Hypericum alpestre extract affects the activity of the key antioxidant enzymes in microglial BV-2 cellular models

Javrushyan H., Ginovyan M., Harutyunyan T., Gevorgyan S., Karabekian Z., Maloyan A., Avtandilyan N.

Plants of the Hypericaceae family have been traditionally used for their medicinal properties, including antibacterial, antiviral, and antioxidant activities. Among these, Hypericum alpestre (HA) extracts have shown notable cytotoxicity against various cancer cell lines, drawing attention to their phenolic compounds as potential anticancer agents. Similarly, N(G)-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) activity, has emerged as a promising candidate in cancer therapy. However, the precise molecular mechanisms underlying the anticancer effects of both HA and L-NAME remain unclear. This study aimed to clarify the impact of HA and L-NAME on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway in A549 human lung adenocarcinoma and MDA-MB-231 breast cancer triple-negative cells, with particular emphasis on the tumor necrosis factor-alpha (TNFα)/cyclooxygenase-2 (COX-2) and vascular endothelial growth factor A (VEGFα)/matrix metalloproteinase-2 (MMP-2) pathways. In silico analyses identified compounds within HA extracts with the highest affinity for PI3K/Akt, a finding subsequently confirmed by in vitro experiments. Notably, the combination of HA and L-NAME demonstrated greater efficacy than the combination of HA and 5-fluorouracil (5-FU), as evidenced by enhanced apoptotic activity. Both HA alone and in combination with L-NAME inhibited the TNFα/COX-2 and VEGFα/MMP-2 pathways. These results suggest that the therapeutic effects of HA, especially in combination with L-NAME, may be mediated through the PI3K/Akt signaling pathway. A better understanding of the interaction between HA polyphenols and PI3K/Akt signaling could pave the way for novel therapeutic strategies against cancer, including drug-resistant tumors.

Minasyan A., Pires V., Gondcaille C., Ginovyan M., Mróz M., Savary S., Cherkaoui-Malki M., Kusznierewicz B., Bartoszek A., Andreoletti P., Sahakyan N.

This study focuses on the investigation of the antioxidant and anti-inflammatory activities of alcohol extracts from Ribes nigrum leaves on murine BV-2 microglial Wt and Acyl-CoA oxidase 1 deficient (Acox1−/−) cell line models, useful for the investigation of some neurodegenerative disorders. The extract chemical composition was analyzed via LC-Q-Orbitrap HRMS. Various assays, including DPPH, MTT, and H2DCFDA, were used to assess the extract’s antioxidant capacity, cell viability, and reactive oxygen species (ROS) production. Immunoblotting and RT-qPCR techniques were employed to measure protein expression and gene transcription in treated cells. Statistical analysis was conducted using GraphPad Prism, with significance determined at p < 0.05. Investigations showed the presence of phenolic compounds in this extract, among which flavan-3-ols, flavonols, furanocoumarins, hydroxycinnamates were major components, which are known for their biological activity in various test systems. The MTT test revealed a concentration of 0.125 mg/mL of R. nigrum extract as the highest non-toxic. The investigated extract showed high antioxidant activity in chemical-based tests. The antioxidant potential of the R. nigrum leaf extract was furtherly explored using the BV-2 microglial cell line models. Moreover, the extract was found to alter the activity of the main antioxidant enzyme, catalase and fatty acid oxidation enzyme, Acyl-CoA oxidase 1 (ACOX1) as well as the expression of appropriate genes in Wt and Acox1−/− BV-2 microglial cells such as Cat, iNos, Il-1β, Tnf-α, and Abcd1. In Wt cells, after the 24-hour treatment with R. nigrum leaf extract, ACOX1 activity was downregulated, meanwhile the catalase activity remains unchanged. Further treatment led to the downregulation of catalase and the upregulation of ACOX1 activity. However, in Acox1−/− cells, which represent a model of oxidative stress, an increase in catalase activity was observed only after 48 h of treatment. It was also observed the reduced ROS and NO formation in cells, showing the pronounced antioxidant capacity of R. nigrum extract in the investigated cell-models. Our study demonstrated the protective effects of R. nigrum leaf extracts on BV-2 microglial cells by reducing oxidative and nitrosative stress, decreasing pro-inflammatory gene expression, and normalizing peroxisomal function, highlighting the potential of these extracts as therapeutic agents for managing oxidative stress and inflammation.

Ayvazyan A., Zidorn C.

The rich and diverse flora of Armenia has been used for medicinal purposes for at least 3000 years. The relevant literature in Armenian, English, and Russian revealed a vast array of used medicinal plants, some of them unique to the Caucasus region. The usage of medicinal plants confirms the position of Armenia as a country at the crossroads of Asia and Europe because of its traditional usage of medicinal plants from both continents. Literature data in Armenian, English, and Russian on medicinal plants of Armenia were mainly obtained using various electronic databases. From all available sources, 320 Armenian medicinal plant species were extracted with their botanical and local names and traditional uses. The use of medicinal plants by the Armenian people is systematically compiled, including the used plant organs and preparations and the ailments for which the various taxa are/were used. Medicinal plants of Armenia are represented for both wild and cultivated species. Some of the taxa used are unique to Armenia or the Caucasus region, while many other species are also used in various other countries. Some of the species from traditional Armenian medicine are currently being studied using modern methods.

Minasyan A., Pires V., Gondcaille C., Savary S., Cherkaoui-Malki M., Andreoletti P., Sahakyan N.

Abstract This study focuses on the investigation of the antioxidant and anti-inflammatory activity of extracts from Ribes nigrum leaves on BV-2 microglial Wt and Acyl-CoA oxidase 1 deficient (Acox1−/−) cell line models, useful for the investigation of some neurodegenerative disorders. Investigations showed the presence of high quantity of phenolic compounds in this extract, among which flavan-3-ols, flavonols, furanocoumarins, hydroxycinnamates, quercetin, and quercetin derivatives were major components, which are known for their biological activity in various test systems. The MTT test revealed the 0.125 mg/mL concentration of R. nigrum extract as the highest non-toxic. The investigated extract showed high antioxidant activity in chemical-based tests. The antioxidant potential of the R. nigrum leaf extract was furtherly explored using the BV-2 microglial cell line models. Moreover, the extract was found to alter the activity of the main antioxidant enzyme, catalase and fatty acid oxidation enzyme, Acyl-CoA oxidase 1 (ACOX1) as well as the expression of appropriate genes in Wt and Acox1−/− BV-2 microglial cells such as Cat, iNos, Il-1β, Tnf-α, and Abcd1. In Wt cells, the treatment with R. nigrum leaf extract ACOX1 activity was downregulated after 24 hours of treatment, meanwhile the catalase activity remains unchanged. The further treatment leaded to the downregulation of catalase and the upregulation of ACOX1 activity. However, in Acox1−/− cells, which represent a model of oxidative stress, an increase in catalase activity was observed only after 48 hours of treatment, indicating an alternative mode of action of the extract. It was also observed the reduced ROS and NO formation in cells, indicating the pronounced antioxidant capacity of R. nigrum extract in the investigated cell-models.

Javrushyan H., Ginovyan M., Petrosyan G., Qocharyan M., Harutyunyan T., Gevorgyan S., Karabekian Z., Maloyan A., Avtandilyan N.

AbstractPlants within theHypericaceaefamily have been traditionally used for their medicinal properties, showcasing a wide range of effects such as antibacterial, antiviral, and antioxidant qualities.Hypericum alpestre(HA) extracts have exhibited significant cytotoxicity against various cancer cell lines. The phenolic compounds found in HA extracts have attracted attention for their potential in cancer prevention. L-NAME, known for its ability to inhibit nitric oxide synthase (NOS) activity, has emerged as a promising approach in cancer therapy. However, the precise molecular mechanisms underlying the anticancer effects of HA and L-NAME remain unclear. This study aims to clarify the impact of HA and L-NAME on the PI3K/Akt signaling pathway in A549 lung adenocarcinoma cells, with a specific focus on TNFa/COX-2 and VEGFa/MMP-2 pathways.In silicoanalysis, they identified the compounds with the highest affinity for PI3K/Akt, a finding validated by subsequentin vitroexperiments. Furthermore, the combination of herbs and L-NAME exhibited superior efficacy compared to the herb and 5-FU combination, as evidenced by the promotion of apoptosis. Both the herb alone and the combination of the herb with L-NAME demonstrated inhibitory effects on the TNFa/COX-2 and VEGFa/MMP-2 pathways. This therapeutic approach is hypothesized to operate through the PI3k/Akt cell signaling pathway. A better understanding of the interaction between HA polyphenols and PI3K/Akt signaling could pave the way for novel therapeutic strategies against cancer, including drug-resistant tumors.

Sahakyan N., Nasim M.J., Jacob C.

Oxidative stress, characterized by an imbalance between reactive oxygen species production and the body’s antioxidant defence mechanisms, has been implicated in the pathogenesis of numerous diseases. This review highlights the possible mechanisms of influence of natural polymeric redox modulators in human organism as well as an importance of supplementation with these metabolites as a promising tool to counteract oxidative stress. The first part provides an overview of oxidative stress and its role in the development of various pathological conditions. The second part of the manuscript focuses on natural redox modulators, highlighting their unique properties and diverse sources, including plant-based bioactive polymers. The third section explores the potential therapeutic applications of natural polymeric redox modulators in oxidative stress–related diseases. Furthermore, the potential synergistic effects of natural redox modulators with conventional therapies have also been discussed, suggesting their value in combination treatments. The review article also addresses the challenges and opportunities in the field of natural polymeric redox modulator supplementation, including enhanced bioavailability, stability and safety considerations. Future directions and emerging research areas, such as personalized supplementation, are also explored, underscoring the need for further investigation and optimization of these compounds. Supplementation with natural polymeric redox modulators represents a promising strategy to combat oxidative stress–related diseases. Their unique properties, diverse sources and potential synergistic effects open avenues for novel therapeutic interventions and improved patient outcomes. According to the current-stage investigation, further research and clinical trials are warranted to harness the full potential of these natural compounds.

Ginovyan M., Javrushyan H., Karapetyan H., Koss‐Mikołajczyk I., Kusznierewicz B., Grigoryan A., Maloyan A., Bartoszek A., Avtandilyan N.

AbstractConventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. This study aims to investigate the antitumor capacity of Hypericum alpestre (H. alpestre) extract in vitro and in vivo, either alone or in combination with the inhibitors of the  l‐arginine/polyamine/nitric oxide (NO) pathway, and to characterize its active phytochemicals using advanced chromatographic techniques. Our previous reports suggest beneficial effects of the arginase inhibitor NG‐hydroxy‐nor‐ l‐arginine and NO inhibitor NG‐nitro‐Larginine methyl ester in the treatment of breast cancer via downregulation of polyamine and NO synthesis. Here, the antitumor properties of H. alpestre and its combinations were explored in vivo, in a rat model of mammary gland carcinogenesis induced by subcutaneous injection of 7,12‐dimethylbenz[a]anthracene. The study revealed strong antiradical activity of H. alpestre aerial part extract in chemical (DPPH/ABTS) tests. In the in vitro antioxidant activity test, the H. alpestre extract demonstrated pro‐oxidant characteristics in human colorectal (HT29) cells, which were contingent upon the hemostatic condition of the cells. The H. alpestre extract expressed a cytotoxic effect on HT29 and breast cancer (MCF‐7) cells measured by the MTT test. According to comet assay results, H. alpestre extract did not exhibit genotoxic activity nor possessed antigenotoxic properties in HT29 cells. Overall, 233 substances have been identified and annotated in H. alpestre extract using the LC‐Q‐Orbitrap HRMS system. In vivo experiments using rat breast cancer models revealed that the H. alpestre extract activated the antioxidant enzymes in the liver, brain, and tumors. H. alpestre combined with chemotherapeutic agents attenuated cancer‐like histological alterations and showed significant reductions in tumor blood vessel area. Thus, either alone or in combination with Nω‐OH‐nor‐ l‐arginine and Nω‐nitro‐ l‐arginine methyl ester, H. alpestre extract exhibits pro‐ and antioxidant, antiangiogenic, and cytotoxic effects.

Ginovyan M., Javrushyan H., Karapetyan H., Koss-Mikołajczyk I., Kusznierewicz B., Grigoryan A., Maloyan A., Bartoszek A., Avtandilyan N.

Abstract Background: Conventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. Particularly, much attention is given to finding combined phytochemical/chemotherapeutic treatment models to overcome drug resistance and decrease side effects. Aim of the study: The aim was to investigate the antitumor capacity of Hypericum alpestre herb extract in vitro and in vivo, either alone or combined with the inhibitors of the L-arginine/polyamine/nitric oxide pathway and characterize its active phytochemicals using advanced chromatographic techniques. Methods: The antioxidant capacity of H. alpestre extract was assessed through chemical spectrophotometric tests (DPPH and ABTS) and in biological systems using Cellular Antioxidant Activity assay. The inhibitory effect of H. alpestre extract on the growth of human colorectal (HT29) and breast cancer (MCF-7) cell cultures was explored by the MTT test. The genotoxicity of the tested extract was studied using a comet assay. In vivo, the antitumor properties of H. alpestre and its combinations were explored in a rat mammary gland carcinogenesis model induced by subcutaneous injection of 7,12-dimethylbenz[a]anthracene. The polyphenolic substances present in H. alpestre extract have been characterized using the LC-Q-Orbitrap HRMS system. Results: The H. alpestre extract expressed promising antiproliferative effects on MCF-7 and HT29 cells. The extract did not exhibit genotoxic activity nor possessed antigenotoxic properties. The in vivo rat mammary carcinogenesis model data showed that the H. alpestre extract stimulated the activity of antioxidant enzymes in the liver, brain, and tumors of rats in the experimental groups, demonstrating its antioxidant protective effects. The herb alone and in combination with Nω-OH-nor-L-arginine and Nω-nitro-L-arginine methyl ester exhibited pro-/antioxidant, antiproliferative, anti-angiogenic, and cytotoxic effects. Conclusion: H. alpestre extract alone and combined with L-arginine metabolic regulatory compounds, demonstrates significant potential for the development of novel therapeutic models.

Sahakyan G., Vejux A., Sahakyan N.

Diabetic nephropathy is manifested in more than 10% of people with diabetes. It is a common cause of kidney failure and end-stage kidney disease. Understanding of mechanisms underlying the initiation and development of diabetes-induced kidney injuries will allow for the development of more effective methods of prevention and treatment of the disease. Diabetic nephropathy is a wide-ranging complication of diabetes, and it is necessary to discuss the “weight” of pro-inflammatory pathways and molecules in the progress of renal injuries during the development of the disease. A large spectrum of pro-inflammatory molecules and pathways participate in different stages of the pathophysiological progression of diabetic nephropathy, including pro-inflammatory cytokines, chemokines, their receptors, adhesion molecules, and transcription factors. On the other hand, it is known that one of the consequences of hyperglycemia-induced ROS generation is the up-regulation of pro-inflammatory cascades, which, in turn, activate the transcription of genes encoding cytokines-chemokines, growth factors, and extracellular matrix proteins. It is a proven fact that a variety of plant secondary metabolites, such as tannins, flavonoids, and other polyphenols, demonstrate significant anti-diabetic, redox-modulating properties and effectively modulate the inflammatory response. Thus, this review is discussing the possible role of plant phenols in the prevention and treatment of diabetic nephropathy.