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Comparison of the Phenolic Compound Profile and Antioxidant Potential of Achillea atrata L. and Achillea millefolium L

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In the present study, Achillea atrata L. and A. millefolium L. were compared for the first time with regard to their phenolic compound profile and antioxidant activity by applying the 2,2-diphenyl-picryl hydrazyl radical assay. For this purpose, aerial plant parts were consecutively extracted with solvents of increasing polarity (dichloromethane, n-butanol, ethyl acetate), revealing that the A. atrata ethyl acetate fraction showed the highest antioxidant activity with an IC50 value of 12.2 ± 0.29 µg/mL compared to 17.0 ± 0.26 µg/mL for A. millefolium. Both species revealed the presence of luteolin, apigenin, centaureidin, and nevadensin exclusively in this most polar fraction, which are known as effective 2,2-diphenyl-picryl hydrazyl radical scavengers. The antioxidant capacity of the aforementioned fractions strikingly correlated with their total phenolic contents, which was highest in the ethyl acetate fraction of A. atrata. Characterization of the metabolite profiles of both Achillea species showed only marginal differences in the presence of key compounds, whereas the concentrations of individual compounds appeared to be species-specific. Our results suggest that A. atrata, based on its compound pattern and bioactivity characteristics, has similar qualities for phytotherapy as A. millefolium.
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molecules
Communication
Comparison of the Phenolic Compound Profile and Antioxidant
Potential of Achillea atrata L. and Achillea millefolium L.
Lysanne Salomon 1, Peter Lorenz 1, Marek Bunse 1, Otmar Spring 2, Florian C. Stintzing 1and
Dietmar R. Kammerer 1, *


Citation: Salomon, L.; Lorenz, P.;
Bunse, M.; Spring, O.; Stintzing, F.C.;
Kammerer, D.R. Comparison of the
Phenolic Compound Profile and
Antioxidant Potential of Achillea atrata
L. and Achillea millefolium L. Molecules
2021,26, 1530. https://doi.org/
10.3390/molecules26061530
Academic Editor: Maria Atanassova
Received: 5 February 2021
Accepted: 8 March 2021
Published: 11 March 2021
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Attribution (CC BY) license (https://
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4.0/).
1WALA Heilmittel GmbH, Department of Analytical Development & Research, Section Phytochemical
Research, 73087 Bad Boll, Germany; Lysanne.Salomon@wala.de (L.S.); Peter.Lorenz@wala.de (P.L.);
Marek.Bunse@wala.de (M.B.); Florian.Stintzing@wala.de (F.C.S.)
2Institute of Botany, Hohenheim University, 70599 Stuttgart, Germany; o.spring@uni-hohenheim.de
*Correspondence: Dietmar.Kammerer@wala.de; Tel.: +49-7164-930-6688; Fax: +49-7164-930-7080
Abstract:
In the present study, Achillea atrata L. and A. millefolium L. were compared for the first
time with regard to their phenolic compound profile and antioxidant activity by applying the 2,2-
diphenyl-picryl hydrazyl radical assay. For this purpose, aerial plant parts were consecutively
extracted with solvents of increasing polarity (dichloromethane, n-butanol, ethyl acetate), revealing
that the A. atrata ethyl acetate fraction showed the highest antioxidant activity with an IC
50
value
of
12.2 ±0.29 µg/mL
compared to 17.0
±
0.26
µ
g/mL for A. millefolium. Both species revealed the
presence of luteolin, apigenin, centaureidin, and nevadensin exclusively in this most polar fraction,
which are known as effective 2,2-diphenyl-picryl hydrazyl radical scavengers. The antioxidant
capacity of the aforementioned fractions strikingly correlated with their total phenolic contents,
which was highest in the ethyl acetate fraction of A. atrata. Characterization of the metabolite profiles
of both Achillea species showed only marginal differences in the presence of key compounds, whereas
the concentrations of individual compounds appeared to be species-specific. Our results suggest
that A. atrata, based on its compound pattern and bioactivity characteristics, has similar qualities for
phytotherapy as A. millefolium.
Keywords:
Achillea atrata L.; Achillea millefolium L.; antioxidant activity; DPPH; phenolic metabolome
1. Introduction
Plants are sessile organisms and are exposed to a wide variety of different abiotic
stress factors in constantly changing environments. Water deficiency, contamination of the
soil with heavy metals, salinity, nutrient surplus or deficiency, high and low temperatures,
extreme light, and UV-B radiation are only some of such abiotic stress factors that affect
plants and strongly influence their growth and development [
1
3
]. Abiotic stress promotes
the production of damaging reactive oxygen species (ROS) and nitrogen species within
cells and leads to rapid changes in cellular redox homeostasis, resulting in peroxidation and
destabilization of cellular membranes [
1
3
]. The accumulation of secondary metabolites
in plant tissues such as phenolics is a typical adaptive response of plants to these adverse
environmental conditions [
1
]. Plant phenolics are aromatic compounds with one or more
hydroxyl groups and are biosynthesized in plants from phenylalanine and shikimic acid
through the shikimic acid pathway [
1
,
4
6
]. When a plant is exposed to abiotic stress, the
activity of phenylalanine ammonia lyase (PAL) and other enzymes necessary for pheno-
lic biosynthesis is upregulated, resulting in increased phenol production to ensure plant
survival and increase stress tolerance [
1
,
5
]. These antioxidant and radical scavenging
properties of phenolics are crucial for the plant. Consequently, they are also attracting
increasing interest in the preservation of human health and in preventing physiopathologi-
cal conditions where oxidative damage is a hallmark [
7
9
]. Medicinal plants with a high
level of these bioactive compounds play an important role in the prevention of chronic
Molecules 2021,26, 1530. https://doi.org/10.3390/molecules26061530 https://www.mdpi.com/journal/molecules
Molecules 2021,26, 1530 2 of 11
diseases, slowing down aging processes as well as reducing the risk of cardiovascular and
neurodegenerative diseases [
9
,
10
]. Due to their health benefits, the search for novel sources
of natural antioxidants for pharmaceutical and medicinal purposes is of growing interest.
In particular, the genus Achillea, consisting of more than 140 perennial species native
to the Northern Hemisphere, is characterized by a pronounced antioxidant activity [
11
].
More than twenty Achillea species and subspecies including A. millefolium, which have been
used as medicinal plants, have previously been assessed with regard to their anti-radical
scavenging properties by investigating various extracts recovered with solvents of different
polarities [
11
13
]. However, investigations into the antioxidant activity of the alpine species
A. atrata have not been reported yet. Consequently, the aim of the present study was a
first in-depth investigation of the radical scavenging capacity of A. atrata applying the
2,2-diphenyl-picryl hydrazyl (DPPH) radical
in vitro
assay as a model test system, which
should form a basis for further assessment of the antioxidant potential of A. atrata, both
in vitro
and
in vivo
. Furthermore, the characterization of secondary metabolites with
particular focus on phenolic compounds and the comparison of the compound profile
and bioactivity with A. millefolium obtained from the same habitat should be performed
to broaden our knowledge of Achillea species potentially applicable to pharmaceutical
purposes.
2. Results
2.1. Phytochemical Comparison of A. atrata and A. millefolium
Both Achillea species were fractionated with solvents of different polarity (i.e., with
dichloromethane, acetone/water, ethyl acetate, and n-butanol). Potential correlations
between secondary metabolites and antioxidant activity, but also species-specific metabo-
lites should be identified. The phenolic compounds of the polar acetone/water extracts
and of the ethyl acetate and n-butanol fractions were characterized based on their UV
characteristics, HPLC retention times, specific mass spectra, and comparison with refer-
ence substances or literature data (Table 1, Figures 1and 2). Individual compounds of
the non-polar dichloromethane fractions were analyzed using gas chromatography-mass
spectrometry (GC-MS) and assigned based on their specific mass spectrometric data as
well as retention times in comparison with the NIST MS database (Table 2).
Molecules 2021, 26, x FOR PEER REVIEW 4 of 11
Figure 1. Comparison of total ion chromatograms (TIC) of the Achillea atrata acetone/water extract (A) and the correspond-
ing ethyl acetate (B) and n-butanol (C) fractions. 1: chlorogenic acid; 4: quercetin-O-hexoside I; 5: 4-methyl-3-methoxy-9a-
hydroxyligballinol-O-glucoside (formate adduct); 6: quercetin-3-O-rutinoside; 8: luteolin-hexoside; 9: quercetin-O-hexo-
side II; 10: mearnsetin-hexoside; 11: isorhamnetin-O-hexoside I; 12: kaempferol-3-O-rutinoside II; 13–16: dicaffeoylquinic
acid I-VI; 17: apigenin-7-O-glucoside; 18: isorhamnetin-O-hexoside II; 19: dicaffeoylquinic acid V; 20: dicaffeoylquinic acid
VI; 23: caffeoyl-feruloylquinic acid; 24: luteolin; 25: apigenin; 27: nevadensin.
Figure 2. Comparison of total ion chromatograms (TIC) of the Achillea millefolium acetone/water extract (A) and the Achillea
millefolium ethyl acetate (B) and n-butanol (C) fractions. 1: chlorogenic acid; 2: p-coumaroyl acid derivative; 3: apigenin-
6,8-di-C-hexoside; 4: quercetin-O-hexoside I; 5: 4-methyl-3-methoxy-9a-hydroxyligballinol-O-glucoside (formate adduct);
6: quercetin-3-O-rutinoside; 7: kaempferol-3-O-rutinoside I; 8: luteolin-hexoside; 9: quercetin-hexoside II; 10: mearnsetin-
hexoside; 11: isorhamnetin-O-hexoside I; 12: kaempferol-3-O-rutinoside II; 13–16: dicaffeoylquinic acid I-VI; 17: apigenin-
7-O-glucoside; 18: isorhamnetin-O-hexoside II; 19: dicaffeoylquinic acid V; 20: dicaffeoylquinic acid VI; 21:
dicaffeoylquinic acid VII; 22: cinnamic acid derivative; 23: caffeoyl-feruloylquinic acid; 24: luteolin; 25: apigenin; 26: cen-
taureidin; 27: nevadensin.
Table 2. Gas chromatography-mass spectrometry (GC-MS) analyses of A. atrata and A. millefolium dichloromethane fractions
(DCM).
Peak
No.
Peak
Assignment Rt [min] Calc. Mr
[Da] Characteristic Fragments, m/z (BPI [%]) A. at-
rata
A. mille-
folium
Figure 1.
Comparison of total ion chromatograms (TIC) of the Achillea atrata acetone/water extract
(
A
) and the corresponding ethyl acetate (
B
) and n-butanol (
C
) fractions. 1: chlorogenic acid; 4:
quercetin-O-hexoside I; 5: 4-methyl-3-methoxy-9a-hydroxyligballinol-O-glucoside (formate adduct);
6: quercetin-3-O-rutinoside; 8: luteolin-hexoside; 9: quercetin-O-hexoside II; 10: mearnsetin-hexoside;
11: isorhamnetin-O-hexoside I; 12: kaempferol-3-O-rutinoside II; 13–16: dicaffeoylquinic acid I–
VI; 17: apigenin-7-O-glucoside; 18: isorhamnetin-O-hexoside II; 19: dicaffeoylquinic acid V; 20:
dicaffeoylquinic acid VI; 23: caffeoyl-feruloylquinic acid; 24: luteolin; 25: apigenin; 27: nevadensin.
Molecules 2021,26, 1530 3 of 11
Table 1.
Spectroscopic data (UV, mass spectrometry (MS)) and high performance liquid chromatography (HPLC) retention times (Rt) of secondary metabolites of A. atrata and A. millefolium
[acetone/water extracts (aw), ethyl acetate fractions (EtOAc) and n-butanol fractions (n-but)]. Only the most intense m/z ratios of the collision-induced dissociation (CID) experiments are
illustrated.
Peak
No.
Rt
[min]
Peak
Assignment
UV λmax
[nm]
MSnData [m/z]A. atrata A. millefolium Ref.
MS 1MS 2MS 3aw EtOAc n-but aw EtOAc n-but
122.6
23.0
Chlorogenic acid 216, 326 353 191 173 +++ RS*1/[14]*2
+++
2 24.5 p-Coumaroyl acid derivative 326 387 207 163 - - - + + + [15] *1
3 25.5 Apigenin-6,8-di-C-hexoside 194, 280 593 473 353 - - - + + + [16] *1/[17] *2
4 30.0 Quercetin-O-hexoside I 282, 342 463 301 283 + + + + + + [15] *1/[18] *2
531.8
32.1
4-Methyl-3-methoxy-9a-
hydroxyligballinol-O-glucoside
202, 278
202, 276 565 339 324 +++ [16] *1
+++
6 33.0 Quercetin-3-O-rutinoside 200, 374 609 301 179 + + + + + + RS *1/[19] *2
7 33.4 Kaempferol-3-O-rutinoside I 202, 342 593 285 255 - - - + + + [16] *1/[20] *2
8 34.4 Luteolin-hexoside 266, 348 447 285 255 + + + + + + [21] *1/[22] *2
934.4
34.7
Quercetin-O-hexoside II 204, 328 463 301 151 +++[15] *1/[18] *2
+++
10 35.3 Mearnsetin-hexoside 198, 336 493 331 316 + + - - - - [23] *1/[24] *2
11 36.5 Isorhamnetin-O-hexoside I 204, 328 477 315 300 + + + - - - [16] *1/[18] *2
12 37.9
38.1
Kaempferol-3-O-rutinoside II 266, 342 593 285 255 +++ [16] *1/[20] *2
+ +
13 38.1 Dicaffeoylquinic acid I 204, 324 515 353 191 + + + + + + [16] *1/[12] *2
14 38.4 Dicaffeoylquinic acid II 204, 324 515 353 191 + + + + + + [16] *1/[12] *2
15 38.6 Dicaffeoylquinic acid III 204, 324 515 353 191 + + + + + + [16] *1/[12] *2
16 39.3
39.9 Dicaffeoylquinic acid IV 204, 324 515 353 191 +++[16] *1/[12] *2
+++
17 40.2 Apigenin-7-O-glucoside 268, 338 431 269 225 + + + + + + RS *1/[17] *2
18 40.6 Isorhamnetin-O-hexoside II 200, 332 447 315 300 + + + - - - [16] *1/[18] *2
Molecules 2021,26, 1530 4 of 11
Table 1. Cont.
Peak
No.
Rt
[min]
Peak
Assignment
UV λmax
[nm]
MSnData [m/z]A. atrata A. millefolium Ref.
MS 1MS 2MS 3aw EtOAc n-but aw EtOAc n-but
19 41.4 Dicaffeoylquinic acid V 194, 324 515 353 191 + + - + + + [16] *1/[12] *2
20 43.0 Dicaffeoylquinic acid VI 216, 326 515 353 191 + + + + + + [16] *1/[12] *2
21 43.8 Dicaffeoylquinic acid VII 216, 326 515 353 191 - - - + + + [16] *1/[12] *2
22 44.6 Cinnamic acid derivative - 549 387 369 - - - + + + [23] *1
23 48.4 Caffeoyl-feruloylquinic acid 196, 326 529 367 191 + + - - - - [25] *1/[26] *2
24 50.1 Luteolin 252, 346 285 241 217 + + - + + - RS *1/[19] *2
25 54.0 Apigenin 286, 332 269 225 - + + - + + - RS *1/[17] *2
26 55.9 Centaureidin 234, 314 359 344 329 - - - + + - [27] *1/[28] *2
27 57.9 Nevadensin 332 343 328 313 + + - + + - [29] *1/[30] *2
RS: Reference standard; + detected; - not detected; *1Reference: LC-MSndata; *2Reference: Substance identification in Achillea spec.
Molecules 2021,26, 1530 5 of 11
Molecules 2021, 26, x FOR PEER REVIEW 4 of 11
Figure 1. Comparison of total ion chromatograms (TIC) of the Achillea atrata acetone/water extract (A) and the correspond-
ing ethyl acetate (B) and n-butanol (C) fractions. 1: chlorogenic acid; 4: quercetin-O-hexoside I; 5: 4-methyl-3-methoxy-9a-
hydroxyligballinol-O-glucoside (formate adduct); 6: quercetin-3-O-rutinoside; 8: luteolin-hexoside; 9: quercetin-O-hexo-
side II; 10: mearnsetin-hexoside; 11: isorhamnetin-O-hexoside I; 12: kaempferol-3-O-rutinoside II; 13–16: dicaffeoylquinic
acid I-VI; 17: apigenin-7-O-glucoside; 18: isorhamnetin-O-hexoside II; 19: dicaffeoylquinic acid V; 20: dicaffeoylquinic acid
VI; 23: caffeoyl-feruloylquinic acid; 24: luteolin; 25: apigenin; 27: nevadensin.
Figure 2. Comparison of total ion chromatograms (TIC) of the Achillea millefolium acetone/water extract (A) and the Achillea
millefolium ethyl acetate (B) and n-butanol (C) fractions. 1: chlorogenic acid; 2: p-coumaroyl acid derivative; 3: apigenin-
6,8-di-C-hexoside; 4: quercetin-O-hexoside I; 5: 4-methyl-3-methoxy-9a-hydroxyligballinol-O-glucoside (formate adduct);
6: quercetin-3-O-rutinoside; 7: kaempferol-3-O-rutinoside I; 8: luteolin-hexoside; 9: quercetin-hexoside II; 10: mearnsetin-
hexoside; 11: isorhamnetin-O-hexoside I; 12: kaempferol-3-O-rutinoside II; 13–16: dicaffeoylquinic acid I-VI; 17: apigenin-
7-O-glucoside; 18: isorhamnetin-O-hexoside II; 19: dicaffeoylquinic acid V; 20: dicaffeoylquinic acid VI; 21:
dicaffeoylquinic acid VII; 22: cinnamic acid derivative; 23: caffeoyl-feruloylquinic acid; 24: luteolin; 25: apigenin; 26: cen-
taureidin; 27: nevadensin.
Table 2. Gas chromatography-mass spectrometry (GC-MS) analyses of A. atrata and A. millefolium dichloromethane fractions
(DCM).
Peak
No.
Peak
Assignment Rt [min] Calc. Mr
[Da] Characteristic Fragments, m/z (BPI [%]) A. at-
rata
A. mille-
folium
Figure 2.
Comparison of total ion chromatograms (TIC) of the Achillea millefolium acetone/water
extract (
A
) and the Achillea millefolium ethyl acetate (
B
) and n-butanol (
C
) fractions. 1: chlorogenic
acid; 2: p-coumaroyl acid derivative; 3: apigenin-6,8-di-C-hexoside; 4: quercetin-O-hexoside I; 5: 4-
methyl-3-methoxy-9a-hydroxyligballinol-O-glucoside (formate adduct); 6: quercetin-3-O-rutinoside;
7: kaempferol-3-O-rutinoside I; 8: luteolin-hexoside; 9: quercetin-hexoside II; 10: mearnsetin-
hexoside; 11: isorhamnetin-O-hexoside I; 12: kaempferol-3-O-rutinoside II; 13–16: dicaffeoylquinic
acid I–VI; 17: apigenin-7-O-glucoside; 18: isorhamnetin-O-hexoside II; 19: dicaffeoylquinic acid
V; 20: dicaffeoylquinic acid VI; 21: dicaffeoylquinic acid VII; 22: cinnamic acid derivative; 23:
caffeoyl-feruloylquinic acid; 24: luteolin; 25: apigenin; 26: centaureidin; 27: nevadensin.
Table 2.
Gas chromatography-mass spectrometry (GC-MS) analyses of A. atrata and A. millefolium dichloromethane fractions
(DCM).
Peak
No.
Peak
Assignment
Rt
[min]
Calc. Mr
[Da] (tms) Characteristic Fragments, m/z (BPI [%]) A.
atrata
A.
millefolium
1α-Thujene 6.29 136.15 93 (100), 92 (42), 91 (50), 79 (20), 77 (24), 63 (9) + +
2 Bornylene 6.57 136.15 93 (100), 89 (29), 84 (32), 79 (25), 72 (88), 63 (29) - +
3β-Thujene 6.81 136.15 93 (100), 91 (43), 79 (26), 77 (36), 69 (9) - +
4 Sabinene 6.98 136.15 93 (100), 91 (32), 79 (19), 77 (17), 69 (21), 67 (8) - +
5γ-Terpinene 7.34 136.15 93 (100), 92 (22), 91 (50), 77 (29), 57 (4) + -
6 Eucalyptol 7.86 154.14 111 (36), 108 (59), 84 (89), 81 (100), 71 (68), 67 (32), 55 (33) - +
7β-Terpineol 8.53 154.14 93 (70), 92 (33), 84 (25), 71 (100), 64 (16), 55 (49) - +
8α-Thujone 9.37 152.12 110 (58), 109 (27), 95 (41), 81 (100), 79 (18), 69 (49), 68 (57) - +
9 Borneol 9.61 154.14 95 (100), 77 (94), 74 (30), 72 (51), 69 (25), 65 (30), 57 (56) - +
10 Camphor 10.47 152.12 95 (100), 83 (23), 81 (63), 69 (27), 67 (17), 55 (20) - +
11 (+)-Borneol 11.03 154.14 110 (18), 95 (100), 67 (9) - +
12 α-Terpineol 11.48 154.14 136 (54), 121 (52), 93 (100), 89 (20), 81 (36), 77 (24), 59 (95) - +
13 β-Bisabolol 12.84 222.20 82 (100), 78 (26), 73 (31), 65 (19), 58 (18), 55 (21), 53 (36) - +
14 Isoborneol 13.92 154.14 95 (100), 89 (20), 79 (22), 77 (15), 70 (29), 68 (26), 64 (18) - +
15 Caryophyllene 17.14 204.19 105 (46), 93 (100), 91 (94), 81 (23), 79 (61), 77 (33), 55 (52) + +
16 (+)-Nerolidol 21.83 222.20 107 (39), 93 (100), 81 (39), 79 (22), 71 (43), 67 (36), 55 (31) - +
17 Caryophyllene oxide 22.95 220.18 107 (38), 106 (33), 95 (48), 93 (68), 91 (55), 79 (100), 69 (33) + +
18 α-Eudesmol 24.00 222.20 204 (96), 161 (100), 149 (40), 108 (32), 93 (48), 79 (27), 59 (79) + +
19 Alkane I 40.19 - 113 (7), 99 (90), 85 (83), 71 (94), 57 (100), 55 (22) + +
20 Alkane II 44.27 - 113 (10), 99 (20), 85 (76), 71 (87), 57 (100), 55 (19) + +
21 Alkane III 48.05 - 113 (12), 99 (30), 85 (84), 71 (97), 69 (19), 57 (100), 55 (22) + +
22 Alkane IV 51.57 - 207 (4), 99 (32), 97 (20), 85 (85), 71 (99), 57 (100), 55 (16) + +
23 Alkane V 54.88 - 209(6), 99 (28), 85 (83), 83 (25), 71 (99), 57 (100), 55 (16) + +
A total of 27 phenolic compounds were identified, among which 21 were detected
in the acetone/water extract of A. atrata and 23 in the corresponding extract of A. mille-
Molecules 2021,26, 1530 6 of 11
folium. By comparing the compound profiles of both Achillea species, nine differences were
detected: the p-coumaroyl acid derivative (2), apigenin-6-8-di-C-hexoside (3), kaempferol-
3-O-rutinoside I (7), dicaffeoylquinic acid VII (21), the cinnamic acid derivative (22), and
centaureidin (26) could only be assigned for A. millefolium. In contrast, the occurrence of
mearnsetin-hexoside (10) and of isorhamnetin-O-hexoside I and II (11, 18) was specific for
A. atrata. The remaining compounds (1, 4–6, 8, 9, 12–25, 27) were detected in both Achillea
species, with the main components being caffeic acid derivatives, kaempferol-, luteolin-,
quercetin-, and apigenin-glycosides. All compounds characterized in the acetone/water
extracts were also detected in the respective ethyl acetate fractions. Marked differences
between the ethyl acetate and the n-butanol fractions were found in the occurrence of
luteolin, apigenin, centaureidin, and nevadensin exclusively in the ethyl acetate fraction.
GC-MS analyses of the dichloromethane fractions revealed a total of 23 constituents. How-
ever, only 10 of the latter were detected in the A. atrata dichloromethane fraction including
the monoterpenes
α
-thujene (1),
γ
-terpinene (5), the sesquiterpenes caryophyllene (15),
caryophyllene oxide (17), and α-eudesmol (18) as well as the alkanes I–V (19–23).
2.2. Antioxidant Capacity and Contents of Phenolics and Volatile Compounds
The radical scavenging capacity of the aforementioned extracts and fractions when
applying the DPPH assay is reported in Table 3by specifying the respective IC
50
values,
thus allowing direct comparison. In general, the dichloromethane fractions did not reveal a
radical scavenging potential, even at higher concentrations (750
µ
g/mL). The ethyl acetate
fractions of both Achillea species showed the highest activity compared to the respective
n-butanol fractions. Comparative analyses of the two species revealed the ethyl acetate
fraction of A. atrata to exhibit both the highest antiradical potential with an IC
50
value of
12.2
±
0.3
µ
g/mL and the highest phenolic content of 250
±
2.5 mg GAE/g dry weight
(Table 4). However, the activity of all Achillea fractions examined was inferior to the
reference compound Trolox with an IC50 value of 7.5 ±0.1 µg/mL.
Table 3. Antioxidant capacity of A. atrata and A. millefolium (n= 3; ±SD).
Samples Regression Equation R2IC50 [µg/mL] ±SD
Trolox y = 6.5233x + 1.3342 0.9975 7.5 ±0.1
A. atrata
ethyl acetate fraction y = 4.0888x 0.1440 0.9983 12.2 ±0.3
n-butanol fraction y = 0.6263x + 2.3083 0.9984 76.15 ±0.3
dichloromethane fraction - - -
A. millefolium
ethyl acetate fraction y = 3.0799x 2.2077 0.9964 17.0 ±0.3
n-butanol fraction y = 0.6131x 0.4695 0.9988 82.3 ±0.8
dichloromethane fraction - - -
SD: Standard deviation.
Table 4. Total phenolic content of A. atrata and A. millefolium (n= 3; ±SD).
Samples Phenolic Content (mg GAE/g DW ±SD)
A. atrata
ethyl acetate fraction 250 ±2.5
n-butanol fraction 70 ±1.0
dichloromethane fraction -
A. millefolium
ethyl acetate fraction 175 ±1.0
n-butanol fraction 80 ±1.5
dichloromethane fraction -
GAE: Gallic acid equivalents; SD: Standard deviation; DW: Dry weight.
Molecules 2021,26, 1530 7 of 11
3. Discussion
Medicinal plants with their unmatched chemical diversity provide unlimited oppor-
tunities for the discovery of novel plant-based medicinal products [
24
]. According to the
World Health Organization (WHO), more than 80% of the world’s population still rely on
plant-based traditional medicines for primary health care [
14
,
15
]. However, only for a small
proportion of ethnopharmaceutically applied medicinal plants have bioactivity studies
been performed so far. As an example, among the ~250,000 higher plant species worldwide,
only about 5 to 10% have been analyzed with regard to their constituents and associated
bioactivity [
31
]. The utilization of novel natural antioxidants from medicinal plants, which
might help to mitigate oxidative damage by protecting lipids from oxidation and thus
might also be beneficial to human health, is of particular interest [
11
]. In order to meet the
high demand for herbal medicines and to provide evidence for documented ethnobotanical
applications of medicinal plants, the identification of further potential health-promoting
plant species and the exploration of correlations between chemical composition and bioac-
tivity is of utmost importance. To the best of our knowledge, only three studies on the
bioactivity of A. atrata, also considering the identification of individual components, have
been reported so far [
32
34
]. In contrast to these investigations, our studies report for the
first time the characterization of the phenolic compound profile and antioxidant activity of
the alpine species A. atrata, together with a comparison of the respective traits of A. mille-
folium originating from the same habitat, to exclude any impact of edaphic and climatic
factors.
The highest anti-radical capacity of the extracts was found in the ethyl acetate fractions
of both species. The phytochemical comparison of these two Achillea species confirmed the
findings of our previous study [
34
], indicating the differences in the occurrence of major
phenolic components of aerial parts to be marginal. In contrast, the amounts of individual
phenolic compounds appear to be species-specific. Furthermore, the results presented here
confirm previous studies indicating that extraction with polar organic solvents such as ethyl
acetate results in highest phenolic yields, which goes along with the pronounced antiradical
scavenging potential of such extracts [
11
,
13
,
35
]. Upon direct comparison of different
Achillea species under similar extraction conditions, the following ranking of their IC
50
value can be established according to the literature data: A. aucherii (IC
50
= 844
µ
g/mL) [
10
]
>A. kellalensis (IC
50
= 518
µ
g/mL) [
10
] > A. pachycephalla (IC
50
= 248
µ
g/mL [
10
] > A.
biebersteinii Afan. (IC
50
= 89.90
µ
g/mL) [
28
] > A. millefolium (IC
50
= 16.95
µ
g/mL) [our
results] > A. atrata (IC
50
= 12.19
µ
g/mL) [our results] > A. moschata (IC
50
= 3.18
µ
g/mL) [
13
].
Only the very closely related alpine species A. moschata had a higher antioxidant capacity
than A. atrata. This might be due to the fact that in this study, the aerial parts of A. moschata
were collected from higher altitudes of 2400 m a.s.l. (Rhaetian Alps, Italy) [
13
], whereas our
investigated A. atrata plants were harvested at an altitude of 2100 m a.s.l. (Nufenen Pass,
Switzerland). Altitude might be a decisive factor for the accumulation of phenolics in plant
tissues and an increased antioxidant potential to protect against the damaging influence of
UV-B radiation, which increases with altitude [
36
]. On the other hand, the IC
50
values of
all other Achillea species were inferior to that of A. atrata, with the latter exhibiting a 43-fold
higher activity than A. kellalensis extracts [
13
]. This high antioxidant activity of A. atrata is
presumably attributed to the high content of phenolic compounds (250
±
2.5 mg GAE/g
dry weight) compared to A. millefolium (175
±
1.0 mg GAE/g dry weight). Regardless
of the species, the fractionation and subsequent phytochemical characterization revealed
that luteolin, apigenin, nevadensin, and centaureidin were detected exclusively in the
corresponding ethyl acetate fractions. A correlation between the contents of these phenolic
compounds and an increased antimicrobial activity has already been demonstrated in
our previous study [
34
]. It is also conceivable that an increased antioxidant capacity
goes along with the occurrence of these four compounds, which are known as effective
DPPH radical scavengers [
37
39
]. Future studies are required, aiming at the identification
of further antioxidant components in the most potent ethyl acetate fraction of A. atrata.
Moreover, the DPPH assay is a widely used method for determining antioxidant activity,
Molecules 2021,26, 1530 8 of 11
not only of Achillea extracts [
13
,
28
,
40
], thus allowing direct comparison with our results,
but also of other plant extracts. Nevertheless, further
in vitro
assays should be performed
to confirm our first insights into the high antioxidant potential of A. atrata and overcome
the limitations of the DPPH assay.
4. Conclusions
This study reported, for the first time, the pronounced antiradical activity of A. atrata
together with its high phenolic contents compared to A. millefolium. Consequently, A. atrata
with its high antioxidant potential might be an alternative source of natural antioxidants
in pharmaceutical and medicinal applications in the future. In particular, alpine plant
species characterized by high contents of phenolic compounds, which are accumulated in
plant tissues in response to damaging environmental conditions such as UV-B radiation,
might be used as potential future phytopharmaceuticals, being promising alternatives to
synthetic active substances.
5. Materials and Methods
5.1. Plant Material
Both Achillea species analyzed in the present study (i.e., A. atrata and A. millefolium)
were collected at the flowering stage in July 2019, on the Nufenen Pass (Switzerland) at
an altitude of 2100 m a.s.l. Aerial parts of the plants were air-dried at room temperature
and subsequently stored in paper bags until analysis. Both Achillea species were identified
by Dr. phil. Rhinaixa Duque-Thüs (Institute of Botany, Hohenheim University, Stuttgart,
Germany), and voucher specimens were deposited in the herbarium of the Institute of
Botany at Hohenheim University (A. atrata: HOH-022704; A. millefolium: HOH-022706).
5.2. Chemicals
Dichloromethane, acetone, ethyl acetate, and n-butanol for plant material extrac-
tion were purchased from Merck KGaA (Darmstadt, Germany). Acetonitrile (LC-MS
grade), formic acid (98%), and methanol (LC-MS grade) for LC-MS
n
analyses were ob-
tained from Sigma–Aldrich (Steinheim, Germany). Purified water (0.056
µ
S/cm) from
a Purelab Option-Q system (Elga Berkefeld GmbH, Celle, Germany) was used through-
out. The DPPH photometric assay and the quantitation of total phenolic compounds
were performed with 2,2-diphenyl-1-picryl hydrazyl radical (DPPH), Folin–Ciocalteu’s
phenol reagent, and sodium carbonate from Th. Geyer GmbH & Co. KG (Renningen,
Germany). The following reference substances were used: luteolin and 5-caffeoylquinic
acid (chlorogenic acid) from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany);
quercetin-3-O-rutinoside, apigenin, and apigenin-7-O-glucoside from Carl Roth GmbH &
Co. KG (Karlsruhe, Germany); gallic acid monohydrate from Sigma–Aldrich (Steinheim,
Germany); and Trolox from Cayman Chemical (Ann Arbor, MI, USA).
5.3. Extraction and Fractionation of the Plant Material
Fractionation was performed by applying the DPPH spectrophotometric assay and
aimed at the identification of those substances from the complex natural compound mixture
of A. atrata and A. millefolium, which are responsible for antioxidant activity. For this pur-
pose, air-dried aerial parts of the plants (30 g each) were pulverized with a mortar and pestle
for 15 min. Subsequently, the plant material was defatted with 800 mL dichloromethane. To
prevent oxidation during extraction, the suspension was bubbled with nitrogen for 15 min.
After incubation for 24 h at 4
C under light exclusion, the suspensions were filtered over
Celite
®
through a Büchner funnel by vacuum suction. This fraction was further processed
for volatile compounds analysis. For this purpose, dichloromethane was removed by rotary
evaporation to yield 0.44 g of A. millefolium and 0.94 g of A. atrata extract, respectively.
Subsequently, the solid residues were extracted twice with 800 mL acetone/water (8:2; v/v)
for 24 h at 4
C in the dark. The suspensions were again filtered over Celite
®
through
a Büchner funnel by vacuum suction. Acetone was removed from the filtrates in vacuo
Molecules 2021,26, 1530 9 of 11
by rotovaporation at 34
C. The resulting aqueous phases were subsequently extracted
with ethyl acetate (2
×
200 mL) and n-butanol (2
×
200 mL). The respective phases were
evaporated to dryness to yield 0.71 g residue of the ethyl acetate extract from each species
as well as 0.37 g (A. atrata) and 0.71 g (A. millefolium) residue of n-butanol extract [
34
,
41
]. In
parallel to this consecutive extraction with different solvents, 4 g of comminuted A. atrata
and A. millefolium plant material were extracted twice with 80 mL acetone/water (8:2; v/v)
under exclusion of light for 24 h to obtain a total compound extract. The suspensions were
filtered as above-mentioned and evaporated to dryness, resulting in crude extract yields of
0.45 g for A. atrata and 0.45 g for A. millefolium, respectively.
5.4. LC-MSnAnalyses for Phenolic Compound Characterization
The chromatographic separation and identification of phenolic compounds were
performed with an Agilent 1200 HPLC (Agilent, Waldbronn, Germany) system connected
with an HCT ultra ion trap MS detector interfaced with an electrospray ionization (ESI) ion
source (Bruker Daltonik, Bremen, Germany). A binary gradient elution system described
previously was applied and consisted of 0.1% formic acid (v/v; eluent A) and acetonitrile
(eluent B) [
41
]. Chromatographic separation was performed on a Kinetex
®
C18 reversed-
phase column (2.6
µ
m particle size, 150
×
2.1 mm i.d., Phenomenex Ltd., Aschaffenburg,
Germany) at a flow rate of 0.21 mL/min. UV absorption of the column eluates was
recorded at 210, 254, 280, and 366 nm. All experiments were performed in triplicate. The
software Agilent Chemstation (Rev. B.01.03 SR1) (Agilent, Waldbronn, Germany) and
Bruker Daltonik esquire control (Version 6.1) (Bruker Daltonik GmbH, Bremen, Germany)
were used for data acquisition and processing [34,41].
5.5. Gas Chromatography-Mass Spectrometry (GC-MS) Analyses for Volatile Compound
Assessment
GC/MS analyses were carried out using a PerkinElmer Clarus 500 gas chromatograph
(PerkinElmer Inc., Waltham, MA, USA) equipped with split injection (split ratio: 30:1,
injection volume: 1.0
µ
L) coupled to a mass spectrometer. The column used was a Zebron
ZB-5ms capillary column (60 m
×
0.25 mm inner diameter
×
0.25
µ
m film thickness,
5% phenylpolysiloxane, and 95% dimethylpolysiloxane coating; Phenomenex, Torrance,
CA, USA). Helium with a flow rate of 1 mL/min was the carrier gas. The column oven
temperature program was 100–320
C at 4
C per min with a final hold time of 30 min.
The mass spectrometer was run in electron ionization (EI) mode and set at 70 eV. The
software Turbomass version 5.4.2 (PerkinElmer Inc., Waltham, MA, USA) was used for
data acquisition and processing. All experiments were carried out in triplicate. Individual
compounds of the dichloromethane fraction of A. atrata and A. millefolium were assigned
based on their specific MS data as well as retention times in comparison with the NIST MS
database (NIST Mass Spectral Library, NIST2011, V 2.0, PerkinElmer Inc., Waltham, MA,
USA) and reference compounds [34,41].
5.6. 2,2-Diphenyl-Picryl Hydrazyl (DPPH) Spectrophotometric Assay for Assessing Radical
Scavenging Capacity
The 2,2-diphenyl-picryl hydrazyl (DPPH) radical scavenging capacity of the Achillea
fractions was determined according to a protocol of Heinrich et al. [
42
] with some modifi-
cations. In brief, 200
µ
L aliquots of each fraction and of the reference antioxidant Trolox,
at five different concentrations for each extract (ethyl acetate: 2.5–20
µ
g/mL; n-butanol:
20–100
µ
g/mL; dichloromethane: 50–750
µ
g/mL; Trolox: 1.5–12.5
µ
g/mL) were added to
1800
µ
L of DPPH solution (100
µ
M) and incubated at 37
C in the dark. After a reaction
time of 30 min, the decrease in absorbance was measured at 516 nm, using a UV/VIS
spectrophotometer (Lambda 35, Perkin Elmer, Rodgau-Juedesheim, Germany). The ex-
periments were performed in triplicate. IC
50
values, indicating a 50% decrease of DPPH
solution absorbance referred to the blank, were calculated by plotting absorbance at 516
nm against the corresponding concentrations and subsequent regression analyses.
Molecules 2021,26, 1530 10 of 11
5.7. Folin–Ciocalteu Method for Total Phenolics Quantitation
The amount of total phenolic compounds in the Achillea extracts and fractions was
determined by the Folin–Ciocalteu method using the protocol of Giorgi et al. [
9
]. Gallic
acid was used as a reference substance. The quantitation was performed on the basis of
the gallic acid standard calibration curve, and the results were reported as mg gallic acid
equivalents (GAE) per g dry weight. All experiments were carried out in triplicate.
Author Contributions:
Study conception and design: L.S., D.R.K., M.B. and P.L.; Data acquisition:
L.S.; Data analysis and interpretation: L.S.; Drafting of manuscript: L.S. and D.R.K.; Critical revision
throughout the study: D.R.K., F.C.S. and O.S. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in manuscript.
Acknowledgments:
The authors wish to thank Beatrix Waldburger for providing Achillea atrata L.
plant material from Switzerland.
Conflicts of Interest: The authors declare no conflict of interest.
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... Namely, Georgieva et al. determined a chlorogenic acid content of 0.78 mg g −1 DW [12], while Tadić et al. reported content of 0.28 mg g −1 DW [14]. Vitalini et al. reported two DCQA isomers that were not quantified [8], while Salomon et al. confirmed the presence of three DCQA isomers in the acetone/water mixture, N-butanol, and ethanol fractions [51]. ...
Article
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In this study, the extraction efficiency of natural deep eutectic solvents (NADES) based on choline chloride as a hydrogen bond acceptor (HBA) and five different hydrogen bond donors (HBD; lactic acid, 1,4-butanediol, 1,2-propanediol, fructose and urea) was evaluated for the first time for the isolation of valuable bioactive compounds from Achillea millefolium L. The phytochemical profiles of NADES extracts obtained after ultrasound-assisted extraction were evaluated both spectrophotometrically (total phenolic content (TPC) and antioxidant assays) and chromatographically (UHPLC-MS and HPLC-UV). The results were compared with those obtained with 80% ethanol, 80% methanol, and water. The highest TPC value was found in the lactic acid-based NADES (ChCl-LA), which correlated with the highest antioxidant activity determined by the FRAP analysis. On the other hand, the highest antiradical potential against ABTS+• was determined for urea-based NADES. Phenolic acids (chlorogenic acid and dicaffeoylquinic acid isomers), flavones (luteolin and apigenin), and their corresponding glucosides were determined as the dominant individual phenolic compounds in all extracts. The antibacterial and antifungal properties of the extracts obtained against four bacterial cultures and two yeasts were evaluated using two methods: the agar dilution method to obtain the minimum inhibitory concentration (MIC) and the minimum bactericidal or fungicidal concentration (MBC or MFC), and the disc diffusion method. ChCl-LA had the lowest MIC and MBC/MFC with respect to all microorganisms, with an MIC ranging from 0.05 mg mL−1 to 0.8 mg mL−1, while the water extract had the weakest inhibitory activity with MIC and MBC/MFC higher than 3.2 mg mL−1.
... Two flavones bearing a methoxy group attached to C-7 or C-3 were reported once, namely 7-O-methyl apigenin (3) (from A. ligustica) [35] and chrysoeriol (20) (from A. millefolium) [40], respectively. Moreover, other methylated flavonoids, such as cirsiliol (22) and cirsimaritin (4), were reported for A. fragrantissima [31,33]; salvigenin (5) for A. millefolium [41] and A. wilhelmsi [50]; and 8-hydroxysalvigenin (6) was identified for A. lycaonica A. monocephala and A. vermicularis [36,42,49]. Moreover, eupatorin (21) was reported for A. alpina and A. tenuifolia [17,48], while eupatilin 7-methyl ether (5-demethylsinensetin) (23) for A. wilhelmsii [25]. ...
... More precisely, decoctions of this plant were used to treat arthritis, cardiovascular diseases, congestions, gastrointestinal disorders, gout, and malaria, and as a diuretic, anthelmintic, and purgative agent [79]. Antioxidant and antimicrobial effects have been reported for this plant [22,105]. ...
... Moreover, in the folk medicine of the Middle East, this plant is reputed for its antidiabetic properties [31], and more specifically, in the Arabia region, A. fragrantissima is used for the treatment of respiratory diseases and gastrointestinal disturbances [81]. Additionally, cirsiliol (22) isolated from A. fragrantissima caused the relaxation of contracted rats' proximal aortas, tracheas, urinary bladders, and uteruses [106]. Furthermore, the ethanolic extract of the plant was tested for its anti-inflammatory effects on lipopolysaccharide-activated primary cultures of brain microglial cells, suggesting that phytochemicals from this extract could be beneficial in preventing/treating neurodegenerative diseases [81]. ...
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Knowledge within the field of phytochemistry research has accelerated at a tremendous speed. The excess of literature reports featuring plants of high ethnopharmacological importance, in combination with our interest in the Asteraceae family and traditional medicine, led us to acknowledge the value of the Achillea L. genus. In a broad context, the various Achillea species are used around the globe for the prevention and treatment of different diseases, including gastrointestinal problems, haemorrhages, pneumonia, rheumatic pains, diuresis, inflammation, infections, and wounds, as well as menstrual and gynaecologic abnormalities. The present review aims to provide and summarize the recent literature (2011–2021) on the phytochemistry of the Achillea genus. In parallel, this study attempts to bridge the reports on the traditional uses with modern pharmacological data. Research articles that focused on secondary metabolites, traditional uses and pharmacological activities were collected from various scientific databases such as Pubmed, ScienceDirect, Reaxys and Google Scholar. This study revealed the presence of 141 phytochemicals, while 24 traditionally used Achillea spp. were discussed in comparison to current data with an experimental basis.
Thesis
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Phenolic compounds are an important class of plant secondary metabolites which play crucial physiological roles throughout the plant life cycle. Phenolics are produced under optimal and suboptimal conditions in plants and play key roles in developmental processes like cell division, hormonal regulation, photosynthetic activity, nutrient mineralization, and reproduction. Plants exhibit increased synthesis of polyphenols such as phenolic acids and flavonoids under abiotic stress conditions, which help the plant to cope with environmental constraints. Phenylpropanoid biosynthetic pathway is activated under abiotic stress conditions (drought, heavy metal, salinity, high/low temperature, and ultraviolet radiations) resulting in accumulation of various phenolic compounds which, among other roles, have the potential to scavenge harmful reactive oxygen species. Deepening the research focuses on the phenolic responses to abiotic stress is of great interest for the scientific community. In the present article, we discuss the biochemical and molecular mechanisms related to the activation of phenylpropanoid metabolism and we describe phenolic-mediated stress tolerance in plants. An attempt has been made to provide updated and brand-new information about the response of phenolics under a challenging environment.
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Aerial parts of Achillea moschata Wulfen (Asteraceae) growing wild in the Italian Rhaetian Alps were investigated to describe, for the first time, their phenolic content, as well as to characterize the essential oil. Inspection of the metabolic profile combining HPLC-DAD and ESI-MS/MS data showed that the methanol extract contained glycosylated flavonoids with luteolin and apigenin as the main aglycones. Among them, the major compound was 7-O-glucosyl apigenin. Caffeoyl derivates were other phenolics identified. The essential oil obtained by steam distillation and investigated by GC/FID and GC/MS showed camphor, 1,8-cineole, and bornylacetate as the main constituents. The antioxidant capacity of three different extracts with increasing polarity and of the essential oil was evaluated by employing ABTS·+ and DPPH· radical scavenging assays. The methanolic extract was the only significantly effective sample against both synthetic radicals. All samples were also tested against Gram-positive (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa) bacterial species using the disk diffusion assay. The non-polar extracts (dichloromethane and petroleum ether) and the essential oil possessed a broad spectrum of antimicrobial activity expressed according to inhibition zone diameter (8-24 mm).
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Various Achillea species are rich in bioactive compounds and are important medicinal plants in phytotherapy. In the present study, Achillea millefolium L., Achillea moschata Wulfen, and Achillea atrata L. were compared with respect to their phenolic profile and antibacterial activity against gram-positive bacteria strains (Staphylococcus, Propionibacterium). Particular focus was given to A. atrata, which has hardly been studied so far. Based on the metabolite profile, A. atrata exhibited more similarities to A. moschata than to A. millefolium. The former two only differed in the occurrence of four compounds. The flavonols syringetin-3-O-glucoside and mearnsetin-hexoside, not reported for an Achillea species before, have been detected in A. atrata and A. moschata. All Achillea species reduced growth of the tested bacteria. A. atrata demonstrated highest activity against Propionibacterium acnes and Staphylococcus epidermidis, both being involved in the pathogenesis of acne vulgaris. Furthermore, A. atrata has a pronounced anti-methicillin-resistant Staphylococcus aureus potential. Bioassay-guided fractionation revealed that only the most polar fraction of A. moschata displayed antimicrobial activity, which was attributed to phenolics such as apigenin, centaureidin, and nevadensin, being present in high amounts in A. atrata. Thus, this alpine species shows promising antimicrobial activity and might be a potential source for developing novel dermal/topical drugs.
Chapter
Plant Phenolics or polyphenols, the aromatic compounds with one or more hydroxyl groups are produced by plants mainly for protection against stresses. Phenolics are secondary natural metabolites emerging from shikimate/phenylpropanoid pathway or polyketide acetate/malonate pathway, producing monomeric and polymeric phenols and polyphenols, which participate in a wide range of physiological activities in plants. Plants are known to have synthesized thousands of different phenolic compounds throughout the course of evolution to cope up with constantly changing environments. Plants accumulate phenolic compounds in their tissues as an adaptive response to adverse environmental conditions and have a key role in the regulation of various environmental stresses, such as high light, low temperatures, pathogen infection, herbivores, and nutrient deficiency. This chapter details with the biosynthesis, role and regulation of plant phenolics in response to various abiotic stresses.
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Seeds of Hypericum perforatum and H. tetrapterum were extracted with dichloromethane and methanol and investigated by chromatographic and mass spectrometric methods. Both species yielded a fatty oil fraction amounting to 30.5% and 18.0% of the seed weight, respectively. Linoleic acid (C18:2n-6) was shown to be the predominant fatty acid constituent. Moreover, xanthone derivatives, i.e. tetrahydroxyxanthones (THX), xanthone-glycosides and xanthone-sulfonates, were assigned in methanolic extracts. For structure elucidation, one representative xanthone, namely 1,3,6,7-THX, was synthesized and analyzed via HPLC-DAD-MS(n) and GC-MS. Total THX contents were quantitated applying a validated HPLC-DAD method, resulting in 1.25 g/kg (H. perforatum) and 0.27 g/kg (H. tetrapterum), respectively. Moreover, the free radical scavenging capacity of the methanol extracts was tested using the DPPH antioxidant assay. Both, H. perforatum (IC50 = 8.7 mg/L) and 1,3,6,7-THX (IC50 = 3.0 mg/L), exhibited good DPPH free radical scavenging activity compared to Trolox (IC50 = 6.6 mg/L). This article is protected by copyright. All rights reserved.