Variation of the Arbutin Content in Different Wild Populations of Arctostaphylos uva-ursi in Catalonia, Spain

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1 This article was downloaded by: [University of Barcelona] On: 01 December 2011, At: 08:55 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Herbs, Spices & Medicinal Plants Publication details, including instructions for authors and subscription information: Variation of the Arbutin Content in Different Wild Populations of Arctostaphylos uva-ursi in Catalonia, Spain Irene Parejo a, Francesc Viladomat a, Jaume Bastida a & Carles Codina a a Department of Natural Products, Plant Biology and Edaphology, Faculty of Pharmacy, University of Barcelona, Av. Diagonal, 643, 08028, Barcelona, Catalonia, Spain Available online: 12 Oct 2008 To cite this article: Irene Parejo, Francesc Viladomat, Jaume Bastida & Carles Codina (2002): Variation of the Arbutin Content in Different Wild Populations of Arctostaphylos uva-ursi in Catalonia, Spain, Journal of Herbs, Spices & Medicinal Plants, 9:4, To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Variation of the Arbutin Content in Different Wild Populations of Arctostaphylos uva-ursi in Catalonia, Spain Irene Parejo Francesc Viladomat Jaume Bastida Carles Codina SUMMARY. The crude drug of Arctostaphylos uva-ursi consists mainly of three groups of pharmaceutically relevant compounds, arbutin being the main phenolic constituent. In this study, four wild populations of A. uva-ursi were evaluated for their arbutin content in two different seasons, spring and autumn, coinciding with two periods of the vegetative cycle. The obtained results showed that autumn was a better period than spring to collect the plant material, and that leaves of A. uva-ursi from the site called Adraén Alt were found to contain the highest concentration of arbutin (9.1% of dry weight). No significant variations in Irene Parejo, Francesc Viladomat, Jaume Bastida, and Carles Codina are affiliated with the Department of Natural Products, Plant Biology and Edaphology, Faculty of Pharmacy, University of Barcelona. Av. Diagonal, 643, Barcelona, Catalonia, Spain. This work was financed by the LIFE programme of the European Commission (project ENV/E/000260). [Haworth co-indexing entry note]: Variation of the Arbutin Content in Different Wild Populations of Arctostaphylos uva-ursi in Catalonia, Spain. Parejo, Irene et al. Co-published simultaneously in Journal of Herbs, Spices & Medicinal Plants (The Haworth Herbal Press, an imprint of The Haworth Press, Inc.) Vol. 9, No. 4, 2002, pp ; and: Breeding Research on Aromatic and Medicinal Plants (ed: Christopher B. Johnson, and Chlodwig Franz) The Haworth Herbal Press, an imprint of The Haworth Press, Inc., 2002, pp Single or multiple copies of this article are available for a fee from The Haworth Document Delivery Service [1-800-HAWORTH 9:00 a.m. - 5:00 p.m. (EST). address: getinfo@haworthpressinc. com] by The Haworth Press, Inc. All rights reserved. 329

3 330 BREEDING RESEARCH ON AROMATIC AND MEDICINAL PLANTS the arbutin content were observed within any of the studied natural sites. [Article copies available for a fee from The Haworth Document Delivery Service: HAWORTH. address: <getinfo@haworthpressinc.com> Website: < by The Haworth Press, Inc. All rights reserved.] KEYWORDS. Arbutin, Arctostaphylos uva-ursi, bearberry, Ericaceae, HPLC, geographical variations, seasonal variations INTRODUCTION Leaves of Arctostaphylos uva-ursi (L.) Sprengel (Ericaceae), also known as bearberry, are used as a urinary antiseptic and an astringent (1) because of their content of arbutin (hydroquinone-β-d-monogluco-pyranoside). This crude drug has been found to contain levels of arbutin of 5.95% (2) and 7.16% (4). The variations within and among natural populations of bearberry, however, have not been reported. In this work several wild populations of bearberry have been evaluated for their arbutin content in two periods of the vegetative cycle, spring and autumn, to check possible variations depending on the collection time and the geographical location. MATERIALS AND METHODS Plant Material. Leaves of Arctostaphylos uva-ursi were collected in four natural populations growing at different altitudes in the Catalan Pyrenees of Spain. Table 1 shows geographical and some ecological data concerning the different characteristics of every natural site. The ontogenic cycle of the bearberry is characterized by two growth periods interrupted by a summer dormancy. The plant begins to sprout from the end of April till the beginning of May, and the second growth period takes place from August to September (3). The plant material was collected in autumn 1997 and spring For each geographical site, samples from 5 different areas of the population were collected in order to check possible variations in the arbutin content. Analytical Method. The levels of arbutine were determined by HPLC. Fifty milligram of dried powdered plant material was extracted with 25 ml of a mixture of water and methanol (95/5) at 25 C in an ultrasound bath for 30 minutes. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was adjusted to 25 ml in a measuring flask. Every sample was extracted by triplicate,

4 Biodiversity and Conservation of Medicinal and Aromatic Plants Resources 331 TABLE 1. Geographical characteristics of the four studied wild populations of Arctostaphylos uva-ursi. Population Altitude (m) Slope Exposition Subtrate Tree occurrence Adraén Alt % NW Calcareous 0-20% Pinus sylvestris P. uncinata Adraén Baix % NW Calcareous 20-30% P. sylvestris Cloterons % S-SE Calcareous 0-10% P. uncinata Guils de Cerdanya % NE Siliceous 50-60% P. uncinata A. uva-ursi density 60-80% % % 15-20% and for each replicate two vials were filled, which were each injected twice. Therefore, the values obtained for each sample are the means of 12 analyses. Samples were quantified immediately after extraction to avoid possible chemical alterations. Blanks and standards of known arbutin concentrations were placed between the samples to monitor the quantification. The quantitative analyses were carried out by RP-HPLC using a C8 column ( mm) and a C8 pre-column ( mm) packed with Nucleosil (5 µm; Teknokroma, Barcelona, Spain). Mobile phase was water/methanol (95/5), previously filtered through 0.45 µm filter (Millipore). Other parameters: flow rate: 1 ml/minute; UV detection: 280 nm; and injection volume: 10 µl. The retention time of arbutin was 3.7 minutes. The concentration of arbutin was determinated using a calibration curve established with 6 dilutions of standard arbutin (cristalline arbutin C 12 H 16 O 7, Sigma-Aldrich), from 25 to 400 µg/ml. The correlation coefficient was r = Methanol and water used for both extraction and chromatographic separation were of HPLC grade (Scharlau). The method offers a good reproducibility (±2.8%, calculation based on the peak of arbutin during the period of analysis) and precision (RSD = 0.34%). Statistical Analysis. The results were subjected to the statistical analysis of variance (Statistics for Windows 4.0, ANOVA two ways) to assess the observed differences in the arbutin levels between the collection period and the geographical sites. Differences were considered to be statistically significant when p value was less than Then individual means were compared by using a Student-Newman-Keuls test. The coefficients of variations were also calculated to check the uniformity of the natural populations.

5 332 BREEDING RESEARCH ON AROMATIC AND MEDICINAL PLANTS RESULTS AND DISCUSSION Period of collection. The levels of arbutin corresponding to each wild population are shown in Table 2. In general, the arbutin content was higher in autumn than in spring, and the differences were found to be statistically significant. These differences were much higher in the sites called Adraén Alt and Adraén Baix, and lower in Cloterons. Comparing the levels of arbutin of each wild population between the two seasons, only the site called Guils de Cerdanya (the only site with a siliceous substart, and with the lowest bearberry density) showed no significant difference. Differences among populations. The content of arbutin in the different sites was not the same in the two periods of collection (Table 2). Thus, while in spring the highest levels of arbutin were found in plants of the Cloterons site (8.05%), the lowest ones corresponded to the Adraén Baix site (6.30%). Nonetheless, in autumn the highest arbutin content was found in the site called Adraén Alt (9.10%) and the lowest in the Guils de Cerdanya one (6.97%). After applying the Student-Newman-Keuls test, two groups of populations (Adraén Alt and Cloterons) can be statistically separated from the other two (Adraén Baix and Guils de Cerdanya) on the base of their arbutine content. According to these results, the Adraén Alt and Cloterons sites are the best to collect the bearberry leaves, independently of the period of collection. Differences within populations. To evaluate the possible uniformity of every studied wild population in relation to the arbutin content, the coefficients of variation were calculated separately for the two periods of collection (Table 3). TABLE 2. Levels of arbutin in leaves of bearberry collected in different time collections and wild populations. Values are expressed as percentage of dry weight, and represent the mean of 12 analyses ± SD. Season Sample Adraén Alt Adraén Baix Cloterons Guils de Cerdanya Autumn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05 x 9.10 ± ± ± ± 0.30 Spring ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.05 x 6.69 ± ± ± ± 0.45

6 Biodiversity and Conservation of Medicinal and Aromatic Plants Resources 333 TABLE 3. Coefficients of variation (%) corresponding to the arbutin content within each natural site and collection time. Adraén Alt Adraén Baix Cloterons Guils de Cerdanya Autumn Spring These coefficients varied from 1.43% (Adraén Baix in Spring) to 9.42% (Adraén Alt in spring). In general, the different natural sites can be considered quite homogeneous, as the coefficients of variation were lower than 10%. The different populations seemed to be more uniform in autumn than in spring in what concerns the levels of arbutin (Table 3). In summary, the analytical determination of arbutin in leaves of bearberry indicates that the arbutin content depends significantly on the season of the year. The levels of arbutin observed in the studied populations (from 6.3% to 9.1%) were found to be quite significant in relation to previous reports. On the basis of the obtained results, the collection of bearberry leaves should be carried out preferably during the autumn period because of their higher arbutin content. This concentration, however, can be different according to the characteristics of the sites where the plants grow. REFERENCES 1. Karikas, G.A., M.R. Euerby, and R.D. Waigh Isolation of piceoside from Arctostaphylos uva-ursi. Planta Med. 53: Matsuda, H., M. Higashino, Y. Nakai, M. Iinuma, M. Kubo, and F.A. Lang Studies of cuticle drugs from natural sources. IV. Inhibitory effects of some Arctostaphylos plants on melanin biosynthesis. Biol. Pharm. Bull. 19: Jahodár, L., M. Sovová, and P. Klemera The effect of ionizing irradiation on the growth of Arctostaphylos uva-ursi and the production of arbutin. Folia Pharmaceutica 10: Kenndler, F., C. Schewer, B. Fritsche, and M. Pöhm Determination of arbutin in uvae-ursi folium (bearberry) by capillary zone electrophoresis. J. Chromatogr. 514: