! " # # $% 004/2009. SpeedExtractor E-916

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2 004/2009 SpeedExtractor E-916! " # # $% The Genépi plant (Artemisia umbelliformis) grows in alpine areas. It is also cultivated and used to produce a herb liquor. Costunolide is a sesquiterpene lactone and the main compound responsible for the bitterness of the Genépi plant. Ground Genépi was extracted with the SpeedExtractor E-916 using an alcohol-water mixture and subsequently analyzed on HPLC. The determined costunolide content in the samples was 9.52 mg/g and corresponds to the values reported in literature [2], [3]. ) The Genépi plant (Artemisia umbelliformis) grows in alpine areas between 2000 and 3700 meters above sea level. The plant is cultivated and used to produce a herb liquor, a local specialty in the region of the Valais Alps of Switzerland, Northern Italy and Western France. Figure 1: Genépi (A. umbelliformis) Figure 2: Costunolide Costunolide is a sesquiterpene lactone and the main compound responsible for the bitterness of the Genépi plant and much desired for its distinguished taste. Costunolide has also been reported to have an anti-cancer effect [1] and as a remedy for stomach cramps and loss of appetite [2]. The amount of costunolide found in Genépi varies based on the time of harvest [3]. Instrumentation: SpeedExtractor E-916, ultra centrifugal mill, HPLC system with UV detector (210nm) The dried and ground plants (<1 mm) were mixed with diatomaceous earth and extracted with the SpeedExtractor E-916 using the parameters shown in Table 1. The sample was extracted in triplicate. Table 1: Extraction method of SpeedExtractor E-916 Temperature 50 C Pressure 100 bar Solvent Water 60%, Ethanol 40% Cells Vials Cycles 3 Heat-up Hold Discharge Flush with solvent Flush with gas 40 ml 240 ml 1 min 9 min 3 min Costunolide was quantified by HPLC (equipped with UV detector at 210 nm), using a calibration based on an external standard. The results (see Table 2) correspond to the values reported in literature from 5.6 mg/g up to 29.1 mg/g [2], [3]. The value of sample two is not an outlier according to the Grubbs test (significance 95%). Table 2: Determined amount of costunolide Costunolide [mg/g] Sample Sample Sample Mean value 9.52 rsd % 6.10 % The extraction of Genépi plants using Speed Extractor E-916 represents a very powerful method to prepare extracts for the quantification of important plant compounds such as costunolide. The values determined are in correspondence with literature. The short total extraction time of approx. 1 h 10 min and the small solvent volume used of approx. 60 ml are further benefits of this procedure. +& " We acknowledge the University of Applied Science of Western Switzerland in Sion/Valais namely Dr. Wilfried Andlauer and Julien Héritier, for the analysis and their support inr the development of this application note. [1] Choi, S. et al (2005) Inhibitory effects of costunolide on the telomerase activity in human breast carcinoma cells. Cancer Lett. 227(2), [2] Rey, Ch.; Slacanin, I. (1997) Domestication du genépi blanc. Revue Suisse Vitic. Arboric. Hortic. Vol.29 (3) I- VIII. [3] Simmonet, X. et al. (2006) Stade phénologique et qualité des hampes florales du genépi blanc. Revue Suisse Vitic. Arboric. Hortic. 38(3) SpeedExtractor E-916 operation manual For more detailed information, refer to Application note 004/2009 &&&'#' $ ((#

3 ) The Genépi plant (Artemisia umbelliformis) grows in alpine areas between 2000 and 3700 meters above sea level. The plant is cultivated and used to produce a herb-liquor, a local specialty in the region of the Valais alps of Switzerland, Northern Italy and Western France. Other Artemisia species are also commercially used, for example for the production of alcoholic beverages as Absinth and Vermouth (Artemisia absinthium). The plants of the Artemisia species contain the neurotoxic compound thuyone. Therefore, the liquors made of these plants were longtime forbidden in many countries. Nowadays, the cultivated plants contain only very small amounts of thuyone, due to the selection during plant breeding. Costunolide is a sesquiterpene lactone and the main compound responsible for the bitterness of the Genépi plant and much desired for the typical taste. Costunolide has also been reported to have an anti-cancer effect [1] and have healing power on stomach cramps and loss of appetite [2]. The amount of costunolide found in Genépi varies upon the time of harvest; they are highest before being in flower and lowest after fading [3]. An efficient extraction method by SpeedExtractor E-916 as preparation before the quantification of costunolide by HPLC is presented. Figure 1: Structure of costunolide, responsible for the bitterness of Genépi, SpeedExtractor E-916, with 40 ml cells Ultra centrifugal mill ZM100 (Retsch, Germany) Analytical balance (reading precision 1 mg) 1200 series HPLC system with UV detector at 210 nm (Agilent technologies, Germany) Volumetric flask, 100 ml Funnel - %# Ethanol absolute >99.8% 1, Fluka (02883) Methanol HPLC quality, Lab-scan analytical sciences (C17) Costunolide, Extrasynthese (7502) Deionized water Diatomaceous earth, Buchi (053201) HPLC column CC 125/3 Nucleosil C8 cc, Macherey-Nagel, Switzerland 2 ml plastic syringe, VWR ( ) 0.45 µm-25 Exapure syringe filter, Alys Technologie (ATSY25TF4) 2 ml glass vials, VWR ( ) with caps, VWR ( ) 1 It is important that the ethanol does not contain any ketones as denaturing agents, because they can react with constituents of the plants. No tests were carried out with ethanol, denatured with methanol; this might work as well. Application Note 004/2009 Version A, Copyright 2009 Büchi Labortechnik AG 3/7

4 Genépi (dried, moisture < 10%) Cut the plants into small pieces (ca. 3 cm) and then grind the sample at rpm in an Ultra Centrifugal mill (Retsch, ZM100) using a 1 mm sieve. (See Figure 1 and 2) Figure 1: Pieces of Genépi Figure 2: Ground Genépi. / The extraction and quantification of Costunolide in Genépi includes the following steps: Grinding of sample Preparation of cells Extraction with SpeedExtractor E-916 Dilution of the extracts to 100 ml and filtration Quantification of costunolide by HPLC (UV detector at 210 nm)..' / # Weigh in approx. 3 g (+/-1 mg) of ground Genépi using an analytical balance. Mix the sample with approximately 7 g of diatomaceous earth (see Figure 3) and fill in the mixture into the 40 ml cells using a funnel. Figure 3: Ground Genépi mixed with diatomaceous earth Application Note 004/2009 Version A, Copyright 2009 Büchi Labortechnik AG 4/7

5 .' & # Carry out the extraction, using the parameters of Table 1. The sample was extracted in triplicate. The total time for the extraction was approx. 1h 10 min. Table 1: Extraction method of Speed Extractor E-916 Parameter Value Temperature 50 C Pressure 100 bar Solvent Water 60% Ethanol 40% Cells Vials Cycles 3 Heat-up Hold Discharge Flush with solvent Flush with gas 40 ml 240 ml 1 min 9 min 3 min.'- 0 # Transfer the extracts quantitatively to 100 ml volumetric flask. Carefully rinse the vials with deionized water and transfer the rinsing water also to the volumetric flask. Complete the flask to volume with deionized water. Filter the extract through a syringe filter (0.45 µm) transferring it into a glass vial..' $ (1/2% The filtered extracts are analyzed on a HPLC system equipped with an UV detector at 210 nm. The mobile phases were water (A) and methanol (B), with the gradient presented in Table 2. Table 2: Applied gradient for the analysis of costunolide Time [min] (A) (B) The flow rate was 0.6 ml/min. The column was equilibrated for between the injections. The injection volume was 20 µl. The calibration curve was established by using standard solutions of costunolide at concentrations of 0.03, 0.06, 0.11, 0.28 and 0.55 mg/ml. ' % 3 The values of the quantification of costunolide are summarized in Table 3 and the calibration curve is plotted in Figure 3. Application Note 004/2009 Version A, Copyright 2009 Büchi Labortechnik AG 5/7

6 Table 3: Determined peak areas of the costunolide standard solutions Costunolide [mg/ml] Peak area [maus] Peak area [maus] Costunolide [mg/ml] Figure 3: Calibration curve (y=126170x , R 2 = ) ' $# In Figure 4, a typical chromatogram of extracts of Genépi is shown. Figure 4: Chromatogram of Genépi extract, with the peak of costunolide The results of the analysis of the extracts are presented in Table 4. Application Note 004/2009 Version A, Copyright 2009 Büchi Labortechnik AG 6/7

7 Table 4: Determined costunolide contents in Genépi extracts Sample weight [g] Peak area [maus] Costunolide [mg/g] Sample Sample Sample Mean value 9.52 rsd % 6.10 The value of sample 2 is considerably lower than the two others, but is no outlier according to the outlier test of Grubbs (α=5%). The determined amount of costunolide in Genépi corresponds to the values found in literature of 5.6 mg/g (after fading) up to 29.1 mg/g (before blooming) [2], [3]. 4 % The extraction of Genépi plants using SpeedExtractor E-916 represents a very powerful method to prepare extracts for the quantification of interesting and important plant compounds as costunolide. The determined contents were in correspondence with literature. The short total extraction time of approx. 1 h 10 min and the small solvent volume used of approx. 60 ml are further benefits of this procedure. 5 +& " We greatly acknowledge the University of Applied Science of Western Switzerland in Sion/Valais namely Dr. Wilfried Andlauer and Julien Héritier for the analysis and their support for the development of this application note. [1] Choi, S. et al (2005) Inhibitory effects of costunolide on the telomerase activity in human breast carcinoma cells. Cancer Lett. 227(2), [2] Rey, Ch.; Slacanin, I. (1997) Domestication du genépi blanc. Revue Suisse Vitic. Arboric. Hortic. Vol.29 (3) I-VIII. [3] Simmonet, X. et al. (2006) Stade phénologique et qualité des hampes florales du genépi blanc. Revue Suisse Vitic. Arboric. Hortic. 38(3) Manuals: Operation manual of SpeedExtractor E-914/916 BÜCHI Labortechnik AG CH-9230 Flawil 1/Switzerland T F &&&'#' $ ((# Application Note 004/2009 Version A, Copyright 2009 Büchi Labortechnik AG 7/7