Phytochemical Studies of Fractions and Compounds Present in Vernonanthura Patens with Antifungal Bioactivity and Potential as Antineoplastic

The wonderful plant diversity of South America and more specifically from the Amazon region has around 30-50% of the worlds biodiversity therafor it is an important source for this type of study. Beside the significant undiscovered resources from these regions, ancestral knowledge of indigenous peoples is another relevant and complementary source for biodiscovery programs. Traditional healers guard centuries of accumulated knowledge about natural medicinal resources of this region. These ancient “physicians” hold the key to discovering new drugs that could benefit millions of people around the world. The Amazon forest has contributed dozens of substances to western medicine. Among the best known are the “curare”; a key component of modern anesthetics and quinine, the first contribution of "natural medicine" to treat malaria1.


Introduction
Phytochemical research is closely related to the needs of finding new and effective pharmaceuticals. Searching for plant substances that are capable forbeing used to develop new therapeutic drugs against catastrophic recognized illnesses such as cancer, diabetes and AIDS is one of the main topics that researchers around the world have been focusing.
The wonderful plant diversity of South America and more specifically from the Amazon region has around 30-50% of the worlds biodiversity therafor it is an important source for this type of study. Beside the significant undiscovered resources from these regions, ancestral knowledge of indigenous peoples is another relevant and complementary source for biodiscovery programs. Traditional healers guard centuries of accumulated knowledge about natural medicinal resources of this region. These ancient "physicians" hold the key to discovering new drugs that could benefit millions of people around the world. The Amazon forest has contributed dozens of substances to western medicine. Among the best known are the "curare"; a key component of modern anesthetics and quinine, the first contribution of "natural medicine" to treat malaria 1 .
Study of new plant species and the structural elucidation of its bioactive molecules are the most important aims of phytochemical research which is in constant technological development.
Initial phytochemical screening and further isolation, purification and identification of molecules structure have made a major breakthrough with the development of new methods of chromatography and spectroscopy. The establishment of new and more effective bioassays is also one of the essential aspects that support biodiscovery programs today.
This chapter contains the main results on the phytochemical study of Vernonanthura patens leaves which according to ancestral knowledge, have been used to treat different diseases in humans.

Botanical classification, general characteristics and ethnobotanical knowledge on Vernonanthura patens
Vernonanthura patens is a wild plant broadly distributed throughout America. It grows from 0 to 2200 meters above sea level in the Ecuadorian coastal region. Folk medicine uses its leaves cooked to combat malaria, postpartum treatment and for healing infected wounds of animals by washing with a plant mixture which includes V. patens leaves (Blair, 2005).
It is also used against headaches, to clean and heal wounds (Kvist et al., 2006); treatment of leishmaniasis (Gachet et al., 2010); preparation of antivenom (Tene et al., 2007) and as a poultice of leaves to combat athlete's foot (Valadeau et al., 2009). Its usefulness for treating certain types of cancer has also been referred by indigenous healers. There is however there are few chemical studies about this species

Vernonanthura patens (Kunth) H. Rob. botanical classification and general characteristics
Species V. patens belongs to the Asteraceae family, quoting 60 synonyms and one basionym (Vernonia patens Kunth) (ARS- GRIN, 2009). Referred to Vernonia patens HBK in the list of lignocellulose species investigated in Ecuador, it is a source of raw material for pulping and papermaking (Acuña, 2000). It is also commercially important in the beekeeping industry, and is ranked as one of the most important honeybee plants from Tundo, Olmedo and Loja (Camacho, 2001) for its excellent production and availability of nectar and pollen (Ramirez et al., 2001).
In the Ecuadorian province of Zamora it is one of four ecologically important species belonging to the typical families of disturbed forests that are been regenerated (Camacho, 2001;REMACH, 2004). It is now registered as representative tree species of secondary forests in Ecuadorian coastal zone (Aguirre, 2001).
The species has the following synonyms (Blair, 2005):

Habitat
V.patens grows wild in the inter-Andean forest located in the south of Ecuador; its maximum height is 3-6 meters and its altitudinal distribution is between 0 and 2000 meters above sea level (Tobías, 1996;León, 2006). This species has been identified in the vegetal community of dry forests at the south-west of Ecuador 3 .
This species is sometimes grown or kept in farms after its spontaneous appearance. Generally it can be found near the forest trail and on the edge of the rivers. Flowering and fruiting occurs between May and October.

Botanical information
V. patens (Figure 1), is a small branched shrub, growins up to six meters high with furrowed stems and ferruginous trichomes. Alternate leaves are petiolate, narrowly lanceolate, petiole tomentose with ferruginous trichomes, 4-11mm long; the leaves are entirely or weakly serrate, rounded base with a sharp or acuminate apex leaves are 7-15 cm long and 1.3 -1.2 cm wide, the adaxial surface is bright and the abaxial is pubescent or puberulent, subcoriaceous, penninerved. Inflorescence is paniculate, terminal, extended branched with the endings scorpioid, provided with leaves and bracts, capitates sessile and very shortly pedicellate, with numerous bell-shaped flowers, 8 mm long, 4-5 sets bracts imbricated, tomentose and of dark brown color, corolla glabrous, about 5 mm long, weakly pubescent achenes, pappus hairs-layered irregular shaped edges that are about 7 mm long. A detailed description of the botanical characteristics of this species has been published by Blair (2005).

Ethnomedical information
In Ecuador the inhabitants of the south-west of Loja and the Marcabelí region of El Oro province recognize both its healing power and analgesic action. They use the leaves of V. patens to wash wounds and to relieve headaches. It is also employed as anti-inflammatory to soothe coughs and against certain types of cancers. In addition, a veterinary practice is described as it can heal infected wounds by washing with a mixture of plants that includes leaves from this species (Blair, 2005). Other interesting uses have been also reported. Gacheta et al., (2010) informed its usefulness for leishmanianis treatment; Tene et al (2007) indicating its use in the preparation of antivenon and the use of "laritaco" leaves in poultices to combat athlete's foot is referred by Valadeau et al., (2009).
Different uses of V. patens have been registered in other South American countries. In the Bolivian community of Tacama, the juice of the plant stem is applied against conjunctivitis (Tacana, 1999) and in Colombia the watery brews of the aerial parts mixed with "panela" 4 , white wine and rosemary are used against malaria. It is also used to relieve pain due to labor and to purge (Blair, 2005).

Biological and chemical activity
There are very few biological and chemical studies of the specie V. patens. The only results published so far refer to the antimalarial activity against Plasmodium falciparum, Itg2 strain (Blair, 2005) ,anti-Leishmania activity (Valadeau et al., 2009) of the leaves of this species and no antiprotozoal activity against different strains of Leishmania (Fournet, 1994). On the chemical composition of the species, reports lack of sesquiterpene lactones and sesquiterpenes present in the aerial parts (Mabry, 1975;Jakupovic, 1986). There are some references on genus Vernonanthura that show the presence of diterpenes compounds (Portillo et al., 2005;Valadeau et al., 2009), flavonoids (Borkosky et al., 2009;Mendonça et al., 2009), triterpenes (Tolstikova et al., 2006, Gallo et al., 2009, saponins (Borkosky et al., 2009) and sesquiterpene lactones. In addition, different biological activities have been described assuming that certain chemical groups could be responsible for the therapeutic properties attributed to species of this genus (Pollora et al., 2003(Pollora et al., , 2004Portillo et al., 2005;Bardon et al., 2007).
These were the main factors that led to the Laboratorio Bioproductos Centro de Investigaciones Biotecnológicas del Ecuador to undertake a chemical-pharmacological study of Vernonanthura patens leaves from plants growing in Ecuadorian areas. Such investigations are part of the Biodiscovery Program developed by this center.

Phytochemical screening
As an initial step of thephytochemical screening research allows to determine qualitatively the main groups of chemical constituents present in a plant. This screening can guide the subsequent extraction and / or fractionation of extracts for the isolation of groups of interest. The phytochemical screening routine is performed by extraction with suitable solvents of increasing polarity and the application of color reactions (Miranda & Cuellar, 2001).
These reactions are characterized by their selectivity to types or groups of compounds, their simplicity, short time consuming and capacity to detect small amount of compounds using a minimum requirement of laboratory equipment. The results are recorded by the presence (+) or absence (-) of the color reactions.
The general outline of steps followed for performing the phytochemical screening of V. patens' leaves is presented in Figure 2, while the analysis of the extracts obtained at different polarities is schematically shown in Figure 3. This methodology has been referred previously (Miranda & Cuellar, 2000;Manzano et al., 2009). The plant material of adult leaves of Vernonanthura patens (laritaco) were used from plants at the vegetative state which were growing in the citadels "July 25", "Imbabura" and "June 24" and all belonget to the Canton Marcabelí, province El Oro, Ecuador. Leaves were collected at early morning at different dates during the months of December to February in 2009 and 2010.
Botanical identification was performed and voucher specimens of the herbs were prepared and deposited at the National Herbarium of Ecuador (QCNE) and a duplicated sample (CIBE37) was kept as herbal witness in the laboratory of the CIBE-ESPOL Bioproducts. Prior www.intechopen.com Fig. 3. Chemical reactions carried out in each type of V. patens' leaf extracts obtained from using solvents of different polarity.
consent was obtained and authorized by the corresponding agencies of the government. The fieldwork and data collection were conducted in accordance with the institutional, national and international principles and guidelines for using and conserving plant biodiversity.
For conducting the phytochemical screening, extraction and fractionation, leaves samples were dried using an automatic dryer (45 °C, 8 hours) and then pulverized in a blender and screened. The fraction that remained in the sieve of 2 mm in diameter was collected and kept in polyethylene bags of low density at 24 ˚C.
The result of phytochemical screening is presented in Table 2. This reveals moderate to low concentration of essentials oils, alkaloids, reducing compounds, phenols, tannins, flavonoids, quinones, saponins, triterpenes and steroids. Some of these chemical compounds have been associated to antibacterial, antifungal, antiprotozoal and citotoxicity properties and thus have a potential therapeutic use (Nweze et al., 2004;Reuben et al., 2008;Vital et al., 2010).

Plant extracts, fractions and compounds
The dry plant material (67 g of leaves of V. patens) was subjected to successive extractions with HPLC grade methanol by maceration in a closed container and in the absence of light. The extraction time was eight days and was conducted until total depletion of plant material; agitator and a rotary evaporator were used for solvent recovery. The extract was evaporated to dryness, yielding 7g (10.44%) of methanol extract. The methanol residue was subjected to fractionation by successive column chromatography (CC) packed with activated silica from 60 to 200 mesh; elution was performed with solvents of increasing polarity using mixtures of hexane and ethyl acetate (10, 9:1 , 8:2, 3:7, 10) ( Table 3). The extracts were analyzed by thin layer chromatography (TLC) on 60 F254 silica gel cromatofolios (Merck) with fluorescent indicator and a solvent system hexane / ethyl acetate (9:1). Plates were observed under UV light at 254 and 366 nm wavelengths.   The isolated fractions with different solvents from methanol extract of leaves of V. patens by column chromatography, have not been referred to this species, resulting in a high mass in the hexane fraction (79mg) compared with other extracted fractions. Nevertheless, methanol, ethyl acetate and hexane extracts from other plant species had showed a relevant antimicrobial activity (Ramya et al., 2008).

Bioassays
Assays for screening the bioactivity of natural products has had an impressive history of development and is one of the keys for discovering new natural bioactive compounds.
In this study, a qualitative preliminary evaluation of the antifungal capacity of fractions and pure compounds isolated were conducted in order to select the most active. Those selected were re-evaluated to quantify their ability to inhibit fungal growth.
The diffusion method (Avello et al., 2009) in potato dextrose agar (PDA) was used to determine the antifungal activity of fractions and pure compounds isolated from V. patens leaves at 100 and 200 µg mL -1 . Dilutions were made with dimethylsulfoxide (DMSO) 10%.

Strains of Fusarium oxysporum and Penicillium notatum, isolated from infected Pinus radiata
and Citrus sinense fruits and maintained in the Collection of Fungi at University of Concepcion were used.
Holes of 5 mm Ø were made in the agar with a sterile cork borer and filled with 20 µL of each concentration of fractions and pure compounds. DMSO 10% was used as negative control in each plate. A disc (5 mm Ø) of already grown fungus was placed in the center of Petri dishes and incubated at 22 °C. Evaluations were made during two weeks.
Experimental design was completely randomized and each assay was performed in triplicate. Descriptive statistics of the experimental data was made in order to represent and point out its most important features.
Most relevant antifungal activity was observed in fraction 1 (100% hexane) and pure compounds 1 and 3 at the both concentrations tested.
The hexane fraction inhibited the growth of both fungal species tested. Highest inhibition exerted against Penicillium notatum (80.2%) and Fusarium oxysporum (81.5%) occurred when using 200 g mL -1 of this fraction. Statistical differences (P≤0,05) with negative controls indicated that DMSO did not influence the results of biological evaluation.
Pure compounds showed selective inhibition properties and a certain concentration dependence in its antifungal activity. Compound 1 showed a rate of inhibition of 50 and 90% (100 and 200 µg mL 1 respectively) against Penicillium notatum while compound 3 was capable to inhibit 80 and 100% of the Fusarium oxysporum growth for each assayed concentrations.
Screening for antifungal activity of fractions and pure compounds of V. patens has been conducted for the first time. The potential of these results is relevant.

Chemical characterization of the fraction with antifungal activity
The isolated fraction with antifungal activity were analyzed for structural identification by gas chromatography-mass spectrometry (GC-MS) using an Agilent 7890A gas chromatograph with an Agilent 5975 detector (Avondale, PA.USA) equipped with a column HP-5MS of 5m long (0.25 mm in diameter and 0.25 cm inside diameter). Helium was used as the carrier gas; the analytical conditions were: initial temperature: 100 ° C (increasing 8 ° C per minute to a final temperature of 250 º C); inlet temperature and mass detector: 250 o C and 300 °C respectively. The mass detector was used in scan mode ("scan") with a range of 100 to 400 amu.
According to this technique and the analytical conditions described, this chromatogram was obtained and is as shown in Figure 8.
Using the library computer and taking into consideration those compounds that exceeded the 90% of confidence, structures of 33 components could be assigned (Table 4).
The compounds identified are mostly hydrocarbons, a logical result given the solvent used. There was a relative abundance of possible bicyclical sesquiterpenos (peaks 1-5) and of the acyclic triterpeno squalene (peak 30). For the sesquiterpenos exist antecedents of antimicrobial activity (Gregori et al ., 2005) and for the escualeno reports of activity antioxidant, antitumor and antimicrobial activities, in addition to its beneficial effect for preventing cardiovascular diseases by reducing cholesterol and triglycerides (Garcia et al., 2010). For this reason, it is possible to hypothesis that antifungal activity of V. patens against F. oxysporum and P. notatum which has been determined could be directed related to the squalene presence despite not being the main component of the fraction tested. The remaining compounds, individually or collectively, could also be involved in the bioactivity demonstrated. The results described here have not been reported previously for V. patens.

Structural identification of isolated compounds
The structures of the three compounds isolated from the hexane soluble fraction by column chromatography were identified by their spectroscopic patterns as compared with references. These pure compounds were identified as Lupeol (compound 1), Acetyl Lupeol (compound 2) and Epi Lupeol (compound 3) (Figure 9).
Spectroscopy was performed in the Laboratory of Organic Chemistry at the University of Lund. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) were recorded at room temperature with a Bruker DRX500 spectrometer with an inverse multinuclear 5 mm probe head equipped with a shielded gradient coil. The spectra were recorded in CDCl 3 , and the solvent signals (7.26 and 77.0 ppm, respectively) were used as reference. The chemical shifts (δ) are given in ppm, and the coupling constants (J) in Hz. COSY, HMQC and HMBC experiments were recorded with gradient enhancements using sine shaped gradient pulses. For the 2D heteronuclear correlation spectroscopy the refocusing delays were optimized for 1 J CH =145 Hz and n J CH =10 Hz. The raw data were transformed and the spectra were evaluated with the standard Bruker XWIN-NMR software (rev. 010101).
The results that are shown in this chapter are unpublished and have not been previously registered for the species V. patens. Even though, the elucidated structures of the pure compounds have been found in other vegetal species, and recognize their diverse biological activity which includes antineoplastic action against certain types of cancer (Gallo & Sarachine, 2009

Concluding remarks
Phytochemical screening of V. patens has showed the presence of essentials oils, alkaloids, reducing compounds, phenols, tannins, flavonoids, quinones, saponins, triterpenes and steroids, of which some have been previously associated to important biological activities.
Fractions and pure compounds of this species were screened for the first time for antifungal activity. Hexane fraction and two pure compounds further identif i e d a s L u p e o l a n d Epilupeol, were active against two important fungal pathogens at high rate (80-100%). Hexane fraction reduced the growth of Fusarium oxysporum in 80% and Epilupeol completely inhibited the Fusarium oxysporum growth.
Thirty-three chemical compounds in the hexane fraction from V. patens leaves were determined, Of which must are hydrocarbons. Antifungal activity of this fraction can be related to presence of squalene and/or combined activity of others identified compounds. Further research must be done for determining specific bioactivity of identified compounds.
Chemical structures of three isolated compounds were elucidated, corresponding to Lupeol, Acetyl Lupeol and Epi Lupeol. These compounds are recognized for their significant and diverse biological activities, including antimicrobial and antineoplastic actions.
Results of this study show that V. patens can be considered as important potential candidate for further chemical and biological researches and justify its inclusion in the biodiscovery program of CIBE.

Acknowledgements
This study was supported by grants from SENESCYT and ESPOL (Ecuador)

References
Acuña O. (2000). Valoración de las características físico químicas de especies lignocelulósicas y subproductos agroindustriales en la obtención de pulpa y elaboración www.intechopen.com Phytochemicals are biologically active compounds present in plants used for food and medicine. A great deal of interest has been generated recently in the isolation, characterization and biological activity of these phytochemicals. This book is in response to the need for more current and global scope of phytochemicals. It contains chapters written by internationally recognized authors. The topics covered in the book range from their occurrence, chemical and physical characteristics, analytical procedures, biological activity, safety and industrial applications. The book has been planned to meet the needs of the researchers, health professionals, government regulatory agencies and industries. This book will serve as a standard reference book in this important and fast growing area of phytochemicals, human nutrition and health.