency.

Application of solid-phase extraction for the concentration of chromophores, fluorophores, and photosensitizers from lens protein digests.

Solid-phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid-phase extraction was capable of concentrating the advanced glycation end-products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.

New biological properties of coffee melanoidins.

Water-soluble melanoidins isolated from roasted coffee induced ex vivo changes in the bioelectric and contractile activity of rat circular gastric smooth muscle tissues. They provoked a depolarization of smooth muscle cellular membranes and an increase in Ca²âº-influx as evidenced by the increase in the frequency and amplitude of Ca²âº-generated spike potentials. In the presence of 1 × 10⁻6 mol L⁻¹ acetylcholine and 1 × 10⁻6 mol L⁻¹ arecoline the melanoidin-evoked contraction was significantly reduced. M-cholinergic receptor blocking agents atropine, ipratropium, pirenzepine, and 4-DAMP also significantly reduced the melanoidin-provoked contraction. Nonspecific N-cholinergic receptor blockers hexamethonium and decamethonium (1 × 10⁻5 mol L⁻¹ each) did not influence the melanoidin-induced mechanical reaction. The melanoidins did not affect the strength of contractions evoked by adrenaline and dopamine (1 × 10⁻6 mol L⁻¹ each). The results obtained support the assumption that melanoidin-evoked contraction is a result of activation of muscarinic-type cholinergic receptors.

Antibacterial electrospun poly(ɛ-caprolactone)/ascorbyl palmitate nanofibrous materials.

The one-step incorporation of ascorbyl palmitate (AP), a widely used derivative of vitamin C, into nanofibrous mats of poly(ɛ-caprolactone) (PCL) by electrospinning was demonstrated. The incorporation of AP was attested by IR spectroscopy; the AP content was determined by thermogravimetric analysis (TGA); and the surface composition of the mats: by X-ray photoelectron spectroscopy (XPS). The possibility for deposition of silver nanoparticles onto PCL/AP mats using the ability of AP to reduce silver ions was demonstrated. The silver content was determined by TGA, and the silver nanoparticles were observed by transmission electron microscopy (TEM). The nanoparticles were composed of elemental silver, as verified by XPS analyses. The UV-vis spectrophotometric analyses, study on quenching of the free 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and microbiological tests against the pathogenic microorganism Staphylococcus aureus showed that AP preserved its stability and its antioxidant and antibacterial activity when incorporated in the nanofibrous mats.

Sensitizing effect of tacrine on M-cholinergic receptors in gastric smooth muscle of rats.

AIM: To find if tacrine exerts a sensitizing effect on the cholinergic receptors of gastric smooth muscles, and study some of the mechanisms inducing it and measure the relative intensity of tacrine's effects on contractile activity. MATERIAL AND METHODS: Isometric recording of the mechanical activity of gastric smooth muscle preparations; determination of acetyl-cholinesterase activity in smooth-muscle tissue homogenates using Ellman's method. RESULTS: We found that the threshold concentration for tacrine not reducing the acetylcholinesterase activity and not having an effect on the smooth muscle preparations was 1 x 10(-8) mol/l. This concentration, however, significantly increased the acetylcholine-induced contraction compared with the controls, after the smooth-muscle tissue was incubated for 60 or 100 min. Treating smooth-muscle preparations with tacrine in a concentration of 5 x 10(-6) mol/l triggered a contraction induced by the drug's anti-cholinesterase activity. A secondary contraction was induced after 38.6 +/- 5.6 min. There was no secondary contraction after the control acetylcholine-induced effect. Atropine (1 x 10(-6) mol/l) inhibits this effect. Preliminary treatment of smooth muscle preparations with hexamethonium (1 x 10(-6) mol/l) did not change significantly the intensity of the first phase of tacrine-induced contraction and shifted in time the appearance of the second contractile phase. CONCLUSION: Tacrine has a sensitizing effect on M-cholinergic receptors; it occurs after a long incubation of the gastric smooth muscles with the drug and is manifested as a secondary contraction which is shifted in time and is significantly inhibited by atropine.

Testing biological activity of model Maillard reaction products: studies on gastric smooth muscle tissues.

Water-soluble Maillard reaction products obtained from five different model systems were investigated for their effects upon the mechanical activity of rat gastric smooth muscle. Most of the total Maillard reaction products applied at concentration of 1.5 mg/ml evoked contractions; among them the product obtained from arginine and glucose (Arg-Glc) produced the most powerful contractions. The product obtained from glycine and ascorbic acid (Gly-AsA) was the only one that brought about relaxation response. The high molecular weight fractions (>3,500 Da) isolated from the reaction systems Arg-Glc and Gly-AsA demonstrated effects similar in type and amplitude to those evoked by non-fractioned reaction products. The results obtained suggest that moieties of molecules acting upon the muscle tonus originate mainly from lysine and arginine residues; that these structures are available in both low and high molecular pools in similar concentrations, and most likely these fragments act upon membrane-located cellular structures involved in calcium transport.

High-nitrate levels in drinking water may be a risk factor for thyroid dysfunction in children and pregnant women living in rural Bulgarian areas.

Nitrate inhibits the accumulation of iodide in thyroid gland. The aim of present study was to evaluate the influence of this ion on the iodine status of two risk population groups. Subjects of study were pregnant women and children aged between 3 and 6 years from two villages in Bulgaria with high- and low-nitrate levels in drinking water. The relative risk of thyroid disorders for the pregnant women living in the village with high-nitrate levels in drinking water expressed as an odds ratio was 5.294 (95% confidence intervals 1.003-27.939; P=0.0454) and was considered as significant. Statistically significant differences were found between the goiter rate in exposed and non-exposed pregnant women. The relative risk of thyroid dysfunction for the children exposed to a high-nitrate level, expressed as an odds ratio was 2.333 (95% confidence intervals 0.8491-6.412; P=0.1396) and was considered as not significant; the goiter prevalence in the exposed children was also not statistically different. The results of the study confirmed the role of high-nitrate level in drinking water as a risk factor for thyroid dysfunction in vulnerable population groups.

Iodine status and goitre prevalence in nitrate-exposed schoolchildren living in rural Bulgaria.

Nitrate and perchlorate are well-known water pollutants that are harmful to human health. They may inhibit the accumulation of iodide in the thyroid gland. The present study investigated iodine status and goitre prevalence in schoolchildren aged 7-14 years from two villages in Bulgaria with high and low levels of nitrate, and negligible levels of perchlorate in drinking water. Comparison between the mean and median urinary iodine concentrations of the exposed and non-exposed children showed a statistically significant difference for the boys. A statistically significant difference was also found for goitre prevalence between the exposed and non-exposed children. The relative risk for schoolchildren exposed to a high level of nitrate in drinking water, expressed as an odds ratio, was 3.014 (95% confidence intervals 1.293-7.027; P=0.0105) and this was considered to be significant. This study confirmed that a high level of nitrate in drinking water is a risk factor for thyroid dysfunction in this population.

Evaluation of endemic goiter prevalence in Bulgarian schoolchildren: results from national strategies for prevention and control of iodine-deficiency disorders.

Iodine deficiency is a major health problem worldwide. The environment of the Balkan countries, including Bulgaria, is distinguished for its low iodine content. In 1994, the strategies for the prevention and control of iodinedeficiency disorders were actualized in Bulgaria and universal salt iodization and supplementation for the risk population groups (schoolchildren, pregnant women) were introduced. The aim of this study was to assess the effectiveness of the iodine prophylaxis in schoolchildren, living in an endemic for goiter area after the introduction of salt iodization in Bulgaria. For this purpose, the goiter prevalence and iodine status in 483 schoolchildren (274 boys and 209 girls) aged between 8 and 15 yr, living in an endemic for goiter area in Bulgaria were evaluated. Despite the normalization of iodine supply, mild iodine deficiency on the basis of goiter prevalence (16.15%) and urinary iodine excretion was found. These data indicate the need for reevaluation of the national strategy for prevention of iodine deficiency.

Influence of model melanoidins on calcium-dependent transport mechanisms in smooth muscle tissue.

Melanoidins obtained from L-arginine and D-glucose (MW > 3500 Da) were tested for their ability to influence the contractility of gastric smooth muscles. A study within the range 0.1-10 mg/mL revealed that at low concentrations, the melanoidins provoked concentration-dependent contraction, whereas a muscle relaxation was registered at high concentrations. The contraction was preceded by changes in the calcium membrane current as measured by single sucrose-gap method and significantly attenuated by the calcium channel blockers D-600 and nifedipine. Measurements with Ca(2+)-selective electrode showed that the melanoidins decreased the concentration of ionized Ca(2+ )in tissue bath in concentration-dependent manner. Experiments carried out in solutions with lower than normal Ca(2+) concentration and using melanoidins preliminary saturated with Ca(2+ )confirmed that the calcium chelation by melanoidins was a key contributing cause for the development of relaxant response. The results obtained showed that the melanoidins could influence the contractility of smooth muscles through at least two pathways: at low concentrations they caused depolarization and activation of L-type calcium channels, stimulated the Ca(2+ )influx, and provoked contraction, whereas at high concentrations calcium binding by melanoidins led to significant depletion of extracellular calcium ions and contributed to the relaxation process observed.