Actividad antitumoral de plantas de Andalucía: Aislamiento de principios activos de corema album (l.) D. DON
- Autores: Antonio José León González
- Directores de la Tesis: Carmen Martín Cordero (dir. tes.), Miguel López Lázaro (dir. tes.), María Jesús Ayuso González (dir. tes.)
- Lectura: En la Universidad de Sevilla ( España ) en 2012
- Idioma: español
- Tribunal Calificador de la Tesis: Maria Dolores García Gimenez (presid.), María de los Ángeles Fernández Arche (secret.), Francesco Epifano (voc.), Luis Goya Suárez (voc.), Nuria Acero Mesa (voc.)
- Materias:
- Texto completo no disponible (Saber más ...)
- Resumen
Antitumor activity of plants from Andalusia. Isolation of active principles of Corema album (L.) D. Don Introduction Cancer kills over seven million people worldwide every year [1]. The mortality rate of this disease has not changed much in the past few decades even in developed countries as the United States [2]. Although cancer therapy in the form of surgery or radiotherapy is effective when the disease is early detected, many cancers are still diagnosed when cells from a primary tumor have already metastasized to other parts of the body. The main form of treatment at this point is chemotherapy, which consists of delivering drugs systemically so that they can reach and kill the tumor cells. But most of these drugs cause severe side effects in patients and, therefore, need to be used at suboptimal levels. The low efficacy of chemotherapy in patients with advanced cancers is reflected in the low 5-year survival rates observed in these patients [2]. For example, cancer statistics show that the most commonly diagnosed cancer in the world is lung cancer [1], that approximately 50% of patients diagnosed with this type of cancer have distant metastasis [2] and that only 3% of these patients manage to survive more than 5 years [2]. The low efficacy of cancer therapy for the treatment of patients with metastasis makes the development of novel chemotherapeutic agents necessary.
In the search for new antitumor molecules, vegetal species have provided a large structural diversity that currently employ in chemotherapy (e.g. taxol, etoposide, topotecan, or vincristine), in fact, about 40% of current drugs are directly or indirectly from plants [3]. Andalusian vascular flora consists of a wide variety of plant species. There are areas of high biodiversity, where many species have not yet undergone any chemical or pharmacological study.
Moreover, it is estimated that two out of three cancers are preventable. Chemoprevention, defined as "use of chemicals to prevent, stop or reverse the carcinogenesis process" is presented as an alternative to traditional treatments. Cumulative experimental data indicate that reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide (H2O2), play a key role in the formation of cancer, and fruits and vegetables containing antioxidant agents are able to inhibit, prevent or reverse carcinogenesis. Berry fruits are nowadays one of the most studied foods in the field of chemoprevention [4-7].
Aim The overall objective of this study was to explore potential chemopreventive or antitumor agents in plant biodiversity of Andalusia by cytotoxicity and antiradical activity screening methods. After selecting a species, we set to analyze their berries and leaves phytochemical composition, and to evaluate the potential chemopreventive or cytotoxic properties of different extracts and isolated compounds. Cytotoxic and antiradical activity plant screening The aim of the first part of this study was to evaluate the antioxidant and cytotoxic activities of ethyl acetate extracts obtained from plants collected in Andalucía in order to select a potential novel antitumor species.
The dried plant material of 25 species collected in Andalucía was subjected to ultrasound-assisted extraction with ethyl acetate for 45 minutes, and was then filtered and concentred in a rotary evaporator. The antioxidant activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and the cytotoxic activity was assessed in HT-29 colon adenocarcinoma cells using the sulforhodamine B (SRB) assay.
The extract from the leaves of Corema album (Portuguese crowberry) was one of the most cytotoxic and antioxidant extracts, and also the extract from the fruits of this plant showed antirradicalary activity. This and the fact that no phytochemical or pharmacological study had been done of this endemism of the Iberian Peninsula, we selected the specie Corema abum to investigate the phytochemical composition and to do a pharmacological study, centred in the chemopreventive and antioxidant activity of the fruits and the cytotoxic activity of the leaves.
Corema album (L.) D. Don (Empetraceae) is a dioecious endemic shrub which berry fruits are traditionally consumed fresh or made into acid-tasting lemonades and are used in folk medicine to treat fevers and pinworm infections [8].
Study of the phytochemical composition of Corema album leaves Corema album leaves were extracted with ethyl acetate and the extract was fractionated by silicagel chromatography column. Two dihydrochalcones, 2¿, 4¿-dihydroxydihydrochalcone (CA1) and 2¿-methoxy-4¿hydroxydihydrochalcone (CA4), a chalcone 2¿-4¿dihydroxychalcone (CA3), and a flavanone, pinocembrin (CA2) were isolated. Their structural identification was based on 1H-NMR and 13C-NMR data, including 2D NMR, and mass spectrometry.
The triterpenes ursolic acid and oleanolic acid have also been identified in this extract.
Study of the phytochemical composition of Corema album berries We have identified in this berry fruit, by gas chromatography coupled to mass spectrometry (GC / MS), free sugars such as glucose, fructose and sucrose; organic acids such as malic acid and citric acid; and triterpenes as ursolic acid and oleanolic acid.
Phenolic compounds were extracted from freeze dried berries with aqueous acetone, according with previous reports [9] and the total phenolic content was determined by the Folin Ciocalteu method [10]. Corema album berries phenolic content was 1214.4±122.7 mg GAE/ kg FW or. This content is lower than that of other berries reported in the literature, such as blackberry, blueberry, or strawberry [11]. This could be explained, at least in part, by the fact that Corema album fruit is a white or pinkish white berry that has very low amounts of anthocyanins, which represent a main contribution in total polyphenol content in coloured berries from genus Vaccinium sp. (Ericaceae), Fragaria sp. (Rosaceae), or Ribes sp. (Saxifragaceae) [12]. The high proportion of the seeds weight (54.97 % of dry weight), also explains that this fruits have lower edible part than other berries.
The most important phenolic compounds in Corema album were tentatively identified by their UV spectra and their molecular weights and their fragments, by using high resolution liquid chromatography equipped with diode-array detection coupled with mass spectrometry (HPLC¿DAD¿MS/MS). Six phenolic acids were present in this berry, being chlorogenic acid the most abundant one. In addition, other phenolic acids were detected in minor proportions, such as, neochlorogenic, caffeic, p-hydroxybenzoic, ferulic, and p-coumaric acid. We also identified and quantified the flavonols myricetin 3-O-galactoside, myricetin 3-O-glucoside, quercetin 3-O-glucoside, rutin, quercetin 3-O-arabinoside and kaempferol 3-O-glucoside. Other phenolic compounds identified in the fruit were the flavanone pinocembrin and the anthocyanins delphinidin 3-O-glucoside, cyanidin 3-O-glucoside and cyanidin 3-O-arabinoside; however, these total flavonols were detected in a minor proportion in the HPLC chromatograms, while phenolic acids were the main constituents.
We have also separated phenolic acids into their free and bound forms, and identified the liberated phenolic acids by GC coupled to MS [9]. Eleven hydroxybenzoic and hydroxycinnamic acid derivatives (benzoic, salicylic, t-cinnamic, p-hydroxybenzoic, vanillic, gentisic, syringic, p-coumaric, gallic, ferulic and caffeic acids) were identified in their free and bound forms on the basis of GC retention times and simultaneously recorded mass spectra. Phenolic acids liberated from esters form comprised a 77% of total phenolic content, of these, caffeic acid was the main one.
Protective effect of phenolic extracts from Corema album berries against oxidative stress on HepG2 cells.
Because of the growing interest on other berries of the same family as functional foods, we evaluated the protective effect of three phenolic extracts, obtained from Corema album berries, against oxidative stress on HepG2 cells. This human hepatoma cell line is a model that reproduces the human liver and has been widely used to evaluate the effects of various naturally occurring compounds in vitro [13].
Characterization of the acetone, ethyl acetate and aqueous berry extracts on phenolics compounds was achieved by liquid chromatography-mass spectrometry (LC-MS). Human hepatoma HepG2 cells were pretreated with 1-40 µg/mL of berry extract for 20 hours and then exposed to oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Several biomarkers of cellular redox status were evaluated: cell viability (lactate dehydrogenase leakage), generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH) and activity of glutathione reductase (GR) and glutathione peroxidase (GPX). Levels of malondialdehyde (MDA) and carbonyl groups were also analyzed to evaluate lipid peroxidation and protein oxidative damage, respectively.
Pretreatment of HepG2 cells with Corema album berry extract showed a significant reduction of lactate dehydrogenase leakage, oxidative damage to lipids and proteins and ROS levels. The extracts also partially prevented the decrease of GSH and modulated the changes in GR and GPx activity induced by t-BOOH. Further experiments are required to determine the potential beneficial effect of C. album berry consumption in the prevention of oxidative stress-related diseases.
Cytotoxic activity of Corema album leaves flavonoids.
The cytotoxic activity of 2¿, 4¿-dihydroxydihydrochalcone (CA1), pinocembrin (CA2) and 2¿-methoxy-4¿hydroxydihydrochalcone (CA4) was determined on human colon adenocarcinoma cell line HT-29 using the sulforhodamine B (SRB) assay [14]. Their cytotoxic activity was dose-dependent and was comparable to that of the positive control 5-Fluorouracil (5-FU), a drug currently used in the treatment of colon cancer. The IC50 (± SEM) values were 1.79 ± 0.43 ¿M for compound (CA1); 7,62 ± 0,88 ¿M for compound (CA2); 8.53 ±2.09 ¿M for compound (CA4) and 8.68 ± 4.0 ¿M for the positive control 5-FU.
The DPPH assay was used to assess the possible antioxidant activity of these compounds. Neither compound showed activity at concentrations up to 100 ¿M; the antioxidant flavonoid quercetin was used as positive control (IC50 = 9.63 ¿M).
Evidence suggests that the anticancer activity of several anticancer drugs commonly used in clinic (e.g. cisplatin, doxorubicin, arsenic trioxide, bortezomib, procarbazine, etoposide, etc) is mediated, at least in part, by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) [15-16]. For instance, although it has been known for many years that the anticancer effect of paclitaxel (taxol) is mediated by its activity on the microtubule protein tubulin, recent experiments have shown that H2O2 plays an important role in paclitaxel-induced cancer cell death [15, 17]. Knowing that the induction of oxidative stress by pro-oxidant agents is emerging as an attractive anticancer strategy [16-19], the possible involvement of ROS in the cytotoxic activity of isolated compounds was evaluated. By incubating the cells with the antioxidants, N-acetylcysteine and Mn(III) Tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride, there was a decrease in the cytotoxic activity of the tested flavonoids, suggesting that the generation of reactive oxygen species are involved in their cytotoxicity. Experimental data have revealed that the induction of the ROS H2O2 by pro-oxidant agents may lead to the selective killing of cancer cells [16, 18]. Our results show that the cytotoxic activity of flavonoides isolated from Corema album leaves may be mediated in part by their pro-oxidant activity. Further experiments are required to establish whether or not these compounds can kill cancer cells selectively. Chalcones have been increasingly studied as chemopreventive agents. We examined the antiproliferative effects of a flavonic fraction (Fr3) of the ethyl acetate extract of Corema album leaves, constituted by a mixture of the chalcone 2¿-4¿dihydroxy-chalcone (CA3) and the flavanone, pinocembrin (CA2 ) on two colon cancer cell lines: HCT-116 and HT-29.
Cell viability at 24 and 48 hours was determined using an ATPLite kit according to the manufacturer¿s instructions: HCT-116 IC50 (48h)= 7.2±0.7 µg/mL; HT-29 IC50 (48h)= 6.8±1.2 µg/mL. The ATPLite assay shows that Fr3 causes a significant decrease of the cellular viability in a time and dose dependent manner. Annexin V-FITC/PI staining and flow cytometry were used to determine the effects of DHC on the induction of apoptosis in HCT-116 and HT-29 colon cancer cells. Treatment of colon cancer cells with Fr3 increased the percentage of cells in apoptosis. The cell cycle analysis shows that the loss in viability of both colon cell lines was due, at least in part, to G2/M arrest.
References [1] Jemal, A., Bray, F., Center, M.M., Ferlay, J., Ward, E., Forman, D. (2011) Global cancer statistics. CA Cancer J Clin; 61: 69-90.
[2] Jema,l A., Siegel, R., Xu, J., Ward, E. Cancer statistics, (2010). CA Cancer J Clin 2010; 60: 277-300.
[3] Cragg, G.M., Grothaus, P.G., Newman, D.J. (2009) Impact of natural products on developing new anti-cancer agents. Chem Rev; 109: 3012-43.
[4] Seeram, N.P. (2008) Berry Fruits for Cancer Prevention: Current Status and Future Prospects. J. Agric. Food Chem. 56: 630¿635 [5] Johnson, I. T. (2007). Phytochemicals and cancer. The Proceedings of the Nutrition Society, 66(2), 207-215.
[6] Jepson, R.C., Craig, J.C. (2007) A systematic review of the evidence for cranberries and blueberries in UTI prevention. Mol. Nutr. Food Res. 51: 738 ¿ 745 [7] Pappas, E., Schaich, K.C. (2009) Phytochemicals of Cranberries and Cranberry Products: Characterization, Potential Health Effects, and Processing Stability, Critical Reviews in Food Science and Nutrition, 49:9, 741 ¿ 781 [8] Font Quer, P. (1990). Plantas Medicinales. In: El Dioscórides Renovado (12th ed.), Ed. Labor, Barcelona, p. 1033 .
[9] Zadernowski, R., Czaplicki, S., Naczk, M. (2009) Phenolic acid profiles of mangosteen fruits (Garcinia mangostana). Food Chem. 112, 685-689 [10] Waterhouse, A. (2009). Folin-Ciocalteau micro method for total phenol in wine. http://waterhouse.ucdavis.edu/phenol/folinmicro (last accessed: February, 2012) [11] Jakobek, L., Šeruga, M., Novak, I., Medvidovi¿- Kosanovi¿, M. (2007). Flavonols, phenolic acids and antioxidant activity of some red fruits. Deutsche Lebensmittel-Rundschau, 103, 369¿378.
[12] Määttä-Riihinen, K.R., Kamal-Eldin, A., Mattila, P.H., González-Paramás, A.M., Törrönen, A.R. (2004) Distribution and contents of phenolic compounds in eighteen Scandinavian berry species. Journal of Agricultural and Food Chemistry, 52, 4477-4486.
[13] Goya, L., Martin, M. A., Ramos, S., Mateos, R., Bravo, L. (2009). A Cell Culture Model for the Assessment of the Chemopreventive Potential of Dietary Compounds. Current Nutrition & Food Science, 5, 56-64.
[14] Monks, A et al. (1991) Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J. Int. Cancer Inst. 83 (11): 757-766.
[15] Alexandre, J., Batteux, F., Nicco, C., Chereau, C., Laurent, A., Guillevin, L., Weill, B., & Goldwasser, F. (2006). Accumulation of hydrogen peroxide is an early and crucial step for paclitaxel-induced cancer cell death both in vitro and in vivo. International journal of cancer.Journal international du cancer, 119(1), 41-48.
[16] Lopez-Lazaro,M. (2007). Dual role of hydrogen peroxide in cancer: Possible relevance to cancer chemoprevention and therapy. Cancer Lett. 252, 1-8.
[17] Alexandre,J., Hu,Y., Lu,W., Pelicano,H., and Huang,P. (2007). Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species. Cancer Res. 67, 3512-3517.
[18] Pelicano,H., Carney,D., and Huang,P. (2004). ROS stress in cancer cells and therapeutic implications. Drug Resist. Updat. 7, 97-110.
