In Vitro Assessment of the Cytotoxic and Antiproliferative Profile of Natural Preparations Containing Bergamot, Orange and Clove Essential Oils.

Medicinal plants and essential oils (EOs), in particular, were intensively studied in recent years as viable alternatives for antiproliferative chemical synthetic agents. In the same lines, the present study focuses on investigating the effects of natural preparations (emulsions) based on EOs obtained from Citrus bergamia Risso (bergamot-BEO), Citrus sinensis Osbeck (orange-OEO), and Syzygium aromaticum Merill et L. M. Perry (clove-CEO) on different healthy (human immortalized keratinocytes-HaCaT and primary human gingival fibroblasts-HGF) and human tumor cell lines (human melanoma-A375 and oral squamous carcinoma-SCC-4) in terms of the cells' viability and cellular morphology. The obtained results indicate that the CEO emulsion (ECEO) induced a dose-dependent cytotoxic in both healthy (HaCaT and HGF) and tumor (A375 and SCC-4) cells. OEO emulsion (EOEO) increased cell viability percentage both for HaCaT and A375 cells and had an antiproliferative effect at the highest concentration in HGF and SCC-4 cells. BEO emulsion (EBEO) decreased the viability percentage of SCC-4 tumor cells. By associating OEO with CEO as a binary mixture in an emulsified formulation, the inhibition of tumor cell viability increases. The E(BEO/OEO) binary emulsion induced an antiproliferative effect on oral health and tumor cells, with a minimal effect on skin cells. The non-invasive tests performed to verify the safety of the test compound's emulsions at skin level indicated that these compounds do not significantly modify the physiological skin parameters and can be considered safe for human skin.

Melissa officinalis L. Aqueous Extract Exerts Antioxidant and Antiangiogenic Effects and Improves Physiological Skin Parameters.

Melissa officinalis (MO) is a medicinal plant well-known for its multiple pharmacological effects, including anti-inflammatory, anticancer and beneficial effects on skin recovery. In this context, the present study was aimed to investigate the in vitro and in vivo safety profile of an MO aqueous extract by assessing cell viability on normal (HaCaT-human keratinocytes) and tumor (A375-human melanoma) cells and its impact on physiological skin parameters by a non-invasive method. In addition, the antioxidant activity and the antiangiogenic potential of the extract were verified. A selective cytotoxic effect was noted in A375 cells, while no toxicity was noticed in healthy cells. The MO aqueous extract safety profile after topical application was investigated on SKH-1 mice, and an enhanced skin hydration and decreased erythema and transepidermal water loss levels were observed. The in ovo CAM assay, performed to investigate the potential modulating effect on the angiogenesis process and the blood vessels impact, indicated that at concentrations of 100 and 500 µg/mL, MO aqueous extract induced a reduction of thin capillaries. No signs of vascular toxicity were recorded at concentrations as high as 1000 µg/mL. The aqueous extract of MO leaves can be considered a promising candidate for skin disorders with impaired physiological skin parameters.

Plant-Derived Anticancer Compounds as New Perspectives in Drug Discovery and Alternative Therapy.

Despite the recent advances in the field of chemically synthetized pharmaceutical agents, nature remains the main supplier of bioactive molecules. The research of natural products is a valuable approach for the discovery and development of novel biologically active compounds possessing unique structures and mechanisms of action. Although their use belongs to the traditional treatment regimes, plant-derived compounds still cover a large portion of the current-day pharmaceutical agents. Their medical importance is well recognized in the field of oncology, especially as an alternative to the limitations of conventional chemotherapy (severe side effects and inefficacy due to the occurrence of multi-drug resistance). This review offers a comprehensive perspective of the first blockbuster chemotherapeutic agents of natural origin's (e.g. taxol, vincristine, doxorubicin) mechanism of action using 3D representation. In addition is portrayed the step-by-step evolution from preclinical to clinical evaluation of the most recently studied natural compounds with potent antitumor activity (e.g. resveratrol, curcumin, betulinic acid, etc.) in terms of anticancer mechanisms of action and the possible indications as chemotherapeutic or chemopreventive agents and sensitizers. Finally, this review describes several efficient platforms for the encapsulation and targeted delivery of natural compounds in cancer treatment.

Thermosensitive Betulinic Acid-Loaded Magnetoliposomes: A Promising Antitumor Potential for Highly Aggressive Human Breast Adenocarcinoma Cells Under Hyperthermic Conditions.

PURPOSE: Breast cancer presents one of the highest rates of prevalence around the world. Despite this, the current breast cancer therapy is characterized by significant side effects and high risk of recurrence. The present work aimed to develop a new therapeutic strategy that may improve the current breast cancer therapy by developing a heat-sensitive liposomal nano-platform suitable to incorporate both anti-tumor betulinic acid (BA) compound and magnetic iron nanoparticles (MIONPs), in order to address both remote drug release and hyperthermia-inducing features. To address the above-mentioned biomedical purposes, the nanocarrier must possess specific features such as specific phase transition temperature, diameter below 200 nm, superparamagnetic properties and heating capacity. Moreover, the anti-tumor activity of the developed nanocarrier should significantly affect human breast adenocarcinoma cells. METHODS: BA-loaded magnetoliposomes and corresponding controls (BA-free liposomes and liposomes containing no magnetic payload) were obtained through the thin-layer hydration method. The quality and stability of the multifunctional platforms were physico-chemically analysed by the means of RAMAN, scanning electron microscopy-EDAX, dynamic light scattering, zeta potential and DSC analysis. Besides this, the magnetic characterization of magnetoliposomes was performed in terms of superparamagnetic behaviour and heating capacity. The biological profile of the platforms and controls was screened through multiple in vitro methods, such as MTT, LDH and scratch assays, together with immunofluorescence staining. In addition, CAM assay was performed in order to assess a possible anti-angiogenic activity induced by the test samples. RESULTS: The physico-chemical analysis revealed that BA-loaded magnetoliposomes present suitable characteristics for the purpose of this study, showing biocompatible phase transition temperature, a diameter of 198 nm, superparamagnetic features and heating capacity. In vitro results showed that hyperthermia induces enhanced anti-tumor activity when breast adenocarcinoma MDA-MB-231 cells were exposed to BA-loaded magnetoliposomes, while a low cytotoxic rate was exhibited by the non-tumorigenic breast epithelial MCF 10A cells. Moreover, the in ovo angiogenesis assay endorsed the efficacy of this multifunctional platform as a good strategy for breast cancer therapy, under hyperthermal conditions. Regarding the possible mechanism of action of this multifunctional nano-platform, the immunocytochemistry of the MCF7 and MDA-MB-231 breast carcinoma cells revealed a microtubule assembly modulatory activity, under hyperthermal conditions. CONCLUSION: Collectively, these findings indicate that BA-loaded magnetoliposomes, under hyperthermal conditions, might serve as a promising strategy for breast adenocarcinoma treatment.

Mechanistic investigations of antitumor activity of a Rhodamine B­oleanolic acid derivative bioconjugate.

Cancer remains a major health problem worldwide due to its high mortality rate. New therapeutic options highlight the importance of discovering new compounds that target the tumor microenvironment, interrupt angiogenesis and act selectively. The present study assessed the antitumor effect and investigated the mechanism of action of a rhodamine B­conjugated oleanolic acid derivative (RhodOA). Consequently, the compound was tested on different human tumor cell lines (A375 melanoma, A549 lung adenocarcinoma and MDA­MB­231 breast adenocarcinoma) and on a non­tumor cell line HaCaT human keratinocyte. RhodOA produced a dose­dependent decrease in tumor cell viability especially in the melanoma cells while affecting the keratinocytes less. In melanoma cells, RhodOA reduced cell migration and produced condensation of cell nuclei and of actin fibers. Furthermore, an impairment in melanoma cell mitochondrial function was observed, while the mitochondrial function of keratinocytes was left intact. In the in ovo chorioallantoic membrane model, RhodOA elicited antiangiogenic effect, without showing irritation effect on the membrane. The study provides information on the selective antitumor effect of the derivative and its ability to inhibit cellular respiration, therefore RhodOA can be classified as 'MITOCAN'.

Lemon Balm Extracts Prevent Breast Cancer Progression In Vitro and In Ovo on Chorioallantoic Membrane Assay.

Breast cancer is the most frequently diagnosed malignant pathology, representing the primary cause of cancer death in women. Natural products are an appealing strategy to limit the progression of the disease. Targeting angiogenesis in breast cancer may positively impact on poor prognosis of breast cancer. As source of natural compounds, we investigated the leaves of Melissa officinalis L. (MO), known as lemon balm, an aromatic plant that spontaneously grows in the South and Western areas of Romania, being traditionally recommended as anxiolytic, antispasmodic, or as digestive remedy. Our aim was to investigate the phytochemical profiling and the antiangiogenic and chemopreventive bioactivity of MO from Banat region, on breast cancer. Two ethanolic extracts of MO (MOE96 and MOE70) and one methanolic extract (MOM80) were subjected to polyphenol and triterpene profiling by HPLC-MS, and the antioxidant capacity was evaluated. The antiangiogenic potential was investigated using the chorioallantoic membrane assay (CAM). The MTT(3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) assay was used to investigate the cytotoxic effects on MCF-7 and MDA-MB-231breast cancer cells, as well as on MCF-10A normal breast epithelial cells, while apoptosis was performed by DAPI staining. Rosmarinic acid (RA) and ursolic acid (UA) were revealed as dominant phytocompounds. The highest concentration in phytochemicals were found in MOM80; MOE96 was more concentrated in UA, while MOE70 extracted more RA. MOE96 inhibited cancer progression and angiogenesis in the in ovo CAM model using MDA-MB-231 cells, inhibiting breast cancer progression and angiogenesis for the MDA-MB-231 breast cancer cell line; no secondary tumoral areas were registered, indicative for a preventive effect against breast tumor cell invasiveness. The highest cell inhibitory activity was also exhibited by MOE96, in particular against the estrogen receptor positive MCF7 breast cancer cell line, with no cytotoxic effect on healthy cells. The estrogen receptor positive MCF7 cell line proved to be more sensitive to the extract antiproliferative activity than the triple negative MDA-MB-231 breast cancer cell line. Nevertheless, the chemopreventive potential of MOE96 extract is phenotype-dependent and is rather related to the apoptosis and antiangiogenic effects suggesting a multitargeted mechanism of action due to its multiple compound composition next to a concentration ratio of RA : UA in favor of UA.

A Comparative Study of Melissa officinalis Leaves and Stems Ethanolic Extracts in terms of Antioxidant, Cytotoxic, and Antiproliferative Potential.

Melissa officinalis L. has attracted an increased interest in recent years due to its multiple pharmacological effects. This study aimed to compare two M. officinalis ethanolic extracts, obtained from leaves and stems, with regard to their antioxidant activity, total phenolic content, and cytotoxic effects. M. officinalis ethanolic extracts showed a very good antioxidant activity in the DPPH test, correlated with the content in total phenols: higher in the case of M. officinalis from leaves extract (32.76 mg GAE/g) and lower for M. officinalis from stems extract (8.4 mg GAE/g). The lemon balm extracts exerted a cytotoxic effect on breast cancer cells (MDA-MB-231) even at low concentrations (100 µg/mL), whereas, in the case of healthy HaCat cells, M. officinalis leaves extract only displayed cytotoxicity at much higher concentrations (500 and 1000 µg/mL) and M. officinalis stems extracts were highly cytotoxic (starting at 100 µg/mL). In addition, the extracts exerted inhibitory effects on cell migration and proliferation. These results provide information that confirms the high potential of M. officinalis as a source of chemopreventive agents. Moreover, these data can be considered a solid background for further in vivo studies involving mice bearing breast tumors.

Mistletoe and Garlic Extracts as Polyurethane Carriers - A Possible Remedy for Choroidal Melanoma.

BACKGROUND: Melanoma is known as the most dangerous form of skin cancer; whereas the malignant choroidal melanoma is an orphan disease known as the most common primary intraocular malignancy in adults. Literature suggests that the consumption of garlic and mistletoe leads to a reduced risk of developing cancer. OBJECTIVE: The aim of this study was the obtaining and the characterization of polymer structures containing mistletoe or garlic extract. METHODS: The structures were obtained in a polyaddition process combined with a spontaneous emulsification; they were characterized by pH, size, Zeta potential and DSC measurements, evaluation of encapsulation efficacy, penetrability through membranes and in vitro cytotoxicity tests. RESULTS: The microstructures present sizes between 1.05 and 2.60 µm and Zeta potentials between -7 and +36 mV. A good encapsulation was observed on different evaluations (88-92%). It was determined that approx. 30% of polymer microstructures containing vegetal extracts pass through an artificial membrane in 4 days. An in vitro cytotoxicity test revealed that these products are safe for administration. The analysis of antitumor efficacy indicates that garlic extracts have important effects after 48 and 72 hours on A375 cells; however, polymer microstructures with herbal extracts did not reveal antiproliferative activities on A375 cells because these polymer structures present a slow degradation. CONCLUSION: Sterile eye drops solutions based on polymer microstructures containing garlic or mistletoe extracts were obtained; the sample based on garlic extracts may be used in the pharmaceutical field as drug carrier with an antiproliferative effect which occurs after a prolong period.

Germinated and Ungerminated Seeds Extract from Two Lupinus Species: Biological Compounds Characterization and In Vitro and In Vivo Evaluations.

In recent years, nutraceuticals attracted a great amount of attention in the biomedical research due to their significant contribution as natural agents for prevention of various health issues. Ethanolic extracts from the ungerminated and germinated seeds of Lupinus albus L. and Lupinus angustifolius L. were analyzed for the content in isoflavones (genistein) and cinnamic acid derivatives. Additionally, the extracts were evaluated for antimicrobial, antiproliferative, and anti-inflammatory properties, using in vitro and in vivo tests. Germination proved to be a method of choice in increasing the amount of genistein and cinnamic acid derivatives in both Lupinus albus L. and Lupinus angustifolius L. seeds. Biological evaluation of all vegetal extracts revealed a weak therapeutic potential for both ungerminated and germinated seeds.

Evaluation of phenolic profile, antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells.

BACKGROUND: Curcuma longa Linnaeus and Zingiber officinale Roscoe are two main representatives of Zingiberaceae family studied for a wide range of therapeutic properties, including: antioxidant, anti-inflammatory, anti-angiogenic, antibacterial, analgesic, immunomodulatory, proapoptotic, anti-human immunodeficiency virus properties and anticancer effects. This study was aimed to analyse the ethanolic extracts of Curcuma rhizome (Curcuma longa Linnaeus) and Zingiber rhizome (Zingiber officinale Roscoe) in terms of polyphenols, antioxidant activity and anti-melanoma potential employing the B164A5 murine melanoma cell line. RESULTS: In order to evaluate the total content of polyphenols we used Folin-Ciocâlteu method. The antioxidant activity of the two ethanolic extracts was determined by DPPH assay, and for the control of antiproliferative effect it was used MTT proliferation assay, DAPI staining and Annexin-FITC-7AAD double staining test. Results showed increased polyphenols amount and antioxidant activity for Curcuma rhizome ethanolic extract. Moreover, 100 µg/ml of ethanolic plant extract from both vegetal products presented in a different manner an antiproliferative, respectively a proapoptotic effect on the selected cell line. CONCLUSIONS: The study concludes that Curcuma rhizome may be a promising natural source for active compounds against malignant melanoma.

Evaluation of phenolic profile, antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells

BACKGROUND: Curcuma longa Linnaeus and Zingiber officinale Roscoe are two main representatives ofZingiberaceae family studied for a wide range of therapeutic properties, including: antioxidant, anti-inflammatory, anti-angiogenic, antibacterial, analgesic, immunomodulatory, proapoptotic, anti-human immunodeficiency virus properties and anticancer effects. This study was aimed to analyse the ethanolic extracts of Curcuma rhizome (Curcuma longa Linnaeus) and Zingiber rhizome (Zingiber officinale Roscoe) in terms of polyphenols, antioxidant activity and anti-melanoma potential employing the B164A5 murine melanoma cell line. RESULTS: In order to evaluate the total content of polyphenols we used Folin-Ciocâlteu method. The antioxidant activity of the two ethanolic extracts was determined by DPPH assay, and for the control of antiproliferative effect it was used MTT proliferation assay, DAPI staining and Annexin-FITC-7AAD double staining test. Results showed increased polyphenols amount and antioxidant activity forCurcuma rhizome ethanolic extract. Moreover, 100 μg/ml of ethanolic plant extract from both vegetal products presented in a different manner an antiproliferative, respectively a proapoptotic effect on the selected cell line. CONCLUSIONS: The study concludes that Curcuma rhizome may be a promising natural source for active compounds against malignant melanoma.