Phytochemical and Pharmacological Investigation of Spiraea
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Kiss et al. BMC Res Notes (2017) 10:762 https://doi.org/10.1186/s13104-017-3013-y BMC Research Notes RESEARCH NOTE Open Access Phytochemical and pharmacological investigation of Spiraea chamaedryfolia: a contribution to the chemotaxonomy of Spiraea genus Tivadar Kiss1,2, Kristóf Bence Cank1, Orsolya Orbán‑Gyapai1, Erika Liktor‑Busa1, Zoltán Péter Zomborszki1,2, Santa Rutkovska3, Irēna Pučka3, Anikó Németh4 and Dezső Csupor1,2* Abstract Objective: Diterpene alkaloids are secondary plant metabolites and chemotaxonomical markers with a strong biological activity. These compounds are characteristic for the Ranunculaceae family, while their occurrence in other taxa is rare. Several species of the Spiraea genus (Rosaceae) are examples of this rarity. Screening Spiraea species for alkaloid content is a chemotaxonomical approach to clarify the classifcation and phylogeny of the genus. Novel phar‑ macological fndings make further investigations of Spiraea diterpene alkaloids promising. Results: Seven Spiraea species were screened for diterpene alkaloids. Phytochemical and pharmacological investiga‑ tions were performed on Spiraea chamaedryfolia, the species found to contain diterpene alkaloids. Its alkaloid-rich fractions were found to exert a remarkable xanthine-oxidase inhibitory activity and a moderate antibacterial activity. The alkaloid distribution within the root was clarifed by microscopic techniques. Keywords: Phytochemistry, Alkaloids, Spiraea, Antibacterial, Xanthine-oxidase, Chemotaxonomy Introduction (Ranunculaceae) are known to be characterized by the Plant metabolism, driven by photosynthesis, provides presence of diterpene alkaloids. Although such alkaloids a huge number and a wide variety of natural products. have also been reported from some Inula (Asteraceae), Tese compounds are of great importance for their ben- Garrya (Garryaceae), Erythrophleum (Fabaceae) and efcial biological activities in humans. Te investigation Spiraea (Rosaceae) species [2, 3], the occurrence of dit- for specifc plant metabolites is also a useful tool for the erpene alkaloids in these taxa is sporadic. Since diterpene clarifcation of taxonomical uncertainties. alkaloids are considered as chemotaxonomic markers [4], Diterpene alkaloids are secondary metabolites belong- their presence in species other than those belonging to ing to pseudoalkaloids [1]. Tis group of molecules the Ranunculaceae family might have an important role includes numerous compounds with diverse skeletons in plant taxonomy. and substitution patterns. Tese compounds can be clas- Te Spiraea genus, comprising approximately 100 spe- sifed according to the number of carbon atoms in the cies, belongs to the Rosaceae family. Phytochemical con- skeleton as bisnor-(C18), nor-(C19) and diterpene (C20) tents of 28 Spiraea taxa have been extensively studied. alkaloids. Aconitum, Delphinium and Consolida genera Mono-, di-, sesqui- and triterpenes have been isolated besides favonoids, lignans, neolignans and other phenyl- propane derivatives. Interestingly, only 9 of the investi- *Correspondence: [email protected]‑szeged.hu gated taxa were found to contain diterpene alkaloids (S. 1 Department of Pharmacognosy, University of Szeged, Eötvös u. 6, formosana Hayata, S. fritschiana var. parvifolia Liou, S. Szeged 6720, Hungary Full list of author information is available at the end of the article japonica L.f., S. japonica var. acuta Yu, S. japonica var. © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kiss et al. BMC Res Notes (2017) 10:762 Page 2 of 6 fortunei (Planchon) Rehder, S. japonica var. glabra (Regel) (Sepsibükszád and Alsórákos, Hungary). S. nipponica Koidz, S. japonica var. incisa Yu, S. japonica var. ovalifolia Maxim (SZTE-FG 852), S. x vanhouttei (Briot) Zabel Zuo, S. japonica var. stellaris). All of the reported 65 dit- (SZTE-FG 853) and S. x billardii hort. ex K. Koch (SZTE- erpene alkaloids bear hetisine- and atisine-type C20 basic FG 854) were collected and identifed by Anikó Németh skeletons (Additional fle 1: Spiraea diterpene alkaloids). (Botanical Garden of University of Szeged, Szeged, Hun- Although only marginal ethnomedicinal use of Spiraea gary). S. media Schmidt. (DAU 0 31 147 009) and Spiraea species has been documented in North-America and chamaedryfolia L. (DAU 0 31 145 023) were harvested Asia, pharmacological studies have reported noteworthy in Daugavpils (Latvia), and identifcation was performed activities of Spiraea extracts and isolated compounds [5]. by Santa Rutkovska (University of Daugavpils, Latvia). Te recent classifcation and clarifcation of Spiraea Voucher specimens were deposited at the herbarium of phylogeny is based mainly on molecular analyses [6–9]. the Department of Pharmacognosy of the University of Te phytochemical analysis is also considered as a useful Szeged and at that of the University of Daugavpils. Herb tool to support plant classifcation. and root of the plant material were separated, dried and Phytochemical studies on Spiraea genus are promis- stored at room temperature until processing. ing, because of their possible utilization as source of pharmacons. On the other hand, screening of this genus Extraction and identifcation of the alkaloid content for diterpene alkaloid content may contribute to the Dried and crushed herb materials were extracted conse- clarifcation of Spiraea phylogeny. Tese considerations quently with methanol (MeOH), chloroform (CHCl3) and motivated our research, aiming to improve the current 2% aqueous HCl, by ultrasonication at room temperature phytochemical knowledge on Spiraea species. (Fig. 1). Te applied drug-solvent ratio was 1:5 in each case. Te drug was dried before each extraction phase. Main text Moistening with 5% aqueous NaOH solvent was applied Materials and methods prior to extraction with chloroform. Plant material Te methanol extract was acidifed with 2% aque- Seven Spiraea species were analysed. S. crenata L. ous HCl and was then extracted with chloroform. Frac- (SZTE-FG 850) and S. salicifolia L. (SZTE-FG 851) tion M1 was obtained by collecting and evaporating the were collected and identifed by Gusztáv Jakab (Szent organic phase. Te pH of the aqueous phase was ren- István University, Budapest, Hungary) in Hungary dered to alkaline (pH 12) with 5% aqueous NaOH and Fig. 1 Alkaloid contents and pharmacological activities of S. chamaedryfolia fractions Kiss et al. BMC Res Notes (2017) 10:762 Page 3 of 6 was then extracted with chloroform. Te chloroform After drying, these loaded flter paper discs were placed phase yielded fraction M2. on the plates containing the bacterial suspensions. Paper Te chloroform extract was further extracted with discs impregnated with 20 µL of pure solvent were used 2% aqueous HCl. Te organic phase was evaporated as a negative control. Te plates were then incubated at and used as fraction L1. Te pH of the aqueous phase 37 °C for 24 h under aerobic conditions. Diameters of was made alkaline and extracted with chloroform. Te the inhibition zones produced by the plant extracts were organic phase was evaporated to yield fraction L2. measured and recorded (as the diameter of the inhibition Te acidic extract was subjected to solvent–solvent zone plus the diameter of the disc) at 24 h. partitioning with chloroform, after adjusting the pH to alkaline. Te dry residue of the organic phase was Xanthine oxidase assay labelled as S1. Te pH of the aqueous phase was rendered Te method is based on a continuous spectrophotomet- to acidic with 2% aqueous HCl and was then extracted ric rate determination: the absorbance of xanthine oxi- with chloroform. Te organic phase was evaporated to dase (XO) enzyme induced uric acid production from produce fraction S2. xanthine was measured at 290 nm for 3 min. Te enzyme- Fractions were screened for alkaloid content by thin inhibitory efect of our plant extracts was determined on layer chromatography (TLC), carried out at room tem- the basis of the decrease in uric acid production. Rea- perature on silica gel (SiO2 60 F254, Merck 1.05554.0001) gents used included: 50 mM potassium bufer, pH 7.5 and toluene/acetone/ethanol/cc.NH3 70:50:18:4.5 was with 1 M KOH, 0.15 mM xanthine solution, pH 7.5, pre- applied as mobile phase. Detection was performed in two pared using xanthine, XO enzyme solution 0.2 Units/mL steps: (1) dry plates were sprayed with Dragendorf’s rea- prepared using XO. Te test solutions applied included: gent; and (2) after drying, the plates were sprayed again S. chamaedryfolia fractions 12 g/mL, 600 µg/mL diluted with 5% aqueous NaNO2. Te alkaloids appeared as per- in DMSO solution. Te fnal reaction mixture of 300 µL manent brown spots. well contained: 100 µL xanthine, 150 µL bufer and 50 µL XO for enzyme-activity. Allopurinol was dissolved in Screening for antibacterial activity DMSO and used as positive control (100% inhibition was Plant extracts were tested for antibacterial activity considered at 10 μg/mL concentration of allopurinol). using the following microorganisms as test