Eleutherine Plicata – Quinones and Antioxidant Activity

reported activity. The isolation and characterization of isoeleutherol and isoeleutherine, which show well-described pharmacological activities, justify the potential of E. plicata Herb. to originate a phytomedicine to face neglected diseases, such as Amoeba infection, and probably explain the reason why this plant finds a wide popular use in Amazonian countries.

The experimental characterization of the antiamoebic activity of the decoction prepared with bulbs of E. plicata contributes to validate its alleged popular use. The detection of isoeleutherine in the analyzed decoction can explain partially the reported antiamoebic activity, which can be attributed to the pro-oxidant activity of the substance [7].

Collection and identification of botanical material
The plant material purchased at the Ver-O-Peso Market, Belem, Pará State, Brazil was collected in October 2007 in the same region. The botanical identification occurred by comparison of the prepared exsiccata ( Figure 3) to a voucher deposited at the Herbarium of the Emilio Goeldi Museum registered under the number 10543 2 .

Processing of plant material and extraction
Approximately 5 kg of fresh bulbs were sliced and after washing, aeration and selection, dehydrated at room temperature for 2 days. Drying was completed under forced hot air circulation at about 40° C. The dried material was ground in a Wiley knives mill to yield 1.20 kg of herbal drug.
An ethanol extract (EE) was obtained by successive macerations, using 500 g of the herbal drug and Ethanol 96 o GL, until total drug exhaustion. Thereafter, the solvent was removed under reduced pressure in a rotary evaporator.

Phytochemical screening
Chemical tests were performed on EE, HF, CF, EAF, and RF based on the Guide for Phytochemical Analysis of Plant Extracts [11] in order to verify the presence of 18 classes of secondary metabolites.

Thin Layer Chromatography (TLC) analyses
TLC analyses aiming to corroborate the phytochemical results on EE, HF, CF, EAF, and RF were performed employing silica gel as stationary phase and as eluents hexane/acetone (80:20), chloroform/methanol (90:10), chloroform/methanol/water (70:25:05), chloroform/acetone (99:03) and (99:01), thus the corresponding chromatographic profiles were defined. The obtained chromatograms were observed under visible and ultraviolet light at 254 nm and 365 nm and then sprayed with KOH 10% in methanol to make possible the detection of quinones. The chromatographic profiles were obtained by determining the retention factor (Rf) and describing the color of each chemical constituent observed as a zone in the chromatograms.

Characterization of 1 and 2
About 20 mg of 1 and 2 were dissolved in deuterated chloroform (CDCl 3 ) to be analyzed in a Variant brand Plus NMR Spectrometer. The characterization of these substances was achieved by comparison of their 1 H-(300 MHz) and 13 C-NMR (75MHz) spectra to those obtained from the same naphthoquinones isolated from other species of Eleutherine and reported in the scientific literature.

LC profile of EE, CF, 1, and 2
The LC-DAD profile was recorded using a LaChrom 7000 Merck HITACHI ® chromatograph hyphenated to a DAD equipped with a LiChrospher100 Agilent column (250 × 4.6 mm). The mobile phase consisted of ultrapure water and acetonitrile (ACN) as described in Table 1 and was pumped at 1 mL/min. The oven temperature was 26°C (± 1°C) and the detection occurred between 200 nm and 500 nm, this method was adapted from Paramapojn et al. (2008) [12]. Aliquots of 50 µL were applied at the following concentrations: 1, 500 µg/mL; 2, 1 mg/mL; CF, 2,500 µg/mL; and crude EE, 1 mL.

Antioxidant activity of EE, 1, and 2
The antioxidant capacity of EE, Ep1, and Ep2 was evaluated using the free radical 2,2diphenyl-1-picrylhydrazyl ([DPPH] ⋅+ ) and as reference the substance butylhydroxytoluene (BHT). The reaction was accompanied by color change and the activity is monitored by the decrease in absorbance of the mixture at 517 nm relative to the solvent as blank [13].

Botanical identification
The characterization of an exsiccata containing herborized plant material of Eleutherine plicata Herb. confirmed the identity of the investigated herbal drug. According to Tropicos®, Eleutherine bulbosa is an accepted name for E. plicata.

Extraction and fractionation
The ethanol extract, EE, weighed 63 g, from which circa 30g provided four fractions by solid/ liquid partition: HF = 2.103 g, 7%; CF = 3,224 g, 11%; EAF = 5,551 g, 18%; and RF = 12.494 g, 41%. The process generates a loss in mass of about 25%, partially due to the solubility of the constituents of the extract in the employed solvents and the partition coefficient of them; and to the evaporation of volatile substances inherent to the methods used to obtain and concentrate the fractions.

Phytochemical screening
The positive results of the phytochemical approach of EE and fractions considering the presence of 18 classes of secondary metabolites are shown in Table 2.
The metabolites were detected in fractions according to the polarity of the solvents used in the fractionation, such as steroids and triterpenes, azulenes, anthraquinones and naphthoquinones in HF and CF, which are solvents and metabolites of low polarity. Moreover, saponins, tannins and phenols, and coumarin derivatives present in EAF and RF show middle to high polarities. Reducing sugars, detected only in EE, are metabolites of very high polarity, which in liquid-liquid partition do not migrate to organic layer. saponin, coumarin derivatives, and tannins and phenols were not detected in EE, probably due to their concentration in the crude extract or the occurrence of interference on the reagent used.

Thin Layer Chromatography (TLC) analyses
TLC analyses are used to define the chromatographic profile of extracts and fractions, thereby contributing to the quality control of herbal drugs and their derivatives. In present case, different chromatographic systems were tested to obtain the chromatographic profile of EE, HF, and CF, wherein normal phase silica gel and chloroform/acetone (99:1) produced chromatograms with very good resolution. This chromatogram also shows, in EE and fractions, brown colored areas with Rfs 0.25, 0.31, and 0.44, respectively, indicating the presence of naphthoquinones after treatment with KOH 10% in methanol ( Figure 4C), which is a reagent to detect quinones [14]. Additionally, three rose colored spots with Rfs 0.5, 0.62, and 0.87, respectively, can be seen in HF.

Isolation of major chemical constituents
Sample A1 (83 mg) appears as an isolated chemical substance when analyzed by TLC; it was named 1 and can be observed in HF fraction with Rf 0.62 ( Figure 4A, B, C). From C-4, a sample reacting like a naphthoquinone could be purified by preparative TLC yielding 51 mg of a substance that was named 2 that in the TLC analyses showed Rf 0.44 and can also be observed in Figure 4A, B, C.

Isoeleutherol
The 1 H-NMR spectral data of 1 listed in Table 3  The signal observed as a singlet (δ = 4.108 ppm) refers to the hydrogen atoms of the methoxy group attached to the aromatic ring. There is also a signal of a methyl group (δ = 1.736 ppm), which is attached to C-1 of the furan ring, coupling with H-1 (δ = 5.718ppm), thus appearing as a doublet.
The hydrogen at C-1 (δ = 5.718ppm) couples with the hydrogen atoms of the methyl group at the same position, generating a quartet. Finally, a phenolic hydrogen appears as a singlet at δ = 9.644.

Phytochemicals -Isolation, Characterisation and Role in Human Health
The 13 C-NMR spectral data of 1 listed in Table 4 shows the presence of 14 carbon atoms. The signals with δ = 19.34 and δ = 56.77 are characteristic of methyl carbon atoms of C-11 and C-10, respectively, and the signal at δ = 76.80 is characteristic of carbon C-1. The signals at δ = 126.79, δ = 123.82, δ = 116.67, and δ = 106.44 correspond to the carbons C-4, C-5, C-6 and C-7, respectively. The carbon C-3 of furan ring that is double bonded to an oxygen atom shows a signal at δ = 170.69 and the resonance of non-substituted aromatic carbon atoms such as C-4a and C-8a appears at δ = 137.37 and δ = 117.66. The resonance at δ = 156.42 refers to C-8 where a methoxy group is attached, while the signal at δ = 149.20 corresponds to C-9 bonded to the hydroxyl group. Finally, the carbon atoms C-3a, C-9a of the furan residue condensed with an aromatic ring resonate at δ = 126.04 and δ = 128.07, respectively.  The very close correspondence of the 1 H-and 13 C-NMR spectral data of 1 to those found in the literature (Tables 3 and 4) allows to infer that the isolated substance is isoeleutherol ( Figure  5), which has been isolated from Eleutherine americana Merr. et Heyne by Hara et al. (1997) [15] being this the first report of its occurrence in E. plicata Herb. Hara et al. (1997) [15] found that isoeleutherol did not inhibit the enzyme topoisomerase II DNA dependent, but significantly hinders the HIV replication in H9 lymphocytes.

CARBON/ POSITION
Since isoeleutherol seems to be very stable and the major chemical constituent in EE, it could be used as a chemical marker of E. plicata and its derivatives.

Isoeleutherine
Substance 2 was analyzed by 1 H-NMR and its structure was characterized by comparison of the obtained spectral data to those reported in the literature.   [15] found that isoeleutherol did not inhibit the enzyme topoisomerase II DNA dependent, but significantly hinders the HIV replication in H9 lymphocytes.
Since isoeleutherol seems to be very stable and the major chemical constituent in EE, it could be used as a chemical marker of E. plicata and its derivatives.

ISOELEUTHERINE
Substance 2 was analyzed by 1 H-NMR and its structure was characterized by comparison of the obtained spectral data to those reported in the literature. Table 5  The hydrogen atoms of the methyl group attached to C-1 appear as a doublet at δ = 1:54 (J H1-CH3 =6.7Hz) and that one bonded to C-3 at δ = 1:33 (J H3-CH3 =6.1Hz).  Figure 5. Chemical structure of isoeleutherol.

Phytochemicals -Isolation, Characterisation and Role in Human Health
The spectral data listed in Table 5 when compared with that from the literature permit to deduce that 2 corresponds to isoeleutherine ( Figure 6), a naphthoquinone already isolated from Eleutherine bulbosa Mill [9] and from E. americana Merr. by Hara et al. (1997) [15]. This is the first report of the occurrence of isoeleutherine in E. plicata. Figure 6. Chemical structure of isoeleutherine.

Chromatographic profile by LC-DAD
The LC-DAD profile of EE and CF was obtained using the method developed by Paramapojn et al. (2008) [12] with modifications and the best chromatograms were registered at 250 nm.     CF chromatogram shows two peaks with high intensity at 19.12 min (Peak 1) and 21.18 min (Peak 2), with areas of 7,813,739 and 1,900,571 and purity of 98.29% and 99.75%, respectively ( Figure 8).
The isolated isoeleutherol was also analyzed by LC-DAD under the same conditions as EE and CF, generating a peak at 21.71 min with 3,641,711 area and purity of 99.92% (Figure 9). The isolated isoeleutherolwas also analyzed byLC-DADunder the same conditionsa ing a peak at21.71minwith 3,641,711areaand purity of99.92% (Figure 9).   The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-DAD profile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity of 99.14% is registered.
The reverse survey feature in the library of the chromatograph shows correlations of 97.40% and 99.89% between the UV spectrum of the peak 01 in EE ( Figure 7) and in CF ( Figure 8) and that of isoeleutherine. Similarly, the peak 02 in EE ( Figure 7) and in CF ( Figure 8) showed a correlation of 99.84% and 99.98%, respectively, between the UV spectra of both the peaks and that of isoeleutherol.  The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC-D ofile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity of 99.14% istered.   Figure 11 and Table 6 show the evaluation of the antioxidant activity of EE, isoeleutherol, and isoeleutherine on DPPH in comparison to the results obtained for BHT used as standard. The antioxidant activity of isoeleutherol (Ep1) appears in concentrations up from 4 µg/mL; for EE, 5 µg/mL; and isoeleutherine (Ep2), 6 µg/mL; while BHT showed activity in concentrations above 1 µg/mL. Figure 11 and Table 6 show the evaluation of the antioxidant activity of EE, isoeleutherol, and isoeleutherine on DPPH in comparison to the results obtained for BHT used as standard. The antioxidant activity of isoeleutherol (Ep1) appears in concentrations up from 4 μg/mL; for EE, 5 μg/mL; and isoeleutherine (Ep2), 6 μg/mL; while BHT showed activity in concentrations above 1 μg/mL. Figure 11. Evaluation of antioxidant activity of EE, and isoeleuterol, and isoeleutherine against DPPH.

Antioxidant activity of EE, isoeleutherol, and isoeleutherine
Among the tested samples, isoeleutherol showed the best antioxidant activity considering its Inhibition Concentration value (   The same procedure was adopted to analyze the obtained isoeleutherine, producing the LC ofile, showed in Figure 10, where a peak at 18.13min with area 24,727,851 and purity of 99. gistered.  Among the tested samples, isoeleutherol showed the best antioxidant activity considering its Inhibition Concentration value ( The antioxidant activity of isoeleutherol, higher than that observed for EE and isoelutherine, may be attributed to the hydroxyl group at C-9; this phenolic residue may act as free radical scavenger and sometimes as chelating agent of metal ion with effective action, mainly in preventing lipid oxidation, acting both on the initiation step as on the propagation step of this oxidative process. Table 6 shows that EE has a higher IC 50 than isoeleutherol, but lower than that of isoeleutherine. This fact can find explanation in the presence of tannins in EE, which are polyphenols that have adequate chemical structure for the capture of free radicals, contributing to an effective antioxidant capacity of the sample. It is noteworthy to mention that the presence of naphthoquinones in EE may antagonize the activity of tannins since these secondary metabolites can induce oxidative stress or show pro-oxidant capacity, leading EE to present a very low antioxidant activity. Indeed, isoeleutherine presents a negligible antioxidant activity as expected from its structural characteristics, but its occurrence in aqueous extract may be a reason for the antiamoebic activity detected in a previous work [5] and described for E. bulbosa, a synonym of E. plicata according to TROPICOS ®3 , 30 years before today [16].

Conclusions
Isoeleutherol and isoeleutherine described before in other Eleutherine species were isolated from the chloroform fraction of EE and characterized by 1 H-and 13 C-NMR. Their HPLC analyses allow confirmation that both are present in the herbal drug, dried bulbs of E. plicata. Isoeleutherol seems to be the major chemical constituent in EE and thus can be indicated as a chemical marker for the quality control of this plant species and its derivatives. The occurrence of isoeleutherine in aqueous antiamoebic extract indicates this substance to be used in the quality control of the extract and derivatives, mainly if the substance can be linked to the reported activity. The isolation and characterization of isoeleutherol and isoeleutherine, which show well-described pharmacological activities, justify the potential of E. plicata Herb. to originate a phytomedicine to face neglected diseases, such as Amoeba infection, and probably explain the reason why this plant finds a wide popular use in Amazonian countries.

Author details
Luiz Claudio