Advances in Studies of Vernonanthura patens (Kunth) H. Rob. Growing in Ecuador

Various chemical components have been reported for the genus: sesquiterpene lactones of glaucolide type, hirsutinolide, vernomargolide, eudesmanolide, cardinanolide, and elephan‐ topus, reported for most species, with some exceptions [5-11]. Moreover, diterpenes derived from ent-kaurano and kaurano, pentacyclic triterpenoids from oleanane and ursane, phytos‐ terols, and flavonoids have been isolated and identified [12-15], while the antimicrobial and molluscicidal pharmacological activities have been the most investigated [15-17].


Introduction
Asteracea family (Asteraceae) includes more than 23,000 species and is the group of Angiosperms with the richest biodiversity existing in the world. Vernonia (Vernonanthura) is the most representative gene among 1000 species [1 -4].
Within the genus Vernonia, Vernonanthura patens has been one of the less studied species. It is a shrub that grows wild in Ecuador and is widely used by people in rural areas, who attribute various medicinal properties among them: analgesic, anti-inflammatory, anticancer, and antileishmanial. However, medicinal use, studies on the chemical composition, and pharmacological activities until 2010 were very few. The first information collected on the chemical composition of the species reported the absence of sesquiterpene lactones [18], which constituted a chemical marker of gender. Nevertheless, in 1986, the aerial parts were studied, isolated and 10 lactones of a species were identified, existing contradictions on the composition [19]. Jakupovic and Schmedia-Hirschmann, With respect to biological activity, less information exists. The first, was the effect demonstrated by a methanol extract of the leaves against Artemia salina, the inertness to the potato disc tumor produced by the introduction of Agrobacterium tumefaciens, and the lack of cytotoxicity against cell lines V79 [20]. The antimalarial activity of aqueous extract of leaves has been described and a mean inhibitory concentration (IC 50 ) of 38.7 ± 10.1 µg/ml against Plasmodium falciparum has been reported [21], whereas others authors have reported that an aqueous extract of leaves, got antileishmanial activity against strains of Leishmania amazonensis with an IC 50 >100 µg/ml, being these the only reports found for the species [22].

Studies in the species V. patens in the Ecuadorian coast
In an earlier chapter of this book, has been published the results for the fractionation of a methanol extract of the species, the study of fractions as antifungal against strains of Fusarium oxysporum and Penicilium notatum, and the chemical characterization by gas chromatography/ mass spectrometry of the fraction that showed that activity. The hexane fraction presented antifungal activity against these strains, and 33 compounds were possible to identify. Three pentacyclic triterpenoids (lupeol, epilupeol, and lupeol acetate), presented as major components, were also isolated and characterized for fractions [23] Unlike other species of Vernonanthura and the own V. patens living in other regions of South America, the one that grows in Ecuador presents as major components pentacyclic triterpenoids and no sesquiterpene lactones. These results forced to perform a genetic characterization of the genes from the plastid rbcL and matK of leaf chloroplast DNA fraction, and it confirmed that the species was V. patens [24,25] This research group has continued with studies to verify the identity of the species, obtaining the identification of 53 compounds from leaves of a methanolic extract within the terpene compounds are highlighted (oxygenated sesquiterpenoids and triterpenoids), aliphatic hydrocarbons, fatty acids, and their methyl and ethyl esters and sugars [26,27].
In others studies, two types of waxes of leaves and fat fractions of stems and flowers were isolated and were identified 29 fatty acids and 8 triterpenoids as components of the lipid fractions of these organs, which had not been previously reported (Tables 1 and 2) [28]. Figure  1 presents some of the chemical structures identified in this species.
Regarding biological studies, the antileishmanial evaluation of ethanolic extract of the leaves and stems was performed, proving the traditional use of the leaves of the species as antileishmanial. The ethanol extract of stems was highly toxic. Leaves extract showed a higher selectivity index than pentamidine, used as reference drug [29].

Materials and methods
Leaves, flowers, and stems of the species in the phenological stage of flowering were used and collected around the Biotechnology Research Center of Ecuador located at Km. 30.5 via Perimeter province of Guayas, Ecuador. A sample of the plant material was taken for botanical identification, which was botanized at the National Herbarium of Ecuador (QCNE), Quito, with the key CIBE37a.

Extraction
The extraction from the aerial parts of the species (leaves, flowers, and stems) was performed in water and ethanol by triplicate in an ultrasonic bath VWR of 35 KHz power [30]. In all cases, 10 g of sample was extracted in the solvent (water or ethanol), in the following time intervals: 5, 15, 30, 45, and 60 min.

Determination of antioxidant activity
The determination of antioxidant activity was performed based on the stability of 1,1diphenyl-2-picrylhydrazyl (DPPH) radical to alcoholic and aqueous extracts obtained from the aerial parts. A total of 800 µl of 0.1 N DPPH and 200 µl of the extracts were taken, and the absorbance was measured at 517 nm after 30 min using 200 µl of ethanol and 800 µl of DPPH as control. Determinations were performed in triplicate.

Determination of total polyphenols
The total polyphenol content was measured by the Folin-Ciocalteu method in a spectrophotometer (BioTek), at 760 nm using gallic acid monohydrate (CAS 149-91-7) as patron of the calibration curve. Results were expressed as milligrams of gallic acid per gram of sample (mg GA/g sample). A total of 250 µl of sample and 350 µl of Folin-Ciocalteu 1 N were mixed; 5 min after, 350 µl of 20% of sodium carbonate was added. After the 90-min incubation at room temperature, the absorbance was measured at 760 nm. Determinations were performed in triplicate.

Chromatographic profile of the extracts by HPLC
Chromatographic profiles by high-resolution liquid chromatography (HPLC) were performed in all extracts obtained at different extraction times to determine whether the extraction time produces chemical changes in the extracts. HPLC analysis was performed with Perkin Elmer Series 2000 HPLC with TotalCrom Software operating system. The phenolic compounds were detected at 280 nm with a flow rate of 1 ml/min. C18 column was used at a temperature of 30°C. Separations were carried out in a pumping system by varying the proportion of 2.5% (v/ v) acetic acid in water (mobile phase A) and 70% methanol in water (mobile phase B). The solvent gradient elution program was as follows: 10% to 26% B (v/v) in 10 min, to 70% B at 20 min, and finally to 90% B at 25 to 31 min. The injection volume for all samples was 10µL.

Statistical analysis
A factorial design 2 × 3 × 5 was used involving the categorical factors: extraction solvent (water and alcohol), plant organ (leaves, flowers, and stems), and extraction time (5,15,30,45, and 60 min), with antioxidant activity and total polyphenol content as response variables.

Antileishmanial activity
This test was performed with aqueous extracts obtained as described.

Aqueous extract
Extraction was carried out by decoction of plant materials in proportion of 10% in water during 20 min. The aqueous extract was evaporated to 87ºC and 400 mmHg. Finally, the concentrated extract was lyophilized to 120 × 10 -3 mbar and 47ºC below zero.

Antileishmanial activity
The activity of the extracts against intracellular amastigotes was evaluated as described previously. The peritoneal macrophages were harvested and plated at 106/ml in 24-well Lab-Tek (Costarâ, USA) and incubated at 37ºC under an atmosphere of 5% CO 2 for 2 h. Nonadherent cells were removed by washing with prewarmed phosphate-buffered saline (PBS). Stationaryphase L. amazonensis promastigotes were added at a 4:1 parasite/macrophage ratio, and the cultures were incubated for further 4 h. The cell monolayers were washed three times with prewarmed PBS to remove free parasites. Then 999 ml of RPMI completed medium and 1 ml of the different products dissolved in DMSO were added in duplicate for further 48 h. The cultures were then fixed in absolute methanol, stained with Giemsa, and examined under a light microscope. The number of intracellular amastigotes was determined by counting the amastigotes in 100 macrophages per each sample. In addition, the percentage of infected macrophages was calculated. The results were expressed as percent of reduction of the infection rate in comparison to that of the controls, where the infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages [31]. The IC 50 value was determined from the linear regression of concentrationresponse curves.
The IC 50 of the extracts for the viability of mouse peritoneal macrophages was determined.

Determination of antioxidant activity
The percentage of antioxidant activity by the method of DPPH of the aqueous and the alcoholic extracts of different plant organs, which were obtained at different times of ultrasonic extraction, were determined and differences in time of extraction were observed.
The highest percentage of antioxidant activity was obtained by employing an extraction time of 5 min for the aqueous extracts of leaves and flowers. However, the highest percentage of activity was obtained at 15 min of extraction for the aqueous extract of stems.
Alcoholic extracts of leaves and stems showed no activity at any time of extraction; in contrast, a greater activity at 45 and 60 min of extraction was observed in flowers of V. patens with no differences of significance between these times (p > 0.05) ( Table 3, Figure 2). Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (p > 0.05).

Table 3. Antioxidant activity (%) of aerials parts of Vernonanthura patens
There is only one antecedent of antioxidant activity for the Vernonia genre, for the species Vernonia tweedieana, with no previous studies for V. patens [33].

Determination of total polyphenols
The part of the plant, the extraction time, and the solvent employed have influence in the content of polyphenols.
No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the spectrophotometric determination of polyphenols in the extract.
When water is used as solvent, there is a direct relationship between the extraction time and the concentration of polyphenols; that is, the longer the extraction, the higher the concentration independent of the plant organ used (Figure 3).
In all cases, the highest percentage of total polyphenols was obtained at 45 min of aqueous extraction without significant differences (p > 0.05) with 60 min of extraction. At this time of extraction (45 min), the leaves of the species exhibited the highest percentage of polyphenols, followed by flowers. Stems exhibited the lowest percentage of polyphenol (Table 4).
There are several reports regarding the presence of phenolic compounds in the different plant organs for the Vernonanthura (Vernonia) genre. Flavonoids have been designated as constituents of gender [34]. Flavonoids, tannins, and phenolics are reported for leaves of V. cinerea [35], whereas others authors have reported the presence of flavonoids [14]. For flowers of the same species, flavonoids, tannins, and phenolic compounds have been identifiedas well [35]. Flavonoids and tannins has been detected for leaves of Vernonia amigdalina, [36,37]. Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (p > 0.05). There is only one antecedent of antioxidant activity for the Vernonia genre, for the species Vernonia tweedieana, with no previous studies for V. patens [33].

Determination of total polyphenols
The part of the plant, the extraction time, and the solvent employed have influence in the content of polyphenols.
No polyphenol could be quantified when ethanol was used as solvent, possibly because this solvent removes other colored chemical compounds such as chlorophyll, carotene, and other pigments that interfere with the spectrophotometric determination of polyphenols in the extract.

Phytochemicals -Isolation, Characterisation and Role in Human Health
When water is used as solvent, there is a direct relationship between the extraction time and polyphenols; that is, the longer the extraction, the higher the concentration independent of the plan 3).    Mean values in the same column or row followed by the same capital or lowercase letters, respectively, are not significantly different (p > 0.05) Table 4. Total polyphenol content (mg GA/g) of aerial parts of Vernonanthura patens using water as solvent.
Phenolic compounds, tannins, and flavonoids among other compounds in the leaves, stems, and flowers of the species V. patens of Ecuadorian coast have been reported [38]. This may explain the total polyphenol content found in different plant organs studied, although in not a very high proportion.

Chromatographic profile of the extracts by HPLC
Considering the fact that the extraction method using ultrasound sometimes causes changes in the chemical composition of the extracts, a chromatographic profile was performed to the aqueous extracts obtained at 5 and 45 min of extraction.
Some changes in the chromatographic profile of the aqueous extract of leaves at 45 min are observed (Figure 4), especially in the baseline, although the highest chromatographic peak intensity observed at about 56.9 min has no variation. Chromatographic profile changes are minor in the aqueous extract of the flowers; a major chromatographic peak around 56.6 min is also seen ( Figure 5).
Nevertheless, the aqueous extract of the stems suffered no change in the chromatographic profile at different extraction times analyzed (5,15, and 45 min). The retention time of the major peak was found at 56.9 min (Figure 6).
Only the study for alcoholic extracts of flowers was done because it was the only one that showed antioxidant activity. Minimal changes were observed in the chromatographic profile of 5 and 45 min extraction, which may be due to the transmission of the ultrasonic waves in the ethanol solvent, which are lower than when water is used [38,39]. A chromatographic peak around 57 min was also observed in this extract (Figure 7).
The results lead us to believe that in all plant organs of the species obtained with water and the alcoholic extract of the flowers, there is a major component or a mixture thereof, which eluted at a similar retention time by HPLC, in the studied conditions, which may or may not be responsible for the antioxidant activity found for these compounds. However, it is important to note that no correlation between the polyphenol content of the extracts and the antioxidant activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the percentage of total polyphenolics ( Figure 8).
These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to the presence of polyphenolic compounds that remain to determine the chemical composition of these extracts to determine which one or more compounds are influential in this activity.  The results lead us to believe that in all plant organs of the species obtained with water and the alcoholic flowers, there is a major component or a mixture thereof, which eluted at a similar retention time by studied conditions, which may or may not be responsible for the antioxidant activity found for these However, it is important to note that no correlation between the polyphenol content of the extracts and th activity was found. In all cases, the higher the percentage of antioxidant activity, the lower the perce polyphenolics ( Figure 8). These results indicate that the antioxidant activity of aqueous extracts of the species is not due solely to th polyphenolic compounds that remain to determine the chemical composition of these extracts to determine more compounds are influential in this activity.

Evaluation of antileishmanial activity
Aqueous extracts of leaves and stems showed activity and selectivity against L. amazonensis (Table 5). Th selectivity present in the aqueous extract of the stems showed a different behavior to that reported for the e

Evaluation of antileishmanial activity
Aqueous extracts of leaves and stems showed activity and selectivity against L. amazonensis ( Table 5). The activity and selectivity present in the aqueous extract of the stems showed a different behavior to that reported for the ethanol extract of this plant organ [28]. The evaluation was reported as not detectable because it caused a minimal toxicity in the concentrations tested, destroying the cell monolayer.
The results obtained for the aqueous extract support the traditional use of the species and are highly relevant as these extracts would enhance the usefulness of a possible low-cost source for the development of an effective herbal drug for the treatment against Leishmania.  Table 5. Antileishmanial activity and cytotoxicity of aqueous extracts of Vernonanthura patens against L. amazonensis IC 50 , half maximal inhibitory concentration, is expressed as the concentration of extract (mg/ml) that inhibits 50% of the parasite growth. CC50, median cytotoxic concentration, is expressed as the concentration of extract (mg/ml) causing 50% of parasite mortality. X (S), mean (standard deviation). SI, selectivity index: CC50 macrophages / IC50 Leishmania. SI ≤ 5 more selective for the cell, SI ≥5 more selective for parasite.

Conclusion
The extraction time ultrasound influences the concentration of polyphenolic compounds in all organs tested, when water is used as solvent. Ethanol extracts are unable to determine the concentration of phenolic compounds due to the possible interference of colored compounds extracted with this solvent. Aqueous extracts of the leaves and flowers and the alcoholic extract of flowers showed higher antioxidant activity than the aqueous extracts of the stems. Antioxidant activity in the alcoholic extracts of leaves and stems was not observed.
The HPLC chromatographic profiles of all extracts tested showed a majority chromatographic peak between 56 and 57 min, which could correspond to a mixture of compounds responsible for the antioxidant activity found.
No correlation between antioxidant activity and polyphenol content was found, so presumably they are not solely responsible for the antioxidant activity found.
Aqueous extracts of the leaves and stems of the species V. patens showed higher activity and selectivity than the alcoholic extracts, against L. amazonensis, corroborating the traditional use of the species.

Future directions
The results obtained in the chemical and biological study of the species V. patens show its potential as antimicrobial, antioxidant, and particularly antileishmanial, antiparasitic diseases of high incidence in Ecuador and other countries. However, inclusion in the therapeutic still requires further studies among which may be mentioned toxicology to demonstrate the safety of their preparations and as a development of a suitable dosage form to the pathology to be treated, aspects that should motivate future research of our work group.