Hydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence

doi:10.1016/j.phymed.2018.06.008

Phytomedicine

Volume 46, 15 July 2018, Pages 1-10

https://doi.org/10.1016/j.phymed.2018.06.008 Get rights and content

Abstract

Background

Ultraviolet light exposure generates, in human tissues, radical species, which represent the main cause of photo-aging, DNA damage and skin cancer onset. On the other hand, Mediterranean plants, being continuously subjected to high solar radiation levels, are naturally adapted to take on this type of abiotic stress, thanks to the production of antioxidant secondary metabolites. For these reasons, several plant extracts were documented to be excellent antineoplastic drugs.

Purpose

We investigated the potential antitumor activity of the flower extract obtained by Spartium junceum L., a Mediterranean shrub, correlating it with the plant metabolic profile.

Study design

After selecting the best extraction method to obtain as more secondary metabolites as possible from S. junceum flowers, we characterized the extract metabolic content. Then, by in vitro analyses, the antioxidant profile and the antineoplastic activity on B16-F10 murine melanoma cell of our extract were investigated.

Methods

Spectrophotometric assays, HPLC-DAD and GC-MS analyses provided us information about flower extract composition and antioxidant activity. MTT assay and Trypan Blue exclusion test were performed to assess the extract toxicity and the viability, after treatments, of B16-F10 cancer cells and of C2C12 murine myoblasts. In vitro experiments (i.e. cytofluorimetry, protein analysis and qPCR) allowed us to analyze the effect of the plant extract on B16-F10 cell redox state, melanogenesis and cell cycle. Senescence induction was investigated by using a specific kit.

Results

We observed that the hydroalcoholic extract of S. junceum flowers (HFE) strongly inhibited B16-F10 murine melanoma cell proliferation, while just a feeble effect was observed on C2C12 murine myoblasts. Moreover, we found that HFE exerted a pro-oxidant activity on melanoma cells, inhibited melanogenesis and caused cell cycle arrest in G2/M phase, inducing senescence. These anti-cancer properties of HFE could be related to the rich metabolic profile of the extract that we characterized by HPLC-DAD and GC-MS analyses.

Conclusion

This evidence suggests that S. junceum phytocomplex can be used as a selective, nontoxic, economic and easily available anticancer drug.

Graphical abstract

Introduction

A lot of pharmaceutical products used to cure cancer, infections, chronic and age-related diseases, metabolic and immune disorders are natural substances or their derivatives (Newman and Cragg, 2016). Among them, the largest group is represented by phytochemicals, or rather secondary metabolites (i.e. phenolics, alkaloids, terpens) which are synthesized by plants to defend themselves from biotic and abiotic factors and promote their propagation (Gismondi et al., 2017). The application of these molecules in drug design and production is mainly linked to the great biological activity they show. Well-known examples are vancomycin (an antibiotic used against Gram-positive bacterial infections), staurosporine (a protein kinase inhibitor), rapamycin (an immunosuppressant) and taxol (an anti-cancer agent) (Clardy and Walsh, 2004, Paterson and Anderson, 2005).

Several scientific works reported that plant extracts inhibit the growth of a wide range of tumor cell lines (e.g. blood, skin, brain, colon, breast, prostate), both in vitro and in vivo, due to their anti-oxidant, anti-inflammatory, antiproliferative, differentiative or pro-apoptotic properties (Gullett et al., 2010, Lesgards et al., 2014). All this evidence suggests that plant molecules can represent non-toxic, economic and easily available chemopreventive and chemotherapeutic agents.

Mediterranean flora, distributed in geographic areas with high solar radiation level, evolved specific adaptive strategies to sustain such type of environmental pressure, which would induce oxidative stress in plant tissues. In this context, the capacity to produce secondary metabolites with free radical scavenging activity is surely the most prominent (Ramakrishna and Ravishankar, 2011). These compounds, thanks to their chemical structure, essentially characterized by aromatic rings and double bonds, can easily provide electrons to reactive species, disrupting the damaging oxidative chain reactions which occur in cell compartments (Gismondi et al., 2017).

As regards human health, it is widely documented that ultraviolet (UV) light exposure generates radical species (e.g. ROS) in skin cells, causing oxidative stress, inflammation, photo-aging, DNA damage and, consequently, malignancy (Sekulic et al., 2008). Therefore, as skin cancer is the most common neoplasia in the World and melanoma is its deadliest form (The American Cancer Society, 2017), antiradical molecules extracted from Mediterranean plants could be used to prevent onset, growth, progression and metastatization of such type of tumor (Działo et al., 2016, Jensen et al., 2010).

Spartium junceum L. (Spanish broom or weaver's broom) is a typical Mediterranean shrub species, belonging to Fabaceae family. Only two literature works demonstrated the anti-neoplastic activity of S. junceum extract (Abusamra et al., 2015, Cerchiara et al., 2012). In detail, Abusamra et al. (2015) tested flower hydromethanolic (80% methanol) extract on U-373 glioblastoma cells, observing a weak cytotoxicity effect, probably associated to a caspase-independent and non-apoptotic cell death. On the other hand, Cerchiara et al., (2012), using S. junceum flower aromatic water (obtained by vacuum distillation), performed a preliminary cytotoxicity screening on several cell lines (RPMI 7932, K562, MCF7-Bart, MCF7-ICLC, SW480 and NCTC 2544, as non-tumor control). In general, they observed a reduction of tumor cell growth, while no toxic effect was detected on NCTC 2544 cells.

Taking into account these promising data and considering that no further work investigated the bioactivity of this Mediterranean plant, the present research aimed to increase the knowledge about the medicinal properties of this species. Therefore, metabolic profile and antitumor effect of S. junceum flower hydroalcoholic extract were studied in depth. In addition, the bioactivity of two other Mediterranean plant species, Pittosporum tobira (Thunb.) W.T.Aiton and Rosa canina L. was evaluated.

Section snippets

Plant name and parts used for extract preparation

Spartium junceum L. flowers were collected from plants grown in the Botanical Garden of the University of Rome “Tor Vergata”. Vegetal tissues were powdered with pestle, mortar and liquid nitrogen, resuspended with ddH₂O (aqueous flower extract, AFE; 100 parts of distilled water) or 50% Ethyl Alcohol (hydroalcoholic flower extract, HFE; 50 parts of ethanol 96% V/V and 50 parts of distilled water), at a concentration of 200 mg of plant powder per ml of solvent, and left in agitation for 24 h, at

Hydroalcoholic extraction of S. junceum flowers is preferable than aqueous one

Plant material was processed and subjected to aqueous (AFE) or hydroalcoholic (HFE) extraction, as widely described in Materials and Methods section. In order to understand which solvent was more suitable for isolation of secondary metabolites from S. junceum flowers, both extracts were analyzed by spectrophotometric and chromatographic approaches. Content of phenolic compounds and antiradical power of the extracts were estimated. HFE showed comparable levels of simple phenols

Discussion

In order to face biotic and abiotic stresses typical of Mediterranean areas, such as exposure to high levels of UV light, plants produce huge amounts of secondary metabolites. These molecules, showing an elevated bioactivity even on mammalian systems as pro- or antioxidant agents, are considered of great scientific, medical and pharmaceutical interest. For this reason, in the present work, we investigated the effect of extracts obtained from three Mediterranean plants (P. tobira (Thunb.)

Conclusion

In conclusion, we proved that S. junceum hydroalcoholic flower extract, probably thanks to its rich biochemical profile and pro-oxidant activity, is able to inhibit melanogenesis and induce senescence in B16-F10 murine melanoma cells, preventing hyper-proliferating events. All these data led us to believe that the Spanish broom phytocomplex can represent a promising candidate as anti-melanoma agent. Indeed, it would be an alternative agent to actual toxic chemotherapeutics (e.g. cisplatinum;

Conflict of interest

The authors state no conflict of interest.

Acknowledgments

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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¹: Angelo Gismondi and Antonella Canini are co-last authors.

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Original ArticleHydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence

Abstract

Background

Purpose

Study design

Methods

Results

Conclusion

Graphical abstract

Introduction

Section snippets

Plant name and parts used for extract preparation

Hydroalcoholic extraction of S. junceum flowers is preferable than aqueous one

Discussion

Conclusion

Conflict of interest

Acknowledgments

Phytochemistry

Biomaterials

Semin. Oncol.

Clin. Dermatol.

Lab. Invest.

Mayo Clin. Proc.

Phytochemistry

J. Ethnopharmacol.

Evaluation of putative cytotoxic activity of crude extracts from Onopordum acanthium leaves and Spartium junceum flowers against the U-373 glioblastoma cell line

Pak. J. Pharm. Sci.

Selective cytotoxic activities of two novel synthetic drugs on human breast carcinoma MCF-7 cells

Anticancer Res.

ROS, stress-activated kinases and stress signaling in cancer

EMBO Rep.

Assessing cell and organ senescence biomarkers

Circ. Res.

Exogenous antioxidants-double-edged swords in cellular redox state: health beneficial effects at physiologic doses versus deleterious effects at high doses

Oxid. Med. Cell Longev.

Spartium junceum aromatic water: chemical composition and antitumor activity

Nat. Prod. Commun.

Lessons from natural molecules

Nature

Chrysin 7-gentiobioside from the flowers of Spartium junceum

Phytochemistry

The potential of plant phenolics in prevention and therapy of skin disorders

Int. J. Mol. Sci.

Senescence from G2 arrest, revisited

Cell Cycle

Transglutaminase-dependent antiproliferative and differentiative properties of nimesulide on B16-F10 mouse melanoma cells

Amino Acids

Original Article
Hydroalcoholic extract of Spartium junceum L. flowers inhibits growth and melanogenesis in B16-F10 cells by inducing senescence