Optimization of the extraction process from Thymus serpyllum L. herb, biological activities and encapsulation of extracts

Врста Thymus serpyllum L. (Lamiaceae) или мајчина душица представља вишегодишњу зељасту биљку богату полифенолима који показују антиоксидативни, антимикробни, антихипертензивни, спазмолитични, антиинфламаторни, антиканцерогени, антиалергијски и анксиолитични ефекат. Оптимизација процеса екстракције представља први корак у добијању максималног приноса активних принципа из биљних извора као безбедне и природне алтернативе синтетским компонентама. Нестабилност полифенола у току процеса производње, дистрибуције и чувања намирница, као и у условима гастроинтестиналног тракта, ограничава њихову активност и потенцијалне корисне ефекте на здравље људи. Инкапсулација полифенола доводи до превазилажења поменутих недостатака, ублажавања горког укуса полифенола и побољшања њихове биорасположивости и полувремена елиминације...Thymus serpyllum L. (Lamiaceae), well-known as wild thyme, is a perennial subshrub, abundant in polyphenols compounds, which possess antioxidant, antimicrobial, antihypertensive, antispasmodic, antiinflammatory, anticarcinogenic, anti-allergic and anxiolytic properties. The optimization of extraction represents the first step in obtaining the maximum yield of active compounds from plant sources, which are safe and natural alternative to synthetic components. Polyphenols instability during food processing, distribution or storage, and in the gastrointestinal tract, limits their activity and potential health benefits. The encapsulation of polyphenols can overcome the drawbacks of their instability, alleviate unpleasant tastes or flavors, and improve the bioavailability and half-life of the compounds..

Estradiol signalling pathways involved in the regulation of inducible nitric oxide synthase and sodium-potassium-ATPase in the heart of obese female rats

Gojaznost je oboljenje povezano sa nizom patoloških stanja kao što su: rezistencija na insulin (IR), kardiovaskularne bolesti (KVB) i Diabetes Mellitus tipa 2 (DMT2). Povećana ekspresija i aktivnost inducibilne azot-monoksid-sintaze (iNOS; engl. Inducible Nitric Oxide Synthase) u srcu u stanju gojaznosti, moţe dovesti do apoptoze kardiomiocita i hipertrofije srca, dok sa druge strane gojaznost zdruţena sa IR doprinosi smanjenoj aktivnosti Na+/K+-ATPaze, što dovodi do smanjenja kontraktilnosti vaskulature i razvoja sistemske hipertenzije. Endogeni estradiol svojim delovanjem sprečava nastanak IR i hiperglikemije i ostvaruje pozitivne efekte na kardiovaskularni sistem (KVS), ali sinteza i kardioprotektivni uticaj estradiola mogu biti smanjeni usled razvoja gojaznosti. Estradiol ostvaruje kardioprotektivne tako što utiče na smanjenje ekspresije i aktivnosti iNOS, kao i povećanje ekspresije i aktivnosti Na+/K+-ATPaze, posredstvom različitih signalnih molekula i kinaza, kao što su: supstrat receptora za insulin 1 (IRS-1; engl. Insulin Receptor Substrate)/ fosfatidil-inozitol-3-kinaza (PI3K; engl. Phosphatidylinositide 3-Kinase)/ protein kinaza B (Akt; engl. Protein Kinase B), ekstracelularnim signalima regulisane kinaze 1 i 2 (ERK1/2; engl. Extracellular Signal-Regulated Protein Kinases 1 i 2) kao i RhoA (engl. Ras homolog gene family, member A)/ROCK (engl. Rho-associated protein kinase). Pored direktnog efekta na srce, estradiol posredno reguliše i njegovu funkciju tako sto utiče na metabolizam i transport glukoze i SMK, preko transportera glukoze (GLUT; engl. Glucose Transporters) i translokaze masnih kiselina (FAT; CD36 ; engl. Fatty Acid Translocase). Za izradu ove doktorske disertacije je korišćeno 16 adultnih ţenki pacova soja Wistar, podeljenih u dve eksperimentalne grupe. Prva grupa pacova je tokom 10 nedelja hranjena standardnom laboratorijskom hranom za pacove, dok je druga grupa pacova tokom 10 nedelja hranjena standardnom laboratorijskom hranom obogaćenom sa 42% masti (HF reţim ishrane). Nakon 10 nedelja pacovi su ţrtvovani, sakupljena je krv i izolovan je serum, a srca su ekstrahovana i delovi tkiva su korišćeni za izolovanje proteina i RNK. U serumu pacova je odreĎivana koncentracija estradiola, dok su u lizatu srca pacova odreĎivane koncentracije L-Arginina (L-Arg), NO i slobodnih masnih kiselina (SMK). Metodom qRT-PCR odreĎivan je nivo iRNK iNOS u srcu pacova...Obesity is associated with several pathological conditions such as: insulin resistance (IR), cardiovascular disease (CVD) and Diabetes Mellitus type 2 (DMT2). Obesity related inducible nitric oxide synthase (iNOS) overexpression could lead to cardiac hypertrophy, while on the other hand obesity induced IR contributes to the reduced Na+/K+-ATPase activity, leading to vascular contractility impairment and development of systemic hypertension. Endogenous estradiol prevents IR and hyperglycaemia and has protective effects on the cardiovascular system (CVS), but the synthesis and cardioprotective effect of estradiol can be reduced due to the development of obesity. Estradiol exerts cardioprotective effects by reducing the expression and activity of iNOS, as well as by increasing the expression of Na+/K+-ATPase activity by regulating the activity of signaling molecules and kinases, such as: insulin receptor substrate 1 (IRS-1)/phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt, PKB), extracellular signal regulated kinase 1 and 2 (ERK1/2) as well as RhoA (Ras homolog gene family, member A)/ROCK (Rho-associated protein kinase). Additionally, estradiol indirectly regulates CVS function by influencing the metabolism and transport of glucose and free fatty acids (FFA), through glucose transporter (GLUT) and FAT; CD36 (Fatty Acid Translocase). In this doctoral dissertation 16 adult female Wistar rats were used and divided into two experimental groups. The first group of rats was fed a standard chow for laboratory rats for 10 weeks, while the second group of rats was fed a standard chow for laboratory rats enriched with 42% fat (HF diet) for 10 weeks. After 10 weeks, the rats were sacrificed, blood was collected and the serum isolated, the hearts were collected, and its tissue fragments were used to isolate proteins and RNA. The concentration of estradiol was measured in serum, while concentrations of L-Arginine (L-Arg), NO and free fatty acids (FFA) were measured in the heart lysates. qRT-PCR method was used to measure the level of iNOS mRNA in rats’ hearts. Na+/K+-ATPase was measured in plasma membranes of rats’ hearts. Western blot was used to measure the level of the following proteins: iNOS, NFkB-p50, ERα, p85 and p110 subunits of PI3K, NDRG2, RhoA, ROCK1 and ROCK2 in heart lysates. The level of phosphorylation and expression of Akt and ERK1/2 kinases, as well as the IRS-1/PI3K associations, were determined in heart lysates..

COMPARATIVE ANALYSIS OF ML AND MAP DETECTORS FOR PAM CONSTELLATIONS IN AWGN CHANNEL

In this paper we perform a comparative performances analysis of “maximum a posteriori” (MAP) and “maximum likelihood” (ML) detectors for one-dimensional constellation in the adaptive white Gaussian noise (AWGN) channel. More precisely, error probabilities per symbol for the aforementioned detectors are compared for the case when the pulse amplitude modulation (PAM) constellation with the equidistant and non-equiprobable constellation points is used as one-dimensional constellation. We perform analysis for different distributions of the constellation point probabilities and different values of the signal-to-noise ratio (SNR). The analysis indicates which detector can be adequate choice for the certain distribution of constellation point probabilities and the SNR. Besides this, for the straightforward performance assessment of the MAP detector we derive a formula for the symbol error probability. Our analysis also points out that the nonuniform distribution of the constellation points probabilities does not necessarily improve the symbol error probability. With the aim to decrease the symbol error probability we propose a method for defining constellation point probabilities. The presented results show that PAM constellation designed by utilizing the method we propose significantly outperforms the conventional PAM constellation in terms to the symbol error probability

From Aloe vera Leaf Waste to the Extracts with Biological Potential: Optimization of the Extractions, Physicochemical Characterization, and Biological Activities

In the study, the optimization of the extraction from Aloe vera leaf waste was performed via varying solid-to-solvent ratio, solvent type, extraction time, and technique (maceration, heat-, ultrasound-, and microwave-assisted extractions—HAE, UAE, and MAE, respectively). The optimal extraction conditions for achieving the highest polyphenol content are a 1:30 ratio, 70% ethanol, and 30 min of HAE. Total flavonoid and protein contents were significantly higher in the extract from MAE, while total condensed tannin content was the highest in HAE. LC-MS analysis quantified 13 anthraquinone and chromone compounds. The variations in the FT-IR spectra of the extracts obtained by different extraction procedures are minor. The influence of extraction conditions on the antioxidant ability of the extracts depended on applied antioxidant assays. The extracts possessed medium inhibition properties against Staphylococcus aureus and weak inhibitory activity against Enterococcus feacalis. The extracts had stimulative effect on HaCaT cell viability. Regarding the extraction yield, there was a significant difference between the used extraction techniques (MAE > HAE > maceration and UAE). The presented study is an initial step in the production of polyphenol-rich extracts from A. vera leaf waste aimed to be used for the potential preparation of pharmaceutical and cosmetic formulations for the skin

Pharmacological potential of Thymus serpyllum L. (wild thyme) extracts and essential oil: A review

In traditional medicine Thymus serpyllum L. (wild thyme) herb represents a part of a large number of herbal medicinal formulations such as syrups, tinctures, infusions, teas, and decoctions. In recent years, there is a growing interest in testing the biological properties of wild thyme, because the plant is a high quality raw material, rich in essential oil and pharmacologically active polyphenolic compounds, which can be included in various formulations in the pharmaceutical, cosmetic, food, and chemical industries. Wild thyme extracts and essential oil have shown significant nitric oxide, ABTS and DPPH radicals scavenging potential, ferrous ion chelating property, reducing activity and ability to inhibit peroxidation of deoxyribonucleic acid (DNA), proteins and lipids. Wild thyme extracts exerted antibacterial activity against Bacillus cereus, Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica, Yersinia enterocolitica, as well as Lactic acid bacteria. Wild thyme essential oil has shown antibacterial potential against E. coli, L. monocytogenes, P. aeruginosa, S. enteritidis, S. aureus, Streptococcus salivariu, S. mutans S. sanguinis, S. pyogenes, E. feacalis, B. cereus, B. subtilis, B. pumilis, Lactobacillus acidophilus, Proteus mirabilis, Klebsiella pneumoniae, Salmonella choleraesuis, and Salmonella poona. T. serpyllum extract possesses antifungal activity against Fusarium solani, F. moniliforme, Aspergillus flavus, Microsporum canis, Alternaria species, Candida albicans, and C. glabarata. The essential oil efficiently inhibited the growth of C. albicans, C. glabrata and Aspergillus fungi. Wild thyme extract significantly and concentration-dependent relaxed spontaneous contractions, acetylcholine-, potassium chloride-, barium chloride- and calcium chloride-induced contractions of isolated rat ileum. The extract significantly reduced the expression of the inducible enzyme cyclooxygenase-2 and inhibited acetylcholinesterase, myeloperoxidase and α-glucosidase activity. T. serpyllum extract has shown cytotoxic activity on human breast cancer cell lines, while essential oil has shown the antitumor potential in human cell lung cancer, colon, cervical, hepatocellular, prostate, and breast adeno-carcinoma

Rosehip Extract-Loaded Liposomes for Potential Skin Application: Physicochemical Properties of Non- and UV-Irradiated Liposomes

In the present study, rosehip (Rosa canina L.) extract was successfully encapsulated in phospholipid liposomes using a single-step procedure named the proliposome method. Part of the obtained liposomes was subjected to UV irradiation and non-treated (native) and UV-irradiated liposomes were further characterized in terms of encapsulation efficiency, chemical composition (HPLC analysis), antioxidant capacity, particle size, PDI, zeta potential, conductivity, mobility, and antioxidant capacity. Raman spectroscopy as well as DSC analysis were applied to evaluate the influence of UV irradiation on the physicochemical properties of liposomes. The encapsulation efficiency of extract-loaded liposomes was higher than 90%; the average size was 251.5 nm; the zeta potential was -22.4 mV; and the conductivity was found to be 0.007 mS/cm. UV irradiation did not cause a change in the mentioned parameters. In addition, irradiation did not affect the antioxidant potential of the liposome-extract system. Raman spectroscopy indicated that the extract was completely covered by the lipid membrane during liposome entrapment, and the peroxidation process was minimized by the presence of rosehip extract in liposomes. These results may guide the potential application of rosehip extract-loaded liposomes in the food, pharmaceutical, or cosmetic industries, particularly when liposomal sterilization is needed

‐sitosterol and gentisic acid loaded 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine liposomal particles

The aim of the present study was the examination of the impact of -sitosterol andgentisic acid on the characteristics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) liposomal particles: (a) bilayer permeability (fluorescence spectroscopy),(b) particle size, polydispersity index (PDI) and zeta potential (photon correlationspectroscopy) and (c) thermal properties (differential scanning calorimetry). -sitosterol induced the increase of liposomal bilayer rigidity, due to rearranging ofthe phospholipid chains, while gentisic acid enhanced the membrane fluidity, dueto the reduced orderliness and the increase of phospholipid dynamics. The inclusionof -sitosterol in liposomes caused a significant increase in particle diameterand PDI, while the encapsulation of gentisic acid did not have influence on particlesize distribution. Apart from that, the presence of -sitosterol resulted in thesignificant zeta potential increase, and thus a better stability of liposomal spheres(in the absence and in the presence of gentisic acid). -sitosterol decreased maintransition temperature (Tm) and phase transition enthalpy (H), and caused thedisappearance of the pre-transition peak as well, whereas the presence of gentisicacid produced a slight decrease in Tm and increase of H. Therefore, gentisic acidhad more favourable, stabilizing interactions with phospholipids than -sitosterol.Thus, it can be concluded that -sitosterol is located in the bilayer interior betweenphospholipids acyl chains, and gentisic acid is incorporated near the outer leaflet ofthe phospholipid membrane, next to the polar head groups. -sitosterol and gentisicacid loaded DPPC liposomal particles have a potential to be used in food andpharmaceutical products, due to the important individual and possible synergisticbeneficial health properties of -sitosterol and gentisic acid

Vaccinium myrtillus leaf waste extracts with natural deep eutectic solvent

Vaccinium myrtillus leaf waste extracts were prepared using lactic acid+amonium acetate and maceration, heat- and ultrasound-assisted extractions (HAE and UAE, respectively). The obtained extracts were characterized via analysis of total polyphenol content (TPC), pH, zeta potential, conductivity, density, surface tension, viscosity, and antioxidant potential. The TPC was the highest in the extract prepared in HAE (53.0±0.9 mg GAE/g), whereas the extracts from maceration and UAE possessed significantly lower TPC (50.4±0.7 and 49.5±0.4 mg GAE/g). The ABTS radical scavenging potential was the highest in the extract prepared in HAE (38.4±1.1 µmol TE/g), followed by the extract obtained using UAE (33.7±1.5 µmol TE/g) and maceration (30.2±1.7 µmol TE/g). The DPPH antioxidant capacity followed the same trend: HAE>UAE>maceration. The zeta potential was low in all extracts (2.66±0.48 mV for macerate, 2.57±0.20 mV for HAE, and 3.17±0.13 mV for UAE), while the conductivity was in the range of 1.87±0.13 and 1.84±0.11 mS/cm (maceration and HAE) to 2.10±0.11 mS/cm (UAE). The density varied from 1.105±0.009 g/mL for macerate to 1.122±0.006 and 1.117±0.001 g/mL for HAE and UAE extracts. There were no statistically significant differences in the surface tension and viscosity (~23.5 mN/m and ~5.5 mPa∙s). The highest TPC and antioxidant potential were measured in the extract obtained using HAE, whereas the extract prepared by ultrasound waves possessed the highest zeta potential and conductivity. Therefore, the extraction technique should be chosen depending on the future application of V. myrtillus extract

Physicochemical characterisation of Thymus serpyllum extracts prepared using natural deep eutectic solvents

Thymus serpyllum L. (wild thyme) extracts were prepared using 1 g of plant material and three natural deep eutectic solvents (30 mL, malic acid+maltose, lactic acid+menthol, or citric acid+glycerol with 50% of water) in maceration (90 min). The extracts were characterized in terms of total polyphenol content (TPC), total protein content, extraction yield, zeta potential, conductivity, pH, density, surface tension, and viscosity. TPC was the highest in citric acid+glycerol extract (35.06±1.13 mg gallic acid equivalents (GAE)/g of plant material), whereas malic acid+maltose and lactic acid+menthol extracts possessed similar TPC (29.62±1.11 and 28.94±1.15 mg GAE/g, respectively). Total protein content amounted to 5.55±0.20 mg/g in citric acid+glycerol extract, while significantly lower values were determined in malic acid+maltose and lactic acid+menthol extracts (1.22±0.30 and 1.73±0.18 mg/g, respectively). The extraction yield was the highest for citric acid+glycerol extract, 1.57±0.11%, followed by malic acid+maltose and lactic acid+menthol extracts, 1.28±0.10 and 1.27±0.13%, respectively. Zeta potential (absolute value, as a predictor of potential application in water treatment) was low in all extracts (0.86±0.06 mV for citric acid+glycerol, -0.35±0.09 mV for lactic acid+menthol, and -0.17±0.05 mV for malic acid+maltose extract). The conductivity (as a predictor of antioxidant capacity) was in the range of 2.35±0.21 and 2.71±0.29 mS/cm (citric acid+glycerol and malic acid+maltose extracts) to 5.70±0.23 mS/cm (lactic acid+menthol extract). pH ranged from 1.44 in citric acid+glycerol extract to 1.78 and 1.85 in malic acid+maltose and lactic acid+menthol extracts. Density varied from 1.067±0.003 g/mL for lactic acid+menthol extract to 1.169±0.002 and 1.195±0.005 g/mL for citric acid+glycerol and malic acid+maltose extracts. Surface tension was the highest in citric acid+glycerol extract (38.0±0.4 mN/m), followed by malic acid+maltose and lactic acid+menthol extracts (31.5±0.4 and 25.9±0.1 mN/m, respectively). The viscosity of lactic acid+menthol extract was significantly lower (1.83±0.30 mPa•s) in comparison to malic acid+maltose and citric acid+glycerol extracts (6.64±0.15 and 7.84±0.10 mPa•s, respectively). The highest TPC, total proteins, and extraction yield were measured in citric acid+glycerol extract, while lactic acid+menthol extract possessed the highest conductivity and the lowest density, surface tension, and viscosity. Therefore, the constitution of natural deep eutectic solvent should be optimized depending on the future application of T. serpyllum extract

Comparison of maceration and ultrasound-assisted extraction of antioxidant compounds from Vaccinium myrtillus L.

Vaccinium myrtillus L.(Ericaceae), a perennial, wild, and small deciduous shrub that grows in the mountains and forests of Europe, contains anthocyanins, phenolic acids, flavonoids, fatty acids, stilbenes, iridoid glycosides, dietary fibers, vitamins, and minerals. The leaves' extracts are widely used in traditional medicine due to their astringent, antiseptic, antioxidant, anti-inflammatory, skin-rejuvenating, lipid-lowering, hypolipidemic, and hypoglycemic activities. The novel extraction techniques, including ultrasound-assisted extraction, provide various benefits, such as reducing solvent consumption and extraction time and increasing extraction yield, and quality. Hence, in the present study, V. myrtillus extracts were prepared using dried leaves (0.66 g), 50% ethanol as the extraction solvent (20 mL), and maceration (60 min) or ultrasound-assisted extraction (ultrasound probe, amplitude of 60% for 5 min). The obtained extracts were examined in terms of total polyphenol content (TPC) and antioxidant activity (ABTS, DPPH, FRAP, and CUPRAC assays). The TPC of the extract prepared using maceration was 55.2±0.7 mg gallic acid equivalents (GAE)/g of plant material, while the TPC of the extract prepared using an ultrasound probe was 55.6±1.0 mg GAE/g. The DPPH radical scavenging activities of the extracts correlated with the TPC and amounted to 1.81±0.05 mg/mL for macerate and 1.79±0.02 mg/mL for the extract obtained using an ultrasound probe, whereas ABTS antioxidant capacity did not correlate with the polyphenol concentration, 31.4±0.9 µmol Trolox equivalents (TE)/g for the macerate and 43.4±1.1 µmol TE/g for the extract from the ultrasound-assisted extraction. According to the results of FRAP and CUPRAC assays, the antioxidant potential was similar for both extracts (15.3±0.2 and 15.5±0.2 µmol Fe2+/g and 45.7±0.5 and 46.0±0.7 µmol TE/g, respectively). Due to higher ABTS radical scavenging potential and significantly shorter extraction time, V. myrtillus extract prepared using an ultrasound probe was favored. The present research was an initial step in the preparation of V. myrtillus extracts which can be potentially implemented in food, pharmaceutical, and cosmetic formulations