An overview on the cellular mechanisms of anthocyanins in maintaining intestinal integrity and function.

Structural and functional changes of the intestinal barrier, as a consequence of a number of (epi)genetic and environmental causes, have a main role in penetrations of pathogens and toxic agents, and lead to the development of inflammation-related pathological conditions, not only at the level of the GI tract but also in other extra-digestive tissues and organs. Anthocyanins (ACNs), a subclass of polyphenols belonging to the flavonoid group, are well known for their health-promoting properties and are widely distributed in the human diet. There is large evidence about the correlation between the human intake of ACN-rich products and a reduction of intestinal inflammation and dysfunction. Our review describes the more recent advances in the knowledge of cellular and molecular mechanisms through which ACNs can modulate the main mechanisms involved in intestinal dysfunction and inflammation, in particular the inhibition of the NF-κB, JNK, MAPK, STAT3, and TLR4 proinflammatory pathways, the upregulation of the Nrf2 transcription factor and the expression of tight junction proteins and mucins.

Low concentrations of antimony impair adipogenesis and endoplasmic reticulum homeostasis during 3T3-L1 cells differentiation.

Antimony (Sb) is a metalloid widely present in plastics used for food contact packaging, toys and other household items. Since Sb can be released by these plastics and come into contact with humans, health concerns have been highlighted. The effect of Sb on human tissues is yet controversial, and biochemical mechanisms of toxicity are lacking. In the present study, the effect of very low nanomolar concentrations of Sb(III), able to mimicking chronic human exposure, was evaluated in 3T3-L1 murine cells during the differentiation process. Low nanomolar Sb exposure (from 0.05 to 5 nM) induced lipid accumulation and a marked increase in C/EBP-ß and PPAR-γ levels, the master regulators of adipogenesis. The Sb-induced PPAR-γ was reverted by the estrogen receptor antagonist ICI 182,780. Additionally, Sb stimulated preadipocytes proliferation inducing G2/M phase of cell cycle and this effect was associated to reduced cell-cycle inhibitor p21 levels. In addition to these metabolic dysfunctions, Sb activated the proinflammatory NF-κB pathway and altered endoplasmic reticulum (ER) homeostasis inducing ROS increase, ER stress markers XBP-1s and pEIF2a and downstream genes, such as Grp78 and CHOP. This study, for the first time, supports obesogenic effects of low concentrations exposure of Sb during preadipocytes differentiation.

Oncogenic IDH mutations increase heterochromatin-related replication stress without impacting homologous recombination.

Oncogenic mutations in isocitrate dehydrogenases 1 and 2 (IDH1/2) produce 2-hydroxyglutarate (2HG), which inhibits dioxygenases that modulate chromatin dynamics. The effects of 2HG have been reported to sensitize IDH tumors to poly-(ADP-ribose) polymerase (PARP) inhibitors. However, unlike PARP-inhibitor-sensitive BRCA1/2 tumors, which exhibit impaired homologous recombination, IDH-mutant tumors have a silent mutational profile and lack signatures associated with impaired homologous recombination. Instead, 2HG-producing IDH mutations lead to a heterochromatin-dependent slowing of DNA replication accompanied by increased replication stress and DNA double-strand breaks. This replicative stress manifests as replication fork slowing, but the breaks are repaired without a significant increase in mutation burden. Faithful resolution of replicative stress in IDH-mutant cells is dependent on poly-(ADP-ribosylation). Treatment with PARP inhibitors increases DNA replication but results in incomplete DNA repair. These findings demonstrate a role for PARP in the replication of heterochromatin and further validate PARP as a therapeutic target in IDH-mutant tumors.

Hypoxia Potentiates the Inflammatory Fibroblast Phenotype Promoted by Pancreatic Cancer Cell-Derived Cytokines.

Cancer-associated fibroblasts (CAF) are a major cell type in the stroma of solid tumors and can exert both tumor-promoting and tumor-restraining functions. CAF heterogeneity is frequently observed in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by a dense and hypoxic stroma that features myofibroblastic CAFs (myCAF) and inflammatory CAFs (iCAF) that are thought to have opposing roles in tumor progression. While CAF heterogeneity can be driven in part by tumor cell-produced cytokines, other determinants shaping CAF identity and function are largely unknown. In vivo, we found that iCAFs displayed a hypoxic gene expression and biochemical profile and were enriched in hypoxic regions of PDAC tumors, while myCAFs were excluded from these regions. Hypoxia led fibroblasts to acquire an inflammatory gene expression signature and synergized with cancer cell-derived cytokines to promote an iCAF phenotype in a HIF1α-dependent fashion. Furthermore, HIF1α stabilization was sufficient to induce an iCAF phenotype in stromal cells introduced into PDAC organoid cocultures and to promote PDAC tumor growth. These findings indicate hypoxia-induced HIF1α as a regulator of CAF heterogeneity and promoter of tumor progression in PDAC. SIGNIFICANCE: Hypoxia in the tumor microenvironment of pancreatic cancer potentiates the cytokine-induced inflammatory CAF phenotype and promotes tumor growth. See related commentary by Fuentes and Taniguchi, p. 1560.

Cyanidin-3-O-glucoside protects intestinal epithelial cells from palmitate-induced lipotoxicity.

CONTEXT: Increased free fatty acids (FFAs) levels, typical in obesity condition, can contribute to systemic lipotoxicity and inflammation adversely influencing Inflammatory Bowel Disease development and progression. Anthocyanins possess health promoting properties mainly associated to the induction of Nrf2-regulated cytoprotective proteins. OBJECTIVE: Using a novel experimental model, we evaluated the in vitro intracellular mechanisms involved in FFAs modulation of intestinal epithelial lipotoxicity and the protective effects of cyanidin-3-O-glucoside (C3G) in Caco-2 cells. RESULTS: Caco-2 exposed to palmitic acid (PA) in the serosal (basolateral) side showed a combined state of epithelial inflammation, inducing NF-κB pathway and downstream cytokines, that was reverted by C3G apical pre-treatment. In addition, PA altered intracellular redox status and induced reactive oxygen species that were reduced by C3G via the redox-sensitive Nrf2 signalling. DISCUSSION AND CONCLUSION: Results suggest that anti-inflammatory properties of anthocyanins, mediated by Nrf2, could represent an interesting tool for intestinal inflammatory disorders.

Phenolic Profiles, Antioxidant and Anti-Inflammatory Activities of Hydrodistillation Wastewaters from Five Lamiaceae Species.

Distillation is the most widely used method to obtain an essential oil from plant material. The biomass used in the process is returned as a solid residue together with variable amounts of water rich in water-soluble compounds, which currently are not addressed to any further application. The scope of this work was to evaluate the phytochemical composition of wastewaters coming from hydrodistillation (DWWs) of five aromatic plants belonging to the Lamiaceae family, and to assess their in vitro antioxidant and anti-inflammatory activities. The phenolic profiles of the DWWs were determined by HPLC-DAD and HPLC-ESI/MS. Free radical scavenging ability, oxygen radical antioxidant capacity and superoxide dismutase mimetic activity of the samples under study were measured. Moreover, to investigate the anti-inflammatory activity of the DWWs, an in vitro experimental model of intestinal inflammation was used. The DWW samples' phytochemical analysis allowed the identification of 37 phenolic compounds, all exhibiting good antioxidant and anti-inflammatory activity. Our study contributes to the knowledge on the polyphenolic composition of the DWWs of five aromatic plants of the Lamiaceae family. The results highlight the presence of compounds with proven biological activity, and therefore of great interest in the pharmaceutical and nutraceutical fields.

The p-Phthalates Terephthalic Acid and Dimethyl Terephthalate Used in the Manufacture of PET Induce In Vitro Adipocytes Dysfunction by Altering Adipogenesis and Thermogenesis Mechanisms.

Public health concerns associated with the potential leaching of substances from Polyethylene terephthalate (PET) packaging have been raised due to the role of phthalates as endocrine-disrupting chemicals or obesogens. In particular, changes in the environment such as pH, temperature, and irradiation can improve contaminant migration from PET food packaging. In this study, the in vitro effects of p-phthalates terephthalic acid (TPA) and dimethyl terephthalate (DMT) on murine adipocytes (3T3-L1) were evaluated using concentrations that might be obtained in adult humans exposed to contaminated sources. TPA and, in particular, DMT exposure during 3T3-L1 differentiation increased the cellular lipid content and induced adipogenic markers PPAR-γ, C/EBPß, FABP4, and FASN, starting from low nanomolar concentrations. Interestingly, the adipogenic action of TPA- and DMT-induced PPAR-γ was reverted by ICI 182,780, a specific antagonist of the estrogen receptor. Furthermore, TPA and DMT affected adipocytes' thermogenic program, reducing pAMPK and PGC-1α levels, and induced the NF-κB proinflammatory pathway. Given the observed effects of biologically relevant chronic concentrations of these p-phthalates and taking into account humans' close and constant contact with plastics, it seems appropriate that ascertaining safe levels of TPA and DMT exposure is considered a high priority.

Anti-Inflammatory Activity of an In Vitro Digested Anthocyanin-Rich Extract on Intestinal Epithelial Cells Exposed to TNF-α.

BACKGROUND: The consumption of foods rich in anthocyanins (ACN) have been associated with beneficial properties in chronic inflammatory disorders such as intestinal bowel diseases (IBD). These effects were attributed not only to a direct antioxidant mechanism but also to the modulation of cell redox-dependent signaling. However, ACN bioavailability is low for their poor stability in the digestive tract, so ACN gastrointestinal digestion should be considered. METHODS: To have a more realistic knowledge of the effects of ACN, we performed an in vitro simulated gastrointestinal digestion of an ACN-rich purified and standardized bilberry and blackcurrant extract (BBE), followed by an evaluation of ACN composition modification (HPLC-DAD and pH differential method) and antioxidant activity (FRAP assay). Then, we studied the effects of BBE gastrointestinal extract on Caco-2 exposed to TNF-α. RESULTS: The results confirmed the high instability of ACN in the mild alkaline environment of the small intestine (17% recovery index). However, the digested BBE maintained part of its bioactivity. Additionally, BBE gastrointestinal extract inhibited the TNF-α-induced NF-κB pathway in Caco-2 and activated the Nrf2 pathway. CONCLUSIONS: Although ACN stability is affected by gastrointestinal digestion, the anti-inflammatory and antioxidant activity of digested extracts were confirmed; thus, the loss of ACN can probably be counterweighed by their metabolites. Then, ACN introduced by diet or food supplements could represent an approach for IBD prevention.

Recent Advances in Glycyrrhetinic Acid-Functionalized Biomaterials for Liver Cancer-Targeting Therapy.

Liver cancer is one of the most common causes of cancer mortality worldwide. Chemotherapy and radiotherapy are the conventional therapies generally employed in patients with liver tumors. The major issue associated with the administration of chemotherapeutics is their high toxicity and lack of selectivity, leading to systemic toxicity that can be detrimental to the patient's quality of life. An important approach to the development of original liver-targeted therapeutic products takes advantage of the employment of biologically active ligands able to bind specific receptors on the cytoplasmatic membranes of liver cells. In this perspective, glycyrrhetinic acid (GA), a pentacyclic triterpenoid present in roots and rhizomes of licorice, has been used as a ligand for targeting the liver due to the expression of GA receptors on the sinusoidal surface of mammalian hepatocytes, so it may be employed to modify drug delivery systems (DDSs) and obtain better liver or hepatocyte drug uptake and efficacy. In the current review, we focus on the most recent and interesting research advances in the development of GA-based hybrid compounds and DDSs developed for potential employment as efficacious therapeutic options for the treatment of hepatic cancer.

In Vitro Protective Effects of a Standardized Extract From Cynara Cardunculus L. Leaves Against TNF-α-Induced Intestinal Inflammation.

Inflammatory bowel disease (IBD) represents a group of progressive disorders characterized by recurrent chronic inflammation of the gut. New unconventional therapies based on plant derived compounds capable of preventing and/or reducing acute or chronic inflammation could represent a valid alternative for the treatment or prevention of IBDs. Cynara cardunculus L. leaves, considered a food-waste suitable as a rich source of bioactive polyphenols including luteolin and chlorogenic acid, has been reported for its positive effects in digestive tract. The aim of the present work was to evaluate the in vitro molecular mechanisms of beneficial effects of a standardized polyphenol-rich extract obtained from the leaves of Cynara cardunculus L (CCLE) against acute intestinal inflammation induced by TNF-α on intestinal epithelial Caco-2 cells. CCLE prevented TNF-α-induced NF-κB inflammatory pathway and the overexpression of IL-8 and COX-2. In addition, CCLE was able to improve basal intracellular antioxidant power in both TNF-α-unexposed or -exposed Caco-2 cells and this effect was associated to the activation of Nrf2 pathway, a master regulator of redox homeostasis affecting antioxidant and phase II detoxifying genes, stimulating an adaptive cellular response. In conclusion, our data clearly evidenced that, although considered a waste, Cynara cardunculus leaves may be used to obtain extracts rich in bioactive polyphenols potentially useful for prevention and treatment of inflammatory intestinal diseases.

A pinitol-rich Glycyrrhiza glabra L. leaf extract as functional supplement with potential in the prevention of endothelial dysfunction through improving insulin signalling.

Background: Glycyrrhyza glabra L. is one of the most popular medicinal plant in the world, its roots having been used since ancient times in many traditional medicines. On the contrary, scarce attention has been dedicated to liquorice aerial parts. Previous studies showed the presence of a large group of polyphenols and a consistent amount of d-pinitol in the leaf extract.Methods: The methanolic extract from G. glabra leaves was profiled for its content in polyphenols; the amount of d-pinitol was also measured with two independent methods (HPLC-ELSD and NMR). The extract was tested for its in vitro protective effects against insulin resistance-related endothelial dysfunction in human umbilical vein endothelial cells exposed to palmitic acid, which is the most prevalent saturated free fatty acid in circulation.Results: Methanolic extract from liquorice leaves has a protective effect against the lipotoxicity-associated alterations of insulin pathway in human endothelial cells, similarly to what observed with pure d-pinitol.Conclusions: Liquorice leaves are to be considered a waste product which gives a phytocomplex endowed with interesting potential therapeutic properties, moreover the use of a liquorice leaves phytocomplex rather than a pure compound allows avoiding a series of isolation/purification procedures and can be easily scaled up for industrial applications.

Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment.

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.

Natural Product-Based Hybrids as Potential Candidates for the Treatment of Cancer: Focus on Curcumin and Resveratrol.

One of the main current strategies for cancer treatment is represented by combination chemotherapy. More recently, this strategy shifted to the "hybrid strategy", namely the designing of a new molecular entity containing two or more biologically active molecules and having superior features compared with the individual components. Moreover, the term "hybrid" has further extended to innovative drug delivery systems based on biocompatible nanomaterials and able to deliver one or more drugs to specific tissues or cells. At the same time, there is an increased interest in plant-derived polyphenols used as antitumoral drugs. The present review reports the most recent and intriguing research advances in the development of hybrids based on the polyphenols curcumin and resveratrol, which are known to act as multifunctional agents. We focused on two issues that are particularly interesting for the innovative chemical strategy involved in their development. On one hand, the pharmacophoric groups of these compounds have been used for the synthesis of new hybrid molecules. On the other hand, these polyphenols have been introduced into hybrid nanomaterials based on gold nanoparticles, which have many potential applications for both drug delivery and theranostics in chemotherapy.

Comparison of Phytochemical Profile and Bioproperties of Methanolic Extracts from Different Parts of Tunisian Rumex roseus.

The genus Rumex (Polygonaceae) is distributed worldwide and the different species belonging to it are used in traditional medicine. The present study aimed at the evaluation of the phytochemical profile and the biochemical properties of methanolic extracts from different parts (roots, stems, and leaves) of Rumex roseus, a wild local Tunisian plant traditionally used as food. The phytochemical analysis on the extracts was performed using standard colorimetric procedures, HPLC-DAD, and HPLC-DAD-ESI-MS; then, several in vitro cell-free assays have been used to estimate their antioxidant/free radical scavenging capability (TAC-PM, DPPH, TEAC, FRAP, ORAC, SOD-like activity, and HOCl-induced albumin degradation). Additionally, anti-inflammatory effect of these extracts was evaluated in an in vitro model of acute intestinal inflammation in differentiated Caco-2 cells. The results showed that the methanolic extracts from stems and, especially, leaves contain substantial amounts of flavones (apigenin and luteolin, together with their derivatives), while the extract from roots is characterized by the presence of tannins and quinic acid derivatives. All the extracts appeared endowed with excellent antioxidant/free radical scavenging properties. In particular, the extract from roots was characterized by a remarkable activity, probably due to its different and peculiar polyphenolic composition. Furthermore, both Rumex roseus roots and stems extracts demonstrated an anti-inflammatory effect in intestinal epithelial cells, reducing TNF-α-induced gene expression of IL-6 and IL-8. In conclusion, R. roseus methanolic extracts have shown to be potential sources of bioactive compounds to be used in the prevention and treatment of pathologies related to oxidative stress and inflammation.

Hydrogels for the Delivery of Plant-Derived (Poly)Phenols.

This review deals with hydrogels as soft and biocompatible vehicles for the delivery of plant-derived (poly)phenols, compounds with low general toxicity and an extraordinary and partially unexplored wide range of biological properties, whose use presents some major issues due to their poor bioavailability and water solubility. Hydrogels are composed of polymeric networks which are able to absorb large amounts of water or biological fluids while retaining their three-dimensional structure. Apart from this primary swelling capacity, hydrogels may be easily tailored in their properties according to the chemical structure of the polymeric component in order to obtain smart delivery systems that can be responsive to various internal/external stimuli. The functionalization of the polymeric component of hydrogels may also be widely exploited to facilitate the incorporation of bioactive compounds with different physicochemical properties into the system. Several prototype hydrogel systems have been designed for effective polyphenol delivery and potential employment in the treatment of human diseases. Therefore, the inherent features of hydrogels have been the focus of considerable research efforts over the past few decades. Herein, we review the most recent advances in (poly)phenol-loaded hydrogels by analyzing them primarily from the therapeutic perspective and highlighting the innovative aspects in terms of design and chemistry.

Anthocyanins As Modulators of Cell Redox-Dependent Pathways in Non-Communicable Diseases.

Chronic Noncommunicable Diseases (NCDs), mostly represented by cardiovascular diseases, diabetes, chronic pulmonary diseases, cancers, and several chronic pathologies, are one of the main causes of morbidity and mortality, and are mainly related to the occurrence of metabolic risk factors. Anthocyanins (ACNs) possess a wide spectrum of biological activities, such as anti-inflammatory, antioxidant, cardioprotective and chemopreventive properties, which are able to promote human health. Although ACNs present an apparent low bioavailability, their metabolites may play an important role in the in vivo protective effects observed. This article directly addresses the scientific evidences supporting that ACNs could be useful to protect human population against several NCDs not only acting as antioxidant but through their capability to modulate cell redox-dependent signaling. In particular, ACNs interact with the NF-κB and AP-1 signal transduction pathways, which respond to oxidative signals and mediate a proinflammatory effect, and the Nrf2/ARE pathway and its regulated cytoprotective proteins (GST, NQO, HO-1, etc.), involved in both cellular antioxidant defenses and elimination/inactivation of toxic compounds, so countering the alterations caused by conditions of chemical/oxidative stress. In addition, supposed crosstalks could contribute to explain the protective effects of ACNs in different pathological conditions characterized by an altered balance among these pathways. Thus, this review underlines the importance of specific nutritional molecules for human health and focuses on the molecular targets and the underlying mechanisms of ACNs against various diseases.

Role of Herpes Simplex Envelope Glycoprotein B and Toll-Like Receptor 2 in Ocular Inflammation: An ex vivo Organotypic Rabbit Corneal Model.

It has been recently reported, using in vitro studies, that the herpes simplex virus 1 (HSV-1) encoded envelope glycoprotein B (gB1) interacts with cell surface toll-like receptor 2 (TLR2) and induces the secretion of interleukin-8 (IL8), a representative marker of inflammatory cytokine activation. The purpose of this study is to investigate the role of gB1 in activating host inflammatory responses by using a secreted form of gB1 (gB1s) and an ex vivo organotypic rabbit corneal model. Abraded corneas exposed to gB1s alone or to the recombinant protein mixed with anti gB polyclonal antibody were cultured in an air-liquid interface. The corneas exposed to gB1s show the appearance of mydriasis and high levels of TLR2 and IL-8 mRNAs transcripts were detected in the superficial layer of corneal epithelial cells. Histological stain and immunohistochemical analyses revealed morphological changes in the epithelium of the treated corneas and variations in expression and localization of TLR2. Collectively these findings provide new insight into the pathogenesis of HSV-1 ocular infection by demonstrating the leading role of gB in activating an inflammatory response and in the appearance of mydriasis, a sign of HSV-1 anterior uveitis.

Curcumin potentiates the antitumor activity of Paclitaxel in rat glioma C6 cells.

BACKGROUND: Glioma is the most common primary cancer in central nervous system, especially in brain. Paclitaxel (PTX) is a microtubule stabilizing agent with anticancer potential, but its clinical application to brain tumours is limited by drug resistance, side effects, and lower brain penetration. PURPOSE: Herein we explored the in vitro effects, in glioma C6 cells, of the combination of PTX with curcumin, a natural compound with chemotherapeutic activity, in order to improve cytotoxic effects and overcome PTX limitations. RESULTS: Our data confirmed PTX antiproliferative activity that was improved by curcumin. These effects were confirmed by clonogenic assay and G0/G1 cell cycle arrest. PTX significantly promoted generation of intracellular reactive species (RS), while curcumin did not affect RS production; the combination of the two drugs resulted in a slight but significant increase in RS levels. Furthermore, we found a constitutive activation of NF-κB in C6 cell line that was inhibited by PTX and curcumin. Interestingly, combination of the drugs totally inhibited NF-κB nuclear translocation and reduced IκB phosphorylation. Our results also supported the involvement of p53-p21 axis in the anticancer effects of curcumin and PTX. The combination of the two drugs further increased p53 and p21 levels enhancing the antiproliferative effects. Furthermore, PTX plus curcumin most impressively activated caspase-3, effector of apoptosis pathways, and reduced the expression of the anti-apoptotic protein Bcl-2. CONCLUSION: In conclusion, our findings demonstrated that combination of PTX and curcumin exerts a potentiated anti-glioma efficacy in vitro that may help in reducing dosage and/or minimizing side effects of cytotoxic therapy.

Phytochemical profiles, phototoxic and antioxidant properties of eleven Hypericum species - A comparative study.

Hypericum is one out of the nine genera belonging to the botanical family Clusiaceae Lindl (syn. Hypericaceae Juss.; APG III, 2009). The genus contains 484 species spread worldwide, one of which, Hypericum perforatum, is largely used in folk medicine. The aim of this study was to evaluate the chemical composition, along with the antioxidant and phototoxic activity, of 11 Hypericum species grown in Sicily (H. perforatum L., H. aegypticum L., H. androsaemum L., H. calycinum L., H. hircinum L., H. hirsutum L., H. montanum L., H. patulum Thunb., H. perfoliatum L., H. pubescens Boiss., H. tetrapterum Fr.). Samples of flowering tops collected from these Hypericum species were extracted and analysed by high performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD-MS) to determine their content of main polyphenols, acylphloroglucinols, and naphthodianthrones. The extracts were also subjected to a photocytotoxic assay using murine fibroblast (NIH/3T3), and their antioxidant activity evaluated by means of Folin-Ciocalteau, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and oxygen radical antioxidant capacity assays. Phytochemical analysis allowed us to identify and quantify 20 metabolites, each of them possessing a well-known biological activity. Furthermore, all examined species showed a good cytotoxic and antioxidant/radical scavenging activity. These results indicate that in addition to the well-known H. perforatum, at least other three species (H. tetrapterum, H. pubescens, and H. montanum) represent potential sources of biologically active compounds, and at least other two species (H. perfoliatum and H. tetrapterum), due to their phototoxicity are candidates for application in photodynamic therapy.