Sonia Apostolova

Background and Objectives: Periodontitis is a chronic multifactorial inflammatory disease characterized by progressive destruction of the tooth-supporting tissues. Although periodontal diagnosis is primarily based on clinical and radiographic parameters, salivary biomarkers may provide additional biological information on inflammatory activity and host-response regulation. Matrix metalloproteinase-9 (MMP-9) is involved in extracellular matrix degradation and periodontal tissue destruction, whereas melatonin is a circadian-related molecule with antioxidant, anti-inflammatory, and immunomodulatory properties. However, evidence regarding the simultaneous assessment of salivary MMP-9 and melatonin using standardized pre-sleep saliva collection remains limited. This pilot study aimed to evaluate salivary melatonin and MMP-9 concentrations in periodontal health and stage III periodontitis, to assess their interrelationship, and to explore their preliminary in-sample ability to distinguish periodontal health from stage III periodontitis. Materials and Methods: This cross-sectional pilot study included 18 systemically healthy non-smoking adults: 9 periodontally healthy participants and 9 patients with stage III periodontitis. Pre-sleep unstimulated saliva was collected between 23:00 and 24:00 h or immediately before sleep, following a standardized protocol. Salivary MMP-9 and melatonin concentrations were determined using enzyme-linked immunosorbent assay. Periodontal diagnosis was established according to the 2017 World Workshop classification. Group comparisons, correlation analyses, and receiver operating characteristic curve analyses were performed as exploratory analyses. Results: Salivary melatonin concentrations were significantly lower in patients with stage III periodontitis than in periodontally healthy participants (p = 0.019), with a large effect size (Hedges’ g = 1.261). Salivary MMP-9 concentrations were higher in the periodontitis group, but the difference was not statistically significant (p = 0.465). No significant correlation was found between salivary melatonin and MMP-9 levels. Receiver operating characteristic analysis suggested preliminary in-sample discriminatory ability for melatonin (AUC = 0.827; p = 0.019), whereas MMP-9 showed limited and non-significant discriminatory ability (AUC = 0.593; p = 0.508). Conclusions: In this pilot sample, lower pre-sleep salivary melatonin levels were associated with stage III periodontitis, whereas MMP-9 showed limited standalone discriminatory ability. These findings should be interpreted as preliminary and hypothesis-generating. The present study does not establish a clinically applicable diagnostic cut-off or validated diagnostic utility for salivary melatonin. Larger, independently validated studies are needed to confirm these observations.

The existing body of literature, in conjunction with our recent studies, shows that melatonin dysfunction can accelerate the aging process, with this effect depending on the specific age of the subject. The present study aims to ascertain the impact of pinealectomy on sphingolipid (SL) turnover in young adult (3-month-old), middle-aged (14-month-old), and old (18-month-old) rats. Ceramide (Cer) levels, neutral (NSMase) and acid sphingomyelinase (ASMase), acid ceramidase (ASAH1), and sphingosine-1-phosphate (S1P) levels in hippocampus and/or plasma, were evaluated by enzyme-linked immunosorbent assay. The accumulation of Cer and its metabolite second messenger S1P in the hippocampus and plasma was associated with increased levels and activity of hippocampal NSMase in the hippocampus and plasma. However, no such association was observed for hippocampal ASMase, whose levels and activity were reduced in middle-aged and old rats compared to young adult rats. Pinealectomy-induced melatonin deficiency in young adult rats showed an increase in hippocampal Cer content compared to the sham group. However, in contrast to young adult rats, pinealectomy had an inverse effect on age-related changes in hippocampal Cer, NSMase, and ASMase in middle-aged rats. Furthermore, pinealectomy exacerbated the age-related increase in S1P in the hippocampus of 18-month-old rats. Collectively, the results of the present study suggest that melatonin deficiency may influence the aging process by modulating SL turnover in an age-specific manner.

Background/objectives: Propolis (bee glue) is a valuable bee product widely used as a natural remedy, a cosmetic ingredient, a nutritional value enhancer and a food biopreservative. The present research aims to investigate the phenolic content, antioxidant activity and in vitro anti-inflammatory and antitumor potential of six propolis samples from three regions of Bulgaria (Vidin, Gabrovo and Lovech). Methods: the analysis of propolis phenolic compounds was determined by high-performance liquid chromatography (HPLC); the antioxidant activity of ethanolic propolis extracts was assessed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and ferric-reducing antioxidant power (FRAP) assay; the in vitro anti-inflammatory activity was assessed by the inhibition of albumin denaturation method; the in vitro antitumor activity was determined in human metastatic breast cancer cell line MDA-MB-231 using 3-(4,5-Dimethyl -2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results: The ethanolic propolis extracts exhibited the total phenolic content from 190.4 to 317.0 mg GAE/g, total flavonoid content from 53.4 to 79.3 mg QE/g and total caffeic acid derivatives content from 5.9 to 12.1 mg CAE/g. The studied propolis extracts showed significant antioxidant capacity (between 1000.3 and 1606.0 mM TE/g determined by the DPPH assay, and between 634.1 and 1134.5 mM TE/g determined by the FRAP assay). The chemical composition analysis indicated high concentrations of caffeic acid benzyl ester, chrysin, pinocembrin and pinobanksin-3-O-propionate, predominantly in three of the propolis samples originating from Gabrovo and Lovech regions. All propolis samples demonstrated promising in vitro anti-inflammatory activity, expressed as the inhibition of thermally induced albumin denaturation by 73.59% to 78.44%, which was higher than that of the conventional anti-inflammatory drugs Aspirin (58.44%) and Prednisolone Cortico (57.34%). The propolis samples exhibited high in vitro cytotoxicity against cancer cells MDA-MB-231 with IC₅₀ values ranging between 9.24 and 13.62 µg/mL as determined by MTT assay. Conclusions: Overall, we can suggest that the high phenolic content of Bulgarian propolis from respective areas contributes to its enhanced antioxidant, anti-inflammatory and antitumor activity. Taken together, our results support the beneficial properties of Bulgarian propolis, with potential application as a promising therapeutic agent.

The conversion of betulinic acid (BA) to organic salts is a strategic approach to modulate its physicochemical properties and biological activity. In our previous study, we demonstrated the enhanced cytotoxicity of certain amino acid ethyl ester betulinates ([AAOEt][BA]) compared to BA against hormone-dependent breast cancer cells (MCF-7). In this study, we extended our investigation to evaluate the cytotoxic response and thermodynamic properties of hormone-independent breast cancer cells (MDA-MB-231) following 72 h of treatment with the same series of betulinates. Our data reveal a lower cytotoxic response in MDA-MB-231 cells, indicated by higher half-maximal inhibitory concentration (IC₅₀) values, which ranged between 31 and 109 μM. Differential scanning calorimetry analysis supported these findings, showing negligible changes in the thermodynamic parameters of the treated MDA-MB-231 cells. However, consistent with our previous observations, [LysOEt][BA]₂, exhibited the highest cytotoxicity and induced the most pronounced morphological alterations in the cancer cells. Overall, our results suggest that MDA-MB-231 cells are less sensitive to [AAOEt][BA] compared to MCF-7 cells, likely due to their distinct phenotypic and genotypic profiles and differences in oncogenic signalling pathways. Nonetheless, the fact that [LysOEt][BA]₂ enhances the cytotoxic activity of BA even in hormone-independent breast cancer cells underscores its therapeutic potential and warrants further investigation, particularly in the context of adjuvant breast cancer therapy.

Data from the literature and our recent studies have shown that dysfunction/deficiency of melatonin accelerates aging or delays lifespan in an age-dependent manner. In the present study, we aimed to determine the effects of pinealectomy on sphingolipid turnover in young adult (3-month-old), middle-aged (14-month-old), and old (18-month-old) rats. Ceramide levels, neutral and acid sphingomyelinase, acid ceramidase and sphingosine-1-phosphate levels in hippocampus and/or plasma, were evaluated by enzyme-linked immunosorbent assay Accumulation of Ceramide and its metabolite second messenger sphingo-sine-1-phosphate in the hippocampus and plasma was associated with increased levels and activity of neutral sphingomyelinase in the hippocampus and plasma, respectively, but not of acid sphingomyelinase, which levels and activity in the hippocampus were reduced in middle-aged and old rats compared to young adult rats. Similar to the aging process, pinealectomy-induced melatonin deficiency increased Ceramide content in the hippocampus of young adult rats compared to the sham group. Pinealectomy also exacerbated an age-related increase in sphingosine-1-phosphate in the hippocampus of 18-month-old rats. However, compared to young adult rats, pinealectomy exerted the opposite effect on age-related changes in Ceramide, neutral, and acid sphingomyelinases in the hippocampus in middle-aged rats. Our results suggest that melatonin deficiency may modulate aging by altering sphingolipid turnover in an age-specific manner.

Diabetic neuropathy (DN) is a common complication of long-lasting type 1 and type 2 diabetes, with no curative treatment available. Here, we tested the effect of the incretin mimetic liraglutide in DN in mice with early-stage type 1 diabetes bred in a standard laboratory or enriched environment. With a single i.p. injection of streptozotocin 150 mg/kg, we induced murine diabetes. Liraglutide (0.4 mg/kg once daily, i.p. for ten days since the eighth post-streptozotocin day) failed to decrease the glycemia in the diabetic mice; however, it alleviated their antinociceptive behavior, as tested with formalin. The second phase of the formalin test had significantly lower results in liraglutide-treated mice reared in the enriched environment vs. liraglutide-treated mice under standard conditions [2.00 (0.00–11.00) vs. 29.00 (2.25–41.50) s, p = 0.016]. Liraglutide treatment, however, decreased the threshold of reactivity in the von Fray test. A significantly higher neopterin level was demonstrated in the diabetic control group compared to treatment-naïve controls and the liraglutide-treated diabetic mice (p < 0.001). The glutamine/glutamate ratio in both liraglutide-treated groups, either reared under standard conditions (p = 0.003) or an enriched environment (p = 0.002), was significantly higher than in the diabetic controls. This study demonstrates an early liraglutide effect on pain sensation in two streptozotocin-induced diabetes mouse models by reducing some inflammatory and oxidative stress parameters.

Betulinic acid (BA) is a natural pentacyclic triterpene with diverse biological activities. However, its low water solubility limits its pharmaceutical application. The conversion of pharmaceutically active molecules into ionic liquids (ILs) is a promising strategy to improve their physicochemical properties, stability, and/or potency. Here, we report the synthesis and characterization of 15 novel ILs containing a cation ethyl ester of a polar, non-polar, or charged amino acid [AAOEt] and an anion BA. Except for [ValOEt][BA], we observed preserved or up to 2-fold enhanced cytotoxicity toward hormone-dependent breast cancer cells MCF-7. The estimated IC50 (72 h) values within the series varied between 4.8 and 25.7 µM. We found that the most cytotoxic IL, [LysOEt][BA]₂, reduced clonogenic efficiency to 20% compared to that of BA. In addition, we evaluated the effect of a 72 h treatment with BA or [LysOEt][BA]₂, the most cytotoxic compound, on the thermodynamic behavior of MCF-7 cells. Based on our data, we suggest that the charged amino acid lysine included in the novel ILs provokes cytotoxicity by a mechanism involving alteration in membrane lipid organization, which could be accompanied by modulation of the visco–elastic properties of the cytoplasm.

Diabetes mellitus is one of the most common chronic diseases in the world. Metabolomic studies have demonstrated altered blood levels of glutamate and glutamine during type 1 diabetes and type 2 diabetes. Riboflavin is a precursor of flavin adenine dinucleotide and flavin mononucleotide, which are coenzymes necessary for the function of enzymes involved in various biochemical reactions, including those affecting amino acid metabolism. In this study, we investigated the effects of riboflavin on serum glutamate and glutamine levels in rats with streptozotocin-induced type 1 diabetes. Diabetic rats received riboflavin (25 mg, 50 mg, or 100 mg) dissolved in the drinking water daily for 2 weeks. Our results showed that riboflavin supplementation did not affect serum glutamate levels but increased serum glutamine levels in diabetic rats. We speculate that increased serum glutamine levels resulting from riboflavin supplementation may have beneficial effects during diabetes.

Fluorescent micellar carriers with controlled release of a novel anticancer drug were developed to enable intracellular imaging and cancer treatment simultaneously. The nanosized fluorescent micellar systems were embedded with a novel anticancer drug via the self-assembling behavior of well-defined block copolymers based on amphiphilic poly(acrylic acid)-block-poly(n-butyl acrylate) (PAA-b-PnBA) copolymer obtained by Atom Transfer Radical Polymerization (ATRP) and hydrophobic anticancer benzimidazole-hydrazone drug (BzH). Through this method, well-defined nanosized fluorescent micelles were obtained consisting of a hydrophilic PAA shell and a hydrophobic PnBA core embedded with the BzH drug due to the hydrophobic interactions, thus reaching very high encapsulation efficiency. The size, morphology, and fluorescent properties of blank and drug-loaded micelles were investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent spectroscopy, respectively. Additionally, after 72 h of incubation, drug-loaded micelles released 3.25 μM of BzH, which was spectrophotometrically determined. The BzH drug-loaded micelles were found to exhibit enhanced antiproliferative and cytotoxic effects on MDA-MB-231 cells, with long-lasting effects on microtubule organization, with apoptotic alterations and preferential localization in the perinuclear space of cancer cells. In contrast, the antitumor effect of BzH alone or incorporated in micelles on non-cancerous cells MCF-10A was relatively weak.