Phytochemical and pharmacological investigation of Spiraea chamaedryfolia: A contribution to the chemotaxonomy of Spiraea genus

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 classification and phylogeny of the genus. Novel pharmacological findings make further investigations of Spiraea diterpene alkaloids promising. ResultsSeven Spiraea species were screened for diterpene alkaloids. Phytochemical and pharmacological investigations 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 clarified by microscopic techniques.

Full text

Kiss et al. BMC Res Notes (2017) 10:762
https://doi.org/10.1186/s13104-017-3013-y
RESEARCH NOTE
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 classification and phylogeny of the genus. Novel phar‑
macological findings 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 clarified by microscopic techniques.
Keywords: Phytochemistry, Alkaloids, Spiraea, Antibacterial, Xanthine‑oxidase, Chemotaxonomy
© 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.
Introduction
Plant metabolism, driven by photosynthesis, provides
a huge number and a wide variety of natural products.
ese compounds are of great importance for their ben-
eficial biological activities in humans. e investigation
for specific plant metabolites is also a useful tool for the
clarification of taxonomical uncertainties.
Diterpene alkaloids are secondary metabolites belong-
ing to pseudoalkaloids [1]. is group of molecules
includes numerous compounds with diverse skeletons
and substitution patterns. ese compounds can be clas-
sified according to the number of carbon atoms in the
skeleton as bisnor-(C18), nor-(C19) and diterpene (C20)
alkaloids. Aconitum, Delphinium and Consolida genera
(Ranunculaceae) are known to be characterized by the
presence of diterpene alkaloids. Although such alkaloids
have also been reported from some Inula (Asteraceae),
Garrya (Garryaceae), Erythrophleum (Fabaceae) and
Spiraea (Rosaceae) species [2, 3], the occurrence of dit-
erpene alkaloids in these taxa is sporadic. Since diterpene
alkaloids are considered as chemotaxonomic markers [4],
their presence in species other than those belonging to
the Ranunculaceae family might have an important role
in plant taxonomy.
e Spiraea genus, comprising approximately 100 spe-
cies, belongs to the Rosaceae family. Phytochemical con-
tents of 28 Spiraea taxa have been extensively studied.
Mono-, di-, sesqui- and triterpenes have been isolated
besides flavonoids, lignans, neolignans and other phenyl-
propane derivatives. Interestingly, only 9 of the investi-
gated taxa were found to contain diterpene alkaloids (S.
formosana Hayata, S. fritschiana var. parvifolia Liou, S.
japonica L.f., S. japonica var. acuta Yu, S. japonica var.
Open Access
BMC Research Notes
*Correspondence: csupor.dezso@pharm.u‑szeged.hu
1 Department of Pharmacognosy, University of Szeged, Eötvös u. 6,
Szeged 6720, Hungary
Full list of author information is available at the end of the article

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Kiss et al. BMC Res Notes (2017) 10:762
fortunei (Planchon) Rehder, S. japonica var. glabra (Regel)
Koidz, S. japonica var. incisa Yu, S. japonica var. ovalifolia
Zuo, S. japonica var. stellaris). All of the reported 65 dit-
erpene alkaloids bear hetisine- and atisine-type C20 basic
skeletons (Additional file1: Spiraea diterpene alkaloids).
Although only marginal ethnomedicinal use of Spiraea
species has been documented in North-America and
Asia, pharmacological studies have reported noteworthy
activities of Spiraea extracts and isolated compounds [5].
e recent classification and clarification of Spiraea
phylogeny is based mainly on molecular analyses [6–9].
e phytochemical analysis is also considered as a useful
tool to support plant classification.
Phytochemical studies on Spiraea genus are promis-
ing, because of their possible utilization as source of
pharmacons. On the other hand, screening of this genus
for diterpene alkaloid content may contribute to the
clarification of Spiraea phylogeny. ese considerations
motivated our research, aiming to improve the current
phytochemical knowledge on Spiraea species.
Main text
Materials andmethods
Plant material
Seven Spiraea species were analysed. S. crenata L.
(SZTE-FG 850) and S. salicifolia L. (SZTE-FG 851)
were collected and identified by Gusztáv Jakab (Szent
István University, Budapest, Hungary) in Hungary
(Sepsibükszád and Alsórákos, Hungary). S. nipponica
Maxim (SZTE-FG 852), S. x vanhouttei (Briot) Zabel
(SZTE-FG 853) and S. x billardii hort. ex K. Koch (SZTE-
FG 854) were collected and identified by Anikó Németh
(Botanical Garden of University of Szeged, Szeged, Hun-
gary). S. media Schmidt. (DAU 0 31 147 009) and Spiraea
chamaedryfolia L. (DAU 0 31 145 023) were harvested
in Daugavpils (Latvia), and identification was performed
by Santa Rutkovska (University of Daugavpils, Latvia).
Voucher specimens were deposited at the herbarium of
the Department of Pharmacognosy of the University of
Szeged and at that of the University of Daugavpils. Herb
and root of the plant material were separated, dried and
stored at room temperature until processing.
Extraction andidentication ofthe alkaloid content
Dried and crushed herb materials were extracted conse-
quently with methanol (MeOH), chloroform (CHCl3) and
2% aqueous HCl, by ultrasonication at room temperature
(Fig.1). e applied drug-solvent ratio was 1:5 in each
case. e drug was dried before each extraction phase.
Moistening with 5% aqueous NaOH solvent was applied
prior to extraction with chloroform.
e methanol extract was acidified with 2% aque-
ous HCl and was then extracted with chloroform. Frac-
tion M1 was obtained by collecting and evaporating the
organic phase. e pH of the aqueous phase was ren-
dered to alkaline (pH 12) with 5% aqueous NaOH and
Fig. 1 Alkaloid contents and pharmacological activities of S. chamaedryfolia fractions

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Kiss et al. BMC Res Notes (2017) 10:762
was then extracted with chloroform. e chloroform
phase yielded fraction M2.
e chloroform extract was further extracted with
2% aqueous HCl. e organic phase was evaporated
and used as fraction L1. e pH of the aqueous phase
was made alkaline and extracted with chloroform. e
organic phase was evaporated to yield fraction L2.
e acidic extract was subjected to solvent–solvent
partitioning with chloroform, after adjusting the pH
to alkaline. e dry residue of the organic phase was
labelled as S1. e pH of the aqueous phase was rendered
to acidic with 2% aqueous HCl and was then extracted
with chloroform. e organic phase was evaporated to
produce fraction S2.
Fractions were screened for alkaloid content by thin
layer chromatography (TLC), carried out at room tem-
perature on silica gel (SiO2 60 F254, Merck 1.05554.0001)
and toluene/acetone/ethanol/cc.NH3 70:50:18:4.5 was
applied as mobile phase. Detection was performed in two
steps: (1) dry plates were sprayed with Dragendorff’s rea-
gent; and (2) after drying, the plates were sprayed again
with 5% aqueous NaNO2. e alkaloids appeared as per-
manent brown spots.
Screening forantibacterial activity
Plant extracts were tested for antibacterial activity
using the following microorganisms as test strains in
the screening assays: 3 different Gram-positive strains,
namely Bacillus subtilis (ATCC 6633), Staphylococcus
aureus (ATCC 29213), and Streptococcus pneumoniae
(ATCC 49619) plus one Gram-negative strain, namely
Moraxella catarrhalis (ATCC 25238). In addition, the
multi-resistant strain, methicillin-resistant S. aureus
(MRSA, ATCC 43300) was used to test whether the frac-
tions have a specific antibacterial effect on a strain of
high public health priority. e test organisms were cul-
tured on standard Mueller–Hinton agar plates or Colum-
bia agar + 5% sheep blood (COS) plates (bioMérieux)
at 37°C. e bacterial cultures were maintained in their
appropriate plates at 4°C throughout the experiment and
were used as stock cultures.
Antibacterial activities of our plant extracts were evalu-
ated by the disc-diffusion method. e bacterial isolates
for screening assay were prepared by picking single col-
ony from 24h old plates and it was suspended in sterile,
isotonic saline solution (5 mL) to reach 0.5 McFarland
standard of optical turbidity, resulting in a suspension
containing approximately 1–2×108CFU/mL. e bac-
terial suspension was spread on appropriate sterile plates
using a sterile cotton swab. Sterile filter paper discs
(6mm of diameter) were loaded with the extracts, using
20μL of dried extracts redissolved in a mixture of ethanol
and water (40/60 v/v) at a concentration of 50mg/mL.
After drying, these loaded filter paper discs were placed
on the plates containing the bacterial suspensions. Paper
discs impregnated with 20µL of pure solvent were used
as a negative control. e plates were then incubated at
37 °C for 24h under aerobic conditions. Diameters of
the inhibition zones produced by the plant extracts were
measured and recorded (as the diameter of the inhibition
zone plus the diameter of the disc) at 24h.
Xanthine oxidase assay
e method is based on a continuous spectrophotomet-
ric rate determination: the absorbance of xanthine oxi-
dase (XO) enzyme induced uric acid production from
xanthine was measured at 290nm for 3min. e enzyme-
inhibitory effect of our plant extracts was determined on
the basis of the decrease in uric acid production. Rea-
gents used included: 50 mM potassium buffer, pH 7.5
with 1M KOH, 0.15mM xanthine solution, pH 7.5, pre-
pared using xanthine, XO enzyme solution 0.2Units/mL
prepared using XO. e test solutions applied included:
S. chamaedryfolia fractions 12g/mL, 600µg/mL diluted
in DMSO solution. e final reaction mixture of 300µL
well contained: 100µL xanthine, 150µL buffer and 50µL
XO for enzyme-activity. Allopurinol was dissolved in
DMSO and used as positive control (100% inhibition was
considered at 10 μg/mL concentration of allopurinol).
e reaction mixture for inhibition: 100 µL xanthine,
140µL buffer, 10µL test and 50µL XO.
Microscopical analysis
Specimens of the plant material were softened by ultra-
sonication in hot water for 1h. Unembedded material
was sectioned on a sledge microtome producing sections
of 100μm thickness. Observations were carried out on
unstained sections. For histological characterisation 1%
aqueous toluidine blue was used, and Dragendorff’s rea-
gent was applied for alkaloid localisation. Transverse sec-
tions were mounted with water/glycerol 1:1. e sections
were observed under light microscope and photographic
images were captured using a digital camera.
Results
Phytochemical screening revealed alkaloid content in S.
chamaedryfolia roots, while all the other six Spiraea spe-
cies were alkaloid-free. e solvent–solvent partitioning
of methanolic, acidic and alkaline extracts of S. chamae-
dryfolia yielded alkaloid-rich ethyl acetate (EtOAc), chlo-
roform and methanol fractions (Fig.1). e most apolar
fraction prepared with n-hexane (hex) was alkaloid-free.
e attempt to isolate diterpene alkaloids have failed due
to the low stability of the compounds.
e fractions were screened for in vitro antibacterial
and xanthine oxidase inhibitory activity. e ethyl acetate

Page 4 of 6
Kiss et al. BMC Res Notes (2017) 10:762
fraction was found to be the most potent xanthine oxi-
dase inhibitor, exerting over 70% of inhibition compared
to allopurinol (Fig.1 and Table1).
ree fractions were found to exert antibacterial activ-
ity against S. aureus (ATCC 29213), B. subtilis (ATCC
6633), S. pneumoniae (ATCC 49619), and M. catarrhalis
(ATCC 25238), while one fraction exerted antibacterial
activity against methicillin-resistant S. aureus (MRSA)
(ATCC 43300) (Fig.1 and Table1).
Examining the transverse section of the root of S.
chamaedryfolia, structures characteristic of second-
ary root were observed (Fig.2). e periderm, primary
and secondary cortex, and xylems with medullary rays
could be observed in the unstained sections. Primary
and secondary cortex with fibers in the primary cortex
became visible after staining with toluidine blue. Dragen-
dorff’s reagent revealed the presence of alkaloids in the
secondary cortex and secondary xylem, while in the pith
no signs of alkaloid content was observed.
Discussion
Plants may contain alkaloids in two forms: either as free
base or as salts of organic acids. e compounds pre-
sent in the free base form can be extracted with organic
solvents, while those in the salt form can be extracted
using diluted inorganic acids. Diterpene alkaloids, and
especially esters, may be unstable, thus they require
special handling. For this reason alcoholic extraction is
considered to be the most cautious method. However,
the diverse structure and the substitution pattern of dit-
erpene alkaloid molecules might require acidic and alka-
line extraction as well. According to the literature, only
alcoholic extraction was applied in previous phytochemi-
cal screening studies of Spiraea species, which might
Table 1 Antibacterial andxanthine oxidase inhibitory activities ofS. chamaedryfolia fractions
Fractions with activity (●) and fractions with no activity (○). (EtOAc ethyl acetate, MeOH methanol)
Fractions Bacillus
subtilis Staphylococcus
aureus Streptococcus pneu-
moniae Moraxella catarrhalis Staphylococcus aureus
MRSA XO inhibition %
M1‑EtOAc ● ● ● ● ● ●
L1‑MeOH ○ ○ ○ ● ○ ○
L1‑EtOAc ○ ○ ○ ○ ○ ●
S2‑EtOAc ● ● ● ● ● ●
Fig. 2 Transverse section of the root of S. chamaedryfolia. Transverse sections of the secondary root of Spiraea chamaedryfolia, unstained (I), stained
with 1% toluidine blue (II) and treated with Dragendorff reagent (III). (P periderm, C cortex, PC primary cortex, SC secondary cortex, X xylem, SX
secondary xylem, MR medullary ray)

Page 5 of 6
Kiss et al. BMC Res Notes (2017) 10:762
have resulted in an incomplete extraction. To prevent
the decomposition of the alkaloid content, the order of
extraction was determined to be started by methanol,
and followed by organic and acidic extraction steps. e
application of all these three extraction methods yielded
fractions with a diverse alkaloid profile.
Unfortunately, although 4.0kg of dried roots was used
for the preparative phytochemical work, our efforts to
isolate pure alkaloids were unsuccessful. After purifica-
tion with adsorption chromatography (i.e. column chro-
matography and centrifugal planar chromatography)
and gel filtration chromatography, the polarity and the
molecular size of alkaloids and matrix compounds were
similar within the obtained fractions, rendering separa-
tion impossible. Beside the notable amount of matrix
compounds the highly unstable manner of alkaloids was
also an obstacle to isolate pure compounds.
Fractions of S. chamaedryfolia were found to exert
noteworthy biological activities. Xanthine oxidase
inhibitory activity of S. chamaedryfolia fractions was
remarkable, and the fractions also exerted a moderate
antibacterial activity.
Proving the presence of alkaloids in S. chamaedryfolia
is noteworthy, since only few taxa are known to have the
ability to produce diterpene alkaloids: it has previously
been reported for S. japonica 64 [10–29], S. fritchiana 2
[12, 16], S. koreana [30] and S. formosa 1 [31] only. No
other types of alkaloids have been reported for the Spi-
raea genus. e alkaloid content of S. chamaedryfolia
and the lack of alkaloids for S. crenata, S. media, S. salici-
folia, S. nipponica, S. x vanhouttei and S. x billardii is first
reported by our research group, making our phytochemi-
cal analyses pioneering in this field.
Limitations
Only TLC detection methods were applied to confirm
the alkaloid content, the subtypes of these alkaloids was
not elucidated by LC–MS or NMR techniques. However,
since no other alkaloid types have been reported from
the Spiraea genus, this finding suggests the presence
(or absence) of diterpene alkaloids in the investigated
species.
Abbreviations
C: cortex; CHCl3: chloroform; EtOAc: ethyl acetate; hex: hexane; LC–MS: liquid
chromatography–mass spectroscopy; MeOH: methanol; MR: medullary ray;
NMR: nuclear magnetic resonance; P: periderm; PC: primary cortex; SC: sec‑
ondary cortex; SX: secondary xylem; TLC: thin layer chromatography; X: xylem;
XO: xanthine oxidase.
Additional le
Additional le1. Spiraea diterpene alkaloids. Diterpene alkaloids
reported from Spiraea genus.
Authors’ contributions
TK and CD conceived and designed the experiments. SR, IP and AN provided
and identified the plant material. CK and TK performed phytochemical experi‑
ments. Pharmacological investigations were performed by OO, EL, ZZ. TK, CK
and CD analysed the data. Funding acquisition by CD. All authors contrib‑
uted in drafting of the manuscript. All authors read and approved the final
manuscript.
Author details
1 Department of Pharmacognosy, University of Szeged, Eötvös u. 6, Sze‑
ged 6720, Hungary. 2 Interdisciplinary Centre for Natural Products, University
of Szeged, Eötvös u. 6, Szeged 6720, Hungary. 3 Department of Chemistry
and Geography, Daugavpils University, Parādes st. 1, Daugavpils 5401, Latvia.
4 Botanical Garden, University of Szeged, Lövölde u. 42, Szeged 6726, Hungary.
Acknowledgements
The authors thank Dora Bokor PharmD for proofreading the manuscript.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The dataset supporting the conclusions of this research is included in the
article.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Spiraea chamaedryfolia and Spiraea chamaedryfolia were collected in Dau‑
gavpills (Latvia). Spiraea crenata was collected in Alsórákos (Hungary) and
Spiraea salicifolia in Sepsibükszád (Hungary). Spiraea nipponica, Spiraea x van-
houttei and Spiraea x billardii were collected in Botanical Garden, University of
Szeged in Szeged (Hungary). None of the plant species used are endangered
at their harvesting place, thus according to the country of origin, there was no
need for permission or licence. Plant material was collected on public territory.
Funding
This work was supported by TÁMOP 4.2.4.A/2‑11‑1‑2012‑0001 ‘National Excel‑
lence Program’ (ÚNKP‑ÚNKP‑16‑2 “New national excellence program of the
Ministry of Human Capacities”); Hungarian Academy of Sciences (János Bolyai
Research Scholarship); National Research, Development and Innovation Office
(115796); GINOP‑2.3.2‑15‑2016‑00012 (New ways in the natural product‑based
drug discovery—system metabolomics approaches to discover biologically
active terpenoids of herbal and microbial origin); TÁMOP 4.2.4.A/2‑11‑1‑2012‑
0001 ‘National Excellence Program’ (ÚNKP‑ÚNKP‑16‑2 “New national excellence
program of the Ministry of Human Capacities”).
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
Received: 18 July 2017 Accepted: 28 November 2017
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