Investigation of common butterbur (Petasytes hybridus L., Asteraceae) non-polar extract Dušanka Runjaić-Antić 1 , Vanja Tadić 2 , Ivana Arsic 3 , Zorica Vujić 3 2 Alternativa medica, Manufacturer of phytopreparations, Loznica, Serbia; 4 Institute for Medicinal Plant Research „Dr Josif Pančić“, Belgrade, Serbia; 1 Faculty of Medicine,Department of Pharmacy, University of Niš, Serbia; 3 Faculty of Pharmacy, University of Belgrade, Serbia; Common butterbur (Petasytes hybridus L., Asteraceae) has numerous applications and expanding clinical interest in the treatment of migraines, allergic rhinitis, and asthma (spasmolytic action). The combined anti-spasmodic, anti- inflammatory and calcium-channel blocking effects of P. hybridus proved a rationale base for its use in the prophylaxis of migraine. The rhizomes (Petasitidis rhizoma), and leaves (Petasitidis folium) are used for medicinal purposes. Butterbur extracts contain sesquiterpene esters, polifenolic acids, flavonoids, lignans, essential oils and in the minor concentration pyrrolizidine alkaloids. The literature data revealed that the main constituents of butterbur are sesquiterpene esters of petasin and isopetasin. The plant material was collected on the mountain Jagodnja (Serbia). The extraction procedure was performed with methylene chloride, and obtained extract was analyzed for total polifenolic compounds (mg GA/g dry mass), flavonoids (%), tannins (%) and petasine (%) content. The total phenolics content-TF was determined by the Folin-Ciocalteu method. The percentage content of flavonoids CF was calculated using the method described in DAB 10. The percentage content of tannins-TC ware investigated using the method described in the Ph. Eur. 6.0. (Table 1). The total sesquiterpene content were determined according to Ph. Eor. 6.0, taking S-petasin as reference standard. HPLC fingerprint of the extract (Figure 2 and 3) was achieved by HPLC (Agilent Technologies 1200). Detection was performed using Diode Array Detector (DAD), and the chromatograms were recorded at λ = 260 and 325 nm (for sesquiterpene lactones – petasines, and poliphenolic compounds, respectively). HPLC separation of components was achieved using a LiChrospher 100 RP 18e (5 μm), 250 × 4 mm i.d. column, with a flow rate of 1 mL/min and mobile phase, A [500 mL of H2O plus 9.8 mL of 85% H3PO4 (w/w)], B (MeCN), elution, combination of gradient mode: 90-75% A, 0-25 min; isocratic 75% A, 25-30 min; 75-55% A, 30-46 min). The sample was prepared dissolving 118.6mg of the extract in 10 mL of MeOH, filtered through 0.2 μm PTFE filters prior to HPLC analysis. The injected volume was 4 μL. Standard solution of S-petasin was prepared at a final concentration of 0.01 mg/mL. Figure. 2. Comparative HPLC spectra of S-petasin (a), MeOH (b) and CH 2 Cl 2 (c) common butterbur extracts (from 55- 65min). UV spectra of S-petasin (d), and the compounds with Rt close to Rt of S- petasin (e and f, from MeOH and CH 2 Cl 2 extracts respectively) Conclusion Further investigation should proceed in aim to determine the main sesquiterpene lacones present in the species from this geografic region. Besides, the profile of phenolic compounds would be a challenge to determined, as well. For more information please do not hesitate to contact us at: E-mail: [email protected] Phone: +381 11 7120 982 Fax.: + 381 11 3120 381 The aim of our investigation was to determine the chemical profile of Petasitides rhizoma non-polar extract regarding the content of total polifenolic compounds, including flavonoids and tannins and total petasine content. Investigated extract subjected to HPLC analysis revealed the absence of S- petasine, as well as in the MeOH extracts, made to check the obtained results (Figure 2). m in 56 58 60 62 64 mAU 0 200 400 DAD1 C ,S ig=260,8 Ref=600,100 (VMT \VT_01042013 2013-04-01 14-14-25\PETASH000100.D) 57.935 58.383 59.457 59.848 m in 56 58 60 62 64 mAU 0 25 50 DAD1 C ,S ig=260,8 Ref=600,100 (VMT \VT_01042013 2013-04-01 14-14-25\PETASH000106.D) 55.272 56.530 57.067 58.182 58.892 59.614 60.461 61.020 62.366 62.819 64.448 m in 56 58 60 62 64 mAU 0 50 100 DAD1 C ,S ig=260,8 Ref=600,100 (VMT \VT_01042013 2013-04-01 14-14-25\PETASH000112.D) 54.501 55.155 56.408 57.134 57.732 58.234 58.980 59.207 59.599 60.058 60.536 61.109 62.004 62.482 63.021 63.357 64.530 nm 250 300 350 400 450 500 550 600 mAU 0 50 100 150 200 DAD1, 57.886 (192 mAU, - ) of PETASH000100.D DAD1, 57.933 (235 mAU, - ) of PETASH000100.D a b c d e f Introduction Aim Material and method Results Petsytidis rhizoma (Petasytes hybridus L., Asteraceae) Total phenols (mg GA/g ekstrakta) 29.00 Flavonoids content (%) 0.10 Tannins content (%) 0.89 Sesquiterpene lactones content 2.10 Table 1. Total phenolic, tannins, flavonoid and sesquiterpene lactones content in non-polar extract of common butterbur root (Petasites hybridus, L., Asteraceae). The results were expressed by dry weight. Literature: A. Bodensieck, O. Kunert, E. Haslinger, R. Bauer. Helv Chim Acta (2007), 90, 183-194; 2. R. Chizzola, T. Langer, C. Franz. Planta Med. 2006, 72, 1254-1256 Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature. 181, 1199- 1200. European Pharmacopoeia. 6th edition. 2004. Strasbourg: Council of Europe. Weißdornblätter mit Blüten. In: Deutsches Arzneibuch, Band 2, Monographien R-Z; Amlitche Ausgabe Deutcher Apotheker Verlag. Stuttgart, Frankfurt: Govi-Verlag GmbH; 1991 min 10 20 30 40 50 60 mAU 0 20 40 60 80 100 120 DAD1A, S ig=260,4Ref=550,100(VMT\VT_020420132013-04-0213-12-42\PETASH000132.D) min 10 20 30 40 50 60 70 mAU 0 50 100 150 200 250 300 350 DAD1B, S ig=325,4Ref=600,100(VMT\VT_010420132013-04-0114-14-25\PETASH000106.D) Figure. 1. HPLC fingerprint of P. hybridus root non-polar extract recorded on 260 and 325 nm HPLC chromatogram of the investigated extract poinetd that the P. hybridus root extract was rich in sesquiterpene lactopnes (Figure 1). The preliminary investigation indicated phenolic compounds presence, as well. Namely, moderate phenolic (mg GA/g) and tannins (%) content were found (29.0 and 0.89, respectively). Flavonoid content was low (0.1%). Contrary to this, high sesquiterpene lactones content was revealed (Table 1).