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Pollen and plant macrofossil analyses supported with radiocarbon dating were conducted on a Holocene core from the peat bog Beliya Kanton (1547 m a.s.l.) located in the Western Rhodope Mountains (South Bulgaria). In the early Holocene until ~10 000 cal. BP the mountain slopes were covered by Pinus , admixed with Betula , Juniperus and shrubland of Ephedra among herb communities dominated by Poaceae, Cichorioideae, Achillea , Artemisia , Brassicaceae species. Subsequently, the afforestation continued with the expansion of broadleaved oak forests with Tilia , Ulmus and Corylus which reached their maximum distribution ~8800 cal. BP. Gradually, these forests began to retreat, replaced at many places first by Corylus and later on by Picea abies , Abies alba and Fagus . The formation of the contemporary coniferous belt with the dominance of Pinus – Picea abies and fragmented mixed coniferous-deciduous communities in the late Holocene was attributed not only to climate change but also to diverse human interference in the natural forest cover. During the last 2000 years the continuous presence of Juniperus , Juglans , Rumex , Cirsium , P lantago lanceolata and Hordeum pollen indicated intensive stock-breeding, grazing and crop cultivation. The fragments of charred wood testify to tree felling and fire clearances on flat ground to obtain new pasture land.

The potential of Marrubium vulgare to alleviate scopolamine (Sco)-induced deficits in spatial working memory has drawn considerable scientific interest. This effect is partly attributed to its potent antioxidant and acetylcholinesterase inhibitory (AChEI) activities. This study examined the effects of M. vulgare extract, standardized to marrubiin content, on recognition memory in healthy and Sco-treated rats. Male Wistar rats (200–250 g) were divided into four groups. The extract was orally administered for 21 days and Sco (2 mg/kg) was intraperitoneally injected for 11 consecutive days. Memory performance was assessed using the novel object recognition test. Levels of acetylcholine (ACh), noradrenaline (NA), serotonin (Sero), and brain-derived neurotrophic factor (BDNF) and the phosphorylation of cAMP response element-binding protein (p-CREB) were evaluated in the cortex and hippocampus via ELISA. BDNF and CREB expression levels were assessed using RT-PCR. The results showed that M. vulgare significantly alleviated Sco-induced memory impairment, preserved cholinergic function in the hippocampus, increased NA levels in the brain, and restored pCREB expression in the cortex following Sco-induced reduction. In healthy rats, the extract upregulated BDNF, pCREB, and Bcl2 expression. Our findings indicate that the neuroprotective effects of M. vulgare may be linked to the modulation of cholinergic function, regulation of NA neurotransmission, and influence on key memory-related molecules.

Sideritis scardica Griseb. is a critically endangered Balkan endemic species, known for its antioxidant, neuroprotective and anti-inflammatory properties. The aim of the present study was to detail an efficient protocol for the micropropagation of S. scardica. In vitro cultures were initiated from the shoot tips of 40 days-old in vivo seedlings and the effects of different plant growth regulator treatments were examined. A Murashige and Skoog nutrient medium (MS) containing 1 mg/L zeatin and 0.1 mg/L indole-3-acetic acid (IAA) proved to be the most efficient for shoot multiplication as it produced quality, vigorous shoots with a mean number of six shoots per explant. For the first time, the antioxidant and antitumor activities of extracts from in vitro-obtained plants were evaluated. In vitro cultivated plants grown in the field revealed a higher total polyphenol content (3929.1 ± 112.2 mg GAE/100 g vs. 3563.5 ± 52.8 mg GAE/100 g) and higher ORAC antioxidant activity (1211.6 ± 27.3 µmol TE/g vs. 939.9 ± 52.4 µmol TE/g) than in situ cultivated plants. A comparison of the antitumor activities of extracts from in vitro propagated shoots, field-grown in vitro-obtained plants and in situ plants on HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) human cancer cell lines showed that in vitro propagated shoots had a significant concentration-dependent cytotoxic effect on the cervical adenocarcinoma cell line HeLa, while the field-grown in vitro-obtained and in situ-collected samples induced the highest reduction in the viability of the mammary carcinoma cell line MCF-7. In both cases, the cells of the control non-tumor cell line, BALB/3T3, were significantly less affected. The results showed that the in vitro multiplication protocol ensured the obtainment of numerous plants with antioxidant and antitumor potential.

Alzheimer’s disease manifests as a complex pathological condition, with neuroinflammation, oxidative stress and cholinergic dysfunction being a few of the many pathological changes. Due to the complexity of the disease, current therapeutic strategies aim at a multitargeted approach, often relying on a combination of substances with versatile and complementary effects. In the present study, a unique combination of α-lipoic acid, citicoline, extracts of leaves from olive tree and green tea, vitamin D3, selenium and an immune-supporting complex was tested in scopolamine-induced dementia in rats. Using behavioral and biochemical methods, we assessed the effects of the combination on learning and memory, and elucidated the mechanisms of these effects. Our results showed that, compared to its components, the experimental combination was most efficient in improving short- and long-term memory as assessed by the step-through method as well as spatial memory as assessed by T-maze and Barnes maze underlined by decreases in AChE activity (p < 0.05) and LPO (p < 0.001), increases in SOD activity in the cortex (p < 0.05) and increases in catalase (p < 0.05) and GPx (p < 0.01) activities and BDNF (p < 0.001) and pCREB (p < 0.05) levels in the hippocampus. No significant histopathological changes or blood parameter changes were detected, making the experimental combination an effective and safe candidate in a multitargeted treatment of AD.

Clinopodium vulgare L. is a valuable medicinal plant used for its anti-inflammatory, antibacterial and wound-healing properties. The present study describes an efficient protocol for the micropropagation of C. vulgare and compares, for the first time, the chemical content and composition and antitumor and antioxidant activities of extracts from in vitro cultivated and wild-growing plants. The best nutrient medium was found to be Murashige and Skoog (MS) supplemented with 1 mg/L BAP and 0.1 IBA mg/L, yielding on average 6.9 shoots per nodal segment. Flower aqueous extracts from in vitro plants had higher total polyphenol content (29,927.6 ± 592.1 mg/100 g vs. 27,292.8 ± 85.3 mg/100 g) and ORAC antioxidant activity (7281.3 ± 82.9 µmol TE/g vs. 7246.3 ± 62.4 µmol TE/g) compared to the flowers of wild plants. HPLC detected qualitative and quantitative differences in phenolic constituents between the in vitro cultivated and wild-growing plants’ extracts. Rosmarinic acid was the major phenolic constituent, being accumulated mainly in leaves, while neochlorogenic acid was a major compound in the flowers of cultivated plants. Catechin was found only in cultivated plants, but not in wild plants or cultivated plants’ stems. Aqueous extracts of both cultivated and wild plants showed significant in vitro antitumor activity against human HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) cell lines. The best cytotoxic activity against most of the cancer cell lines, combined with the least detrimental effects on a non-tumor human keratinocyte cell line (HaCaT), was shown by the leaf (250 µg/mL) and flower (500 µg/mL) extracts of cultivated plants, making cultivated plants a valuable source of bioactive compounds and a suitable candidate for anticancer therapy.

Parkinson’s disease (PD) has emerged as the second most common form of human neurodegenerative disorders. However, due to the severe side effects of the current antiparkinsonian drugs, the design of novel and safe compounds is a hot topic amongst the medicinal chemistry community. Herein, a convenient peptide method, TBTU (O-(benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), was used for the synthesis of the amide (E)-N-(2-methylcinnamoyl)-amantadine (CA(2-Me)-Am; 3)) derived from amantadine and 2-methylcinnamic acid. The obtained hybrid was studied for its antiparkinsonian activity in an experimental model of PD induced by MPTP. Mice (C57BL/6,male, 8 weeks old) were divided into four groups as follows: (1) the control, treated with normal saline (i.p.) for 12 consecutive days; (2) MPTP (30 mg/kg/day, i.p.), applied daily for 5 consecutive days; (3) MPTP + CA(2-Me)-Am, applied for 12 consecutive days, 5 days simultaneously with MPTP and 7 days after MPTP; (4) CA(2-Me)-Am +oleanoic acid (OA), applied daily for 12 consecutive days. Neurobehavioral parameters in all experimental groups of mice were evaluated by rotarod test and passive avoidance test. Our experimental data showed that CA(2-Me)-Am in parkinsonian mice significantly restored memory performance, while neuromuscular coordination approached the control level, indicating the ameliorating effects of the new compound. In conclusion, the newly synthesized hybrid might be a promising agent for treating motor disturbances and cognitive impairment in experimental PD.

Alzheimer’s disease (AD) is a neurodegenerative disease associated with memory impairment and other central nervous system (CNS) symptoms. Two myrtenal–adamantane conjugates (MACs) showed excellent CNS potential against Alzheimer’s models. Adamantane is a common pharmacophore for drug design, and myrtenal (M) demonstrated neuroprotective effects in our previous studies. The aim of this study is to evaluate the MACs’ neuroprotective properties in dementia. Methods: Scopolamine (Scop) was applied intraperitoneally in Wistar rats for 11 days, simultaneously with MACs or M as a referent, respectively. Brain acetylcholine esterase (AChE) activity, noradrenaline and serotonin levels, and oxidative brain status determination followed behavioral tests on memory abilities. Molecular descriptors and docking analyses for AChE activity center affinity were performed. Results: M derivatives have favorable physicochemical parameters to enter the CNS. Both MACs restored memory damaged by Scop, showing significant AChE-inhibitory activity in the cortex, in contrast to M, supported by the modeling analysis. Moderate antioxidant properties were manifested by glutathione elevation and catalase activity modulation. MACs also altered noradrenaline and serotonin content in the hippocampus. Conclusion: For the first time, neuroprotective properties of two MACs in a rat dementia model were observed. They were stronger than the natural M effects, which makes the substances promising candidates for AD treatment.

N-methyl-D-aspartate (NMDA) receptor blockade can improve L-DOPA (l-3,4-dihydroxyphenylalanine)-induced dyskinesias in Parkinson’s disease (PD) patients. Amantadine is a well-tolerated and effective antiparkinsonian agent, recently found to possess NMDA antagonistic properties. Oxidative damage may contribute to dopaminergic (DAergic) neurodegeneration in the substantia nigra of patients with PD. N,N-dimethylglycine (DMG) (also known as vitamin B15 or pangamic acid) acts as an antioxidant, extending the lifespan of animal cells through protection from oxidation. In this study, we synthesized and tested in vivo the newly obtained compound N,N-dimethylglycine-amantadine (DMG-Am) for antiparkinsonian activity. MPTP (1-methyl-4–phenyl-1, 2, 3, 6-tetrahydropyridine) is a widely used neurotoxin to induce an experimental model which mimics Parkinson disease-like symptoms. The neuroprotective capacity of the new amantadine derivative DMG-Am was evaluated by its potential to ameliorate the neuromuscular coordination and behavioral changes worsened by the toxin. Our experimental results showed that DMG-Am applied for 12 consecutive days, 5 days simultaneously and 7 days after MPTP, restored motor and memory performance of the animals to the control level, indication of beneficial protective effect of this compound. In summary, our results reveal the potential of newly synthesized DMG-Am as promising antiparkinsonian agent.

Abstract The results of palynological studies on the late Miocene freshwater deposits of the Tundzha Basin (Southeast Bulgaria, SE Europe) are presented. The basin is relatively well known in terms of geology and palaeogeography. The age of sediments in the Tundzha Basin ranges between the late Miocene to the Pliocene, based on mammal and diatom fossils. We carried out a palynological analysis of clayey sediments interlayered with coal beds from four cores and from one outcrop, aiming to obtain information about the composition and the structure of fossil vegetation. The ratios between the main floristic elements and the composition of the fossil flora are analysed and discussed from a palaeoecological point of view. Several main vegetation palaeocommunities were recorded: swamp forests, mixed mesophytic, communities of aquatic plants, and herbaceous palaeocoenoses. The changes in vegetation and in plant diversity are identified. The palaeoclimate analysis indicates a warm temperature climate with high rainfall and mild winter temperatures, without seasonal drier conditions. The early Pontian climate was about 3–4 °C warmer than today, with rainfalls per year at least 300 mm higher than today. The results of palaeoecological analysis of the flora and of the quantitative palaeoclimate data show that the climate in the Southeast Bulgaria indicates a climate change towards slight cooling and some drying. This event is consistent with the period of accumulation of the upper, undivided part of the Elhovo Formation.

There is growing attention on natural substances capable of stimulating the cholinergic system and of exerting antioxidant effects, as potential therapeutic agents in Alzheimer’s disease (AD). The aim of the present study is to evaluate the expected neuroprotective mechanisms of myrtenal (M) in an experimental model of dementia in rats. Dementia was induced in male Wistar rats by scopolamine (Sc) administration (0.1 mg/kg for 8 days and 20.0 mg/kg on day 9). The animals were divided into 5 groups (1) Controls; (2) Sc; (3) Sc + Myrtenal (40 mg/kg), (4) Sc + Galantamine (1 mg/kg); (5) Sc + Lipoic acid (30 mg/kg). Changes in recognition memory and habituation were evaluated via the Novel Object Recognition and Open Field tests. Acetylcholinesterase (AChE) activity, ACh levels, and changes in oxidative status of the brain were measured biochemically. The histological changes in two brain regions—cortex and hippocampus, were evaluated qualitatively and quantitatively. Myrtenal improved recognition memory and habituation, exerted antioxidant effects and significantly increased ACh brain levels. Histologically, the neuroprotective capacity of myrtenal was also confirmed. For the first time, we have demonstrated the neuroprotective potential of myrtenal in an experimental model of dementia. Our study provides proof-of-concept for the testing of myrtenal, in association with standard of care treatments, in patients affected by cognitive decline.

Three main physico-chemical parameters (moisture content, pH, and electrical conductivity), 19 elements contents (K, Ca, Mg, P, S, Al, As, Cd, Co, Cr, Cu, Fe, Mn, Na, Ni, Pb, Sr, V, and Zn) and microscopic characteristics of 30 honeydew honey samples from Bulgaria were evaluated. The most abundant elements were K, P, Ca and Mg in the honeydew honey investigated and low concentrations of toxic elements and heavy metals were established. The electrical conductivity – one of the most important parameters for honeydew honey, exceeded 0.8 mS*cmˉ¹. The honeydew index in Bulgarian honeydew honey varied widely and its values were often below 3.

The SARS-CoV-2 virus, first reported in December 2019 in China, is the causative agent of the current COVID-19 pandemic that, at the time of writing (1 November 2020) has infected almost 43 million people and caused the death of more than 1 million people. The spectrum of clinical manifestations observed during COVID-19 infection varies from asymptomatic to critical life-threatening clinical conditions. Emerging evidence shows that COVID-19 affects far more organs than just the respiratory system, including the heart, kidneys, blood vessels, liver, as well as the central nervous system (CNS) and the peripheral nervous system (PNS). It is also becoming clear that the neurological and psychological disturbances that occur during the acute phase of the infection may persist well beyond the recovery. The aim of this review is to propel further this emerging and relevant field of research related to the pathophysiology of neurological manifestation of COVID-19 infection (Neuro-COVID). We will summarize the PNS and CNS symptoms experienced by people with COVID-19 both during infection and in the recovery phase. Diagnostic and pharmacological findings in this field of study are strongly warranted to address the neurological and psychological symptoms of COVID-19.

The melissopalynological characteristics, three main physicochemical parameters (water content, pH, and electrical conductivity), and 19 macro- and microelements contents of 15 honey types from throughout Bulgariathat were collected from 2006 to 2009 were evaluated. The main honeys studied came from Robinia pseudoacacia L., Helianthus annuus L., Brassica spp., Tilia spp., and Vicia spp. The botanical origins of unifloral honey samples were identified as Lotus spp., Coriandrum sativum L., Daucus-type, Stachys-type, Salix spp., Prunus spp., Castanea sativa Mill., Paliurus spina-christi Mill., Sophora japonica, and Amorpha spp. Based on the physicochemical parameters and elements contents, one sample with high a percentage of Trifolium spp. pollen was identified as honeydew honey.