Biological activity of (Rheum L.) secondary metabolites - assessment of the effect of Rheum rhabarbarum and Rheum rhaponticum extracts on selected plasma and cell components of the haemostatic system in vitro

Choroby układu sercowo-naczyniowego są główną przyczyną śmiertelności w krajach rozwiniętych. Stąd też rośnie zainteresowanie opracowaniem nowych terapii profilaktycznych i leczniczych, pomocnych w zwalczaniu tych schorzeń. W grupie środków leczniczych przydatnych w działaniach profilaktycznych i terapii chorób cywilizacyjnych, coraz większą rolę odgrywają preparaty pochodzenia roślinnego. Rodzaj Rheum L. (rzewień, rabarbar) od dawna jest znany w różnych regionach świata, nie tylko jako źródło gatunków jadalnych, ale także jako ceniony surowiec zielarski. Wiele aspektów działania tych roślin na organizm człowieka pozostaje jednak słabo opisanych, a ich stosowanie w wielu przypadkach opiera się jedynie na danych pochodzących z medycyny tradycyjnej. Celem prowadzonych badań była ocena wpływu ekstraktów z dwóch gatunków rzewienia - Rheum rhaponticum L. oraz Rheum rhabarbarum L. oraz typowych dla tych roślin stilbenów na funkcjonowanie wybranych osoczowych i komórkowych składników układu hemostazy. Badania skupiały się na aktywnościach biologicznych wskazywanych jako jedne z kluczowych dla kardioprotekcyjnego działania substancji naturalnych i obejmowały działanie przeciwzapalne, antykoagulacyjne/przeciwzakrzepowe oraz przeciwutleniające. Oceniano efektywność działania ekstraktów (frakcje butanolowe) z korzeni oraz ogonków liściowych wyżej wspomnianych gatunków rzewienia oraz obecnych tych roślinach pochodnych stilbenu: rapontygeniny i rapontycyny. Plan badań obejmował zastosowanie modeli eksperymentalnych ściśle związanych z fizjologią układu sercowo-naczyniowego i hemostazą, tj. komórek śródbłonka ściany naczyniowej, leukocytów oraz osoczowych składników układu hemostazy (w tym kluczowych białek kaskady krzepnięcia krwi oraz układu fibrynolitycznego). W celu uzyskania jak najszerszego obrazu aktywności biologicznej badanych substancji, zastosowano zróżnicowany panel metod analitycznych: badania ekspresji genów, testy skriningowe inhibitorów, oznaczenia spektrofotometryczne i fluorymetryczne, kinetyczną analizę aktywności enzymów, monitorowanie procesu krzepnięcia osocza krwi (oznaczenia turbidymetryczne), testy ELISA, mikroskopię fluorescencyjną, elektroforezę 1D oraz profilowanie wielocytokinowe oparte na technice blottingu. Badane substancje roślinne zmniejszały odpowiedź zapalną komórek śródbłonka (HUVECs), działając na różnych poziomach molekularnych (modulacji ekspresji genów, hamowania uwalniania czynników prozapalnych oraz redukcji adhezyjności komórek). Odnotowano istotny spadek uwalniania markerów/białek odpowiedzi zapalnej (np. TNF-α, 25 IL- 2, MMP-9). Wykazano również właściwości antykoagluacyjne badanych ekstraktów, co może przekładać się na efekt przeciwzakrzepowy. Ekstrakty z rzewienia hamowały proces krzepnięcia krwi indukowany czynnikiem tkankowym, stanowiącym główny fizjologiczny szlak aktywacji hemostazy osoczowej. Analizy bezpośredniego wpływu badanych substancji roślinnych na kluczowe proteazy serynowe kaskady krzepnięcia: trombinę i czynnik Xa wskazują, że obserwowany efekt antykoagulacyjny może być wynikiem inhibicji tych czynników. Ponadto, po raz pierwszy wykazano również zdolność badanych ekstraktów z rzewienia i stilbenów do ograniczania uszkodzeń składników osocza, wywołanych stresem oksydacyjnym. Przeprowadzone badania dostarczyły nowych informacji na temat właściwości biologicznych metabolitów rzewienia w kontekście kardioprotekcyjnym, a uzyskane wyniki mogą stanowić podstawę do dalszych prac badawczych.Cardiovascular diseases are the leading cause of mortality in developed countries. Hence, there is a growing interest in the development of new preventive and therapeutic therapies to help combat these diseases. In the group of agents useful in prophylaxis and therapy of civilization diseases, preparations of plant origin play an increasingly important role. The Rheum L. (rhubarb) genus has long been known in various regions of the world, not only as a source of edible species, but also as a valuable herbal material. However, many aspects of the effects of these plants on the human body remain poorly described, and in numerous cases, their use is still based only on data derived from traditional medicine. The aim of this study was to assess the effect of extracts from two species of rhubarb - Rheum rhaponticum L. and Rheum rhabarbarum L. and stilbenes typical for these plants on functions of selected plasma and cellular components of the haemostatic system. The work was focused on biological activities indicated as the key to the cardioprotective effect of natural substances, and included anti-inflammatory, anticoagulant/antithrombotic and antioxidant effects. Effectiveness of extracts (butanol fractions) from the roots and petioles of the above-mentioned rhubarb species and the stilbene derivatives that are present in these plants, i.e. rhapontigenin and rhaponticin, was evaluated. The study design included the use of experimental models closely related to the physiology of the cardiovascular system and haemostasis, i.e. endothelial cells, leukocytes and plasma components of the haemostasis system (including key proteins of the blood coagulation cascade and the fibrinolytic system). To obtain the most comprehensive insight into the biological activity of the examined substances, a diverse panel of analytical methods was used: gene expression studies, inhibitor screening tests, spectrophotometric and fluorimetric assays, kinetic analysis of enzyme activity, monitoring of the blood plasma coagulation process (turbidimetric assays), ELISA, fluorescence microscopy, 1D-electrophoresis and the blotting-based multicytokine profiling. The examined plant substances reduced the inflammatory response of endothelial cells (HUVECs), acting at various molecular levels (modulation of gene expression, inhibition of the release of pro-inflammatory factors and reduction of cell adhesion). There was a significant decrease in the release of inflammatory response markers/proteins (e.g. TNF-α, IL-2, MMP-9). The anticoagulant properties of the examined extracts were also demonstrated, which may result in an antithrombotic effect. Rhubarb extracts inhibited the blood plasma coagulation process, induced by the tissue factor, which is the main physiological pathway of 27 the blood plasma hemostasis activation. Analyzes of direct effects of the examined plant substances on the key serine proteases of the coagulation cascade: thrombin and factor Xa indicated that the observed anticoagulant effect might be a result of inhibition of these factors. Furthermore, for the first time, the ability of the investigated rhubarb extracts and stilbenes to reduce the oxidative stress-induced damage to plasma components was demonstrated as well. The executed experiments provided new information on the biological properties of rhubarb metabolites in the cardioprotective context, and the obtained results may be a basis for further research.NCN: UMO-2018/31/B/NZ9/0123

A Review on Rhubarb-Derived Substances as Modulators of Cardiovascular Risk Factors—A Special Emphasis on Anti-Obesity Action

The currently available anti-obesity therapies encounter many associated risks and side effects often causing the ineffectiveness of treatment. Therefore, various plant-derived substances have been extensively studied as a promising support or even an alternative for existing anti-obesity therapies. This review is dealing with the anti-obesity potential of edible and ethnomedicinal rhubarb species and emerging possible role of the rhubarb-derived extracts or individual compounds in the prevention of obesity and perspectives for their use in an anti-obesity treatment. A special emphasis is put on the most popular edible specimens, i.e., Rheum rhabarbarum L. (garden rhubarb) and Rheum rhaponticum L. (rhapontic rhubarb, Siberian rhubarb); however, the anti-obesity potential of other rhubarb species (e.g., R. officinale, R. palmatum, and R. emodi) is presented as well. The significance of rhubarb-derived extracts and low-molecular specialized rhubarb metabolites of diversified chemical background, e.g., anthraquinones and stilbenes, as potential modulators of human metabolism is highlighted, including the context of cardiovascular disease prevention. The available reports present multiple encouraging rhubarb properties starting from the anti-lipidemic action of rhubarb fibre or its use as purgative medicines, through various actions of rhubarb-derived extracts and their individual compounds: inhibition of enzymes of cholesterol and lipid metabolism, targeting of key molecular regulators of adipogenesis, regulators of cell energy metabolism, the ability to inhibit pro-inflammatory signalling pathways and to regulate glucose and lipid homeostasis contributing to overall in vivo and clinical anti-obesity effects

Valorization of the Photo-Protective Potential of the Phytochemically Standardized Olive (Olea europaea L.) Leaf Extract in UVA-Irradiated Human Skin Fibroblasts

Leaves of Olea europaea are a by-product of the olive oil industry and a dietary supplement with acknowledged antioxidant and anti-inflammatory activity but underestimated photoprotective potential. We investigated the protective effects of the LC-PDA-MS/MS standardized ethanol-water extract of olive leaves (OLE), containing 26.2% total phenols and 22.2% oleuropein, with underlying mechanisms against the UVA-induced oxidative damage in human dermal fibroblasts. Hs68 cells were pre-treated (24 h) with OLE (2.5–25 μg/mL) or the reference antioxidants, quercetin and ascorbic acid (25 μg/mL), followed by irradiation (8 J/cm2). OLE significantly reduced the UVA-induced DNA damage and reactive oxygen species (ROS) overproduction and increased the thioredoxin reductase (TrxR) expression and post-radiation viability of fibroblasts by inhibiting their apoptosis. Both intrinsic and extrinsic apoptotic signaling pathways appeared to be inhibited by OLE, but the activity of caspase 9 was the most reduced. We hypothesized that the TrxR up-regulation by OLE could have prevented the UVA-induced apoptosis of Hs68 cells. In addition, a significant decrease in UVA-induced secretion levels of tumor necrosis factor (TNF-α) and interleukin-2 (IL-2) was shown in human lymphocyte culture in response to OLE treatment. In summary, our results support the beneficial effect of OLE in an in vitro model and indicate its great potential for use in the cosmetic and pharmaceutical industry as a topical photoprotective, antioxidant, and anti-inflammatory agent

Table1_Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro.DOCX

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs).Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation.Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1–50 μg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts’ constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts’ biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs).Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin’s enzymatic activity, reducing its amidolytic and proteolytic functions at 50 μg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 μg/mL) induce alterations within the α-helices’ secondary structure, resulting in noticeable changes in the enzyme’s CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed.Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.</p