Hydroxycinnamates from elecampane Emphasis Type=

Authors: Anna Stojakowska Janusz Malarz Anna K Kiss

Publish Date: 2016/01/22

Volume: 38, Issue: 2, Pages: 41-

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Abstract

Hydroalcoholic extract from Inula helenium callus tissue showed remarkable reducing capacity An HPLC/DAD analysis revealed the presence of numerous hydroxycinnamic acid derivatives including chlorogenic acid 5OCQA 01  dry weight and 35dicaffeoylquinic acid 35DCQA 03  dry weight which were among major constituents of the examined extract Application of a hyphenated chromatographic method—UHPLC/DAD/MSn—allowed identification of sixteen compounds derivatives of caffeic acid Apart from the compounds commonly found in Inula sp the plant material contained eight conjugates of caffeic acid with aldaric acid The aldarates constituted over 50  of the hydroxycinnamate fraction in the examined tissue This is the first report on the occurrence of the caffeoylaldaric acids in Inula sp and in a plant tissue cultureHydroxycinnamic acid derivatives are common plant constituents They are present in plant tissues as free acids ie coumaric ferulic caffeic and sinapic acids their oligomers and as conjugates with other compounds most commonly with quinic acid and glucose Mono di tri and tetracaffeoylquinic acids as well as feruloylquinic and coumaroylquinic acids and conjugates of caffeic acid with tartaric acid are frequently found in Asteraceae Jaiswal et al 2011 Hydroxycinnamates are wellknown radical scavengers and inhibitors of lipid peroxidation Ohnishi et al 1994 Olmos et al 2008 Shahidi and Chandrasekara 2010 The compounds also play a role in plant defense against pathogens and insects Kodoma et al 1998 Leiss et al 2009 They are of potential value in pharmacy as antiviral hepatoprotective and neuroprotective agents Robinson et al 1996 Kim et al 2005 2007 in cosmetics protection against UV and in food industry as bioactive components of the diet and precursors for flavors Inula helenium L a perennial herb of the Inulae tribe Asteraceae is a reputed medicinal plant The species native of Middle Asia widely occurs in Europe Asia and Northern America Underground parts of the elecampane plant collected in the autumn after 2 or 3 years of growth are traditionally used for medicinal purposes for example to treat asthma cough bronchitis lung disorders tuberculosis skin infections and helminthic diseases Seca et al 2014 The herbal product under the name of “Radix Inulae” “Radix Helenii” or “Rhizoma Helenii” is described in several European pharmacopeias eg PF X Ned 5 BHP Roots of I helenium contain up to 5  of essential oil with alantolactone isoalantolactone eudesmanetype sesquiterpene lactones and monoterpenoid thymol derivatives as major constituents Moreover sterols triterpenes and inulin up to 44  were found in the roots Blaschek et al 1998 Literature data concerning hydroxycinnamic acid derivatives present in I helenium are sparse It is only recently that some reports on analysis of extracts from leaves and roots of the plant using high performance liquid chromatography with mass spectrometry HPLC/MS and tentative identification of phenolic compounds have been published Jaiswal et al 2011 Spiridon et al 2013 Callus cultures of I helenium maintained in our laboratory showed high total phenolic content As far as we were aware secondary metabolites produced by callus cultures of elecampane were not studied before Thus the aim of our work was to identify and quantify phenolics accumulated by the tissueSeeds of botanically verified I helenium plants of known wild origin No 1026/2001 were supplied by the Botanical Garden and Botanical Museum BerlinDahlem of the Free University in Berlin Germany Voucher specimen of the donor plant was deposited in the garden herbarium or in the main herbarium of the Botanical Museum The seeds after surface sterilization were aseptically germinated on a halfstrength solidified 08  agar MS medium Murashige and Skoog 1962Leaf explants ca 1 cm long were excised from aseptic seedlings and subsequently inoculated onto a solidified MS medium supplemented with 24d 24dichlorophenoxyacetic acid 452 µM l−1 and kinetin 139 µM l−1 containing 3  sucrose pH 58 before autoclaving The explants were kept at 28 °C under continuous light 40 μmol m−2 s−1 cool white fluorescent tubes to initiate callus proliferation The calli were further maintained on the culture medium of the same composition with an unaltered temperature and illumination regime The tissue was transferred to the fresh nutrient medium every 6 weeks The subculturing was performed by inoculating ca 20 g FW fresh weight of the tissue onto 50 ml of the culture medium For the phytochemical analysis the calli were harvested at the end of 6 week culture period5Ocaffeoylquinic acid chlorogenic acid 5OCQA purity 97  by HPLC and a standard sample of cynarin 13dicaffeoylquinic acid 13DCQA purity 99  by HPLC were bought from Roth Karlsruhe Germany 35DCQA was isolated from L virosa hairy root culture Stojakowska et al 2012 The compound was of 900  purity by HPLC FolinCiocalteu reagent was purchased from SigmaAldrich Co St Louis MO USA Methanol MeOH of analytical grade was purchased from POCh SA Gliwice Poland Water for chromatography was purified by a MiliQ system Milipore Corp Bedford MA USA Acetonitrile MeCN and MeOH of gradient grade for liquid chromatography as well as glacial acetic acid and formic acid HCOOH of analytical grade were bought from Merck Darmstadt GermanyThe “total phenolic content” which reflects reducing capacity of the plant material Huang et al 2005 was analyzed using the FolinCiocalteu colorimetric method as described previously Stojakowska et al 2013 Results means of three measurements were expressed as gallic acid equivalentsThe dry pulverized plant tissue 100 mg was extracted twice for 3 h with 10 ml of 70  MeOH at room temperature on a rotary shaker 100 rpm The combined methanol extracts were evaporated in vacuo to give a dry residue which was redissolved in 1 ml of 70  MeOH and subsequently centrifuged 11340g 5 min just before chromatographic analysis Analytical HPLC separations of the samples were carried out according to the method described by Malarz et al 2013 Quantification was performed using an external standard method The calibration curves were calculated using four concentration levels 0001 001 01 and 10 mg ml−1 of 5OCQA and 13DCQA Peak areas at 325 nm were referred to the corresponding calibration curves Caffeic acid derivatives were quantified as equivalents of 5OCQA monocaffeoyl derivatives and 13DCQA di and tricaffeoyl derivatives

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