Art17 Journal of Applied Botany and Food Quality 82, 99 - 102 (2008) 1Humboldt University Berlin, Institute for Horticultural Science, Berlin Germany, 1aSection of Urban Horticulture, Berlin, Germany 1b Section of Tree Production and Nursery, 2Landesforstanstalt Eberswalde, Section of Forest Development/Monitoring, Eberswalde, Germany Influence of the season on the salicylate and phenolic glycoside contents in the bark of Salix daphnoides, Salix pentandra, and Salix purpurea N. Förster1a, C. Ulrichs1a, M. Zander1b, R. Kätzel2, I. Mewis1a (Received June 15, 2008) Summary Due to the benefits of herbal medicine and their wide range of application for human health, the usage of natural drug products, such as willow bark extract, has increased in the last few years. The principle active compounds of the drugs comprised primarily of willow bark are phenolic glycosides like salicylates. Phenolic glycoside profiles of bark vary among species and between the seasons. To identify and preserve willow clones with high salicylate content for possible commercial usage at a later stage, we have screened three Salix sp. in respect to their chemical profiles. The willow species analysed were: Salix daphnoides, Salix pentandra, and Salix purpurea. These species had distinct phenolic glycoside profiles. The major salicylate of S. daphnoides and S. purpurea clones was salicortin, whereas the main compound of S. pentandra was 2’- O-acetylsalicortin. According to the chemical profiles of 140 clones, seven independent clones of S. daphnoides and S. purpurea as well as four clones of S. pentandra with high phenolic glycoside contents were picked to study seasonal changes in bark chemistry. Overall, the clones of S. daphnoides showed the highest mean salicylate and phenolic glycoside contents, followed by S. pupurea and S. pentan- dra. The secondary metabolite content of willow bark clones de- creased during the vegetative season from March to June 2007 and further from June to July 2007. Our study revealed that for optimum yield of phenolic glycosides the species, the clone, and the time of harvest during the season have to be taken in consideration. Introduction Herbal medicine products are dietary supplements that people take to improve their health. Many herbs have been used for a long time because of their claimed health benefits. In recent years herbal medicine gained more and more interest, even within the scientific community. At the end of the 20th century, many medical studies published proved biotic effects of willow bark extracts. Certain phenolic glycosides of Salix bark, particulary salicin and its esters like tremulacin or salicortin, have been shown to relieve rheumatic disturbances, infections, and headache (BIEGERT et al., 2004; CHRUBASIK et al., 2000; CHRUBASIK et al., 2001; LARDOS et al., 2004). These glycosides have non-inflammable, temperature-reducing, and pain-alleviating effects. Adverse effects, like a negative influence on the aggregation of the thrombocytes or local lesions of the gastric mucosa caused by the synthetic medicine product acetylsalicylic acid (Aspirin), have not been reported for salicin. Phenolic glycosides present in bark vary among Salix species (THIEME, 1965; THIEME, 1971; MEIER et al., 1985a). Commercial extraction of phenolic glycosides from willow bark is only eco- nomical when the content reaches a minimum of 1% total salicin, according to the „European Scientific Cooperative on Phytotherapy“ (ESCOP). Furthermore, it has to be taken into consideration that the phenolic glycosides content varies with the seasons (THIEME, 1965; THIEME, 1971). To identify and preserve willow clones with high salicylate content for possible recovery of certain phenolic com- pounds at a later stage, we have screened clones of Salix daphnoides, Salix purpurea, and Salix pentandra in respect to their chemical profiles. A second object of the study was to analyse the seasonal variability of salicylate and phenolic glycoside contents in the Salix species to identify the optimum harvest time for secondary meta- bolites. Materials and methods Origin and selection of plant material Osier stacks of different willow clones were collected in the north- east of Germany and the north-west of Poland in April and May 2006 and stock planted in Zepernick (Berlin). Bark samples of 140 col- lected clones were taken in December 2006. According to their chemical profiles seven independent clones of S. daphnoides (DA7, DA40, DA52, DA56, DA58, DA61, DA64) and S. purpurea (PU9, PU10, PU11, PU12, PU15, PU24, PU25) as well as four clones of S. pentandra (PE11, PE12, PE13, PE23) with high phenolic glycoside contents were picked for further studies. To determine changes within phenolic glycosides within the beginning vegetation period, willow bark for chemical analysis was collected in March, June, and July 2007. Extraction procedure and analysis The peeled bark was lyophilised in a Christ Alpha 1- 4 at ex- erted pressure of 0.034 bars for two days up to 20°C. After mil- ling of probes (Retsch MM 301, total samples 1- 2 g) for 1.5 min at 20000 Hz the phenolic glycoside samples, 50 mg each, were extracted with 750 µl methanol. 100 µl of standard solution (resorcinol) was added to the first extraction. After conditioning the probes for 10 min in an ultrasonic bath (Bandelin SONOREX SUPER RK 513) and centrifugation for 5 min and 6000 rpm (Eppendorf Centrifuge 5416) the supernatant was collected. The pellet was used for reextraction with methanol (500 µl, 80 %) twice more. The combined supernatants were concentrated in a vacuum concentrator (Speed Vac SC110, Savant Instruments) until a volume of 150 µl was remaining. The extracts were filled up to 1 ml with ultra pure water (MilliQ quality) and filtered by using Spin-X tubes (Corning, The Netherlands). The obtained extracts were analysed by HPLC. 10 µl of willow bark extracts were injected in a Dionex P680A HPLC system equipped with an ASI-100 auto sampler and a PDA-100 photodiode array detector. Phenolic glycosides were separated on a narrow bore column (Acclaim Polar Advantage C16: 3 µm, 120 Å, 2.1 x 150 mm, Dionex). Eluents used for HPLC analysis were: A) 2% tetrahydrofuran and 0.5% phosphoric acid and B) 100% methanol. The eluent programme was: 0% B (0 - 5 min), 15% B (10 min), 25% B (20 min), 35% B (30 min), 50% B (35 min), 100% B (40 - 42 min) and 0% B (44 - 49 min). The flow rate was 0.35 ml/min and the eluent was detected at 270 nm. Qualitative identification of the phenolic glycosides was carried out by using standards and via specific UV- spectra (SHAO, 1991). The quantitative analysis based on peak area 100 N. Förster, C. Ulrichs, M. Zander, R. Kätzel, I. Mewis relative to the standard resorcinol by using respective response factors. For peak evaluation the software Chromeleon Version 6.0 was used. Data were analysed for significant differences by using analysis of variance following the mean comparison test Tukey’s HSD for honest significant difference with SYSTAT 12.0. Results and discussion The chemical compounds identified in willow species were the salicylates salicin, salicortin, 2’-O-acetylsalicin, 2’-O-acetylsali- cortin, and tremulacin and the other phenolic glycosides syrengin, picein, catechin, ampelopsin, vimalin, purpurein, and naringenin-5- glycoside. Furthermore, a not-yet identified salicin derivate (elution at circa 35 min) and an unknown phenolic glycoside (elution at about 18 min) could be detected. Clones of S. daphnoides showed the highest mean salicylate and phenolic glycoside content, followed by S. pupurea and S. pentandra. Every Salix species had a characteristic phenolic glycoside profile. The major salicylate present in S. daphnoides and S. purpurea was found to be salicortin, whereas the main compound of S. pentandra was 2’-O-acetylsalicortin. Type-chromatograms for clones of the three analysed willow species are given in Fig. 1 - 3. Initial total phenolic glycoside content determined in March for S. daphnoides was 8.24%, for S. purpurea 5.67%, and 3.25% for S. pentandra. The clone variability of secondary metabolites in willow species was found to be different with highest variability in S. daph- noides, followed by S. purpurea and S. pentandra (Tab. 1). The secondary metabolites generally decreased from March to June 2007 and further from June to July 2007, whereby the the total phenolic glycosides decreased significantly in all species already from March to June (Fig. 4). Furthermore, the salicylate content of all species was significantly lower in July compared to March 2007. Significant differences were found more frequently between March and June than between June and July. The salicylate and phenolic glycoside contents in clones of S. daphnoides, S. pentandra, and S. purpurea decreased about 30% from March to June (Tab. 1). The salicylate contents of S. daphnoides and S. purpurea decreased further from 4.71% and 3.27%, respectively around 18% from June to July. Also from June to July, the total phenolic glycoside contents decreased in S. daphnoides and S. purpurea around 22% and 14% from initially 5.14% and 4.07%. An increase of 8% from 2.15% phenolic glyco- sides content was detected in S. pentandra from June to July. But the salicylic content remained unchanged in these months in S. pen- tandra. In general it could be seen that the secondary metabolite contents decreased from March to June with more intensity than from June to July. Our data collected for the seasonal variability are proof of the results reported by MEIER et al. (1985a, 1985b). They found that the salicylate and phenolic glycoside content decreased from winter to summer. Furthermore, our results show the same trend as documented by THIEME (1965, 1971). He found that the maximum content of phenolic Fig. 3: Chromatogram of Salix purpurea bark (clone 9, March 2007) 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 -50 500 1.000 1.500 30.04.2007 #58 N201 S. purpurea PU9 (2) UV_VIS_1 mAU min S a lic in R e s o rc in S y re n g in 2 '- O -A ce ty ls a lic in S a lic o rt in P h e n o lg lu c o s id A m p e lo p si n V im a lin 2 '-O -A c e ty ls a lic o rt in P u rp u re in T re m u la ci n S a lic in d e ri v a t W VL:270 nm Fig. 2: Chromatogram of Salix pentandra bark (clone 11, March 2007) 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 -50 500 1.000 30.04.2007 #38 N181 S. pentandra PE11 (2) UV_VIS_1 mAU min S a lic in R e so rc in S yr e n g in 2 '- O -A ce ty ls a lic in C a te ch in S a lic o rt in P h e n o lg lu co si d A m p e lo p si n V im a lin 2 '- O -A ce ty ls a lic o rt in P u rp u re in WVL:270 nm Fig. 1: Chromatogram of Salix daphnoides bark (clone 52, March 2007) 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 -50 500 1.300 30.04.2007 #12 N155 S. daphnoides DA52 (1 UV_VIS_1 mAU min S a lic in R e so rc in S yr e n g in 2 '- O -A c e ty ls a lic in C a te c h in S a lic o rt in P h e n o lg lu co si d A m p e lo p s in V im a lin 2 '- O -A ce ty ls a lic o rt in N a ri n g e n in -5 -G lu co si d P u rp u re in T re m u la ci n S a lic in d e ri v a t WVL:270 nm Phenolic glycosides in Salix bark 101 0 1 2 3 4 5 6 7 8 9 S. daphnoides S. pentandra S. purpurea C o n te n t [% ] SC March SC June SC July PGC March PGC June PGC July a a b A B B a b b A B B a a b A B B glycosides could be detected during spring, in March. The content of phenolic glycosides decreased from spring over summer to come to a minimum in September/October. In autumn and winter, an ac- cumulation of these compounds was found by THIEME (1965, 1971). Further research in our institute about the seasonality of phenolic glycoside contents of willow bark in autumn and winter is ongoing. Several compounds in the three willow species restrained different- ly in their seasonal development. From March to June most of the compounds in S. daphnoides decreased, especially purpurein, tre- mulacin, ampelopsin, catechin, syringin, salicin, 2’-O-acetylsali- cortin. In some clones, a few compounds like tremulacin, catechin or purpurein could not be detected anymore. Naringenin-5-glycoside showed an increase from March to June but a decrease from June to July. Another decreasing compound was salicortin. Syrengin, ampelopsin, purpurein, tremulacin, and 2’-O-acetylsalicortin increased from March to July. In S. pentandra from March to June decreasing concentrations of purpurein, syrengin, 2’-O-acetylsalicin, 2’-O-acetylsalicortin, as well as vimalin and increasing concen- trations of salicin, picein, and salicortin could be detected. From June to July some compounds like 2’-O-acetylsalicortin, syrengin, vimalin, purpurein increased, and some compounds like picein, salicortin, and ampelopsin decreased. In S. purpurea the content of syrengin, picein, 2’-O-acetylsalicin, ampelopsin, vimalin, purpurein, and tremulacin declined from March to June, whereas the content of catechin and 2’-O-acetylsalicortin increased. From June to July decreasing concentrations of 2’-O-acetylsalicortin, purpurein, and salicortin and increasing concentrations of vimalin and tremulacin could be found. A general, clear trend could not be found. Only some compounds like syrengin, 2’-O-acetylsalicin, ampelopsin, vimalin, purpurein, and tremulacin decreased in all three willow species from March to June. From June to July an increasing content of syrengin, vimalin, and tremulacin and a decreasing content of salicortin could be detected. The production of willow bark extracts is very expensive. There- fore, the use of willow species and clones with highest amounts of salicylates is obligatory. Furthermore, we had to select individual clones from which maximum harvests per hectare can be gained, e. g. with fast growth performance (RIIPI et al., 2002). Besides the bark, other plant organs of willow, like the leaves contain phenolic glyco- sides (JULKUNEN-TIITTO, 1992; THIEME 1965). Usage of these parts for phenolic glycoside production could be also considered. There are other factors which influence secondary metabolite content in willow bark, such as the species, the clone (JULKUNEN-TIITTO, 1992), the climate (MEIER et al., 1985b; JULKUNEN-TIITTO, 1985), the nutrient supply of the plant (Hakulinen, 1998; Julkunen-Tiitto et al., 1993), the physiological age (JULKUNEN-TIITTO, 1992; THIEME, 1965; Fig. 4: Seasonal variability of salicylates and phenolic glycosides in the bark of willow species (Different small letters indicate seasonal significant differences of salicylate contents within the species and large letters indicate differences within levels of phenolic glycosides, Tukey‘s HSD test, p < 0.05; salicylate: SC and phenolic glycoside: PGC). Tab. 1: Salicylate (SC) and phenolic glycoside (PGC) contents in Salix daphnoides, Salix pentandra, and Salix purpurea bark in March, June, and July Salicylate and phenolic glycoside content [%] Salix daphnoides Salix pentandra Salix purpurea SC PGC SC PGC SC PGC March 6.41 8.24 2.41 3.25 4.69 5.67 ± 1.71 ± 1.96 ± 0.27 ± 0.14 ± 0.66 ± 0.68 June 4.71 5.14 1.84 2.15 3.27 4.07 ± 1.31 ± 1.48 ± 0.27 ± 0.35 ± 0.74 ± 0.77 July 3.85 4.01 1.80 2.32 2.67 3.52 ± 0.68 ± 0.84 ± 0.16 ± 0.18 ± 0.53 ± 0.49 102 N. Förster, C. Ulrichs, M. Zander, R. Kätzel, I. Mewis THIEME, 1971; SULIMA et al., 2006), the time of day (Thieme, 1971), and the gender (Thieme, 1965). All parameters need to be considered for effective commercial recovery of phenolic glycosides. Acknowledgements The project was funded by BLE (Bundesanstalt für Landwirtschaft und Ernährung). 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Mitteilung: Unter- suchungen über die Gykosidspektren und den Glykosidgehalt der mittel- deutschen Salixarten. Pharmazie 20, 570-574. THIEME, H., 1971: Vorkommen und Verbreitung von Phenolglucosiden in der Familie der Salicaceen. Herba Polonica 17, 248-257. Address of the authors: Dr. Inga Mewis, Prof. Dr. Dr. Christian Ulrichs and Nadja Förster, Institute for Horticultural Science, Section Urban Horticulture, Humboldt University Berlin, Lentzeallee 55-57, D-14195 Berlin, Germany. Dr. Matthias Zander, Institute for Horticultural Science, Section Tree Pro- duction and Nursery, Humboldt University Berlin, Lentzeallee 75, D-14195 Berlin, Germany. Dr. Ralf Kätzel, Eberswalde Forestry Research Institute of the State of Brandenburg, Section Forest Development/Monitoring, Landesforstanstalt Eberswalde, Alfred-Möller-Straße 1, D-16225 Eberswalde, Germany. << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /FRA /ENU (Use these settings to create PDF documents with higher image resolution for improved printing quality. 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