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Apigenin glycoside: an antioxidant isolated from Alchornea coelophylla pax & k. Hoffm.

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.

A great number of ethnopharmacological applications of species belonging to the Alchornea genus is reported in tropical Africa and some regions of central Brazil [6, 7]. The activities are antiulcerogenic, anti-inflammatory, hepatoprotective, antibacterial and cytotoxic, attributed to flavonoids present in Alchornea castaneafolia [8], Alchornea floribunda [7], Alchornea glandubsa [9], Alchornea laxiflora [10], Alchornea triplinervia [11] 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.

Materials and Methods

General Methods

Column chromatography was carried out on silica gel (230-400 mesh, Macherey Nagel, Duren, 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 Technologies, California, United States) and the data acquisition was done using ChemStation (Version B.04.03). Columns RESTEK Ultra AQ C18 (3 [micro]m, 100 x 3.2 mm, RESTEK, Pennsylvania, United States) and Agilent ODS Hypersil (5 [micro]m, 250 x 4 mm, Agilent Technologies, California, United States) were used for analytical purpose. Thin-layer chromatography (TLC) was conducted over pre-coated silica gel 60[F.sub.254] plates (Merck, Darmstadt, Germany) and spot detection was performed under UV light ([lambda] = 254 and 366 nm) and then spraying with 1 % Al[Cl.sub.3] in ethanol (EtOH) (Sigma-Aldrich, Missouri, United States). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-400 NMR Spectrometer (Bruker Biospin Gmbh, Rheistetten, Germany) with solvent residual peaks of dimethyl sulfoxide (DMSO)-[d.sub.6] at [[delta].sub.H] 2.5 and [[delta].sub.C] 39.52 as references.

Antioxidant colorimetric assays using [DPPH.sup.*] and [ABTS.sup.*+] (Sigma-Aldrich, Missouri, United States) and phenolic and flavonoid quantifications, using Folin-Ciocalteu & Al[Cl.sub.3] (Sigma-Aldrich, Missouri, United States), respectively, were assessed on a microplate spectrometer Multiskan GO (Thermoscientific, Massachusetts, Unites States). Infrared Spectroscopic data were acquired in an Agilent Cary 630 FTIR (Agilent Technologies, California, United States) with a Windows compatible version of MicroLab Software (Agilent Technologies, California, United States).

Plant Material

Leaves and twigs of Alchornea coebphylla (Euphorbiaceae) were collected in a natural protected zone known as Bremen-La Popa at the coordinates 4[degrees] 40' 48.6" North and 75[degrees] 37' 32.7" West at 6737 feet above sea level, near to the municipality of Filandia (Quindio, Colombia), this procedure was completed under the contract of access to genetic sources for scientific research without commercial interest number 56 of February 28th of 2016 granted by the Ministry of Embironment. A voucher specimen was deposited at the Herbarium of the Universidad de Antioquia (Medellin, Colombia), labelled as FJR-3969. The botanical identification was done by Professor Francisco Javier Roldan from Universidad de Antioquia, Medellin, Colombia.

Extraction and Isolation

The collected plant material (leaves and twigs) was dried in a laboratory stove at 50[degrees]C and further ground with a hammer mill (Nogueira (0: 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.

Fifteen grams of methanolic leaf extract were subjected to open column chromatography with DIAION HP-20 resin (200 g, 74.5 x 90 mm) and eluted with MeOH-[H.sub.2]O mixtures (0:100, 20:80, 40:60, 60:40, 80:20, 100:0 (v/v) with 1.5, 1.25, 5.55, 4.6, 3.7 and 2 L, respectively). After pooling related fractions according to their TLC profiles, 29 fractions (DHP-1 to DHP-29) were finally obtained. Afterwards, a first exclusion step was applied based on the mass availability of each fraction. In this sense, 17 fractions with a mass greater than 300 mg were selected to continue the bio-guided isolation process. Thus, based on the correlation among mass availability and further antioxidant data, fraction DHP-11 (750 mg) were seeded onto a silica gel column (22.5 g, 20 x 150 mm) and then washed with approximately 10 bed volumes (BV) of chloroform (CH[Cl.sub.3]) and ethyl acetate (EtOAc) to eliminate low polarity impurities, to finally elute the sample with EtOAc-MeOH-AcOH-[H.sub.2]O (70:20:5:5) collecting fractions DHP-11-A to DHP-11-G. The presence of the target compound in the fraction DHP-11-C was confirmed by analytical RP-HPLC using a 100 % MeOH system as the mobile phase at a flow rate of 1 mL. [min.sup.-1] at a retention time of 1.010 min. Spectral data were also recorded.

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.sup.*] and [ABTS.sup.*+], 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. [IC.sub.50] 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 [micro]g x [mL.sup.-1], respectively. A 1,000 [micro]g x [mL.sup.-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.sup.*] Radical Assay

100 [micro]L of a 20 [micro]g x [mL.sup.-1] DPPH solution in MeOH (prepared just before the assay) were thoroughly mixed with 25 [micro]L 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 [micro]L of the sample and 100 [micro]L of MeOH.

[ABTS.sup.*+] Radical Assay

An aqueous 3.5 mM [ABTS.sup.*+] 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 [micro]L of the adjusted solution was added to each well where 6 [micro]L 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 [micro]L of the sample and 294 [micro]L 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 [micro]M to 100 [micro]M, 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 Magalhaes [17]. A. coelophylla extracts and fractions were evaluated at 50 [micro]g x [mL.sup.-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 [micro]L of NaOH 0.35 M were transferred to the same wells where previously 50 [micro]L of the plant extracts and 50 [micro]L 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 [micro]L 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 [micro]g x [mL.sup.-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 [micro]g x [mL.sup.-1] in methanol. First, 7.5 [micro]L of 5 % aqueous NaN[O.sub.2] were transferred to each well where the extracts were going to be evaluated. Then, 20 [micro]L of each plant extract and 115 [micro]L of distilled water were then pipetted. After 5 minutes of reaction, 30 [micro]L of 2.5 % Al[Cl.sub.3] were added while shaking; then, once 5 minutes of reaction took place, 50 [micro]L of NaOH 1 M and 50 [micro]L 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 [micro]g x [mL.sup.-1].

Results

Determination of Antioxidant Activity and Bio-guided Isolation

The dried and grounded plant material of Alchornea coebphylla (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.sup.*] and [ABTS.sup.*+] 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 0.04-0.063 mm/ 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. [IC.sub.50] values of the compound present in fraction DHP-11-C were 7.528 and 379.7 [micro]g x [mL.sup.-1], through [DPPH.sup.*] and [ABTS.sup.*+] assays, respectively.

Structural Elucidation of Isolated Compound

Apigenin-8-C-([alpha]-L-rhamnopyranosyl-(1 [right arrow] 2)-[beta]-D-glucopyranoside). Obtained as a yellowish powder. IR [cm.sup.-1]: 3400, 2970, 2925, 1700, 1650, 1600, 1560, 1500, 1430, 1350, 1300, 1260, 1200, 1175, 1025, 830, 800. [sup.1]H NMR (DMSO-[d.sub.6], 400 MHz) 8: 6.74 (1H, s, H-3), 6.28 (1H, s, H-6), 8.01 (2H, d, J=8.464 Hz, H-2', H-6% 6.93 (2H, d, J=8.475 Hz, H-3', H-55), 4.77 (1H, dJ=10.041 Hz, H-1"), 4.95 (1H, s, H-1""), 2.85-4.10 (10H, m), 0.46 (3H, d, J=5.911 Hz, H-6'"). [sup.13]C NMR (DMSO-[d.sub.6], 100 MHz) [delta]: 164.00 (C-2), 102.43 (C-3), 182.10 (C-4), 161.20 (C-5), 98.29 (C-6), 162.34 (C-7), 104.47 (C-8), 155.83 (C-9), 104.22 (C-10), 121.6 (C-15), 128.99 (C-25), 115.89 (C-37), 160.67 (C-4'), 115.89 (C-5'), 128.99 (C-6'), 70.67 (C-1"), 75.09 (c-2"), 79.90 (C-3"), 70.46 (C-4"), 81.81 (C-5"), 61.17 (C-6"), 100.32 (C-1'"), 70.26 (C-2'"), 68.22 (C-3'"), 71.68 (C-4'"), 71.49 (C-5'"), 17.71 (C-6'").

Discussion

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.sup.*] and [ABTS.sup.*+] methods at 500 [micro]g x [mL.sup.-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.sup.*] assay, 99.75 % of antioxidant activity under the [ABTS.sup.*+] test, 28.49 [micro]g x [mL.sup.-1] of gallic acid equivalents and 0.42 [micro]g x [mL.sup.-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 ([micro]mol x mg-1) were found to be 108.7 and 564.67 for the [DPPH.sup.*] and [ABTS.sup.*+] assays, respectively.

Once the purity of the isolated compound was determined, the median inhibitory concentration ([IC.sub.50]) 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 [micro]g x [mL.sup.-1] for both the [DPPH.sup.*] and [ABTS.sup.*+] methods. [IC.sub.50] values of 7.528 and 379.7 [micro]g x [mL.sup.-1] were obtained for the [DPPH.sup.*] and [ABTS.sup.*+] determinations, respectively. Those results agree with previous investigations in which it was found that the methanolic crude extract of A. coelophylla had an [IC.sub.50] even lower than the reference control (Hydroquinone), which revealed [IC.sub.50] of 41.14 and 151.19 [micro]g. m[L.sup.-1] for both antioxidant colorimetric assays and also that the antioxidant response was notoriously higher when evaluated through the [ABTS.sup.*+] determination than the antioxidant values obtained for the [DPPH.sup.*] determination [19]. Curves constructed for [IC.sub.50] determinations of the isolated compound are in Figure 2.

Structural Elucidation of Isolated Compound

The isolated compound, present in fraction DHP-11-C, was obtained as a yellowish powder in which 1H NMR spectrum showed two singlets at [delta] 6.74 and 6.28 (1H each, H-3 and H-6, respectively), four aromatic protons at [delta] 6.93 (2H, d, J=8.475 Hz, H-3', H-5') and 8.01 (2H, d, J=8.464 Hz, H-2', H-6') indicating para-substitution pattern at B aromatic ring, suggesting the presence of the apigenin moiety (See Figure 3). Additionally, the spectrum displayed two anomeric protons at [delta] 4.77 (1H, d, J= 10.041 Hz, H-1") and 4.95 (1H, s, H-1'") and one methyl signal at [delta] 0.46 (3H, d, J=5.911 Hz, H-6'") indicating the presence of a [beta]-glucosyl and a-rhamnosyl moieties [20]. The [sup.13]C NMR spectrum displayed signals of a carbonyl group at [delta] 182.10 (C-4), 14 aromatic carbons ranging 98.29-162.34, 2 anomeric carbons at [delta] 70.67 (C-1") and 100.32 (C-1'"), 8 oxygenated carbons ranging from [delta] 68.22 to 79.9, one methyl group at [delta] 17.71 (C-6'") and one aliphatic methylene at [delta] 61.17 (C-6") (with negative phase ([down arrow]) in DEPT 135[degrees] spectrum). These data indicated that the compound correspond to an apigenin diglycoside [21, 22]. Shifting of C-8 signal downfield up to 104.47, absence of a H-8 signal and anomeric shifting of glucose suggest that glycosidic linkage is a C-linkage over such position. Finally, these findings could be supported with literature reported for the structure described and related compounds found in other plant species [23, 24, 25, 26].

As displayed in Figure 3, in the heteronuclear multiple-bond correlation (HMBC) spectrum, long range correlations were detected from H-1" ([delta] 4.77) to C-7 ([delta] 162.34), C-8 ([delta] 104.47) and C-9 ([delta] 155.83), from H-1" ([delta] 4.77) and H-1"' ([delta] 4.95) to C-2" ([delta] 75.09). Correlations involving aromatic protons were also observed. Thus, H-3 ([delta] 6.74) correlates to carbonyl ([delta] 182.10) and H-6 ([delta] 6.28) detected C-7, C-5 ([delta] 161.20) and C-4. Also symmetric correlations inside the B aromatic para-substituted system H-2',6' ([delta] 8.01) to H-3',5' ([delta] 6.93).

Finally, based on the spectroscopic data, bibliographic revisions and comparisons with theoretical predictions of the NMR spectra, the structure of the target compound would be apigenin-8-C-([alpha]-L-rhamnopyranosyl-(1 [right arrow] 2)-[beta]-D-glucopyranoside), illustrated in Figure 4. This compound could also receive the name of Apigenin-8-C-neohesperoside and vitexin-2"-0-rhamnoside.

Based on the proposed structure upon NMR spectroscopic data, this article is the first report of isolation of this compound from any Euphorbiaceae species [24, 25, 26, 27, 28, 29, 30].

Conclusions

An apigenin glycoside was isolated from the defatted MeOH leaf extract of Alchornea coelophylla (Euphorbiaceae). Its structure was elucidated by 1D and 2D NMR spectroscopy experiments. The compound corresponds to Apigenin-8-C-([alpha]-L-rhamnopyranosyl-(1[right arrow]2)- [beta]-D-glucopyranoside) which showed moderate to potent antioxidant activity with an [IC.sub.50] of 7.528 and 379.7 [micro]g x [mL.sup.-1] to [DPPH.sup.*] and [ABTS.sup.*+] assays, respectively.

Acknowledgments

The authors thank Professors Luiz Claudio de Almeida Barbosa and Jose Dias de Souza Filho from the Universidade Federal de Minas Gerais (Belo Horizonte, Brazil) for the NMR data collection. This work was supported by the Vice-Rectorate for Research, Innovation and Extension of the Universidad Tecnologica de Pereira (E9-14-3).

Conflicts of Interest

The authors declare no competing financial interests.

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Cesar Augusto Martinez Garcia

Is an Industrial Chemist graduated from Universidad Tecnologica de Pereira. He received a distinguished student diploma based on his academic performance. He works as research assistant at Biotechnology--Natural Products Laboratory accounting 5 years of research experience, and as lecturer adscribed to the School of Chemistry of Universidad Tecnologica de Pereira on General Chemistry Courses.

Oscar Marino Mosquera Martinez

Is Chemist graduated from Universidad del Valle. Since that period he holds the position as Associate Professor ascribed to School of Chemistry from Universidad Tecnologica de Pereira and Director of the Biotechnology--Natural Products Laboratory which performs studies about the organic chemistry of natural products and has been focused in bioprospecting plant species belonging to protected areas from Colombian Coffee Ecoregion.

Jaime Nino Osorio

Holds a BA in Biology and Chemistry from Universidad de Antioquia. Finished his MSc in Eastern Michigan University (US, 1982) and PhD in Plant Biotechnology from Universidad Politecnica de Valencia (Spain, 2005). He is professor from the School of Chemistry of the Universidad Tecnologica de Pereira (UTP) and advisor of Biotechnology--Natural Products Laboratory where has oriented several research projects and thesis of undergraduate students.

Cesar A Martinez (1), *, Oscar M Mosquera (1), Jaime Nino (1)

Edited by

Juan Carlos Salcedo-Reyes (salcedo.juan@javeriana.edu.co)

Geison Modesti Costa (modesticosta.g@javeriana.edu.co)

(1.) Laboratorio Biotecnologia--Productos Naturales, Escuela de Quimica, Facultad de Tecnologias, Universidad Tecnologica de Pereira, Colombia.

* ceaumartinez@utp.edu.co

Received: 14-07-2015

Accepted: 23-08-2016

Published on line: 14-10-2016

doi:10.11144/Javeriana.SC21-3.agaa

Funding:

Vice-Rectorate for Research, Innovation and Extension, Universidad Tecnologica de Pereira (Funding Code: E9-14-3).

Electronic supplementary material: N/A

Caption: Figure 1. Determinations of the first column chromatographic fractions. A) Antioxidant activity through [ABTS.sup.*+]; B) Antioxidant activity through [DPPH.sup.*]; C) Total phenolic content and D) Total flavonoid content.

Caption: Figure 2. [IC.sub.50] assay with DPH-11-C for A) [ABTS.sup.*+] and B) [DPPH.sup.*].

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

Caption: Figure 4. Apigenin-8-C-([alpha]-L-rhamnopyranosyl-(1 [right arrow] 2)-[beta]-D-glucopyranoside) isolated from MeOH extract of Alchorena coelophylla (Euphorbiaceae).
Table 1. Masses and yields for fractions of the first DIAION
HP-20 chromatographic step.

Fraction   Mass     Yield      Fraction       Mass     Yield
            [g]      (%)                       [g]      (%)

DHP-1      1.4575   9.71        DHP-16        0.0694   0.46
DHP-2      0.3108   2.07        DHP-17        0.3963   2.64
DHP-3      0.1995   1.33        DHP-18        0.0363   0.24
DHP-4      0.093    0.62        DHP-19        0.8514   5.68
DHP-5      0.0137   0.09        DHP-20        0.2582   1.72
DHP-6      0.0219   0.15        DHP-21        0.3408   2.27
DHP-7      0.3066   2.04        DHP-22        0.0763   0.51
DHP-8      0.3229   2.15        DHP-23        0.6604   4.40
DHP-9      0.2471   1.65        DHP-24        0.0857   0.57
DHP-10     0.7945   5.30        DHP-25        0.1348   0.90
DHP-11     0.9553   6.37        DHP-26        0.0832   0.55
DHP-12     0.3628   2.42        DHP-27        0.1998   1.33
DHP-13     1.0863   7.24        DHP-28        0.5339   3.56
DHP-14     0.1552   1.03        DHP-29        1.0736   7.16
DHP-15     0.0773   0.52    Total Yield (%)            74.69

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

Code       Mass [mg]   Yield (%)

DPH-11-A     283.3       37.7
DPH-11-B     181.4       24.2
DPH-11-C     114.4       15.2
DPH-11-D     24.3         3.2
DPH-11-E     34.5         4.6
DPH-11-F     34.3         4.6
DPH-11-G     12.8         1.7
Total Yield (%)          91.2
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Title Annotation:ORIGINAL ARTICLE
Author:Martinez, Cesar A.; Mosquera, Oscar M.; Nino, Jaime
Publication:Revista Universitas Scientarum
Date:Sep 1, 2016
Words:4817
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