euphorbia antisyphiliitica a redisues as a new source of ellagic acid

8/3/2019 Euphorbia Antisyphiliitica a Redisues as a New Source of Ellagic Acid

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J. A. Ascacio-Valds et al./Chemical Papers 64 (4) 528532 (2010) 529

the School of Chemistry, Universidad Autnoma deCoahuila, Mxico.

Candelilla stalks were cleaned and cut for wax ex-traction into three parts using the traditional methodreported by De Leon-Zapata et al. (2008). The ex-tracting agent used in this process was sulfuric acid(20 %). Stalks were placed into a stainless steel con-tainer, covered with the extracting agent and the con-tainer was heated to the boiling temperature. The waxbegan to appear as gray foam which was collected ina beaker (500 mL) and left to cool. As soon as thewax cooled off, it was cleaned for the purification bya method known as refinement (washed first with sul-furic acid (20 %) and then with water) to remove un-desirable compounds.

The residual vegetal tissue (stalks after wax ex-traction) was dehydrated in a stove at 60 C for 48 h.

After the tissue dehydration, it was ground to powerin a cleavers mill.

Wax, dehydrated stalks and total stalks (wax +stalk) were hydrolyzed to determine the EA contentby a method previously reported by Ascacio-Valdset al. (2007). In this hydrolytic method, a mixture of190 L of concentrated sulfuric acid (96 %) and 1 mLof methanol was used for the hydrolysis. Samples, 10mg of each, were placed in assay tubes closed witha screw top with a plastic ring for hermetic closing.An aliquot of 1.5 mL of the hydrolysis mixture wasadded to the tubes which were closed with a screwtop; hydrolysis was carried out in a stove at 80 C for

30 h. After the hydrolysis, solvents were evaporatedto remove volatile components; 1.5 mL of water wasadded to the samples, from which were hydrosolublecompounds removed under sonic vibration. After that,the samples were centrifuged at 3300 g for 30 min,the supernatant was discarded, and 1.5 mL of ethanolwas added for EA dissolution. EA quantification wascarried out using HPLC (High Performance LiquidChromatography) equipment, according to the usageconditions reported by Aguilera-Carb et al. (2008).The samples were analyzed in triplicate.

For this determination, candelilla stalks without

wax, dehydrated and pulverized were used. The threeevaluated factors were: time of extraction (A) of 30min, 90 min, and 150 min (0 min as a control); temper-ature (B) of 30C, 60C, and 90C, and mass/volumeratio (C) of 0.33 g mL1, 0.20 g mL1, and 0.14g mL1 respectively. This research was establishedunder a completely randomized experimental designwith factorial arrangement. The dependent variablewas the EA concentration. The samples were analyzedin triplicate.

In this step, the procedure employed was: 1 g ofsample powder and the amount of water according tothe treatment were placed in a beaker. After ellagi-

tannin extractions, 250 L of the sample and 1.5 mLof the sulfuric acid/methanol mixture were placed inassay tubes with a screw top. The samples were placed

EAc

ontent/(mgg1)

Fig. 1. EA mass concentrations found in pomegranate husk(Pm), wax (Cw), dehydrated stalks (Caw), and totalstalks (Cbw) after hydrolysis with a mixture of concen-trated sulfuric acid and methanol.

in a stove at 80 C for 30 h. EA quantification was per-formed in an HPLC equipment as mentioned above.

Total polyphenols of candelilla (TPC) were deter-mined using the best extraction conditions resulting

from our experiments. For this step, 100 g of dehy-drated and pulverized candelilla stalks were placedin a beaker with 500 mL of water. TCP extractionwas performed at 60C for 30 min. After that, the ob-tained extract was filtered using filter paper (What-man #41) to eliminate the biggest residue particles.Column chromatography of the candelilla extract wasperformed using an Amberlite XAD-16. First, waterwas used as the eluent to discard undesirable com-pounds, and then, ethanol was employed as the eluentto obtain a TPC fraction (Seeram et al., 2005). Sol-vent was evaporated from the fractionated extract andTPC was recovered as a fine powder; EA content of

the TPC powder was determined using the methodol-ogy mentioned above.

A Varian HPLC was used for the EA determina-tion under the following operation conditions: 5 mcolumn Optisil ODS, 250 mm 4.6 mm, flow rate of 1mL min1, sample volume of 10 L, with the followingsolvents to the analysis solvent methanol, acetonitrile,and 3 % acetic acid.

During the hydrolysis of vegetal materials, treat-ments under completely randomized design were es-tablished in three replicates. During the optimizationof EA recovery conditions, a completely experimen-

tal design with factorial arrangement was used; sam-ples were analyzed in triplicate. Data were statisti-cally analyzed using an ANOVA procedure (with thesignificance level of p = 0.05) and a Tukey test forthe comparison of mean values. When necessary, thetreatment means were compared using the Tukey mul-tiple range test; data were analyzed using the SigmaPlot software 10.0 version.

Candelilla has not been reported as a source ofEA, this study reports a process for EA extractionusing the wax extracted (Cw), residual stalks afterwax extraction (Caw), and stalks before wax extrac-tion (Cbw). In addition, pomegranate husks (Pm) were

used as a control. EA content was 3.4 times higher inPm than in the candelilla samples (Fig. 1).

EA was not found in candelilla wax or the EA levels


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530 J. A. Ascacio-Valds et al./Chemical Papers 64 (4) 528532 (2010)

EAcontent/(mgg1)

Mass/volumeratio/(gmL1)

Fig. 2. Influence of temperature, time, and mass/volume ratio on EA recovery (mg g1) from candelilla stalks without wax: 30C(a), 60C (b), and 90C (c).

Table 1. Principal EA vegetal sources

Ellagic acidVegetal source content/(mg g1) Reference

Quercus rubus 19 Bianco et al., 1998Euphorbia antisyphilitica 7.9 Present workQuercus alba 3.7 Bianco et al., 1998Rubus idaeus 3.3 Kop onen et al., 2007Rubus occidentalis 1.5 Hakkinen et al ., 2000

could not be detected by the method employed. Thehighest EA concentration was found in residual stalksafter wax extraction (Caw = 7.90 mg g1) and a lower

EA concentration was found in total candelilla stalks(Cbw = 6.06 mg g

1), however, these values were notstatistically different (p < 0.05). These results showthat the traditional process of wax extraction modifiedby Saucedo-Pompa et al. (2008) does not affect EAcontent in stalks after wax extraction.

This is the first report where EA content in can-delilla stalks is analyzed. The obtained values of EAcontent suggest that this plant can be an alternativesource of EA as candelilla EA content is higher thanthose reported in other plants (Table 1). The EA con-tent may give an added value to this endemic plant of

Northern Mexico.Wax extraction residues were not used, but accord-ing to the results of this study, they have the potentialto be used as a new EA source. It was decided to usecandelilla stalks without wax for EA recovery and todefine the best recovery conditions according to themethodology described in the experimental section.Results of EA recovery are shown in Fig. 2.

Fig. 2 shows the influence of time, temperature,and mass/volume ratio on EA extraction. As temper-ature increased from 30C to 60C, EA recovery in-creased. It has been reported that temperature playsa very important role in the phytochemical extraction

process, especially for ellagitannins, anthocyanins andlignans, because it promotes phytochemical solubiliza-tion in the obtained infusions (Feldman et al., 2003).

Fig. 3. Pareto chart showing evaluated factors. Time/min (A),

temperature/C (B), and mass/volume ratio/(g mL1)(C).

Another determinant factor for EA recovery fromcandelilla stalks is the mass/volume ratio. The resultsshow that at the 0.20 g mL1 mass/volume ratio and60C, the recovered levels of EA (18.60 mg g1) weresimilar to those (18.66 mg g1) obtained at the 0.14g mL1 mass/volume ratio and 90C. The results ofthis study show that the extraction time is a factorthat does not affect the EA recovery. Because en-

ergy consumption, time and the amount of organicsolvents used, the conditions of 60C, 0.20 g mL1

mass/volume ratio, and 30 min were chosen as thebest for EA recovery.

The Pareto chart (Fig. 3) shows the effect of thestudied factors on the EA extraction process. Temper-ature (B) and mass/volume ratio (C) affect the EArecovery. However, it was observed that the interac-tion between these two factors is also important (BCinteraction). In this case, if one of the two main factorsis modified or eliminated, the EA recovery process isaffected.

Once the best EA recovery conditions were deter-

mined, EA quantification from the TPC powder wasperformed. The EA concentration in this hydrolyzedpowder was 305 mg g1 demonstrating that when us-


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J. A. Ascacio-Valds et al./Chemical Papers 64 (4) 528532 (2010) 531

EAcontent/(mgg1)

Mass/volumeratio, C/(g mL

1)

Temperature,B/C

Fig. 4. Response surface graph, mass/volume ratio (C), tem-perature (B), and EA mass concentration relationship.

ing the best extraction conditions established in this

study, it is possible to recover the highest EA contentfrom candelilla stalks.

A response surface analysis, carried out consider-ing the B and C factors as variables with significanteffect on the response variable due to factor A (time),did not present the same effect (p < 0.05). Fig. 4 rep-resents the response surface graph demonstrating thezone of maximum accumulation of EA allowing thusto calculate the maximum yield of EA under the testconditions.

A mathematical equation was developed to obtainthe highest amount of EA according to the results ofthis study

Y = 1 + 2A + 3B + 4C+ 5AB+ 6AC+ 7BC+ 8ABC

Y = 8.66 + (0.05)A + 4.42B + 2.77C+ 0.74AB+ 1.33AC+ 2.31BC+ 0.72ABC (1)

where Y corresponds to the dependent variable (EA),i represent the values of coefficients of the ratio ob-tained in the statistical analysis, and A, B, and Crep-resent the evaluated factors for EA extraction. Fromthis complete equation, insignificant factors and their

interactions were discarded

Y = 1 + 3B + 4C+ 7BC

Y = 8.66 + 4.42B + 2.77C+ 2.31BC (2)

Eq. (2) allowed comparing theoretical and exper-imental data, as this model is predictive. For exam-ple, illustration, replacing the corresponding values inthe equation (B = 60C and C = 0.20 g mL1), the-oretically, an EA concentration of 302.13 mg g1 isobtained considering the experimental error and reli-ability of 95 %. Experimentally obtained EA concen-

tration in the TPC powder was 305 mg g1

. Using thepresent equation, it was demonstrated that theoreticaldata were adjusted to experimental ones.

Results show that ellagic acid as equivalents of el-lagitannins is not present in the wax extracted fromcandelilla stalks and that the wax extraction processdoes not affect the ellagic acid concentration in can-delilla stalks. EA content in candelilla stalks offers anadded value to the stalks residues from the candelillawax process. Temperature and proportion between thestalk residue and the extraction liquid (mass/volumeratio) are determinant factors for EA recovery. Sta-tistical analysis showed that time does not influenceEA extraction. The best conditions for EA extractionfrom candelilla stalks (Euphorbia antisyphilitica) are:60C and the 0.20 g mL1 mass/volume ratio.

Acknowledgements. Ascacio-Valdes wants to thank CONA-

CYT for the scholarship assigned to his postgraduate study in

the program of Food Science and Technology, UAdeC. Au-

thors thank the financial support provided by the program

CONACYT-CONAFOR-2008-91633.

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euphorbia antisyphiliitica a redisues as a new source of ellagic acid

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