Chemical Papers 64 (4) 528532 (2010) DOI: 10.

2478/s11696-010-0034-6

SHORT COMMUNICATION

residues as a new source of ellagic acid

Juan A. Ascacio-Valds, b Antonio Aguilera-Carb, a Jos L. Martnez-Hernndez, a Ral Rodrguez-Herrera, a Cristbal N. Aguilar*
a Food

Science and Technology Department, School of Chemistry, Universidad Autnoma de Coahuila, Blvd. Venustiano Carranza. Col. Repblica, 25280 Saltillo, Coahuila, Mxico
b Food

Science and Nutrition Department, Universidad Autnoma Agraria Antonio Narro, Buenavista, 25315 Saltillo, Coahuila, Mxico

Received 27 October 2009; Revised 2 February 2010; Accepted 9 February 2010

Keywords: Euphorbia antisyphilitica Zucc, candelilla, extraction, ellagic acid

Candelilla (Euphorbia antisyphilitica Zucc.) stalks are of great economic importance in Northern Mexico. They are used for candelilla wax extraction, which is used for various purposes in the food industry and also for traditional use in ornaments and therapeutic applications. However, candelilla wax extraction generates 140 tons of waste per year. Previous reports showed that ellagic acid (EA) is present in candelilla stalks (Aguilera-Carb et al., 2008); however, there is no information on studies describing the recovery of EA which can be potentially used in diverse and important industrial sectors. EA (4,4,5,5,6,6-hexahydroxidifenic acid 2,6,2,6dilactone) is a lactone derived from gallic acid and it is generally recognized as the residual product of the hydrolysis of ellagitannins; during this reaction, hexahydroxydiphenoyl (HHDP) ester group spontaneously converts to its characteristic bislactone structure (Quideau & Feldman, 1996). EA is present in many plants, fruits and nuts, and over 500 dierent ellagitannins have been identied in nature (Helm et al., 1999). EA and ellagitannins have important bio-

ut

ho

In this study, a new source of ellagic acid (EA) is reported. Euphorbia antisyphilitica or candelilla was used to extract phenolic dilactone. Cereous layers and brous tissue were analyzed. A completely randomized experimental design with a treatment factorial arrangement was employed. The factors considered were: plant/extracting agent ratio, extraction temperature and time. Candelilla wax does not contain EA. Temperature and the ratio plant/extracting agent were determinant during the EA extraction process. Around 20 mg of free EA per gram of brous tissue were found. Residues of candelilla are a good source of EA. c 2010 Institute of Chemistry, Slovak Academy of Sciences

*Corresponding author, e-mail: cristobal.aguilar@mail.uadec.mx.

rc op y

logical functions as antitumoral, antiviral, antioxidant agents, etc. (Puech et al., 1999). EA is used as a potent antioxidant in dierent industrial sectors. For this reason, there is a continuous search for new sources of this phytochemical. Evidence of EA content in candelilla stalks is of great relevance for the establishment of this vegetal specie as a good EA source. The objectives of this study were to determinate the content and distribution of EA in the candelilla plant and to determine the best conditions for EA extraction. Candelilla stalks were recollected in the Southwest region of the Coahuila State, Mexico; vegetal material was selected as uniform as possible considering morphologic criteria which included stalks size and color. Stalks were cut at the soil level avoiding the damage of the plant roots in order to preserve the plant according to the Mexican norm (SEMARNAT, 1999). Pomegranate husk powder (Pm) was used as the reference sample as it contains a high level of ellagitannins, mainly punicalagin and punicalin. Pm was proportioned by the Laboratory of Natural Products of

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

529

the School of Chemistry, Universidad Autnoma de Coahuila, Mxico. Candelilla stalks were cleaned and cut for wax extraction into three parts using the traditional method reported by De Leon-Zapata et al. (2008). The extracting agent used in this process was sulfuric acid (20 %). Stalks were placed into a stainless steel container, covered with the extracting agent and the container was heated to the boiling temperature. The wax began to appear as gray foam which was collected in a beaker (500 mL) and left to cool. As soon as the wax cooled o, it was cleaned for the purication by a method known as renement (washed rst with sulfuric acid (20 %) and then with water) to remove undesirable compounds. The residual vegetal tissue (stalks after wax extraction) was dehydrated in a stove at 60 C for 48 h. After the tissue dehydration, it was ground to power in a cleavers mill. Wax, dehydrated stalks and total stalks (wax + stalk) were hydrolyzed to determine the EA content by a method previously reported by Ascacio-Valds et al. (2007). In this hydrolytic method, a mixture of 190 L of concentrated sulfuric acid (96 %) and 1 mL of methanol was used for the hydrolysis. Samples, 10 mg of each, were placed in assay tubes closed with a screw top with a plastic ring for hermetic closing. An aliquot of 1.5 mL of the hydrolysis mixture was added to the tubes which were closed with a screw top; hydrolysis was carried out in a stove at 80 C for 30 h. After the hydrolysis, solvents were evaporated to remove volatile components; 1.5 mL of water was added to the samples, from which were hydrosoluble compounds 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 ethanol was added for EA dissolution. EA quantication was carried out using HPLC (High Performance Liquid Chromatography) equipment, according to the usage conditions 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 three evaluated factors were: time of extraction (A) of 30 min, 90 min, and 150 min (0 min as a control); temperature (B) of 30 C, 60 C, and 90 C, and mass/volume ratio (C ) of 0.33 g mL1 , 0.20 g mL1 , and 0.14 g mL1 respectively. This research was established under a completely randomized experimental design with factorial arrangement. The dependent variable was the EA concentration. The samples were analyzed in triplicate. In this step, the procedure employed was: 1 g of sample powder and the amount of water according to the treatment were placed in a beaker. After ellagitannin extractions, 250 L of the sample and 1.5 mL of the sulfuric acid/methanol mixture were placed in assay tubes with a screw top. The samples were placed

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

in a stove at 80 C for 30 h. EA quantication was performed in an HPLC equipment as mentioned above. Total polyphenols of candelilla (TPC) were determined using the best extraction conditions resulting from our experiments. For this step, 100 g of dehydrated and pulverized candelilla stalks were placed in a beaker with 500 mL of water. TCP extraction was performed at 60 C for 30 min. After that, the obtained extract was ltered using lter paper (Whatman #41) to eliminate the biggest residue particles. Column chromatography of the candelilla extract was performed using an Amberlite XAD-16. First, water was used as the eluent to discard undesirable compounds, and then, ethanol was employed as the eluent to obtain a TPC fraction (Seeram et al., 2005). Solvent was evaporated from the fractionated extract and TPC was recovered as a ne powder; EA content of the TPC powder was determined using the methodology mentioned above. A Varian HPLC was used for the EA determination under the following operation conditions: 5 m column Optisil ODS, 250 mm 4.6 mm, ow rate of 1 mL min1 , sample volume of 10 L, with the following solvents to the analysis solvent methanol, acetonitrile, and 3 % acetic acid. During the hydrolysis of vegetal materials, treatments under completely randomized design were established in three replicates. During the optimization of EA recovery conditions, a completely experimental design with factorial arrangement was used; samples were analyzed in triplicate. Data were statistically analyzed using an ANOVA procedure (with the signicance level of p = 0.05) and a Tukey test for the comparison of mean values. When necessary, the treatment means were compared using the Tukey multiple range test; data were analyzed using the Sigma Plot software 10.0 version. Candelilla has not been reported as a source of EA, this study reports a process for EA extraction using the wax extracted (Cw ), residual stalks after wax extraction (Caw ), and stalks before wax extraction (Cbw ). In addition, pomegranate husks (Pm ) were used as a control. EA content was 3.4 times higher in Pm than in the candelilla samples (Fig. 1). EA was not found in candelilla wax or the EA levels

ut

ho

rc op y

EA content/(mg g1)

530

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

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

Table 1. Principal EA vegetal sources Ellagic acid content/(mg g1 ) Reference 19 7.9 3.7 3.3 1.5

Vegetal source

Quercus rubus Euphorbia antisyphilitica Quercus alba Rubus idaeus Rubus occidentalis

Bianco et al., 1998 Present work Bianco et al., 1998 Koponen et al., 2007 Hakkinen et al., 2000

could not be detected by the method employed. The highest EA concentration was found in residual stalks after wax extraction (Caw = 7.90 mg g1 ) and a lower EA concentration was found in total candelilla stalks (Cbw = 6.06 mg g1 ), however, these values were not statistically dierent (p < 0.05). These results show that the traditional process of wax extraction modied by Saucedo-Pompa et al. (2008) does not aect EA content in stalks after wax extraction. This is the rst report where EA content in candelilla stalks is analyzed. The obtained values of EA content suggest that this plant can be an alternative source of EA as candelilla EA content is higher than those reported in other plants (Table 1). The EA content may give an added value to this endemic plant of Northern Mexico. Wax extraction residues were not used, but according to the results of this study, they have the potential to be used as a new EA source. It was decided to use candelilla stalks without wax for EA recovery and to dene the best recovery conditions according to the methodology described in the experimental section. Results of EA recovery are shown in Fig. 2. Fig. 2 shows the inuence of time, temperature, and mass/volume ratio on EA extraction. As temperature increased from 30 C to 60 C, EA recovery increased. It has been reported that temperature plays a very important role in the phytochemical extraction process, especially for ellagitannins, anthocyanins and lignans, because it promotes phytochemical solubilization in the obtained infusions (Feldman et al., 2003).

ho

ut

rc op y
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 from candelilla stalks is the mass/volume ratio. The results show that at the 0.20 g mL1 mass/volume ratio and 60 C, the recovered levels of EA (18.60 mg g1 ) were similar to those (18.66 mg g1 ) obtained at the 0.14 g mL1 mass/volume ratio and 90 C. The results of this study show that the extraction time is a factor that does not aect the EA recovery. Because energy consumption, time and the amount of organic solvents used, the conditions of 60 C, 0.20 g mL1 mass/volume ratio, and 30 min were chosen as the best for EA recovery. The Pareto chart (Fig. 3) shows the eect of the studied factors on the EA extraction process. Temperature (B ) and mass/volume ratio (C ) aect the EA recovery. However, it was observed that the interaction between these two factors is also important (BC interaction). In this case, if one of the two main factors is modied or eliminated, the EA recovery process is aected. Once the best EA recovery conditions were determined, EA quantication from the TPC powder was performed. The EA concentration in this hydrolyzed powder was 305 mg g1 demonstrating that when us-

Mass/volume ratio/(g mL1)

EA content/(mg g )

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

531

Mass/volume ratio, C /(g mL1) Temperature, B/C

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 coecients of the ratio obtained in the statistical analysis, and A, B, and C represent the evaluated factors for EA extraction. From this complete equation, insignicant factors and their interactions were discarded Y = 1 + 3 B + 4 C + 7 BC Y = 8.66 + 4.42B + 2.77C + 2.31BC (2)

Eq. (2) allowed comparing theoretical and experimental data, as this model is predictive. For example, illustration, replacing the corresponding values in the equation (B = 60 C and C = 0.20 g mL1 ), theoretically, an EA concentration of 302.13 mg g1 is obtained considering the experimental error and reliability of 95 %. Experimentally obtained EA concentration in the TPC powder was 305 mg g1 . Using the present equation, it was demonstrated that theoretical data were adjusted to experimental ones.

ut

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

ho

ing the best extraction conditions established in this study, it is possible to recover the highest EA content from candelilla stalks. A response surface analysis, carried out considering the B and C factors as variables with signicant eect on the response variable due to factor A (time), did not present the same eect (p < 0.05). Fig. 4 represents the response surface graph demonstrating the zone of maximum accumulation of EA allowing thus to calculate the maximum yield of EA under the test conditions. A mathematical equation was developed to obtain the highest amount of EA according to the results of this study

rc op y

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

Results show that ellagic acid as equivalents of ellagitannins is not present in the wax extracted from candelilla stalks and that the wax extraction process does not aect the ellagic acid concentration in candelilla stalks. EA content in candelilla stalks oers an added value to the stalks residues from the candelilla wax process. Temperature and proportion between the stalk residue and the extraction liquid (mass/volume ratio) are determinant factors for EA recovery. Statistical analysis showed that time does not inuence EA extraction. The best conditions for EA extraction from candelilla stalks (Euphorbia antisyphilitica) are: 60 C and the 0.20 g mL1 mass/volume ratio.
Acknowledgements. Ascacio-Valdes wants to thank CONACYT for the scholarship assigned to his postgraduate study in the program of Food Science and Technology, UAdeC. Authors thank the nancial support provided by the program CONACYT-CONAFOR-2008-91633.

EA content/(mg g1)

References

Aguilera-Carbo, A. F., Augur, C., Prado-Barragan, L. A., Aguilar, C. N., & Favela-Torres, E. (2008). Extraction and analysis of ellagic acid from novel complex sources. Chemical Papers, 62, 440444. DOI: 10.2478/s11696-008-0042-y. Ascacio-Valds, J. A., Prieto-Nieto, A., Hernndez-Rivera, J. S., Aguilera-Carbo, A., & Aguilar, C. N. (2007). Determination of ellagitannins in native plants of Mexican semiarid zone: relationship between two analytical methods. In Proceedings of the Third Mexican Symposium on Biopolymers: Advances and Perspectives, 1517 March 2007. Monterrey, NL, Mxico. Bianco, M.-A., Handaji, A., & Savolainen, H. (1998). Quantitative analysis of ellagic acid in hardwood samples. The Science of the Total Environment, 222, 123126. DOI: 10.1016/S0048-9697(98)00294-0. De Len-Zapata, M. A., Saucedo-Pompa, S., Contreras-Esquivel, J. C., Jasso-Cantu, D., Saenz-Galindo, A., & Aguilar, C. N. (2008). Alternative method for extraction of candelilla wax (Euphorbia antysiphilitica). In Congress on Food Science and Food Biotechnology in Developing Countries, 14 17 October 2008. Quertaro, QT, Mxico. Feldman, K. S., Iyer, M. R., & Liu, Y. (2003). Ellagitannin chemistry. Studies on the stability and reactivity of 2,4HHDP-containing glucopyranose systems. Journal of Organic Chemistry, 68, 74337438. DOI: 10.1021/jo034495x. Hkkinen, S. H., Krenlampi, S. O., Mykknen, H. M., Heinonen, I. M., & Trrnen, A. R. (2000). Ellagic acid cono o tent in berries: Inuence of domestic processing and storage. European Food Research and Technology, 212, 7580. DOI: 10.1007/s002170000184. Helm, R. F., Zhentian, L., Ranatunga, T., Jervis, J., & Elder, T. (1999). Toward understanding monomeric ellagitannin biosynthesis. In G. G. Gross, R. W. Hemingway, & T. Yoshida (Eds.), Plant polyphenols 2: Chemistry, biology, pharmacology, ecology (pp. 8399). New York, NY, USA: Kluwer Academic/Plenum Publishers. Koponen, J. M., Happonen, A. M., Mattila, P. H., & Trrnen, o o A. R. (2007). Contents of anthocyanins and ellagitannins in selected foods consumed in Finland. Journal of Agricultural and Food Chemistry, 55, 16121619. DOI: 10.1021/jf062897a. SEMARNAT, The Mexican Ministry of Enviroment and Natural Resources (1999). Mexican ocial norm: Procedures, criterions and technical and administrative specications for

532

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

sustainable use of candelilla shrub, transportation and storage. NOM-018-RECNAT. The Mexican Ministry of Enviroment and Natural Resources. Tlalpan, DF, Mxico. (in Spanish) Puech, J. L., Feuillat, F., & Mosedale, J. R. (1999). The tannins of oak heartwood: Structure, properties, and their inuence on wine avor. American Journal of Enology and Viticulture, 50, 469478. Quideau, S., & Feldman, K. S. (1996). Ellagitannin chemistry. Chemical Reviews, 96, 475503. DOI: 10.1021/cr904716a.

Saucedo-Pompa, S., Jasso-Cantu, D., De Len-Zapata, M., Saenz-Galindo, A., & Aguilar, C. N. (2008). Technological improvement to the candelilla wax extraction process and implementation of new technologies that eliminate pollutants emissions. In XXIX National meeting of AMIDIQ, 23 May 2008 (pp. 245252). Mazatlan, SIN, Mxico. Seeram, N., Lee, R., & Herber, D. (2005). Rapid large scale purication of ellagitannins from pomegranate husk, a byproduct of the commercial juice industry. Separation and Purication Technology, 41, 4955. DOI: 10.1016/j.seppur.2004. 04.003.

ut

ho

rc op y