Phenolics in Primula veris L. and P. elatior (L.) Hill Raw Materials

Abstract

Primula veris L. and Primula elatior (L.) Hill represent medicinal plants used for the production of herbal teas and preparations with antioxidant and expectorant activity. Flowers and roots of both species possess the same biological activity. In the presented study, raw materials of wild growing P. veris and P. elatior were compared in terms of the content and composition of phenolic compounds using a fast and simple HPLC-DAD method. The study showed that flowers of both species were rich in flavonoids. However, P. veris flowers were characterized with a distinctly higher content of isorhamnetin-3-O-glucoside, astragalin, and (+)-catechin, whereas P. elatior occurred to be a richer source of rutoside and isorhamnetin-3-O-rutinoside. Hyperoside was found exclusively in P. elatior flowers. Phenolic glycosides (primverin and primulaverin) were identified only in the roots. Their content was about ten times higher in P. veris in comparison with P. elatior underground organs. The obtained results clearly show that both Primula species differ distinctly in terms of the content and composition of phenolic compounds. The compounds differentiating both species to the highest degree (hyperoside, in flowers, as well as primverin and primulaverin, in the roots) may be useful chemical markers in the identification and evaluation of both species.

Full text

Research Article
Phenolics in Primula veris L. and P. elatior (L.)
Hill Raw Materials
Katarzyna Bdczek, JarosBaw L. PrzybyB,MaBgorzata Mirgos, Olga Kosakowska,
Izabela Szymborska-Sandhu, and Zenon Wwglarz
Laboratory of New Herbal Products, Department of Vegetable and Medicinal Plants, Warsaw University of Life Sciences-SGGW,
Nowoursynowska 166, 02-787 Warsaw, Poland
Correspondence should be addressed to Katarzyna Bączek; katarzyna baczek@sggw.pl
Received 3 March 2017; Revised 7 June 2017; Accepted 27 June 2017; Published 1 August 2017
Academic Editor: Ravi Ramasamy
Copyright ©  Katarzyna Bączek et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Primula veris L. and Primula elatior (L.) Hill represent medicinal plants used for the production of herbal teas and preparations with
antioxidant and expectorant activity. Flowers and roots of both species possess the same biological activity. In the presented study,
raw materials of wild growing P. v e r i s and P. e l a t i o r were compared in terms of the content and composition of phenolic compounds
using a fast and simple HPLC-DAD method. e study showed that owers of both species were rich in avonoids. However, P. v e r i s
owers were characterized with a distinctly higher content of isorhamnetin--O-glucoside, astragalin, and (+)-catechin, whereas P.
elatior occurred to be a richer source of rutoside and isorhamnetin--O-rutinoside. Hyperoside was found exclusively in P. e l a t i o r
owers. Phenolic glycosides (primverin and primulaverin) were identied only in the roots. eir content was about ten times
higher in P. v e r i s in comparison with P. e l a t i o r underground organs. e obtained results clearly show that both Primula species
dier distinctly in terms of the content and composition of phenolic compounds. e compounds dierentiating both species to
the highest degree (hyperoside, in owers, as well as primverin and primulaverin, in the roots) may be useful chemical markers in
the identication and evaluation of both species.
1. Introduction
Cowslip (Primula veris L., syn. P. o  c i n a l i s Hill) and oxlip
(Primula elatior (L.) Hill) are small, long-lived perennials
from the family Primulaceae, growing wild in temperate
Europe and Asia []. Cowslip grows on nutrient-poor grass-
lands, herb-rich meadows, and at the edges and in clearings of
warm and bright woodlands. Oxlip prefers moist and shaded
forests,butitalsogrowsinmountainmeadows[,].Both
species produce a rosette of leaves and leaess ower stalks,
up to – cm high. Cowslip owers are fragrant, bright-
yellow with orange spots at the edge of each lobe. ey are
formed at the top of the stalks in an umbel-like inorescence.
In turn, the pale-yellow, almost scentless, owers of oxlip
areproducedonseparatestalks.Inthecentralpartof
these owers an orange ring is visible [, ]. Underground
organs consist of slightly curved, grayish-brown rhizomes
with yellowish-white (P. veris) or brown (P. elatior) roots,
commonly called roots [, ].
Both species have a long history of medicinal use. In the
current (h) edition of the European Pharmacopeia, they
arelistedasasourceofPrimula roots []. However, in the
British Herbal Pharmacopeia [] as well as in Pharmacopee
Franc¸aise [], only P. v e r i s is mentioned as a source of Primula
raw materials.
Primula veris and P. e l a t i o r have mainly been exploited for
the production of herbal teas and preparations that are also
considered dietary supplements []. ey indicate various
pharmacological activities, for example, secretolytic, expec-
torant, anti-inammatory, diuretic, antimicrobial, antifungal,
and sedative [–]. According to EMA, Primula owers and
roots are used against coughs, bronchitis, and catarrhs of the
respiratory tract and also to treat nervousness, headache, or
rheumatism [, ]. In the past, Primula leaves and owers
Hindawi
International Journal of Analytical Chemistry
Volume 2017, Article ID 2871579, 7 pages
https://doi.org/10.1155/2017/2871579

 International Journal of Analytical Chemistry
were also eaten raw or cooked as a source of vitamins
and microelements available in late winter []. Apart from
P. v e r i s and P. e l a t i o r ,otherPrimula species are described
as also revealing some medicinal potential. According to
Demir et al. [], P. v u l g a r i s demonstrates antioxidant activity.
Extracts from P. d e n t i c u l a t a show cytostatic properties, while
P. m a c r o p h y l l a shows antifungal ones [–].
e main active compounds of Primula owers and
roots are triterpene saponins as well as phenolic compounds,
including avonoids (about % in owers), phenolic acids,
and phenolic glycosides [, ]. Saponins are responsible
for secretolytic and expectorant activity. In turn, phenolic
compounds, present especially in Primula owers, reveal
antioxidant, antimicrobial, and cytostatic properties [, ].
Phenolic compounds can be easily separated on a C
reversed-phase (RP) column and detected using a UV or
diode array detector (DAD) [–]. All these substances
contain at least one aromatic ring and thus eciently absorb
UV light. So, the UV spectra obtained by the DAD are a
valuableindicatorinscreeningandpreliminaryqualitative
analyses of the dierent groups of phenolics. For better
structure elucidation of metabolites and/or unambiguous
identication of target compounds, liquid chromatography
mass spectrometry (LC-MS) techniques or even nuclear
magnetic resonance (NMR) detection are used [–].
BasedonEuropeanMedicinesAgency(EMA)andEuro-
pean Pharmacopeia monographs, Primula preparations are
produced exclusively out of P. v e r i s and P. e l a t i o r raw materi-
als, which are considered to possess the same values [, , ].
Due to the developmental and morphological similarities
between both species, they are hard to dierentiate on natural
sites, and aer the drying process the raw materials collected
from them are indistinguishable. Despite the above regu-
lations, some authors report dierences between these two
species in terms of their chemical composition [, , ]. Other
herbal raw materials, even those described in pharmacopeias,
are very oen provided by two or even three plant species. For
example, the lime ower is collected from Tilia platyphyllos
Scop., Tilia cordata Mill, and their hybrid Tilia ×vulgaris [].
Numerous studies conrm that the quality of such materials
may be highly diversied, which is undesirable from an
industrial point of view [–].
e aim of our study was to compare wild growing P.
veris and P. e l a t i o r in terms of the accumulation of phenolic
compounds as chemical markers for species identication
and more accurate assessment of raw materials, in the context
of their potential usage, by a simple, but reliable, analytical
method on a standard HPLC-DAD system.
2. Materials and Methods
2.1. Plant Material. Flowers and roots of P. v e r i s and P.
elatior werecollectedintheeasternpartofPolandfrom
eightwildgrowingpopulationsofeachspecies.eplant
material from one population was used as one replication for
chemical evaluation. Flowers were collected at the stage of full
owering (in May, Figures  and ) from randomly chosen
plants (about  g of fresh owers per population). Roots
F : Cowslip (Primula veris).
F : Oxlip (Primula elatior).
were harvested from the same populations in September, aer
seed setting (about  g of fresh roots per population). ey
were washed and cut into pieces. Both sets of raw materials
were dried at ∘C. Voucher specimens were taken from each
population. ese are stored in the Department of Vegetable
and Medicinal Plants, WULS-SGGW.
2.2. HPLC Analysis. Air-dry, nely powdered, and homog-
enized raw material (. g) was extracted with  ml of
methanol (Sigma-Aldrich, Pozna´
n, Poland, reagent grade) in
aB
¨
uchi Labortechnik AG B- Extraction System. Soxhlet
hot extraction was used with twenty-ve extraction cycles,
ushing and drying. Aer evaporation of solvents, the residue
was dissolved in  ml of methanol. e obtained extracts
were ltered with a Supelco Iso-DiskSyringe Tip Filter
Unit, a PTFE membrane, diameter  mm, pore size . 𝜇m
and injected in triplicate. Separation was achieved using
a modern C reversed-phase, Kinetex. 𝜇m,  mm
×. mm column with a porous outer layer on solid
silica core particles (Phenomenex, USA). e analyses were

International Journal of Analytical Chemistry 
T : Validation parameters of the HPLC-DAD analysis (𝑛=6).
Compound Purity
(%)
Precision
intraday
(CV, %)
Calibration equation Linearity
(𝑟2)
Linear range
(mg/mL)
LOD
(𝜇g/L)
LOQ
(𝜇g/L)
/+/-Catechin , . 𝑦 = 8216.4𝑥 −6069.3 . .–. . .
Luteolin -C-glucoside (orientin) , . 𝑦 = 2407.3𝑥 −2358.1 . .–. . .
Quercetin -O-rutinoside (rutoside) , . 𝑦 = 1434.0𝑥 − 5093.0 . .–. . .
Quercetin -O-galactoside (hyperoside) , . 𝑦 = 3435.5𝑥 −6882.2 . .–. . .
Isorhamnetin--O-rutinoside , . 𝑦 = 2096.1𝑥 − 904.8 . .–. . .
Isorhamnetin--O-glucoside , . 𝑦 = 1940.0𝑥 −897.4 . .–. . .
Kaempferol -O-glucoside (astragalin) , . 𝑦 = 2104.5𝑥− 2426.3 . .–. . .
-O-Caeoylquinic acid (chlorogenic acid) , . 𝑦 = 6517.4𝑥 −12016.6 . .–. . .
Primverin , . 𝑦 = 12488.0𝑥− 3594.7 . .–. . .
Primulaverin , . 𝑦 = 2785.4𝑥 −5313.2 . .–. . .
performed on a Shimadzu chromatograph equipped with an
SIL-A autosampler, an SPD-MA VP PDA photodiode
array detector, and CLASS VP. chromatography soware
(Shimadzu, Kyoto, Japan). e content of the determined
compounds was calculated in mg per  g of dry weight
(DW).
e analysis of ower extracts was carried out using a
binary gradient of deionized water adjusted to pH  with
phosphoric acid (Sigma-Aldrich, Pozna´
n, Poland, reagent
grade) (mobile phase A) and ACN (Sigma-Aldrich, Pozna´
n,
Poland, gradient grade) (mobile phase B) as follows: . min,
.% B; . min, % B; . min, % B; . min, .% B;
 min, stop. e ow rate was . ml/min, oven temperature
∘C and injection volume  𝜇l. Data were recorded at
wavelength of  nm for (+)-catechin,  nm for luteolin
-C-glucoside (orientin), quercetin -O-rutinoside (ruto-
side), quercetin -O-galactoside (hyperoside), isorhamnetin-
-O-rutinoside, isorhamnetin--O-glucoside,  nm for
kaempferol -O-glucoside (astragalin), and  nm for -O-
caeoylquinic acid (chlorogenic acid).
For separation of root extract compounds, a binary
gradient of deionized water adjusted to pH  with phosphoric
acid (Sigma-Aldrich, Pozna´
n, Poland, reagent grade) (mobile
phase A) and ACN (Sigma-Aldrich, Pozna´
n, Poland, gradient
grade) (mobile phase B) was used as follows: . min, % B;
. min, % B; . min, % B; . min, % B; . min,
% B,  min, stop. e ow rate was . ml/min, oven
temperature ∘C, and injection volume  𝜇l. Compounds
were monitored at wavelength of  nm for primverin and
 nm for primulaverin.
Peak identication was conrmed by comparison of re-
tention time and UV spectra with adequate parameters of
standards. Commercially available standards of the investi-
gated compounds (ChromaDex, Irvine, USA) were
separately dissolved with methanol (Sigma-Aldrich, Pozna´
n,
Poland) in a  ml volumetric ask according to the Chro-
maDex’s Tech Tip : Reference Standard Recovery and
Dilutionandthenusedasstandardstocksolutions(https://
ww w.chromadex.com/media//techtip-recoverydilu-
tionprocedures nl pw.pdf). Working standard solutions were
prepared by dilution of , , , , , or  𝜇lstock
solutions of each compound with methanol in  ml
volumetric asks. e working solutions were injected
(. 𝜇l)onacolumninsixreplicates(𝑛=6)usingSIL-A
autosampler (Shimadzu, Kyoto, Japan) to generate a six-point
calibrationcurve.Standardcurveparameterswerecalculated
using Microso Excel  (Table ). e signal-to-noise (S/N)
ratio approach was used to determine LOD (S/N of : ) and
LOQ (S/N of  : ).
2.3. Statistical Analysis. Data were subjected to statistical
analysis using Statgraphics Plus for Windows v. . soware.
e mean values were compared using one-way analysis of
variance (ANOVA) and expressed as means with standard
deviation (SD) and coecients of variation (CV%). e
dierences between individual means were considered to be
signicant at 𝑝 < 0.01.
3. Results and Discussion
According to Wichtl [], the total content of avonoids in
Primula owers is up to about %. To date in P. v e r i s extracts,
quercetin, quercetin--O-rutinoside, quercetin--O-gentio-
bioside, quercetin-trihexoside, kaempferol, kaempferol--O-
diglucoside--O-glucoside, kaempferol--rutinoside, kaemp-
ferol--O-galactoside-rhamnoside--O-rhamnoside, luteolin,
isorhamnetin, isorhamnetin--O-glucoside, isorhamnetin-
-O-rutinoside, limocitrin--O-glucoside, limocitrin--O-
rutinoside, apigenin, catechin, epicatechin, and epigallocat-
echin, as well as some methoxylated avones, have been
identied using LC-MS and HPLC techniques [–, , ].
Data on the composition of P. e l a t i o r owers are much more
scarce. ese indicate the presence of rutoside, kaempferol-
-rutinoside, and isorhamnetin--glucoside [].
e above authors report only the chemical composition
of avonoids isolated from both Primula owers. ere
is little information on the content of those substances.
According to Wichtl [], owers of P. e l a t i o r are characterized
by a higher content of rutoside (.%) than the owers
of P. v e r i s (.%). Our results conrm the presence of six

 International Journal of Analytical Chemistry
T : e content of identied phenolic compounds in P. v e r i s and P. e l a t i o r owers (mg/ g DW).
Identied phenolic compounds P. v e r i s P. e l a t i o r
Mean ±sd CV (%) Mean ±sd CV (%)
(+)-Catechin . ±.1∗∗ . . ±. .
Orientin . ±. ns . . ±. .
Rutoside . ±. . . ±.1∗∗ .
Hyperoside nd . ±. .
Isorhamnetin--O-rutinoside . ±. . . ±.6∗∗ .
Isorhamnetin--O-glucoside . ±.9∗∗ . . ±. .
Astragalin . ±.9∗∗ . . ±. .
Chlorogenic acid . ±. ns . . ±. .
Values are the mean ±standard deviation (𝑛=8); ∗∗𝑝<0.01, ns: insignicant dierence, nd: not detected, and CV: coecient of variation.
Orientin
Rutoside
Astragalin
(+)-Catechin
Isorhamnetin-3-O-rutinoside
Isorhamnetin-3-O-glucoside
Chlorogenic acid
Spectrum max plot
123456789100
(Minutes)
0
100
200
300
400
500
600
(mAu)
0
100
200
300
400
500
600
(mAu)
F : HPLC chromatogram of methanolic extract of the owers
of Primula veris.
avonoid compounds in the owers of both species, namely,
orientin (luteolin--C-glucoside), rutoside (quercetin -
O-rutinoside), isorhamnetin--O-rutinoside, isorhamnetin-
-O-glucoside, astragalin (kaempferol--O-glucoside), and
(+)-catechin. e contents of isorhamnetin--O-glucoside,
astragalin, and (+)-catechin were distinctly higher in the
owers of P. v e r i s , that is, ., ., and . mg/ g
DW,respectively.Inturn,rutosideandisorhamnetin--O-
rutinoside were detected in higher amounts in P. e l a t i o r
(. and . mg/ g DW, resp.). A clear dierence
between both species concerned the presence of hyperoside
(quercetin -O-galactoside), which was only identied in
P. e l a t i o r owers (. mg/ g DW) (Table , Figures 
and ). Among the analyzed substances, the content of this
compound was also the most diversied (CV .%).
According to Kim et al. [], hyperoside indicates anti-
inammatory and antioxidant activities. Results obtained by
Wuetal.[]showthatthiscompoundrevealsantiviral
activity, while Kohlm˝
unzer [] also mentions diuretic and
hypotensiveeects.Inturn,rutosideisknownforitsstrong
antioxidant potential as well as antimicrobial and anti-
inammatory activities []. us, this may explain the appli-
cation of owers of both Primula species in the treatment
of coughs and respiratory tract diseases. e results of this
study show that both hyperoside and rutoside dierentiated
the investigated species to a considerable degree. erefore,
owers of P. e l a t i o r , which are rich in hyperoside and
Orientin
Rutoside
Hyperoside
Astragalin
(+)-Catechin
Isorhamnetin-3-O-rutinoside
Isorhamnetin-3-O-glucoside
Chlorogenic acid
Spectrum max plot
123456789100
(Minutes)
0
100
200
300
400
500
600
(mAu)
0
100
200
300
400
500
600
(mAu)
F : HPLC chromatogram of methanolic extract of the owers
of Primula elatior.
characterized by a higher content of rutoside in comparison
to P. v e r i s , may indicate stronger pharmacological activity.
Unlike in the data presented by Wichtl [], both species
contained isorhamnetin--glucoside in their owers (Table ,
Figures  and ). Similar to hyperoside, the diversity of
the content of both isorhamnetin derivatives was very high.
However, the content of isorhamnetin--O-rutinoside was
diversied to a higher degree for P. v e r i s (CV .%), while
for isorhamnetin--O-glucoside this was seen in P. e l a t i o r
(CV .%). According to Teng et al. [], isorhamnetin
aglycon reveals cytotoxic activity toward human hepatocellu-
lar carcinoma cells. In our study, the presence of one phenolic
acid (chlorogenic acid) in both Primula owers was also
conrmed, and its content was similar in P. v e r i s and P. e l a t i o r
(. and . mg/ g DW, resp.).
Primverin and primulaverin (phenolic glycosides) are
typical compounds of P. v e r i s and P. e l a t i o r underground
organs.epresenceofthesesubstancesinPrimula roots had
been previously conrmed by M¨
ulleretal.[].Accordingto
EMA [], their content in both species is very diversied and
maybeashighas.%.eyareresponsibleforthespecic
odor of the raw material, which appears during the drying
process []. In our study, the content of both compounds
was ten times higher in P. v e r i s (. and . mg/ g
DW, resp.) than in P. e l a t i o r (. and . mg/ g DW,
resp.) roots (Table , Figures  and ). Such a relationship had
previously been reported only for primverin []. In addition,

International Journal of Analytical Chemistry 
T : e content of identied phenolic compounds in P. v e r i s and P. e l a t i o r roots (mg/  g DW).
Identied phenolic compounds P. v e r i s P. e l a ti o r
Mean ±sd CV (%) Mean ±sd CV (%)
Primverin . ±.2∗∗ . . ±. .
Primulaverin . ±.2∗∗ . . ±. .
Values are the mean ±standard deviation (𝑛=8); ∗∗𝑝<0.01, CV: coecient of variation.
Primverin
Primulaverin
0,25
0,50
0,75
1,00
1,25
1,50
1,75
2,00
2,25
2,50
2,75
3,00
3,25
3,50
3,75
4,00
4,25
4,50
4,75
5,00
0,00
(Minutes)
0
20
40
60
80
100
120
140
160
180
200
(mAu)
0
20
40
60
80
100
120
140
160
180
200
(mAu)
002 v
Detector 1–254 nm
F : HPLC chromatogram of methanolic extract of the roots
of Primula veris.
Primverin
Primulaverin
0,25
0,50
0,75
1,00
1,25
1,50
1,75
2,00
2,25
2,50
2,75
3,00
3,25
3,50
3,75
4,00
4,25
4,50
4,75
5,00
0,00
(Minutes)
0
20
40
60
80
100
120
140
160
180
200
(mAu)
0
20
40
60
80
100
120
140
160
180
200
(mAu)
Detector 1–254 nm
001 K
F : HPLC chromatogram of methanolic extract of the roots
of Primula elatior.
the results of our study conrm that the content of primverin
was much less diversied in both species than the content of
primulaverin (Table ).
According to our results, the use of a column with
porous outer layer on solid silica core particles signicantly
reduces the analysis time and mobile phase consumption
in comparison to existing methods [, ]. As a result, the
analysis of phenolics in Primula raw materials can be eected
faster and at lower cost and can be performed on standard
(older) chromatographic systems.
e data concerning the chemical prole of other Primula
species are fragmentary. Hashimoto et al. [] identied
three avonol glycosides in P. s i e b o l d i i owers and leaves,
that is, quercetin and kaempferol derivatives, as well as two
anthocyanins, that is, malvidin and petunidin glycosides,
which were detected only in the owers. In turn, Ozkan et
al. [] used HPLC to assess the content of catechin, rutin,
and some phenolic acids, namely, gallic, protocatechuic, p-
OH benzoic, vanillic, and p-coumaric acids in P. v u l g a r i s
owers. According to this analysis, rutin and p-coumaric
acidseemedtobethemainphenoliccompoundofthisraw
material. In other Primula species, that is, P. d e n t i c u l a t a ,P.
auricular,P. h a l l e r i ,P. m a l a c o i d e s ,andP. marginata,primetin
(,-dihydroxyavone), which is responsible for strong sen-
sitizing properties, was also detected []. Depending on
the chemical composition and content of biologically active
compounds, dierent plant species of the genus have been
used for various medicinal purposes, such as food poisoning,
indigestion, dysentery, and ulcers as well as coughs or
bronchitis, which are typical ailments treated with P. v e r i s and
P. e l a t i o r extracts.
4. Conclusions
Our results show distinct dierences in terms of the content
and composition of phenolic compounds identied in P. v e r i s
and P. e l a t i o r raw materials. Primula elatior owers seem to be
an interesting source of avonoids. ey are rich in rutoside
and hyperoside, which reveal numerous pharmacological
activities, that is, anti-inammatory, antioxidant, and antimi-
crobial. us, they can be considered more interesting for the
herbal medicine industry than P. v e r i s .Inturn,hyperoside
was only found in the owers of P. e l a t i o r ,whichmaybe
used in the identication of Primula species. Flowers of
neither species contained primverin or primulaverin. ese
substances were only identied in the roots. Primula veris was
characterized by a ten times higher content of both phenolic
glycosides in comparison with P. e l a t i o r .
Phenolic compounds identied in our study, especially
hyperoside, primverin, and primulaverin, may be applied as
chemical markers in the identication of Primula species as
well as quality markers for their raw materials sourced from
both natural sites and cultivated ones. e proposed analyt-
ical methods for the determination of these compounds in
plant material are fast and reliable and can be performed on
every standard HPLC system.

 International Journal of Analytical Chemistry
Conflicts of Interest
e authors declare that there are no conicts of interest
regarding the publication of this paper.
Acknowledgments
e studies were supported by the Polish Ministry of Agri-
culture and Rural Development, within the Multiannual
Programme “Creating the Scientic Basis of the Biological
Progress and Conservation of Plant Genetic Resources as a
Source of Innovation to Support Sustainable Agriculture and
Food Security of the Country –.”
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