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Trop J Nat Prod Res, October 2023; 7(10):4242-4248 ISSN 2616-0684 (Print)
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Tropical Journal of Natural Product Research
Available online at https://www.tjnpr.org
Original Research Article
Polyphenolic Compounds, Triterpenes, Carlina Oxide, Antioxidant Activity and
Carbohydrate Profile of Different Vegetal Parts of Carlina vulgaris L., Carlina
acanthifolia All. and Carlina corymbosa L.
Emine N. Saralieva1, Nadezhda Tr. Petkova1*, Ivan G. Ivanov1, Ina Y. Aneva2, Vasil G. Georgiev3, Krastena T. Nikolova4
1Department of Organic Chemistry and Inorganic Chemistry, University of Food Technologies, Plovdiv, Bulgaria
2Institute of Biodiversity and Ecosystem Research Bulgarian Academy of Sciences, Sofia, Bulgaria
3Stephan Angelov Institute of Microbiology, Bulgarian Academy of Sciences. Sofia, Bulgaria
4Department of Physics and Biophysics, Medical University—Varna, 9000 Varna, Bulgaria
Introduction
Carlina L. genus belongs to the Compositae family, to the
tribe Cardueae, subtribe Carlininae. The plants of the Carlina genus are
also known as carline thistles. These species are widely spread across
the Canary Islands and the Mediterranean throughout central Siberia
and northwestern China.1-3 The genus Carlina L. comprises nearly 50
plant species from Europe and West Africa, but scientists reported
observing the highest diversity in the Mediterranean region4. In
Bulgaria, Carlina vulgaris L., Carlina acanthifolia All., Carlina
corymbosa L. and Carlina lanata L. have a widespread distribution. 5-7
Common Carline thistle (Carlina vulgaris L.) is a biennual thistle and
grows well on limestone or calcareous sand. It is natively distributed in
Western, Central and Eastern Europe, and has been introduced to North
America and New Zealand.8 In Bulgaria, it is distributed in all floristic
regions; from 0 to 1500 meters above sea level.5,6 The flowering period
is mainly during the second part of the year between late June and early
August,5-8 whereas seeds germinate from April to June.8
*Corresponding author. E mail: petkovanadejda@abv.bg
Tel: +359888840789
Citation: Saralieva EN, Petkova NT, Ivanov IG, Aneva IY, Georgiev VG,
Nikolova KT. Polyphenolic Compounds, Triterpenes, Carlina Oxide,
Antioxidant Activity and Carbohydrate Profile of Different Vegetal Parts of
Carlina vulgaris L., Carlina acanthifolia All. and Carlina corymbosa L.
Trop J Nat Prod Res. 2023; 7(10):4242-4248.
http://www.doi.org/10.26538/tjnpr/v7i10.18.
Official Journal of Natural Product Research Group, Faculty of Pharmacy,
University of Benin, Benin City, Nigeria
Carlina acanthifolia All. is an annual/biennial herbaceous plant that
grows to a height of 10-50 cm. It is a geographically widespread species
across Bulgaria. It can be found from 0 to 1500 meters above sea level
in dry, sandy, and rocky places, slopes, grassy places, and mountain
pastures. The root is fleshy, with a pleasant smell and it usually reaches
a length of 50 cm and 1 m. The plant does not have an aboveground
stem. The leaves and bracts of the basket are spiny. The flower basket
is very large (up to 12 cm in diameter). The flowers are regular,
bisexual, with tubular corollas. It is used as a medicinal and tanning
plant.5,6,9
Clustered carline thistle (Carlina corymbosa) is found in dry and poor
habitats of the Mediterranean region and it is present in Albania,
Bulgaria, Serbia, Turkey, and Balearic Islands.10 The plant can reach a
height of 10–70 cm. In Bulgaria, it can be found in stony and grassy
places such as the Black Sea coast, Strum Valley, Strandzha mountain,
Rhodopes mountains (Eastern Rhodopes), Tundzha hilly plain, and
Strandzha (0 to 1000 altitude).5,6 The flowering period is between late
June and September. The outer bract of the flower heads is brown-
yellow on the adaxial surface. The leaves are deeply pinnately divided,
lobed at the top with strong spines.10
Wooly carline thistle (Carlina lanata L). is 9–30 cm in height and is
distributed in Bulgaria, especially on the Black Sea coast, Strandzha,
and Rhodopes mountain from 0- 300 –m.5,7
Nowadays, various species of Carlina genus (C. acaulis, C.
acanthifolia, C. utzka (C. acanthifolia subsp. utzka) and C. corymbosa)
are mainly used in traditional medicine of the Balkan countries,
Hungary, Spain, Italy, Poland and Lithuania, largely because of their
cholagogic, diuretic, antibiotic, and cleansing effects.11-14 It is
considered that C. acanthifolia exerts an anti-inflammatory effect on the
digestive system due to the tannin content. The leaves and stems of
Carlina curetum are also used for lowering blood glucose levels and
ART ICL E I NFO
ABSTRACT
Article history:
Received 10 August 2023
Revised 03 October 2023
Accepted 19 October 2023
Published online 01 November 2023
It is known that plants from the Carlina genus possessed many biologic activity due to the
bioactive compounds. The current study investigates the phytochemical constituents and
antioxidant potential of the different vegetal parts of Carlina vulgaris L., Carlina acanthifolia All.
and Carlina corymbosa L. The samples (roots and aerial part) were collected from Bulgaria (Golo
Bardo and Vlahina mountains). Total phenols, flavonoids, individual phenolic compounds,
triterpenes, phytosterols, carlina oxide, fructans, and individual sugars were determined.
Antioxidant potential was evaluated using four methods. The highest total phenolic content was
found in ethanol extract from C. acantifololia All. roots. Three phenolic acids (chlorogenic acid,
ferulic acid, and salicylic acid), three flavonoids (rutin, hesperidin, and quercetin), and triterpenes
(lupeol and betulin) were detected in all samples (mainly in roots). However, p-Coumaric acid
and ursolic acid were detected only in C. vulgaris, while carlina oxide was found only in C.
acantifololia All. roots. The result showed that the roots of C. acanthifolia All. were characterized
by appreciable amounts of total fructans (20 g/100 g dry weight), while inulin represented 18-12
g/100 g of dry weight. Sugars were found in all plant materials. The current study provides data
about the chemical composition of extracts obtained from three members of the Carlina genus and
their use as a source of antioxidants, phenolic compounds, carlina oxide, and inulin-type
prebiotics.
Keywords: Carlina genus, phenolic compounds, antioxidant activity, fructan, inulin, sugars
Copyright: © 2023 Saralieva et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction
in any medium, provided the original author and
source are credited.
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loosing weight. Carlina root decoction is also used in the treatment of
rashes, toothache, skin lesions, and catarrh. 15
Carlina acaulis and Carlina acantifolia are used not only as medical
but also as food plants. In Alpine regions, it is cooked and consumed as
artichoke and its heads are used to prepare liqueurs and snacks.14 In
Italy, thistle rennet or aqueous extracts of Carlina acanthifolia All. were
used for cheese-making. 16-17 A large quantity of the leaves and petals
of Carline thistle (Carlina acaulis) was consumed in Slovakia. 18. In
Bulgaria, Carlina acanthifolia All. flour was used for preparing dark
chocolate bonbons.19
Carlina acaulis is one of the most investigated species, but the fact
remains that its chemical composition has not yet been investigated in
detail. According to earlier studies, inulin (12-20 %),20,21 essential oil
(1-2 %),20,22 sugars, tannins and resinous substances, dyes21 and trace
amounts of lupeol were found in the root of C. acaulis.23 Another study
by Petkova et al.,9 reported that inulin contributed to a large part (nearly
55%) of the total fructan content (12.6 g/100 g dw) of Carlina
acanthifolia. Different flavonoids,20 phenolic acids and pentacyclic
triterpenes (lupeol, lupeol acetate, α-amyrin, β-amyrin, β-amyrin
acetate, betulinic, oleanolic and ursolic acids)23 were found in Carlina
vulgaris. Lupeol, β-amyrin, and α-amyrin were found only in C.
corymbosa var. globosa and C. oligocephala.23 Another study by
Strzemski et al.24 estimated the chlorogenic acid, mineral, total
phenolic, and total flavonoid content of three Polish populations of
Carlina vulgaris L. In the roots of Carlina gummifera L., it was also
detected amino acids, inulin, sugars, latex, essential oil, flavonoid
heterosides, and a triglucosyl derivative of luteolin.25 Even so, scientists
have paid insufficient attention to investigating the phytochemical
composition of many members of Carlina genus, and a useful bit of
information about phytochemicals is still missing. Hence, the current
study aimed at investigating the polyphenolic compounds, triterpenes,
carlina oxide, carbohydrate profile, and the antioxidant activity of
different parts of Carlina vulgaris L., Carlina acanthifolia All. and
Carlina corymbosa L.
Material and Methods
Plant material
The plant material from Carlina acanthifolia, Carlina vulgaris, and
Carlina corymbosa was collected in October 2020 and it was identified
by Assoc. Prof. Ina Aneva from the Institute of Biodiversity and
Ecosystem Research at the Bulgarian Academy of Sciences. Carlina
acanthifolia All. roots and aerial parts were collected from Golo Bardo
mountain. Carlina vulgaris L. was collected from the „Komatinski
rocks“– Vlahina mountain and Carlina corymbosa L. was collected
from the South Struma Valley. The plants were identified by the
references of the Herbarium of the Institute of Biodiversity and
Ecosystem Research - BAS, where a voucher specimen for Carlina
acanthifolia All. (SOM 287349), Carlina vulgaris L. (SOM 287350)
and Carlina corymbosa L. (SOM 287351) were deposited. The samples
were air-dried at room temperature and then finely ground in a
laboratory homogenizer. The dried Carlina radix was purchased from
Dicrassin Ltd. online herbal shop (www.dicrassin-online.com).
Chemicals and reagents
All chemical reagents were of analytical grade. Solvents were
purchased from Merck (Germany) and used as they were received.
Folin-Ciocalteu reagent, Trolox (6- hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), Al(NO3)3, DPPH (1,1-
diphenyl-2-picrylhydrazyl radical), ABTS, gallic acid, quercetin, TPTZ
(2,4,6-tri-(2-pyridyl)-s-triazine), neocuproine, CuCl3, ammonium
acetate, glucose, fructose, sucrose, nystose and 1-kestose were
purchased from Sigma-Aldrich (Steinheim, Germany).
Determination of moisture content
The moisture content was determined by a moisture analyzer KERN
DAB 100-3 (Kern, Germany).
Preparation of extracts
Two grams of aerial parts or roots from three Carlina species were
extracted with 95 % ethanol in a centrifuge plastic tube (50 ml)
employing a solid-to-liquid ratio of 1:15 (v/v) in an ultrasonic bath SIEL
UST 5.7-150 bath (Gabrovo, Bulgaria) with the following parameters:
36 kHz frequency and 240 W ultrasonic power. The extraction was done
in duplicate. The extracts were filtered and combined for further
analysis. The same extraction procedure was repeated as 95 % ethanol
was replaced with distilled water.
Total phenolic content
The total phenolic content in the obtained water and 95% ethanol
extracts was estimated by the method of Folin–Ciocalteu.26 The
absorbance was measured at 765 nm against a blank sample.27 The
results are presented as milligram equivalents of gallic acid per gram
(mg GAE/g dry weight).
Total flavonoids content
The quantity of total flavonoids in carline thistle extracts was evaluated
using Al(NO3)3 reagent.28 The results were presented as milligram
equivalents of mg quercetin (mg QE)/g dw.
Determination of antioxidant activity
DPPH method. Carline thistle extracts (0.15 mL) were mixed with 2.85
mL of freshly prepared DPPH (2,2-diphenyl-1-picrylhydrazyl) (0.1 mM
in methanol). After incubation for 15 minutes at 37°C, the reduction in
the absorbance was measured at 517 nm.29
ABTS method. Carlina extracts (0.15 mL) were mixed with 2.85 mL of
freshly prepared ABTS radical solution. After 15 min at 37ºC, the
adsorption reduction was recorded at 734 nm. 30
FRAP method. The carlina thistle extracts (0.1 mL) were mixed with 3
mL of freshly prepared FRAP reagent. After 5 min the absorption was
recorded at 593 nm.27
CUPRAC method. Carlina thistle extracts (0.1 mL) were added to the
plastic centrifuge tube and mixed with reagents in the following order:
1 mL CuCl2 × 2H2O, 1 mL ethanol solution of Neocuproine, 1 mL 0.1M
ammonium acetate buffer and 1 mL distilled H2O. The absorbance was
recorded at 450 nm after 20 min at 50°С.31
All results from antioxidant activity were presented as mM Trolox
equivalents per g dry weight (mM TE/g dw).
HPLC analysis of phenolic acids and flavonoids.
Individual phenolic acids and flavonoids were analyzed on a HPLC
system equipped with Waters 1525 Binary Pump (Waters, Milford, MA,
USA), Waters 2484 Dual Absorbance Detector (Waters, Milford, MA,
USA), and a C18 column (Supelco Discovery HS, 5 µm, 25cm ×
4.6mm), and Breeze 3.30 software.31 For flavonoids, separation gradient
mode was used with a mobile phase composed of 2.0% (v/v) acetic acid
(solvent A) and methanol (solvent B). The injected volume was 20 μL.32
The results were calculated according to calibration curves.
HPLC-DAD analysis of terpenes, phytosterols, and carlina oxide
The determination of triterpenes, phytosterols, and carlina oxide content
was performed on a Hitachi LaChrom Elite® HPLC System (Hitachi
High Technologies America, Inc., Schaumburg, Illinois, USA), with
diode-array detector (DAD, L-2455) and EZChrom Elite™ software.
The separation of betulin, betulinic acid, oleanolic and ursolic acid, β-
sitosterol and carlina oxide was performed on a reverse-phase column
Supelco, Discovery® HS C18 (5 μm, 25 cm × 4.6 mm) operating at 26
°C. The mobile phase was composed of methanol and 0.1% HCOOH
in a ratio of 92:8 (v/v), (Sigma-Aldrich Chemie GmbH, Darmstadt,
Germany) and the separation was conducted in an isocratic mode with
a flow rate of 0.4 mL/min. The separation of lupeol and α-amyrin
(Extrasynthese, Lyon, France) was done on a reverse-phase column
Waters Spherisorb C8 (5 μm, 15 cm × 4.6 mm) at 26 °C with a mobile
phase acetonitrile:0.1% HCOOH = 92:8 (v/v), (Sigma-Aldrich Chemie
GmbH, Darmstadt, Germany) in a isocratic mode with a flow rate of 0.4
mL/min.32
Analysis of total fructans
The fructan content was determined spectrophotometrically by the
resorcinol-thiourea reagent. The absorbance was measured at 480 nm
against a blank sample prepared with distilled water.9
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HPLC-RID analysis of inulin and sugars
Analysis of inulin and sugars was performed on a HPLC instrument
Elite Chrome Hitachi (Japan), with refractive index detector (RID)
Chromaster 5450 at 35°C, as previously described.33
Statistical analysis.
Statistical analysis was performed using MS Excel 2010. The data were
presented as mean values ± standard deviation (SD) from three
replications. Statistical analysis was done using ANOVA, with Tukey's
range statistically significant at p< 0.05. Different letters within each
column show significant differences according to Tukey’s test at p <
0.05
Results and discussion
Total phenolic and flavonoids content
The content of total phenolics and total flavonoids of the 95% ethanol
and water extracts from different vegetal parts of Carlina species are
shown in Figure 1.
The highest total phenolic content was found in ethanol extract from
Carlina acantifololia All. roots (6.50±0.77 mg GAE/g dw). In general,
ethanol extracts obtained from different vegetal parts were
characterized by the highest values of phenolic and flavonoid content.
Carlina corymposa L. and Carlina vulgaris L. ethanol and water
extracts demonstrated close results for total phenolic content (between
3.79 and 2.50 mg/dw). The lowest values of total phenolic compounds
were detected in water extracts from the aerial part of Carlina vulgaris
L. Strzemski et al. successfully obtained methanolic extracts by
ultrasonic irradiation from leaves, flowers, and root of Carlina vulgaris
L. growing in Poland.24,33 These scientists found 5.8 mg GAE/g total
phenolic content in Carlina vulgaris L. root which is two times higher
than the results reported in the current research. Kaçar reported that the
aerial part of Carlyna corymbose contained a higher amount of total
phenolics 27.3 mg GAE/g dw,34 while, in the current study, we found
considerably lower values in the root of this plant.
The previous research findings showed that water and 70 % ethanolic
extracts had more than three times higher results for the total phenolic
and flavonoids content of Carlina acantifolia roots9 in comparison to
the current results. This item is the first detailed report on the total
phenolic and flavonoids content in three species of Carlina genus.
Antioxidant activity
The antioxidant activity of the obtained extracts from vegetal parts of
Carlina genus was evaluated by four methods, based on different
mechanisms (Table 1). In general, water extracts demonstrated higher
antioxidant potential, especially by the CUPRAC method based on
electron transfer, followed by the ABTS method based on a mixed
mechanism. The obtained data were compared with our previous
observation for C. acannthifolia ethanol and water root extracts9. There
are some studies about the antioxidant potential of Carlina vulgaris
extract,24,36 as ethyl acetate fractions demonstrated the highest activity
by the FRAP method, and the lowest values were found for water
extract36. According to Stremski et al.24 flower head extracts showed the
highest ability to scavenge free radicals, and it was more than 2-fold
higher compared to that for the leaf extract. By contrast, root extracts
exhibited the lowest activity, and it may be explained by the lower
production of antioxidants in the underground part of the plant. In the
current research, there is a tendency for aerial part water extract to have
a higher antioxidant potential than root extract. The ethanol extract
showed higher antioxidant potential by DPPH and FRAP methods
compared to water extracts.
Phenolic compounds in carlina thistle extracts
The polyphenolic and flavonoid composition in ethanolic extracts of
different parts of the three Carlina species was investigated (Table 2).
Chlorogenic acid, ferulic acid, and salicylic acid were identified as
major phenolic acid constituents, but only in the roots of C. vulgaris p-
coumaric acid was found in low concentration (Table 2). The amount of
chlorogenic acid was two times higher in the aerial parts of C.
acathifolia compared to the roots. Similar amounts of chlorogenic acid
were also found in the aerial and root parts of Serbian and Polish
populations. 11,20 Strzemski et al. 24 prove the presence of chlorogenic
acid in the aerial parts (leaves and flowers) and roots of C. vulgaris. The
Bulgarian population of C. vulgaris showed a certain amount of
chlorogenic acid in the aerial parts of this species (0.26±0.02).
Figure 1: Total phenolic content and total flavonoids in different extracts from vegetal parts of Carlina genus representatives
Notes: Values are mean ± standard deviation of three replications. Different letters within each column show significant differences according to Tukey’s
test at p < 0.05
3.79 b
2.89 b
6.50 a
3.09 b
3.79 b
4.33 b
2.59 c 2.41 c 2.54 c
1.92 c
4.44 b
2.29 c
0.76 b 1.03b
1.51a
0.74b
1.66a
1.16b
0.14c 0.43c 0.79b 0.66b
1.17b
0.33c
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Value
Sample
Total phenolic content, mg GAE/g dw Total flavonoid content, mg QE/g dw
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Table 1: Antioxidant activity in different extracts from vegetal parts of Carlina genus, mM TE/g dw
Samples
DPPH
ABTS
FRAP
CUPRAC
Carlina corymbosa roots EtOH
14.86 ± 1.95a
19.44 ± 0.19d
16.10 ± 0.08c
43.74 ± 2.14c
Carlina corymbosa roots Water
3.21 ± 0.33c
101.39 ± 2.11b
3.82 ± 0.15d
197.70 ± 2.14a
Carlina acanthifolia roots EtOH
18.09 ± 0.23a
37.60 ± 7.00c
20.10 ± 0.47b
55.28 ± 1.65c
Carlina acanthifolia roots Water
0.59 ± 0.19e
21.96 ± 0.94d
3.38 ± 0.22d
212.66 ± 11.90a
Carlina acanthifolia aerial parts EtOH
18.78 ± 0.05a
25.30 ± 0.02d
20.21 ± 0.95b
47.32 ± 2.42c
Carlina acanthifolia aerial parts Water
7.59 ± 0.60b
33.76 ± 7.44c
6.40 ± 0.17d
204.73 ± 6.36a
Carlina vulgaris roots EtOH
5.02 ± 0.24c
22.12 ± 1.80d
12.32 ± 0.22c
17.37 ± 1.66d
Carlina vulgaris roots Water
2.65 ± 0.10d
19.51 ± 1.23d
3.60 ± 0.20d
153.18 ± 4.71b
Carlina vulgaris aerial EtOH
10.84 ± 0.07b
6.07 ± 4.45e
15.23 ± 0.27c
35.60 ± 3.00c
Carlina vulgaris aerial Water
7.01 ± 0.40b
136.57 ± 4.65a
5.32 ± 0.02d
159.70 ± 7.53b
Carlinae radix EtOH
8.58 ± 0.03b
44.98 ± 0.19c
34.36 ± 1.13a
38.66 ± 1.27c
Carlinae radix water
1.48 ± 1.50d
18.70 ± 0.32d
2.95 ± 0.28d
203.40 ± 10.52b
Notes: Values are mean ± standard deviation of three replications. Different letters within each column show significant differences according to
Tukey’s test at p < 0.05
Table 2: Phenolic compounds in vegetal parts of Carlina genus
Compound
Concentration, mg/g dw
C. corymbose
C. acanthifolia
C. vulgaris
Carlinae radix
root
root
aerial parts
root
aerial parts
Phenolic acids
Gallic acid
nf
nf
nf
nf
nf
nf
Protocatehuic acid
nf
nf
nf
nf
nf
nf
Chlorogenic acid
0.50 ± 0.01b
0.46 ± 0.01b
0.91 ± 0.02a
nf
0.26 ± 0.02c
0.59 ± 0.02b
Vanillic acid
nf
nf
nf
nf
nf
nf
Caffeic acid
nf
nf
nf
nf
nf
nf
Syringic acid
nf
nf
nf
nf
nf
nf
p-Coumaric acid
nf
nf
nf
0.02 ± 0.01
nf
nf
Ferulic acid
4.50 ± 0.03a
0.17 ± 0.02c
0.21 ± 0.02c
1.05 ± 0.04b
0.84 ± 0.02b
0.06 ± 0.01c
Salicylic acid
1.38 ± 0.02b
2.03 ± 0.03a
0.48 ± 0.02c
0.86 ± 0.02b
0.59 ± 0.02c
0.43 ± 0.01c
Flavonoids
(+)-Catechin
nf
nf
nf
nf
nf
nf
(-)-Epicatechin
nf
nf
nf
nf
nf
nf
Rutin
0.02 ± 0.01c
0.14 ± 0.01b
0.20 ± 0.01a
nf
nf
0.06 ± 0.01c
Hesperidin
nf
1.38 ± 0.02a
nf
0.33 ± 0.01b
nf
0.54 ± 0.01b
Quercetin
0.05 ± 0.01a
0.02 ± 0.01b
nf
0.06 ± 0.01a
nf
nf
Notes: nf – not found. Values are mean ± standard deviation of three replications. Different letters within each column show significant differences
according to Tukey’s test at p < 0.05.
Furthermore, the phenolic acid composition was studied in C.
corymbose. It was evident that three phenolic acids chlorogenic acid
(0.50±0.01 mg/g dw), ferulic acid (4.50±0.03mg/g dw), and salicylic
acid (1.38±0.02 mg/g dw) were quantified and identified for the first
time. In addition, significant concentrations of ferulic acid and salicylic
acid were found in various parts of C. acanthifolia and C. vulgaris. In
the roots of C. acanthifolia, salicylic acid was found in an amount of
2.03±0.03 mg/g dw, while in C. vulgaris ferulic acid reached 1.05±0.04
mg/g dw. Both phenolic acids were observed for the first time in
Bulgarian populations. In Polish populations of C. acantifolia, on the
other hand, only chlorogenic acid and protocatechuic acid11 were found,
while in Serbian only the presence of chlorogenic acid was reported.20
In three studied Polish populations of C. vulgaris, only chlorogenic acid
was detected.24 A relatively high concentration of hesperidin was found
in the roots of the Bulgarian populations of C. acanthifolia (1.38±0.02
mg/g dw) and C. vulgaris (0.33±0.01 mg/g dw).
The quercetin glycoside rutin was found only in C. acanthifolia (aerial
part and root) and C. corymbosa (root). It was found that quercetin was
present in trace amounts in the roots of all three investigated carlina
species.
Triterpenes, phytosterols, and carlina oxide
The results of the triterpenes and phytosterols chromatographic analysis
are summarized in Table 3. It was evident that different constituents
such as betulin, betulinic acid, oleanolic acid, ursolic acid, lupeol, α-
amyrin and β-sitosterol were identified and quantified. Interestingly, a
great diversity of triterpenes and phytosterols was observed in the aerial
parts of Carlina vulgaris and C. acanthifolia (Table 3). Oleanolic acid,
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betulin, and betulinic acid dominated in Carlina vulgaris (5.04±0.06,
4.08±0.08 and 2.92±0.06 mg/g dw, respectively), whereas betulin
(4.44±0.03 mg/g dw) was found in C. acanthifolia. The β-sitosterol
content in Carlina acanthifolia (35.83±0.05 mg/g dw) was nearly
twofold higher than that found in Carlina vulgaris (19.05±0.05 mg/g
dw). It was found that the concentration of triterpenes in the roots of the
investigated species was considerably low. These findings are similar
to those reported in previous studies on Polish cultivated plants. 23,24 In
the current study, for the first time, we found the triterpenes lupeol
(1.39±0.02 mg/g dw) and betulin (0.44±0.01 mg/g dw) in the extracts
of Carlina corymbosе roots. Table 3 contains important data relating to
the levels of polyacetylene carlina oxide. It is interesting to note that the
quantity of carlina oxide was considerably higher in the roots of Carlina
acanthifolia (15.06±0.07 mg/g dw, Carlinae radix - 10.62±0.05 mg/g
dw) by comparison with the aerial parts of the species (2.04±0.01 mg/g
dw). Our results for C. acanthifolia roots are the same as the results for
C. acanthifolia, and Carlina acaulis roots (1-2%) of a previous study,
where the content of carlina oxide accounted for 98-90% of essential
oil.37 Contrary to the report of Sörensen & Sörensen38 in the current
research carlina oxide was not found in Carlina vulgaris. Other
scientists 39,40 have also investigated an essential oil of the roots of C.
vulgaris. Carlina oxide (33.7%) and 13-methoxy carlina oxide (11.5%)
represented a high percentage of the oil.
Correlation between total phenolic content, total flavonoids and
antioxidant activity
Table 4 shows the correlation (r2) between antioxidant activity and the
total phenolic content and total flavonoids (Table 4).
As can be seen, there was a positive linear correlation between
CUPRAC and total phenolic content and total flavonoids (r2>0.85)
suggesting that polyphenols in carlina thistle extracts were responsible
for the high antioxidant activity exhibited by ABTS and CUPRAC
methods. In addition, the highest correlation was also observed between
total phenolic content and metal-reducing method CUPRAC, (r2
>0.9260). The total flavonoids showed the highest correlation with
CUPRAC and DPPH methods (r2 >0.79).
Table 3: Triterpenes and carlina oxide in different vegetal parts of Carlina genus
Compound
Concentration, mg/g dw
C. corymbose
C. acanthifolia
C. vulgaris
Carlinae radix
root
root
aerial parts
root
aerial parts
Triterpenes
Betulin
0.44 ± 0.01b
0.68 ± 0.03b
4.44 ± 0.03a
0.50 ± 0.02b
4.08 ± 0.08a
0.08 ± 0.01c
Betulinic acid
nf
1.05 ± 0.02c
nf
1.76 ± 0.03b
2.92 ± 0.06a
nf
Oleanolic acid
nf
0.20 ± 0.01b
nf
nf
5.04 ± 0.06a
nf
Ursolic acid
nf
nf
nf
nf
0.60 ± 0.03
nf
Lupeol
1.39 ± 0.02a
0.10 ± 0.01b
0.21 ± 0.01b
tr
0.35 ± 0.02b
0.02 ± 0.00c
α-Amyrin
nf
tr
0.02 ± 0.01b
0.03 ± 0.01b
0.06 ± 0.01a
nf
Phytosterols
β-Sitosterol
nf
nf
35.83 ± 0.05a
nf
19.05 ± 0.05b
nf
Polyacetylenes
Carlina oxide
nf
15.06 ± 0.07a
2.04 ± 0.01c
nf
nf
10.62 ± 0.05b
Notes: nf – not found, tr-traces. Values are mean ± standard deviation of three replications. Different letters within each column show significant
differences according to Tukey’s test at p < 0.05
Table 4: Correlation coefficient (r2) between antioxidant activities and total phenolic content, and total flavonoids
Total phenolic content
Total flavonoids
Ethanol
Water
Ethanol
Water
DPPH
0.5425
0.2902
0.7917
0.5161
ABTS
0.7320
0.3474
0.4562
0.2929
FRAP
0.4289
0.5218
0.4999
0.6516
CUPRAC
0.7338
0.9260
0.8571
0.6742
Total flavonoids
0.6616
0.7657
-
-
Table 5: Fructan content and individual sugars in water extracts, g/100 g dw
Sample
Total fructans
Inulin
Nystose
1-Kestose
Sucrose
Glucose
Fructose
Carlina corymbosa roots
3.11 ± 0.18c
1.02 ± 0.36c
0.45 ± 0.06a
nd
0.82 ± 0.20b
0.29 ± 0.01c
0.33 ± 0.14b
Carlina acanthifolia roots
15.47 ± 0.07b
12.14 ± 0.34b
nd
nd
3.03 ± 0.73a
0.65 ± 0.18b
1.56 ± 0.41a
Carlina acanthifolia aerial parts
2.06 ± 0.24a
nd
nd
nd
nd
2.54 ± 0.01a
0.80 ± 0.01b
Carlina vulgaris roots
1.13 ± 0.08c
0.06 ± 0.01c
0.20 ± 0.01b
0.03 ± 0.01
0.25 ± 0.02c
0.25 ± 0.08c
0.14 ± 0.02c
Carlina vulgaris aerial parts
3.31 ± 0.94c
0.28 ± 0.03c
0.58 ± 0.01a
nd
0.35 ± 0.01c
0.29 ± 0.23c
0.57 ± 0.37b
Carlinae radix
20.29 ± 0.07a
18.10 ± 0.05a
nd
nd
1.85 ± 0.42b
1.00 ± 0.13b
1.21 ± 0.46a
Notes: n.d. – not detected, Values are mean ± standard deviation of three replications. Different letters within each column show significant
differences according to Tukey’s test at p < 0.05
Trop J Nat Prod Res, October 2023; 7(10):4242-4248 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
4247
© 2023 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
The total flavonoids were weakly correlated with the ABTS and FRAP
assay. A similar tendency for phenolic content to correlate highly with
antioxidant activity was reported by other researchers.9 A high
correlation existed between TPC/TFC, TPC, and DPPH/ABTS, and
TFC and DPPH/ABTS (r > 0 79, r > 0 61, and r > 0 68) was also
reported.23,24
Total fructans, inulin, and sugar content in Carlina thistles extracts
The results for sugar composition and fructan content in different plant
materials of Carlina corymbosa, Carlina acanthifolia, and Carlina
vulgaris were summarized in Table 5. This item is the first detailed
study that gives information about the sugar and inulin content of the
investigated Carlina species. As shown (Table 3), the roots contained a
higher quantity of inulin polysaccharide, whereas the aerial parts
contained very small amounts (0.3 g/100 g dw). Glucose and fructose
constituted all vegetal parts of the plants. Fructooligosaccharides (1-
kestose and nystose) were detected mainly in the roots. The highest
content of inulin was detected in commercial Carlina radix - 18.10 g/100
g dw, followed by Carlina acanthifolia roots – 12.14 g/100 g dw. In an
earlier study, it was reported, that Calina acaulis roots contained 20 %
of inulin.20 Another study40 it was suggested that inulin is the main
compound of Carlina spp. (18-20 %), while a previous study by
Petkova et al. found that inulin content in commercial Carlina
acanthifolia roots reached 5-6.8 g/100 g dw.9 Furthermore, Table 5
shows that Carlina corymbosa roots contained a considerably lower
amount of inulin in comparison to Carlina acanthifolia roots. The
lowest values of inulin were detected in Carlina vulgaris roots and
vegetal parts (< 0.3 g/100 g dw). The roots of Carlina vulgaris L. and
Carlina corymbosa L., on the other hand, showed a level of inulin below
1%. The last two species contained 0.5 % nystose, while it was
completely missing in Carlina acanthifolia All. There was not any
amount of 1-kestose in most of the samples. Our results indicated that a
majority of sugars were present in the aerial part of the plants than in
the roots, especially for Carlina vulgaris L.
Our findings led to the conclusion that Carlina acanthifolia All. could
serve as a better source of fructans and inulin in comparison with
chicory and Echinacea plants.
Conclusion
This research is the first detailed study about phytochemical
constituents in different plant parts of Carlina vulgaris L., Carlina
acanthifolia All. and Carlina corymbosa L. Extracts of Carlina species
exhibited antioxidant potential mainly by CUPRAC method. In
addition, there was a higher correlation between the total phenolic
compounds and the antioxidant activity values by ABTS and CUPRAC
methods. The detected phenolic acids, flavonoids, and triterpenes were
in the highest concentration in Carlina vulgaris L and Carlina
acanthifolia All., while in Carlina acanthifolia All. roots predominated
carlina oxide and inulin. Our study reveals for the first time the
carbohydrate profile of Carlina vulgaris L., Carlina acanthifolia All.
and Carlina corymbosa L. and evaluated Carlina acanthifolia as a rich
source of inulin-type fructans. Owing to the various phytochemical
compounds, the extracts from the investigated carlina thistles can be
used in food, pharmaceutical, and cosmetic formulations.
Conflict of Interest
The authors declare no conflict of interest.
Authors’ Declaration
The authors hereby declare that the work presented in this article is
original and that any liability for claims relating to the content of this
article will be borne by them.
Acknowledgments
This study was financed by the European Union-NextGenerationEU,
through the National Recovery and Resilience Plan of the Republic of
Bulgaria, the project № BG-RRP-2.004-0009-C02“.
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