DEVELOPMENT OF STANDARDIZATION METHODOLOGY OF ELECAMPANE RHIZOMES AND ROOTS (INULA HELENIUM L.) FOR THE HYDROXYCINNAMIC ACIDS CONTENT

Abstract

The methodologies of rhizomes with roots elecampane standardization were developed through the identification and quantification of hydroxycinnamic acids. To identify the raw materials it was proposed to establish the presence of four hydroxycinnamic acids zones, including chlorogenic and chicoric in TLC profi le. The results of hydroxycinnamic acids content determination for industrial and wild samples of raw materials are in the range of 1.10–1.35 %. The developed methodologies allow distinguishing two types of materials – elecampane rhizome and roots and chicory roots, which have a high content of inulin and are similar in crushed condition.

Full text

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ISSN 2312-0967. Фармацевтичний часопис. 2016. № 2
Аналіз лікарських препаратів
Analysis of drugs
Рекомендована д. фармац. наук, проф. В. А. Георгіянц
UDK 615.322:582]-092.4
DOI
DEVELOPMENT OF STANDARDIZATION METHODOLOGY OF ELECAMPANE
RHIZOMES AND ROOTS (INULA HELENIUM L.) FOR THE HYDROXYCINNAMIC
ACIDS CONTENT
© L. V. Vronska1, A. Ye. Demyd1, M. A. Ezhned2
1I. Horbachevsky Ternopil State Medical University
2Bukovynian State Medical University
Summary: the methodologies of rhizomes with roots elecampane standardization were developed through the identifi cation
and quantifi cation of hydroxycinnamic acids. To identify the raw materials it was proposed to establish the presence of
four hydroxycinnamic acids zones, including chlorogenic and chicoric in TLC profi le. The results of hydroxycinnamic acids
content determination for industrial and wild samples of raw materials are in the range of 1.10–1.35 %. The developed
methodologies allow distinguishing two types of materials – elecampane rhizome and roots and chicory roots, which have
a high content of inulin and are similar in crushed condition.
Key words: Inula helenium, roots and rhizomes, hydroxycinnamic acids, chlorogenic acid, chicoric acid, standardization.
Introduction. The elecampane rhizomes and roots
are an offi cinal medicinal plant raw material (MP), which
belongs to a group of medicines that stimulate expecto-
ration with indication in the instruction for use – for up-
per respiratory tract and lungs diseases (bronchitis, tra-
cheitis, catarrh of the upper respiratory tract); diseases
of the gastrointestinal tract (gastritis, enterocolitis, lack
of appetite, poor digestion). According to the literature
about the analysis and standardization of elecampane
rhizomes and roots, this usage is associated with high
content of inulin and other fruitosans [1–7]. Numerous
studies indicate other kinds of activities of MP and its
various extracts–antidiabetic, antioxidant, bacteriostatic,
antifungal [8–11], which is associated with the content of
polyphenolic compounds, fl avonoids, hydroxycinnamic
acids. Considering insuffi cient level of elecampane rhi-
zomes and roots standardization – absence of a mono-
graph in State Pharmacopoeia of Ukraine and leading
world pharmacopoeias, it is time to revise quality criteria
for this raw material.
Therefore, the object of this study was to develop a
methodology of elecampane rhizomes and roots stan-
dardization for the hydroxycinnamic acids content.
Materials and methods. Research of the quality and
quantity content of hydroxycinnamic acids was carried
out on three industrial series of MP (JSC “Liktravy” Zhy-
tomyr, Ukraine, samples 1–3 correspond to the series
20315, 30915, 10116) and three growing wild samples
of MP (samples 4–6 , collected in Ternopil region).
The methods of thin layer chromatography (TLC) and
absorption spectrophotometry in the UV and visible re-
gion of the spectrum were used in this work. Chromato-
graphic plates Silica gel 60 F254 (“Merсk”, Germany),
chromatographic chamber “CAMAG”, an instrument for
spotting Linomat 5 (“CAMAG”, Switzerland), a lamp for
observing chromatograms in ultraviolet light “CAMAG”
were used for investigations by TLC method.
The study of the quality content of hydroxycinnamic
acids was carried out in methanol extracts of raw ma-
terials. The study was conducted using mobile phases:
formic acid – water – ethyl acetate (6: 9, 90) and anhy-
drous formic acid – water – ethyl acetate (10:10:80). The
chromatograms observation was performed in ultravio-
let light with a wave-length of 365 nm after their sequen-
tial processing with 10 g/l solution of diphenylboric acid
aminoethyl ester and 50 g/l solution of macrogol 400 in
methanol.
Standard samples of hydroxycinnamic acids: chloro-
genic acid (Fluka), caffeic acid (Fluka), rosmarinic acid
(Fluka), chicoric acid (pharmacopoeia standard sample
(SPU CRS)), ferulic acid (Sigma); fl avonoids: rutin and
hyperoside (SPU CRS) were used for identifi cation. Ex-
act masses of standard samples were dissolved in ap-
propriate volumes of methanol.
Quantitative determination of hydroxycinnamic acids
was defi ned by absorption spectrophotometry in the UV
and visible region of the spectrum, using Сarry-50 spec-
trophotometer (Varian, USA).
Reagents of cleanliness qualifi cation were used (ethyl
acetate, formic acid, water, diphenylboric acid aminoeth-
yl ester, macrogol 400) that meets the requirements of
SPU for appropriate methods of analysis, reagent solu-
tions were prepared according to SPU methodologies.
Results and discussion. On the chromatogram of
methanol extract of the elecampane roots four intensive
zones appear, the colour of fl uorescence of which in-
dicates that the corresponding to them substances be-
long to the hydroxycinnamic acids. In particular, on the

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ISSN 2312-0967. Pharmaceutical review. 2016. № 2
Аналіз лікарських препаратів
Analysis of drugs
ly active substances composition of elecampane raw
materials, has been done. In chicory roots chlorogenic
and chicoric acids in signifi cant quantities, caffeic acid
are identifi ed. Between chlorogenic and chicoric acids
zones there is a zone with weak fl uorescence. On the
chromatogram of the test solution for all elecampane
samples in this area there are two intense zones that are
located nearby. Thus, the proposed chromatographic
conditions to identify elecampane raw materials allow to
establish its identity and distinguish it from chicory roots.
According to the authors [10] in ethyl acetate extract
of elecampane roots chlorogenic, caffeic, dicaffeoylqui-
nic, 3-feruloyl-4-caffeoyl-quinic acids were identifi ed,
and their content was relatively high. According to the
Polish authors [11] in 70 % methanol-water extract of
elecampane callus culture chlorogenic and neochlo-
rogenic acids (0.1%), 1.5– and 3.5-di-O-caffeoylquinic
acids (0.3 %) were identifi ed; in total, sixteen acid deriv-
atives of caffeic acid, including four dicaffeoyl aldarates
(0.085 %), three tricaffeoyl aldarates (0.608 %) and tetra-
caffeoyl aldarate (0.077 %) were identifi ed. The results
of chlorogenic acid identifi cation in the studied samples
of raw materials correlate with data [10, 11]. The com-
chromatogram of the test solution a very intense zone
with blue fl uorescence and intense with greenish-blue
fl uorescence appear that are at chlorogenic and chicoric
acids levels respectively on the chromatogram of refer-
ence solution. The two other zones with intense green-
ish-blue fl uorescence are detected in the chromatogram
of test solutions between chlorogenic and chicoric acids
zones. Weak zones of blue fl uorescence in the bottom
of the chromatogram, located below the zone of chlo-
rogenic acid and near the starting line were observed
additionally besides the already described.
The samples of raw materials have been generally
similar in the composition of hydroxycinnamic acids –
the number, position, colour and relative intensity of fl uo-
rescence (within one sample) of hydroxycinnamic acids
zones are similar for all samples, in particular, for some
elecampane samples, and are presented in Figure 1.
The chromatogram of the fi fth sample of raw materials
was slightly different and contained an additional intense
zone of blue fl uorescence at the bottom of the chromato-
gram.
Additionally, the chromatography of methanol extract
of chicory roots, which are the similar to the biological-
1 2 3456789
Fig. 1. The chromatogram in conditions of hydroxycinnamic acids identifi cation, obtained after sequential processing by
solutions of aminoethyl ester of diphenylboric acid and macrogol 400, when viewing in UV light at 365 nm wavelength.
Tracks: 1–4 – test solutions for elecampane samples 1, 2, 5 and 6; 5 – reference solution (rutin, chlorogenic acid,
hyperoside, caffeic acid – a bottom-up); 6 – reference solution of rosmarinic acid; 7 – reference solution of ferulic acid;
8 – reference solution of chicoric acid; 9 – test solution of chicory roots.

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Аналіз лікарських препаратів
Analysis of drugs
position in local elecampane samples of chicoric acid
(dicaffeoyltartaric acid) correlates with the authors’ data
[11] as for the presence of dicaffeoyl aldarates.
Thus, based on the results of chromatographic re-
searches, taking into account the literature data on the
hydroxycinnamic acids composition [1, 5, 10, 11] and
biological activity of aqueous-alcoholic extracts of this
MP [8] and hydroxycinnamic acids, such as [12–15] it
was proposed to identify these substances during the
standardization of rhizomes with roots elecampane raw
materials.
The methodology of hydroxycinnamic acids iden-
tifi cation in elecampane rhizomes with roots.
Test solution. Place 1.0 g of the powdered raw ma-
terial into a 50 ml fl ask and add 25 ml of methanol. The
solution is being refl uxed on a water-bath for 45 min-
utes. Then it is cooled and fi ltered.
Reference solution. 2.5 mg of the standard samples
of chlorogenic, chicoric and caffeic acids is dissolved in
10 ml of methanol.
Plate: TLC silica gel plate.
Mobile phase: formic acid – water – ethyl acetate
(10:10:80 V/V).
Application: 30 μL of the test solution and 5 μL of the
reference solution, as 10 mm bands.
Drying: in air.
Distance that a mobile phase should pass: 12 cm.
Drying: at the temperature of 100–105° C for 10 min-
utes in the drying cabinet.
Detection: the hot plate is sprayed with a 10 g/l di-
phenylboric acid aminoethyl ester methanol solution,
dried in air and then sprayed by 50 g/l solution of mac-
rogol 400 in methanol; then it is dried in air for about
30 min and examined in ultraviolet light at 365 nm of
wavelength.
Results: on the reference solution chromatogram
a fl uorescent blue zone corresponding to chlorogenic
acid, a greenish-blue fl uorescent zone of chicoric acid
and a blue fl uorescent zone corresponding caffeic acid
should be identifi ed (in order of retardation factor (Rf)
increasing).
On the test solution chromatogram two fl uorescent
zones should be detected - at chlorogenic (most in-
tense) and chicoric acid levels on the reference solution
chromatogram, corresponding them by color and fl uo-
rescence as well as two intense zones of greenish-blue
fl uorescence between chlorogenic and chicoric acids
zones. See below the sequence of zones, present in
chromatograms obtained for the reference solution and
the test solution (fi g. 2). Furthermore, others weak zones
may be present in the chromatogram of the test solution.
In electronic absorption spectra of water-alcohol ex-
tracts of different series elecampane rhizomes and roots
the characteristic for hydroxycinnamic acids absorbance
curve with maximum at a wavelength of 325 ± 2 nm was
observed. In particular, the electronic absorption spectra
of 1, 2 and 4 elecampane samples in conditions of the
hydroxycinnamic acids study are presented in Figure 3.
Fig. 2. TLC scheme of chromatogram in conditions of elecampane hydroxycinnamic acids identifi cation.

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Analysis of drugs
It should be mentioned that the electronic absorption
spectrum appearance for the elecampane rhizomes
and roots (Fig. 3) and chicory roots (Fig. 4) are different.
This fact may be another additional identifi cation marker
when establishing identity of the studied raw materials.
The nature of the spectrum, in particular the presence
of clear absorption maximum at 325 nm wavelength,
gives for hydroxycinnamic acid determination by direct
measurement of absorption in the maximum followed by
calculation of content by using specifi c absorption index.
Chlorogenic acid was chosen as the standard for calcu-
lating the amount of hydroxycinnamic acids content, de-
spite the fact that it is present in elecampane rhizomes
with roots both according to the literature data and to the
results of the chromatographic research of industrial and
growing wild raw samples.
In developing the methodology of quantitative deter-
mination of the hydroxycinnamic acids amount it was
found that in order to ensure the fullness of their ex-
traction ethanol should be used as extractant (50 % V
/ V).
The methodology of quantitative determination of
hydroxycinnamic acids amount in elecampane rhi-
zomes with roots.
Stock solution. To 1 g (exact sample) of the pow-
dered sample 60 ml of ethanol (50 % V / V) is added.
The solution is being refl uxed on a water–bath for 45
min. It is cooled and fi ltered into a 100 mL volumetric
fl ask. The extraction is repeated twice, using 20 and 15
ml of ethanol (50 % V / V) R, refl uxing on a water bath
for 15 minutes each time. It is cooled and fi ltered into the
same volumetric fl ask. The fi lter and the fl ask are rinsed
with ethanol (50 % V/V), combining the fi ltrate and the
rinsing solutions and making the volume of the solution
up to 100.0 ml with ethanol (50 % V/V).
Test solution. 2.0 ml of stock solution is made up to
25.0 ml with ethanol (50 % V/V).
Сompensation solution. Ethanol (50 % V/V).
The optical density of the test solution at 325 nm is
measured relatively to the compensation solution.
The hydroxycinnamic acids amount (X, %) expressed
as a chlorogenic acid, is calculated according to the for-
mula:
W)(100mE2
100100A25
X

,
where А – absorbance of the test solution;
m – mass of sample in g;
W – content of wet, in %;
Е – specifi c absorption index of chlorogenic acid at
325 nm (Е = 531).
The investigated samples of elecampane rhizomes
with roots were analyzed with the help of this methodol-
ogy, the results are presented in the table.
As can be seen from these results, the investigated
samples contain the hydroxycinnamic acid in an amount
of slightly more than one percent. These data are gen-
erally lower than reported by the authors [6] – 2 %. The
Fig. 3. The electronic absorption spectra of the test
solution of elecampane rhizomes and roots extracts
(curves 1-3 correspond to 1, 2 and 4 samples of raw
materials, ethanol (50% V/V)).
Fig. 4. The electronic absorption spectrum of the chicory
roots extracts (ethanol (50 % V / V)).

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Аналіз лікарських препаратів
Analysis of drugs
difference in the content can be explained from different
perspectives - the results of hydroxycinnamic acids deter-
mination of wild raw growing in central regions of Ukraine
are shown in the work [6], but the extractant used, the
conditions of sample preparation and method of fi nal de-
termination are not listed. The total content of hydroxycin-
namic acids defi ned in local raw material is comparable
to the content of hydroxycinnamic acids and their deriva-
tives, obtained in elecampane callus culture [11].
Conclusions. As a result of the development of stan-
dardisation methodology of elecampane rhizomes and
roots additional indicators of their quality – the identifi -
cation and quantifi cation of hydroxycinnamic acids are
proposed.
1. It was proposed to identify the investigated raw
material comparing TLC profi les of elecampane meth-
anol extract and standard substances solution. Chloro-
genic and chicoric acids were selected as identifi cation
markers. In the test solution chromatogram two intense
Sample Content of hydroxycinnamic acids (%), expressed as a chlorogenic
acid and dry raw material
Series 20315, JSC "Liktravy" 1.10 ± 0.02
Series 30915, JSC "Liktravy" 1.23 ± 0.03
Series 10116, JSC "Liktravy" 1.30 ± 0.02
Growing wild, Ternopil region, Zbarazh 1.24 ± 0.03
Growing wild, Ternopil region, Berezhany 1.35 ± 0.02
Growing wild, Ternopil region, Podvolochisk 1.20 ± 0.02
Table. The results of determination of the hydroxycinnamic acids amount in elecampane rhizomes with roots
zones of greenish-blue fl uorescence should be detected
between chlorogenic and chicoric acids.
2. The spectrophotometric methodology of hydroxy-
cinnamic acids quantifi cation, calculated into a chloro-
genic acid, was developed. The content of hydroxycin-
namic acids for industrial raw materials and wild samples
is in the range of 1.10–1.35 %. To establish the quality
criterion of the hydroxycinnamic acids content results of
more samples research for elecampane samples from
different areas and different year of growth for taking
into account possible effects of environmental factors on
their content are needed.
3. The application of the proposed methods of identifi -
cation and quantifi cation of hydroxycinnamic acids allow
to distinguish, by comparison of TLC profi les and elec-
tronic absorption spectra, two raw materials that are rich
in inulin, identifi ed by its presence, and in the crushed
state are similar - rhizomes with roots elecampane and
roots of chicory.
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РОЗРОБКА МЕТОДИКИ СТАНДАРТИЗАЦІЇ КОРЕНЕВИЩ І КОРЕНІВ ОМАНУ (INULA HELENIUM L.)
ЗА ВМІСТОМ ГІДРОКСИКОРИЧНИХ КИСЛОТ
Л. В. Вронська1, А. Є. Демид1, М. А. Ежнед2
ДВНЗ «Тернопільський державний медичний університет імені І. Я. Горбачевського МОЗ України» 1
ВДНЗ «Буковинський державний медичний університет» 2
Резюме: розроблено методики стандартизації кореневищ з коренями оману високого шляхом ідентифікації і
кількісного визначення гідроксикоричних кислот. Для ідентифікації сировини було запропоновано встановлювати
наявність чотирьох зон гідроксикоричних кислот, серед яких хлорогенова і цикорієва, у ТШХ-профілі. Результати
визначення вмісту гідроксикоричних кислот для промислових і дикорослих зразків сировини знаходяться в інтервалі
1,10–1,35 %. Розроблені методики дозволяють розрізняти два види сировини, які характеризуються високим вмістом
інуліну й є схожими у подрібненому стані – кореневища і корені оману високого і корені цикорію.
Ключові слова: Inula helenium, корені і кореневища, гідроксикоричні кислоти, хлорогенова кислота, цикорієва
кислота, стандартизація.
РАЗРАБОТКА МЕТОДИКИ СТАНДАРТИЗАЦИИ КОРНЕВИЩ И КОРНЕЙ ДЕВЯСИЛА (INULA
HELENIUM L.) ПО СОДЕРЖАНИЮ ГИДРОКСИКОРИЧНЫХ КИСЛОТ
Л. В. Вронска1, А. Е. Демид1, М. А. Эжнед2
ГВУЗ «Тернопольский государственный медицинский университет имени И. Я. Горбачевского
МЗ Украины»1
ВГУЗ «Буковинский государственный медицинский университет»2
Резюме: разработаны методики стандартизации корневищ с корнями девясила высокого путем идентификации
и количественного определения гидроксикоричных кислот. Для идентификации сырья было предложено
устанавливать наличие четырех зон гидроксикоричных кислот, среди которых хлорогеновая и цикориевая, в ТСХ-
профиле. Результаты определения содержания гидроксикоричных кислот для промышленных и дикорастущих
образцов сырья находятся в интервале 1,10–1,35 %. Разработанные методики позволяют различать два вида
сырья, которые характеризуются высоким содержанием инулина и похожи в измельченном состоянии – корневища
и корни девясила высокого и корни цикория.
Ключевые слова: inula helenium, корни и корневища, гидроксикоричные кислоты, хлорогеновая кислота,
цикориевая кислота, стандартизация.
Отримано 27.04.2016