Clinical Evaluation of a Defined Zeolite-Clinoptilolite Supplementation Effect on the Selected Blood Parameters of Patients.

The natural clinoptilolite material is an inorganic crystal mineral called zeolite. It has been extensively studied and used in industrial applications and veterinary and human medicine due to positive effects on health. Limited data is available in the scientific literature about its effects on the levels of physiologically relevant minerals in the human organism. Accordingly, we performed a comprehensive and controlled monitoring of the relevant mineral and contaminants levels in human subjects supplemented with a certified clinoptilolite material within three clinical trials with different supplementation regimens. Effects of a registered and certified clinoptilolite material PMA-zeolite on selected mineral and metal levels were determined by standard biochemical methods and inductively coupled plasma mass spectrometry (ICP-MS) in the blood of subjects enrolled in three clinical trials: short-term (28 days, Mineral Metabolism and selected Blood Parameters study MMBP), medium-term (12 weeks, Morbus Crohn study), and long-term (4 years, Osteoporosis TOP study) supplementation. Lower concentrations were observed for copper (Cu) in patients with osteoporosis, which normalized again in the long-term supplementation trial, whereas sodium (Na) and calcium (Ca) levels diminished below the reference values in patients with osteoporosis. In the short- and long-term supplementation trials, increased levels of lead (Pb) were observed in PMA-zeolite-supplemented subjects, which decreased in the continued long-term supplementation trial. Increased levels of aluminum (Al) or Pb attributable to eventual leakage from the material into the bloodstream were not detected 1 h after intake in the short-term supplementation trial. Nickel (Ni) and Al were statistically significantly decreased upon long-term 4-year supplementation within the long-term supplementation trial, and arsenic (As) was statistically significantly decreased upon 12-weeks supplementation in the medium-term trial. Alterations in the measured levels for Na and Ca, as well as for Pb, in the long-term trial are probably attributable to the bone remodeling process. Checking the balance of the minerals Cu, Ca, and Na after 1 year of supplementation might be prescribed for PMA-supplemented patients with osteoporosis. Clinical Trial Registration: [https://clinicaltrials.gov], identifiers [NCT03901989, NCT05178719, NCT04370535, NCT04607018].

Synthesis, antiproliferative and antitrypanosomal activities, and DNA binding of novel 6-amidino-2-arylbenzothiazoles.

A series of 6-amidinobenzothiazoles, linked via phenoxymethylene or directly to the 1,2,3-triazole ring with a p-substituted phenyl or benzyl moiety, were synthesised and evaluated in vitro against four human tumour cell lines and the protozoan parasite Trypanosoma brucei. The influence of the type of amidino substituent and phenoxymethylene linker on antiproliferative and antitrypanosomal activities was observed, showing that the imidazoline moiety had a major impact on both activities. Benzothiazole imidazoline 14a, which was directly connected to N-1-phenyl-1,2,3-triazole, had the most potent growth-inhibitory effect (IC50 = 0.25 µM) on colorectal adenocarcinoma (SW620), while benzothiazole imidazoline 11b, containing a phenoxymethylene linker, exhibited the best antitrypanosomal potency (IC90 = 0.12 µM). DNA binding assays showed a non-covalent interaction of 6-amidinobenzothiazole ligands, indicating both minor groove binding and intercalation modes of DNA interaction. Our findings encourage further development of novel structurally related 6-amidino-2-arylbenzothiazoles to obtain more selective anticancer and anti-HAT agents.

Treatment of osteoporosis with a modified zeolite shows beneficial effects in an osteoporotic rat model and a human clinical trial.

The severity of osteoporosis in humans manifests in its high incidence and by its complications that diminish quality of life. A societal consequence of osteoporosis is the substantial burden that it inflicts upon patients and their families. Several bone-modifying drugs have been prescribed to patients with osteoporosis. However, evidence for their anti-fracture efficacy remains inconclusive. To the contrary, long-term use of anti-osteoporotic drugs such as bisphosphonates and Denosumab, an RANKL inhibitor, have resulted in adverse events. We now present an alternative and adjuvant approach for treatment of osteoporosis. The data derive from in vivo studies in an ovariectomized rat model and from a randomized double blind, placebo-controlled human clinical study. Both studies involved treatment with Panaceo Micro Activation (PMA)-zeolite-clinoptilolite, a defined cation exchange clinoptilolite, which clearly improved all bone histomorphometric parameters examined from ovariectomized animals, indicative for increased bone formation. Moreover, intervention with PMA-zeolite-clinoptilolite for one year proved safe in humans. Furthermore, patients treated with PMA-zeolite-clinoptilolite showed an increase in bone mineral density, an elevated level of markers indicative of bone formation, a significant reduction in pain, and significantly improved quality of life compared with patients in the control (placebo) group. These encouraging positive effects of PMA-zeolite-clinoptilolite on bone integrity and on osteoporosis warrant further evaluation of treatment with PMA-zeolite-clinoptilolite as a new alternative adjuvant therapy for osteoporosis.

Assessment of the Biological Activity and Phenolic Composition of Ethanol Extracts of Pomegranate (Punica granatum L.) Peels.

Pomegranate (Punica granatum L.) is a rich source of constituents with confirmed strong biological activities. However, pomegranate peel, which encompasses approximately 30-40% of its weight, is treated as a biological waste. The aim of this paper was to evaluate the potential of pomegranate peel extracts and to propose its functional properties that can be used for development of functional products. Eight ethanol extracts of pomegranate peels (PPEs) were characterized by use of direct infusion quadrupole-time of flight (Q-TOF), and afterwards tested on their antioxidant, antibacterial and antiproliferative activities. Mass spectrometry analysis revealed that the most prevalent compounds in pomegranate peels were punicalagin, granatin and their derivatives. Analysed extracts had high total phenolic contents that ranged from 5766.44 to 10599.43 mg GAE/100 g, and strong antioxidant activity (7551.31-7875.42 and 100.25-176.60 µmol TE/100 g for DPPH and FRAP assays, respectively). The results of biological activity assays showed that all PPEs possessed antibacterial activity, and that S. aureus was the most sensitive specie with minimum inhibitory concentration and minimum bactericidal concentrations ranging from 0.8 to 6.4 mg/mL. Additionally, the analysis of antiproliferative activity revealed high potency of PPEs, as the IC50 values ranged from 0.132 mg/mL to 0.396 mg/mL. Multivariate analysis pointed out the most discriminative metabolites for antioxidant or antiproliferative activity. Overall, the pomegranate peel confirmed to be a highly valuable source of bioactive compounds that could be used to improve the food functional characteristics.

Treatment With Medicinal Mushroom Extract Mixture Inhibits Translation and Reprograms Metabolism in Advanced Colorectal Cancer Animal Model as Evidenced by Tandem Mass Tags Proteomics Analysis.

Colorectal cancer (CRC) is the third most frequent cancer type in both males and females, with about 35% of patients being diagnosed in stage IV metastatic disease. Despite advancements in treatment, life expectancy in patients with metastatic disease is still not satisfying. Due to frequent drug resistance during conventional and targeted cancer treatments, the development and testing of multi-target therapies is an important research field. Medicinal mushrooms specific isolated compounds as well as complex extract mixtures have been studied in depth, and many mushroom species have been proven to be non-toxic multi-target inhibitors of specific oncogenic pathways, as well as potent immunomodulators. In this study, we have performed a tandem mass tags qualitative and quantitative proteomic analyses of CT26.WT colon cancer tumor tissues from Balb/c mice treated with the studied medicinal mushroom extract mixture, with or without 5-fluorouracil. Besides significantly improved survival, obtained results reveal that Agarikon.1 alone, and in combination with 5-fluorouracil exert their anticancer effects by affecting several fundamental processes important in CRC progression. Bioinformatic analysis of up- and downregulated proteins revealed that ribosomal biogenesis and translation is downregulated in treatment groups, while the unfolded protein response (UPR), lipid metabolism and tricarboxylic acid cycle (TCA) are upregulated. Moreover, we found that many known clinical biomarkers and protein clusters important in CRC progression and prognosis are affected, which are a good basis for an expanded translational study of the herein presented treatment.

Modulation of gut microbiota in healthy rats after exposure to nutritional supplements.

INTRODUCTION: Rats are experimental animals, frequently used as model organisms in the biomedical studies, and increasingly used to study the gut microbiota. Specifically, the aim of latter studies is either the elucidation of relationship between intestinal dysbiosis and diseases or the determination of nutrients or pharmaceutical agents which can cause the modulation in the presence or abundance of gut microbiota. AIM: Herein, the research studies conducted on the gut microbiota of healthy rats are presented in a summarized and concise overview. The focus is on studies aimed to reveal the shifts in microbial composition and functional changes after exposure to various types of nutritional supplements. METHODS: We performed the search of PubMed database using the term "rat gut microbiome microbiota" and examined studies aimed to assess the composition of gut microbiota in physiological homeostasis as well as the effect of various nutritional supplements on the gut microbiota of healthy rats.

Green synthesis and biological evaluation of 6-substituted-2-(2-hydroxy/methoxy phenyl)benzothiazole derivatives as potential antioxidant, antibacterial and antitumor agents.

We present a new efficient green synthetic protocol for introduction of substituents to the C-6 position of 2-arylbenzothiazole nuclei. Newly synthesized compounds were designed to study the influence of the hydroxy and methoxy groups on the 2-arylbenzothiazole scaffold, as well as the influence of the type of substituents placed on the C-6 position of benzothiazole moiety on biological activity, including antibacterial, antitumor and antioxidant activity. Modest activity was observed against the tested Gram-positive and Gram-negative bacterial strains for only amidino derivatives 5d and 6d. The tested compounds exhibited moderate to strong antiproliferative activity towards the tumor cell lines tested. The SAR study revealed that the introduction of substituents into the benzene ring of the benzothiazole nuclei is essential for antiproliferative activity, while introduction of the hydroxy group into the 2-aryl moiety of the 2-arybenzothiazole scaffold significantly improved selectivity against tumor cell lines. The observed results revealed several novel 6-substituted-2-arylbenzothiazole compounds, 5b, 5c, 5f and 6f, with strong and selective antiproliferative activity towards HeLa cells in micro and submicromolar concentrations, with the most selective compounds being 6-ammonium-2-(2-hydroxy/methoxyphenyl)benzothiazoles 5f and 6f. The compound 5f bearing the hydroxy group on the 2-arylbenzothiazole core showed the most promising antioxidative activity evaluated by DPPH, ABTS and FRAP in vitro assays. The presence of the amino protonated group attached at the benzothiazole moiety was essential for the antiproliferative and antioxidant activity observed, exerted through a change in the levels of the reactive oxygen species-modulated HIF-1 protein.

Novel 1,2,4-triazole and imidazole derivatives of L-ascorbic and imino-ascorbic acid: synthesis, anti-HCV and antitumor activity evaluations.

Several novel 1,2,4-triazole and imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus (HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lymphoblastic leukaemia cells (CEM/0) with IC(50)s of 10 ± 4 and 7.3 ± 0.1 µM, respectively. Unlike compound 9, compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action of compound 6 accounting for observed cytostatic activity towards haematological malignancies might be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispensable for cell growth and division, which has emerged as an important target for antileukemic therapy. In addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as well. However, observed antiviral effect was most likely associated with the effect that the compound exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclusion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhibitor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts encourage further development of this compound as a lead.

Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

PURPOSE: p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. METHODS: p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. RESULTS: In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. CONCLUSIONS: This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics' modality of action.

Screening of potential prodrugs on cells derived from Dupuytren's disease patients.

Dupuytren's disease (DD) is a fibroproliferative disorder, the cure for which is still limited to surgical excision of the affected fascia, often leading to high recurrence rates. Due to this fact, non-surgical treatments are being investigated, among them those targeting molecular processes of proliferation and differentiation in Dupuytren's cell cultures. Drugs with antiproliferative action may be valuable in DD treatment. Through characterization of changes on DD-specific cells, we, therefore, decided to test the therapeutic potential of new cytostatic drugs for DD treatment and/or for reduction of post-operative recurrence rates. The N-sulfonylpyrimidine derivative, amidino-substituted benzimidazo[1,2-a]quinoline, and amidino dihydrothienothienyl[2,3-c]quinolone hydrochloride, known to affect proliferation processes, were tested for their antiproliferative activity on primary fibroblasts/myofibroblasts cell cultures derived from the palmar fascia of patients with DD. Only amidino dihydrothienothienyl[2,3-c]quinolone hydrochloride acted in a highly specific manner on cells derived from diseased fascia of DD patients and exhibited a low cytotoxic effect. This result might be a consequence of its specific activity on cytoskeleton changes occurring in differentiating cells. A similar short-term differential antiproliferative effect was observed by the N-sulfonylpyrimidine derivative that was, however, completely lost after 6- and 14-day treatments. The amidino-substituted benzimidazo[1,2-a]quinoline exerted a strong non-specific, dose-related antiproliferative activity on cell types.