Tailored Melatonin- and Donepezil-Based Hybrids Targeting Pathognomonic Changes in Alzheimer's Disease: An In Vitro and In Vivo Investigation.

A plethora of pathophysiological events have been shown to play a synergistic role in neurodegeneration, revealing multiple potential targets for the pharmacological modulation of Alzheimer's disease (AD). In continuation to our previous work on new indole- and/or donepezil-based hybrids as neuroprotective agents, the present study reports on the beneficial effects of lead compounds of the series on key pathognomonic features of AD in both cellular and in vivo models. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the anti-fibrillogenic properties of 15 selected derivatives and identify quantitative changes in the formation of neurotoxic ß-amyloid (Aß42) species in human neuronal cells in response to treatment. Among the most promising compounds were 3a and 3c, which have recently shown excellent antioxidant and anticholinesterase activities, and, therefore, have been subjected to further in vivo investigation in mice. An acute toxicity study was performed after intraperitoneal (i.p.) administration of both compounds, and 1/10 of the LD50 (35 mg/kg) was selected for subacute treatment (14 days) with scopolamine in mice. Donepezil (DNPZ) and/or galantamine (GAL) were used as reference drugs, aiming to establish any pharmacological superiority of the multifaceted approach in battling hallmark features of neurodegeneration. Our promising results give first insights into emerging disease-modifying strategies to combine multiple synergistic activities in a single molecule.

The Phytochemical Profiling, In Vitro Antioxidant, and Hepatoprotective Activity of Prenanthes purpurea L. and Caffeoylquinic Acids in Diclofenac-Induced Hepatotoxicity on HEP-G2 Cells.

Oxidative stress is a common phenomenon of many liver disorders; it both affects patient survival and directly influences the applicability, effectiveness, and toxicity of drugs. In the pursuit of reliable natural remedies for hepatoprotection, this study reports on the complete phytochemical characterization, antioxidant, and hepatoprotective activities of the Prenanthes purpurea methanol-aqueous extract in an in vitro model of diclofenac-induced liver injury (DILI). An ultra high-performance liquid chromatography-high-resolution mass spectrometry analysis (UHPLC-HRMS) was conducted, delineating more than 100 secondary metabolites for the first time in the species, including a series of phenolic acid-hexosides, acylquinic, acylhydroxyquinic and acyltartaric acids, and flavonoids. Quinic acid, chlorogenic, 3,5-dicaffeoylquinic and 5-feruloylhydroxyquinic acid, caffeoyltartaric and cichoric acids, eryodictiol-O-hexuronide, and luteolin O-hexuronide dominated the phytochemical profile and most likely contributed to the observed hepatoprotective activity of the studied P. purpurea leaf extract. The potency and molecular basis of cellular protection were investigated in parallel with pure caffeoylquinic acids in a series of pretreatment experiments that verified the antiapoptotic and antioxidant properties of the natural products.

Antioxidant and Hepatoprotective Potential of Echinops ritro L. Extracts on Induced Oxidative Stress In Vitro/In Vivo.

Echinops ritro L. (Asteraceae) is traditionally used in the treatment of bacterial/fungal infections and respiratory and heart ailments. The aim of this study was to evaluate the potential of extracts from E. ritro leaves (ERLE) and flowering heads (ERFE) as antioxidant and hepatoprotective agents on diclofenac-induced lipid peroxidation and oxidative stress under in vitro and in vivo conditions. In isolated rat microsomes and hepatocytes, the extracts significantly alleviated oxidative stress by increasing cell viability and GSH levels and reducing LDH efflux and MDA production. During in vivo experiments, the administration of the ERFE alone or in combination with diclofenac resulted in a significant increase in cellular antioxidant protection and a decrease in lipid peroxidation witnessed by key markers and enzymes. A beneficial influence on the activity of the drug-metabolizing enzymes ethylmorphine-N-demetylase and aniline hydroxylase in liver tissue was found. In the acute toxicity test evaluation, the ERFE showed no toxicity. In the ultrahigh-performance liquid chromatography-high-resolution mass spectrometry analysis, 95 secondary metabolites were reported for the first time, including acylquinic acids, flavonoids, and coumarins. Protocatechuic acid O-hexoside, quinic, chlorogenic and 3, 5-dicaffeoylquinic acid, apigenin; apigenin 7-O-glucoside, hyperoside, jaceosidene, and cirsiliol dominated the profiles. The results suggest that both extracts should be designed for functional applications with antioxidant and hepatoprotective capacity.

A Study on the Safety and Effects of Amorpha fruticosa Fruit Extract on Spontaneously Hypertensive Rats with Induced Type 2 Diabetes.

Metabolic syndrome is characterized by a variety of diagnostic criteria: obesity, dyslipidemia, type 2 diabetes, and arterial hypertension. They contribute to the elevated risk of cardiovascular morbidity and mortality. The potential for Amorpha fruticosa L. (Fabaceae) to improve diabetes and metabolic disease is promising, based on in vitro tests. This is why a further investigation of the species is needed. Additionally, a toxicity review in relation to safety revealed that to date, there are no published data regarding the toxicity of A. fruticosa towards humans. This species could provide abundant and cheap resources because it is an aggressive invasive plant that grows almost unrestrictedly. The objective of this study was to evaluate the acute toxicity of a purified extract of A. fruticosa (EAF), and to assess its antioxidant, antihypertensive, and antihyperglycemic activity in streptozotocin-induced diabetic spontaneously hypertensive rats (SHRs). The EAF was slightly toxic (LD50 = 2121 mg/kg, b.w.) when administered orally, and moderately toxic (LD50 = 316 mg/kg, b.w.) at intraperitoneal administration, both in mice. The oral administration of EAF (100 mg/kg) for 35 days to SHRs caused significant decreases in the systolic pressure, blood glucose levels, and MDA quantity. It also increased the hepatic level of the endogenous antioxidant GSH, not only in diabetic SHRs, but also in the control group. An additional potential benefit to human health might be conferred through the environmental management of A. fruticosa based on its large-scale use for medicinal purposes, as this aggressive invasive species brings problems to natural habitats in many European countries.

Effects of a new 1,2,3-thiadiazole containing hydrazone antimycobacterial agent on serum and liver biochemical parameters in female mice.

Isoniazid (INH), a first-line drug in anti-tuberculosis therapy, is known to be potentially harmful and is associated with numerous side effects especially in the blood and liver. In the course of our previous investigations, 1,2,3-thiadiazole containing hydrazone (compound 3) showed excellent antimycobacterial activity against a referent strain M. tuberculosis H37Rv (MIC value 0.39 µM), low cytotoxicity, and did not have toxic effects when administered by oral or intraperitoneal routes to experimental animals (selectivity index SI > 1979, LD50>2000 mg/kg b.w.) what revealed its suitability for further exploration. In the present study compound 3 was chosen to determine its effects on the liver and kidney functions in female mice. The compound was administered orally for 14 days at three doses (100, 200, and 400 mg/kg b.w.). The quantity of malondialdehyde (MDA), the level of reduced glutathione (GSH), blood hematological and biochemical parameters were assessed, and urine analysis was carried out. As a positive control INH was used orally at a dose of 50 mg/kg b.w. The investigated compound 3 did not affect the urine and serum hematological and biochemical parameters as INH did, compared to those of the control mice. The new compound did not affect significantly the MDA quantity and maintained its level near to the control values, though lower by 36% (p < 0.05) than in the INH treated animals. At the higher doses, 200 and 400 mg/kg, it depleted the GSH content by 25% (p < 0.05), compared to the control. However, its level remained 47% (p < 0.05) higher than in the INH treated animals.

Ex Vivo Antioxidant and Cholinesterase Inhibiting Effects of a Novel Galantamine-Curcumin Hybrid on Scopolamine-Induced Neurotoxicity in Mice.

Oxidative stress is an essential factor in the development and progression of Alzheimer's disease (AD). An excessive amount of reactive oxygen species (ROS) induces the peroxidation of lipid membranes, reduces the activity of antioxidant enzymes and causes neurotoxicity. In this study, we investigated the antioxidant and cholinesterase inhibitory potential of a novel galantamine-curcumin hybrid, named 4b, administered orally in two doses (2.5 mg/kg and 5 mg/kg) in scopolamine (SC)-induced neurotoxicity in mice. To evaluate the effects of 4b, we used galantamine (GAL) (3 mg/kg) and curcumin (CCN) (25 mg/kg) as positive controls. Ex vivo experiments on mouse brains showed that the higher dose of 4b (5 mg/kg) increased reduced glutathione (GSH) levels by 46%, catalase (CAT) and superoxide dismutase (SOD) activity by 57%, and glutathione peroxidase (GPx) activity by 108%, compared with the SC-treated group. At the same time, 4b (5 mg/kg) significantly reduced the brain malondialdehyde (MDA) level by 31% and acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities by 40% and 30%, respectively, relative to the SC-impaired group. The results showed that 4b acted as an antioxidant agent and brain protector, making it promising for further experimental research in the field of neurodegenerative diseases.

A Novel Galantamine-Curcumin Hybrid as a Potential Multi-Target Agent against Neurodegenerative Disorders.

The acetylcholinesterase (AChE) inhibitors are the main drugs for symptomatic treatment of neurodegenerative disorders like Alzheimer's disease. A recently designed, synthesized and tested hybrid compound between the AChE inhibitor galantamine (GAL) and the antioxidant polyphenol curcumin (CU) showed high AChE inhibition in vitro. Here, we describe tests for acute and short-term toxicity in mice as well as antioxidant tests on brain homogenates measured the levels of malondialdehide (MDA) and glutathione (GSH) and in vitro DPPH, ABTS, FRAP and LPO inhibition assays. Hematological and serum biochemical analyses were also performed. In the acute toxicity tests, the novel AChE inhibitor given orally in mice showed LD50 of 49 mg/kg. The short-term administration of 2.5 and 5 mg/kg did not show toxicity. In the ex vivo tests, the GAL-CU hybrid performed better than GAL and CU themselves; in a dose of 5 mg/kg, it demonstrates 25% reduction in AChE activity, as well as a 28% and 73% increase in the levels of MDA and GSH, respectively. No significant changes in blood biochemical data were observed. The antioxidant activity of 4b measured ex vivo was proven in the in vitro tests. In the ABTS assay, 4b showed radical scavenging activity 10 times higher than the positive control butylhydroxy toluol (BHT). The GAL-CU hybrid is a novel non-toxic AChE inhibitor with high antioxidant activity which makes it a prospective multitarget drug candidate for treatment of neurodegenerative disorders.

Mitigating Effects of Tanacetum balsamita L. on Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

The metabolic syndrome and its associated co-morbidities have been recognized as predisposing risk factors for the development of metabolic-associated fatty liver disease (MAFLD). The present study reports on the beneficial effect of the Tanacetum balsamita methanol-aqueous extract (ETB) at 150 and 300 mg/kg bw on biochemical parameters related to oxidative stress, metabolic syndrome, and liver function in rat animal models with induced MAFLD. ETB was found to be non-toxic with LD50 > 3000 mg/kg and did not affect cell viability of hepatic HEP-G2 cells in a concentration up to 800 µg/mL. The pathology was established by a high-calorie diet and streptozotocin. Acarbose and atorvastatin were used as positive controls. At the higher dose, ETB reduced significantly (p < 0.05) the blood glucose levels by about 20%, decreased lipase activity by 52%, total cholesterol and triglycerides by 50% and 57%, respectively, and restored the amylase activity and leukocytes compared to the MAFLD group. ETB ameliorated oxidative stress biomarkers reduced glutathione and malondialdehyde in a dose-dependent manner. At 300 mg/kg, the beneficial effect of the extract on antioxidant enzymes was evidenced by the elevated catalase, glutathione peroxidase, and superoxide dismutase activity by 70%, 29%, and 44%, accordingly, compared to the MAFLD rats. ETB prevents the histopathological changes related to MAFLD. ETB, rich in 3,5-dicafeoylquinic, chlorogenic, and rosmarinic acids together with the isorhamnetin- and luteolin-glucoside provides a prominent amelioration of MAFLD.

Protective effects of a purified saponin mixture from Astragalus corniculatus Bieb., in vivo hepatotoxicity models.

In this study, the in vivo effects of a purified saponin mixture (PSM), obtained from Astragalus corniculatus Bieb., were investigated using two in vivo hepatotoxicity models based on liver damage caused by paracetamol (PC) and carbon tetrachloride (CCl4 ). The effects of PSM were compared with silymarin. Male Wistar rats were challenged orally with 20% CCl4 or PC (2 g/kg) four days after being pre-treated with PSM (100 mg/kg) or silymarin (200 mg/kg). A significant decrease of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase (LDH) activities and glutathione (GSH) levels and an increase of malondialdehyde (MDA) quantity was observed after CCl4 and PC administration alone. PSM pre-treatment decreased serum transaminases and LDH activities and MDA levels and increased the levels of cell protector GSH. Biotransformation phase I enzymes were also assessed in both models. In the CCl4 hepatotoxicity model, pre-treatment with PSM or silymarin resulted in significantly increased activities of ethylmorphine-N-demethylase and aniline 4-hydroxylase activity and cytochrome P450, compared to the CCl4 only group. Neither silymarin nor PSM influenced PC biotransformation. Our results suggest that PSM, obtained from A. corniculatus, Bieb. showed in vivo hepatoprotective and antioxidant activities against CCl4 and PC-induced liver damage comparable to that of silymarin.

Recent Advances in Anti-Tuberculosis Drug Discovery Based on Hydrazide-Hydrazone and Thiadiazole Derivatives Targeting InhA.

Tuberculosis is an extremely serious problem of global public health. Its incidence is worsened by the presence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. More serious forms of drug resistance have been observed in recent years. Therefore, the discovery and/or synthesis of new potent and less toxic anti-tubercular compounds is very critical, especially having in mind the consequences and the delays in treatment caused by the COVID-19 pandemic. Enoyl-acyl carrier protein reductase (InhA) is an important enzyme involved in the biosynthesis of mycolic acid, a major component of the M. tuberculosis cell wall. At the same time, it is a key enzyme in the development of drug resistance, making it an important target for the discovery of new antimycobacterial agents. Many different chemical scaffolds, including hydrazide hydrazones and thiadiazoles, have been evaluated for their InhA inhibitory activity. The aim of this review is to evaluate recently described hydrazide-hydrazone- and thiadiazole-containing derivatives that inhibit InhA activity, resulting in antimycobacterial effects. In addition, a brief review of the mechanisms of action of currently available anti-tuberculosis drugs is provided, including recently approved agents and molecules in clinical trials.

Newly Synthesized Creatine Derivatives as Potential Neuroprotective and Antioxidant Agents on In Vitro Models of Parkinson's Disease.

Oxidative stress is one of the key factors responsible for many diseases-neurodegenerative (Parkinson and Alzheimer) diseases, diabetes, atherosclerosis, etc. Creatine, a natural amino acid derivative, is capable of exerting mild, direct antioxidant activity in cultured mammalian cells acutely injured with an array of different reactive oxygen species (ROS) generating compounds. The aim of the study was in vitro (on isolated rat brain sub-cellular fractions-synaptosomes, mitochondria and microsomes) evaluation of newly synthetized creatine derivatives for possible antioxidant and neuroprotective activity. The synaptosomes and mitochondria were obtained by multiple centrifugations with Percoll, while microsomes-only by multiple centrifugations. Varying models of oxidative stress were used to study the possible antioxidant and neuroprotective effects of the respective compounds: on synaptosomes-6-hydroxydopamine; on mitochondria-tert-butyl hydroperoxide; and on microsomes-iron/ascorbate (non-enzyme-induced lipid peroxidation). Administered alone, creatine derivatives and creatine (at concentration 38 µM) revealed neurotoxic and pro-oxidant effects on isolated rat brain subcellular fractions (synaptosomes, mitochondria and microsomes). In models of 6-hydroxydopamine (on synaptosomes), tert-butyl hydroperoxide (on mitochondria) and iron/ascorbate (on microsomes)-induced oxidative stress, the derivatives showed neuroprotective and antioxidant effects. These effects may be due to the preservation of the reduced glutathione level, ROS scavenging and membranes' stabilizers against free radicals. Thus, they play a role in the antioxidative defense system and have a promising potential as therapeutic neuroprotective agents for the treatment of neurodegenerative disorders, connected with oxidative stress, such as Parkinson's disease.

Evaluation of Acute and Sub-Acute Toxicity, Oxidative Stress and Molecular Docking of Two Nitrofuranyl Amides as Promising Anti-Tuberculosis Agents.

Tuberculosis (TB) remains a widespread infectious disease and one of the top 10 causes of death worldwide. Nevertheless, despite significant advances in the development of new drugs against tuberculosis, many therapies and preventive measures do not lead to the expected favorable health results for various reasons. The aim of this study was to evaluate the acute and sub-acute toxicity and oxidative stress of two selected nitrofuranyl amides with high in vitro antimycobacterial activity. In addition, molecular docking studies were performed on both compounds to elucidate the possibilities for further development of new anti-tuberculosis candidates with improved efficacy, selectivity, and pharmacological parameters. Acute toxicity tests showed that no changes were observed in the skin, coat, eyes, mucous membranes, secretions, and vegetative activity in mice. The histological findings include features consistent with normal histological architecture without being associated with concomitant pathological conditions. The observed oxidative stress markers indicated that the studied compounds disturbed the oxidative balance in the mouse liver. Based on the molecular docking, compound DO-190 showed preferable binding energies compared to DO-209 in three out of four targets, while both compounds showed promising protein-ligand interactions. Thus, both studied compounds displayed promising activity with low toxicity and can be considered for further evaluation and/or lead optimization.

Metabolite profiling and bioactivity of Cicerbita alpina (L.) Wallr. (Asteraceae, Cichorieae).

Cicerbita alpina (L.) Wallr. is a perennial herbaceous plant in the tribe Cichorieae (Lactuceae), Asteraceae family, distributed in the mountainous regions in Europe. In this study, we focused on the metabolite profiling and the bioactivity of C. alpina leaves and flowering heads methanol-aqueous extracts. The antioxidant activity of extracts, as well as inhibitory potential towards selected enzymes, involving in several human diseases, including metabolic syndrome (α-glucosidase, α-amylase, and lipase), Alzheimer's disease, (cholinesterases: AChE, BchE), hyperpigmentation (tyrosinase), and cytotoxicity were assessed. The workflow comprised ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS analysis revealed more than 100 secondary metabolites, including acylquinic, acyltartaric acids, flavonoids, bitter sesquiterpene lactones (STLs), such as lactucin, dihydrolactucin, their derivatives, and coumarins. Leaves showed a stronger antioxidant activity compared to flowering heads, as well as lipase (4.75 ± 0.21 mg OE/g), AchE (1.98 ± 0.02 mg GALAE/g), BchE (0.74 ± 0.06 mg GALAE/g), and tyrosinase (49.87 ± 3.19 mg KAE/g) inhibitory potential. Flowering heads showed the highest activity against α-glucosidase (1.05 ± 0.17 mmol ACAE/g) and α-amylase (0.47 ± 0.03). The obtained results highlighted C. alpina as a rich source of acylquinic, acyltartaric acids, flavonoids, and STLs with significant bioactivity, and therefore the taxon could be considered as a potential candidate for the development of health-promoting applications.

In Vivo Toxicity, Redox-Modulating Capacity and Intestinal Permeability of Novel Aroylhydrazone Derivatives as Anti-Tuberculosis Agents.

The emergence and spread of Mycobacterium tuberculosis strains resistant to many or all anti-tuberculosis (TB) drugs require the development of new compounds both efficient and with minimal side effects. Structure-activity-toxicity relationships of such novel, structurally diverse compounds must be thoroughly elucidated before further development. Here, we present the aroylhydrazone compounds (3a and 3b) regarding their: (i) acute and subacute toxicity in mice; (ii) redox-modulating in vivo and in vitro capacity; (iii) pathomorphology in the liver, kidney, and small intestine tissue specimens; and (iv) intestinal permeability. The acute toxicity test showed that the two investigated compounds exhibited low toxicity by oral and intraperitoneal administration. Changes in behavior, food amount, and water intake were not observed during 14 days of the oral administration at two doses of 1/10 and 1/20 of the LD50. The histological examination of the different tissue specimens did not show toxic changes. The in vitro antioxidant assays confirmed the ex vivo results. High gastrointestinal tract permeability at all tested pH values were demonstrated for both compounds. To conclude, both compounds 3a and 3b are highly permeable with low toxicity and can be considered for further evaluation and/or lead optimization.

Beneficial effects of the fructus Sophorae extract on experimentally induced osteoporosis in New Zealand white rabbits.

Sophora japonica is a source of several flavonol, flavone and isoflavone glycosides that are reported to positively affect menopausal symptoms including osteoporotic complications. In the present study fructus Sophorae extract (FSE) was administered orally for three months at a dose of 200 mg kg-1 in ovariectomized (OVX) New Zealand rabbits. 3D computed tomography scans and histopathological images revealed microstructural disturbances in the bones of the castrated animals. FSE recovered most of the affected parameters in bones in a manner similar to zoledronic acid (ZA) used as a positive control. The aglycones of the main active compounds of FSE, daidzin, and genistin, were docked into the alpha and beta estrogen receptors and stable complexes were found. The findings of this study provide an insight into the effects of FSE on bone tissue loss and suggest that it could be further developed as a potential candidate for the prevention of postmenopausal osteoporotic complications.

Hepatoprotective activity of a purified methanol extract and saponins from the roots of Chenopodium bonus-henricus L.

An ultra-high-performance liquid chromatography-high-resolution mass spectrometry based profiling of a purified MeOH extract (PME) from the roots of Chenopodium bonus-henricus L. (Amaranthaceae) tentatively identified 15 saponins of six sapogenins. The PME exerts hepatoprotective and antioxidant activities comparable to those of flavonoid complex silymarin in in vitro (1 and 10 µg/mL) and in vivo (200 mg/kg/daily for 7 days) models of hepatotoxicity, induced by CCl4. The main constituents of PME, respectively saponins bonushenricoside A (1), 3-O-ß-D-glucuronopyranosyl-bayogenin-28-O-ß-D-glucopyranosyl ester (2), 3-O-ß-D-glucuronopyranosyl-medicagenic acid-28-O-ß-D-xylopyranosyl (1→4)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl ester (3), 3-O-ß-D-glucuronopyranosyl-2ß-hydroxygypsogenin-28-O-ß-D-glucopyranosyl ester (4), 3-O-α-L-rabinopyranosyl-bayogenin-28-O-ß-D-glucopyranosyl ester (6) and bonushenricoside B (8) (3 µg/mL each), compared to silymarin (5 and 50 µg/mL), significantly reduced the cellular damage caused by CCl4 in rat hepatocytes, preserved cell viability and glutathione level, decreased lactate dehydrogenase leakage and reduced lipid damage. The experimental data suggest that the glycosides of phytolaccagenin, bayogenin, medicagenic acid, 2ß-hydroxygypsogenin, 2ß-hydroxyoleanoic acid and oleanoic acid are a promising and safe class of hepatoprotective agents.

In silico and in vivo studies of Astragalus glycyphylloides saponin(s) with relevance to metabolic syndrome modulation.

Triterpenoids are well known modulators of metabolic syndrome. One of the suggested modes of action (MoAs) involves peroxisome proliferator-activated receptor gamma (PPARγ) binding. In this study we aimed to: (i) evaluate in silico potential metabolites and PPARγ-mediated MoA of the sapogenin of the main saponin present in a purified saponins' mixture (PSM) from Astragalus glycyphylloides; (ii) estimate in silico and in vivo PSM's toxicity; and (iii) investigate in vivo antihyperglycaemic, hypolipidaemic, antioxidant and hepatoprotective effects of PSM. Metabolites and toxicity were predicted using Meteor and Derek Nexus expert systems (Lhasa Limited) and PPARγ binding was investigated using the software MOE (CCG Inc.). PSM's acute oral toxicity was evaluated in mice and the pharmacological effects were assessed in streptozotocin-induced diabetic spontaneously hypertensive rats (SHRs). Liver histopathology was studied as well. PPARγ weak partial agonism was predicted in silico for 24 probable/plausible Phase I metabolites which docking poses were clustered in 12 different binding modes with characteristic protein-ligand interactions. PSM's beneficial effects on the levels of blood glucose, triglycerides, and total cholesterol, on oxidative stress markers and liver histology in diabetic SHRs were comparable to those of the PPARγ ligand pioglitazone. PSM's safety profile was confirmed in silico and in vivo.

In vivo toxicity assessment of Clinopodium vulgare L. water extract characterized by UHPLC-HRMS.

Clinopodium vulgare L. (Lamiaceae) was used in the traditional Bulgarian medicine for treatment of wounds, diabetes and gastric ulcers. In this study we aimed at safety assessment of C. vulgare lyophilized water extract (CVE) characterized by ultra high-performance liquid chromatography-Orbitrap high resolution mass spectrometry (UHPLC-HRMS). The acute and sub-acute toxicity of CVE was determined in two rodent species (mice and rats), and two routes of administration - intraperitoneal (i.p.) and oral (p.o.). LD50 (i.p.), were found to be 675 mg/kg (mice) and 500 mg/kg (rats). An acute i. p. administration resulted in central nervous system toxic effects. LD50 (p.o.) was higher than 2000 mg/kg for both species. In sub-acute oral administration, CVE did not exert any toxic effect on hematology, blood and urine biochemistry, and histomorphology in pancreas, liver, spleen and kidney. In addition, based on accurate masses, MS/MS and comparison with standards, a variety of flavonoids, caffeic acid oligomers and saponins were tentatively elucidated in CVE. Rosmarinic acid was the major compound. In conclusion, CVE did not cause hematological, biochemical and histopathological changes after oral administration and it is safe for internal use. The obtained UHPLC-HRMS profile revealed CVE as a new rich source of water soluble caffeic acid oligomers.

Trans-3,5-dicaffeoylquinic acid from Geigeria alata Benth. & Hook.f. ex Oliv. & Hiern with beneficial effects on experimental diabetes in animal model of essential hypertension.

Geigeria alata Benth. & Hook.f. ex Oliv. & Hiern (Asteraceae) is used in Sudanese folk medicine for treatment of diabetes. The study aimed to estimate the acute oral toxicity of trans-3,5-dicaffeoylquinic acid (3,5-diCQA) from G. alata roots and to assess its antihypeglycemic, antioxidant and antihypertensive effects on chemically-induced diabetic spontaneously hypertensive rats (SHRs). The structure of 3,5-diCQA was established by NMR and HRMS spectra. Type 2 diabetes was induced by intraperitoneal injection of streptozotocin. 3,5-diCQA was slightly toxic with LD50 = 2154 mg/kg. At 5 mg/kg 3,5-diCQA reduced significantly (p < 0.05) the blood glucose levels by 42%, decreased the blood pressure by 22% and ameliorated the oxidative stress biomarkers reduced glutathione, malondialdehyde, and serum biochemical parameters. The beneficial effect on antioxidant enzymes was evidenced by the elevated glutathione peroxidase, glutathione reductase, and glutathione S-transferase activitiy in the livers of diabetic animals. 3,5-diCQA prevents the histopathological changes related to diabetes and hypertension. 3,5-diCQA was more potent α-glucosidase inhibitor (IC50 27.24 µg/mL) than acarbose (IC50 99.77 µg/mL). The antihyperglycemic action of the compound was attributed to the α-glucosidase inhibition. The beneficial effects of 3,5-diCQA on streptozotocin-induced diabetic hypertensive rats support the traditional use of G.alata for the management of diabetes.

Bone protective effects of purified extract from Ruscus aculeatus on ovariectomy-induced osteoporosis in rats.

Ruscus aculeatus is a source of steroidal saponins that could mimic sex hormones and could help alleviate the risk of fracture in osteoporotic patients. The aim of the present study was to evaluate the in vitro effects of an extract from R. aculeatus (ERA) on the proliferation of human osteoblast-like SaOS-2 cell line and to investigate the effects of the ERA administered orally for 10 weeks at three doses (50, 100 and 200 mg/kg) on the bone structure of rats with estrogen deficiency induced by bilateral ovariectomy. Bone turnover markers, hormones, histopathological and radiological disturbances were evidenced in the ovariectomized rats. ERA recovered most of the affected parameters in a dose-dependent manner similar to diosgenin and alendronate used as positive comparators. The main active compounds of ERA (ruscogenin and neoruscogenin) were docked into the Vit. D receptor and oestrogen receptors alpha and beta, and stable complexes were found with binding scores equal to those of estradiol and diosgenin. The findings of this study provide for the first time an insight into the effects of ERA on bone structure and suggest that ERA could be developed as a potential candidate for the prevention of postmenopausal osteoporotic complications.