Apigenin glycoside: an antioxidant isolated from Alchornea

An antioxidant flavonoid has been isolated from methanolic leaf extract of Alchornea coelophylla Pax & K. Hoffm. by means of different column chromatography steps with DIAION HP-20 resin and silica gel in combination with analytical high-performance liquid chromatography (HPLC). It was identified as Apigenin-8-C - (α-L-rhamnopyranosyl-(1→2)-β- D -glucopyranoside) on the basis of spectroscopic analysis and by comparison with related values reported in the literature. This compound exhibited high in vitro antioxidant activity through DPPH • and ABTS •+ colorimetric assays with IC 50 values of 7.528 and 379.7 µg. mL -1 , respectively.


Introduction
A great variety of molecules exists in nature with the capacity of scavenging free radicals through different mechanisms.Flavonoids are phenolic compounds generally present in plants and fruits.Sometimes, they are responsible for some flower colours such as red, pink, and purple or violet [1] and play biological roles as preventing damages by continuous exposure to UV radiation [2].
Flavonoids display their noteworthy antioxidant activity by three different mechanisms, (i) hydrogen transfer to radical type compounds [3], (ii) prevention of Fenton type reactions due to metal chelation [4] and (iii) synergist effects with other antioxidant compounds [5].These mechanisms could explain the wide range of biological activities attributed to flavonoids.

original article
Bogotá and Alchornea cordifolia [12,13].Our aim was to investigate the antioxidant properties of Alchornea coelophylla, a wild plant endemic to Colombia with no previous reports on antioxidants and to isolate compounds related to the assessed biological activity.

General Methods
Column chromatography was carried out on silica gel (230-400 mesh, Macherey Nagel, Düren, Germany) and macro-porous resin DIAION HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan).Analytical high performance liquid chromatography (HPLC) was carried out on an Agilent 1100 Series liquid chromatograph equipped with an Agilent DAD-G1315A detector for the UV spectrum acquisition (Agilent

Plant Material
Leaves and twigs of Alchornea coelophylla (Euphorbiaceae) were collected in a natural protected zone known as Bremen-La Popa at the coordinates 4º 40' 48.

Extraction and Isolation
The collected plant material (leaves and twigs) was dried in a laboratory stove at 50 ºC and further ground with a hammer mill (Nogueira (Ø: 5.1 mm)).Afterwards the dried material (1.212 g) was exhaustively extracted at room temperature allowing it to stand overnight using 10 L of n-hexane (HEX), dichloromethane (DCM) and methanol (MeOH), with an increasing polarity order.The concentration of the respective solutions under vacuum produced the respective dried extracts.

Antioxidant Activity
The antioxidant activity was evaluated along the isolation process to ensure the isolation of a biologically active flavonoid.In this sense, biological activities were assessed on the crude extract and fractions of the first chromatographic purification step following the methodology described by Brand-Williams & Berset [14] and Re [15] for the antioxidant assays of DPPH • and ABTS •+ , respectively, with minor modifications in order to perform the experiments in a 96-well microplate.
Measurements were done by triplicate and with two different repetitions in order to obtain statistically homogeneous results.IC50 determinations and calibration curves were constructed in the same way and analysed with GraphPad Prism V. 5.01.
Methanolic crude extract and chromatographic fractions were evaluated at concentrations of 1,000 and 500 µg.mL -1 , respectively.A 1,000 µg.mL -1 methanolic solution of hydroquinone, prepared the same day of the test, was used as positive control.Analogously a photometric blank to each sample extract was employed additionally with the photometric blank (the respective solvent mixture).

DPPH • Radical Assay
100 µL of a 20 µg.mL -1 DPPH • solution in MeOH (prepared just before the assay) were thoroughly mixed with 25 µL of the sample.The reaction was allowed to stand for 30 minutes in the absence of light and then the absorbance was measured at a wavelength of 517 nm.The photometric blank for the plant extracts consisted of 25 µL of the sample and 100 µL of MeOH.

ABTS •+ Radical Assay
An aqueous 3.5 mM ABTS •+ and 1.25 mM potassium persulfate solution was allowed to react 12 hours before the evaluation, then the absorbance of the final solution was adjusted to 0.7 with EtOH.Finally, 294 µL of the adjusted solution was added to each well where 6 µL of the sample extract had been transferred previously.The reaction was allowed to stand for 30 minutes in the darkness and at that time the absorbance was measured at 732 nm.The photometric blank for plant extract consisted in 6 µL of the sample and 294 µL of EtOH.
In order to be able to compare and report antioxidant activity results as equivalents of the same reference standard, calibration curves of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX) were constructed with concentrations ranging from 1 µM to 100 µM, using it as antioxidant reference compound [16].
Additional to the determinations of the biological activity, quantifications of phenolic compounds and flavonoids were considered, in order to determinate which could be the most promising fraction to continue the isolation process.

Quantification of Phenolic Compounds
This determination was completed following the Folin-Ciocalteu method, described by Magalhães [17]. A. coelophylla extracts and fractions were evaluated at 50 µg.mL -1 in methanol.The Folin-Ciocalteu reactive was also diluted in a proportion 1:50 with distilled water to adjust the absorbance of the solution into the range of 0.2 to 0.8.Then, 100 µL of NaOH 0.35 M were transferred to the same wells where previously 50 µL of the plant extracts and 50 µL of the diluted Folin-Ciocalteu reactive had been mixed.Afterwards, the reaction was allowed to stand for three minutes in the absence of light and the absorbance was measured at 760 nm in a microplate spectrometer MultiSkan Go.
In this determination the blank for each sample was also used and the photometric blank corresponded to 200 µL of water.Finally, to express the results as equivalents of some comparable parameter [17], a calibration curve of gallic acid was constructed at concentrations of 0, 2, 4, 8 and 16 µg.mL -1 .

Quantification of Flavonoid Content
The flavonoid content was assessed following the methodology described by Kim [18].The sample extracts were evaluated at a concentration of 100 µg.mL -1 in methanol.First, 7.5 µL of 5 % aqueous NaNO2 were transferred to each well where the extracts were going to be evaluated.Then, 20 µL of each plant extract and 115 µL of distilled water were then pipetted.After 5 minutes of reaction, 30 µL of 2.5 % AlCl3 were added while shaking; then, once 5 minutes of reaction took place, 50 µL of NaOH 1 M and 50 µL of distilled water were added and thoroughly mixed.Finally, 5 minutes after the NaOH addition the absorbance was measured at 500 nm.
Finally, in the same way of the phenolic compounds determination described above and to obtain these results as equivalents of some comparable parameter, a calibration curve with kaempferol was constructed at concentrations of 0, 0.05, 0.1, 0.2, 0.4 and 0.8 µg.mL -1 .

Determination of Antioxidant Activity and Bio-guided Isolation
The dried and grounded plant material of Alchornea coelophylla (1.212 g) subjected to successive extraction by maceration with hexane, dichloromethane and methanol, gave 91.0997 g of methanolic dried extract.
A phytochemical screening of the plant extracts revealed that the major flavonoid content occurred in the most polar methanolic leaf extract, which was fractionated in a first chromatographic partition based on polarity and molecular size with DIAION HP-20 resin, through which 29 fractions (DHP-1 to DHP-29) were collected.Mass and yield fractions are shown in Table 1.
Antioxidant activities were assessed over the 29 fractions through both DPPH • and ABTS •+ assays, in order to determine which fraction could be the most promising for the isolation process.Similarly, total phenolic and flavonoids contents were determined.Figure 1 summarizes the values of above mentioned evaluations for all the chromatographic fractions.After data collection, fraction 11 was selected and subjected to a second Silica gel 0.04-0.063mm/ 230-400 mesh column to obtain 7 fractions (DHP-11-A to DHP-11-G).Finally the target compound was confirmed to be with a high grade of purity (> 98 % based on HPLC data) in fraction DHP-11-C, fraction that was then used in the spectroscopic data acquisition.Masses and yields of fraction of the second column chromatography are presented in Table 2. IC50 values of the compound present in fraction DHP-11-C were 7.528 and 379.7 µg.mL -1 , through DPPH • and ABTS •+ assays, respectively.

Determination of Antioxidant Activity and Bio-guided Isolation
The methanolic crude extract obtained by maceration gave a yield of 7.52 %.
The MeOH leaf extract was dissolved in methanol and fractionated by open column chromatography with DIAION HP-20 resin to obtain 29 fractions (DHP1 to DHP29) which then were classified and selected by mass, yielding 17 fractions with an availability greater than 300 mg.Afterwards, the antioxidant activity of the selected fractions was evaluated by the DPPH • and ABTS •+ methods at 500 µg.mL -1 ; in addition, the above quantifications were assessed to obtain more information that could suggest the most promising fraction to continue the isolation process.
With all the collected information regarding biological activities, and taking into account the amount available of each fraction, the 11th fraction was selected as the most promising to continue the isolation process with a mass of 955.53 mg, with 51.85 % of antioxidant activity under the DPPH • assay, 99.75 % of antioxidant activity under the ABTS •+ test, 28.49 µg.mL -1 of gallic acid equivalents and 0.42 µg.mL -1 of kaempferol equivalents.As a consequence, this fraction was subjected to a silica gel column fractionation yielding fractions DHP-11-A to DHP-11-G, which finally by analytical HPLC revealed the presence of a flavonoid glycoside in the fraction DHP-11-C.
Thus, based on the antioxidant percentage values obtained for fraction DHP-11-C and using the TEAC calibration curves, total equivalents of Trolox (µmol.mg-1) were found to be 108.7 and 564.67 for the DPPH • and ABTS •+ assays, respectively.Once the purity of the isolated compound was determined, the median inhibitory concentration (IC50) was calculated for both of the above mentioned assays.The biological activity was assessed on the isolated compound at concentrations of 500, 250, 100 and 50 µg.mL -1 for both the DPPH • and ABTS •+ methods.IC50 values of 7.528 and 379.7 µg.mL -1 were obtained for the DPPH • and ABTS •+ determinations, respectively.Those results agree with previous investigations in which it was found that the methanolic crude extract of A. coelophylla had an IC50 even lower than the reference control (Hydroquinone), which revealed IC50 of 41.14 and 151.19 µg.mL -1 for both antioxidant colorimetric assays and also that the antioxidant response was notoriously higher when evaluated through the ABTS •+ determination than the antioxidant values obtained for the DPPH • determination [19].Curves constructed for IC50 determinations of the isolated compound are in Figure 2.

Figure 1 .
Figure 1.Determinations of the first column chromatographic fractions.A) Antioxidant activity through ABTS •+ ; B) Antioxidant activity through DPPH • ; C) Total phenolic content and D) Total flavonoid content.

Figure 3 .
Figure 3. 2D NMR interactions observed in HMBC experiment for the isolated compound.

Table 1 .
Masses and yields for fractions of the first DIAION HP-20 chromatographic step.

Table 2 .
Masses and yields for fractions of the second Silica gel chromatographic step.