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Metabolic Profile of Agropyron repens (L.) P. Beauv. Rhizome Herbal Tea by HPLC-PDA-ESI-MS/MS Analysis

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Agropyron repens (L.) P. Beauv. (couch grass) is a world-wide infesting rhizomatous plant with pharmacological applications. Chemical research is focused on its allelopathic and anti-inflammatory components, which are mainly present in the essential oil. Conversely, the aqueous extracts have been sparingly investigated, although the herbal tea is by far the most used formulation. To fill the gap, the metabolic profile of Agropyron repens rhizome herbal tea was investigated by electrospray ionization (ESI) tandem–mass spectrometry (MS/MS); the phenolic profile was investigated by HPLC-PDA-ESI-MS/MS. ESI-MS fingerprinting was provided, evidencing diagnostic ions for saccharides, organic acids and amino acids. The HPLC-PDA-ESI-MS/MS analysis evidenced at least 20 characteristic phenolic compounds, the most representative being caffeoyl and feruloyl quinic esters, followed by coumaric, caffeic and ferulic acids, and hesperidin among flavonoids. In addition, the essential amino acid tryptophan was identified for the first time. The results suggest new perspectives of applications for Agropyron repens rhizome.
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Citation: Bortolami, M.; Di Matteo, P.;
Rocco, D.; Feroci, M.; Petrucci, R.
Metabolic Profile of Agropyron repens
(L.) P. Beauv. Rhizome Herbal Tea by
HPLC-PDA-ESI-MS/MS Analysis.
Molecules 2022,27, 4962. https://
doi.org/10.3390/molecules27154962
Academic Editor: Mercedes Bonfill
Received: 19 July 2022
Accepted: 31 July 2022
Published: 4 August 2022
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molecules
Article
Metabolic Profile of Agropyron repens (L.) P. Beauv. Rhizome
Herbal Tea by HPLC-PDA-ESI-MS/MS Analysis
Martina Bortolami 1, Paola Di Matteo 1, Daniele Rocco 2, Marta Feroci 1, * and Rita Petrucci 1, *
1Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome,
Via del Castro Laurenziano 7, 00161 Rome, Italy
2Department of Ingegneria Meccanica ed Aerospaziale, Sapienza University of Rome, Via Eudossiana 18,
00184 Rome, Italy
*
Correspondence: marta.feroci@uniroma1.it (M.F.); rita.petrucci@uniroma1.it (R.P.); Tel.: +39-649766736 (R.P.)
Abstract:
Agropyron repens (L.) P. Beauv. (couch grass) is a world-wide infesting rhizomatous
plant with pharmacological applications. Chemical research is focused on its allelopathic and anti-
inflammatory components, which are mainly present in the essential oil. Conversely, the aqueous
extracts have been sparingly investigated, although the herbal tea is by far the most used formulation.
To fill the gap, the metabolic profile of Agropyron repens rhizome herbal tea was investigated by
electrospray ionization (ESI) tandem–mass spectrometry (MS/MS); the phenolic profile was investi-
gated by HPLC-PDA-ESI-MS/MS. ESI-MS fingerprinting was provided, evidencing diagnostic ions
for saccharides, organic acids and amino acids. The HPLC-PDA-ESI-MS/MS analysis evidenced
at least 20 characteristic phenolic compounds, the most representative being caffeoyl and feruloyl
quinic esters, followed by coumaric, caffeic and ferulic acids, and hesperidin among flavonoids. In
addition, the essential amino acid tryptophan was identified for the first time. The results suggest
new perspectives of applications for Agropyron repens rhizome.
Keywords:
tryptophan; phenolic acids; flavonoids; antioxidants; ESI-MS fingerprinting; saccharides;
amino acids; small organic acids
1. Introduction
Agropyron repens (L.) P. Beauv. or Elymus repens (L.) Gould (couch grass, quackgrass)
is a well-known perennial rhizomatous plant, native from Europe and Central Asia, but
widespread in the world. Its notoriety is mainly due to its infesting action in crops [
1
]
and to a quite minor extent of its use as a traditional medicinal plant for urinary prob-
lems [
2
4
]. The infesting action is due to its allelopathic effects [
5
] on crops. It is an
aggressive competitor for many plants. Moreover, allelopathic toxins are produced from
plant decaying residues. However, the traditional medicinal use in urinary calculus dis-
ease has received scientific confirmation, including for its other pharmacological effects [
6
].
Among the pharmacological actions, there are the hypoglycemic [
7
], hypolipidemic [
8
], anti-
inflammatory [
9
,
10
], and antidiabetic activities [
11
], effects on motility [
12
], and beneficial
effects in urinary tract infections [13].
For these reasons, the chemical research has mainly focused on the allelopathic com-
pounds and on the anti-inflammatory molecules, primarily found in the essential oil
(volatiles, 0.01–0.05% of the plant mass by distillation). In particular, agropyren (possibly
1-phenylhex-2-en-4-yne, C
12
H
12
) was thought to be one of the active molecules (and thus
named after Agropyron repens) [
14
], but its presence in the essential oil was not confirmed
by later studies [
15
,
16
]. Aqueous extracts were rarely characterized, and seldom a quantita-
tion was reported, while organic extracts as well as alcoholic and hydro-alcoholic extracts
were characterized, and in some cases, a quantitation of active compounds was carried
out [
17
28
]. Early reports failed in the identification of allelopathic compounds [
17
,
18
],
while Weston in 1987 identified tricin (5,7,4
0
-trihydroxy-3
0
,5
0
-dimethoxyflavone) as an
Molecules 2022,27, 4962. https://doi.org/10.3390/molecules27154962 https://www.mdpi.com/journal/molecules
Molecules 2022,27, 4962 2 of 19
allelopathic molecule in water extracts [
19
]. Later, other allelopathic molecules were identi-
fied: 6-hydroxy-1,2,3,4-tetrahydro-
β
-carboline-3-carboxylic acid (6-HT
β
C-3-COOH, toxic
towards slug species) from hydro-alcoholic extracts [
20
], (E)- and (Z)-16-hydroxyhexadecyl
esters of p-hydroxycinnamic acid (mono- and diesters) from organic extracts [
21
,
22
], also
with antiadhesive activity against uropathogenic E. coli [
23
], 5-n-alkylresorcinols from
acetone extracts [24], 3-indoleacetic acid (IAA, plant growth hormone and inhibitor) from
hydro-alcoholic extracts [
25
], 2,4-dihydroxy-2H-1,4-benzoxazin-3-one (DIBOA, phytotoxin),
2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one (DIMBOA, phytotoxin) and 2-hydroxy-
1,4-benzoxazin-3-one (HBOA, phytotoxin) from water extracts [
26
,
27
], 5-hydroxyindole-3-
acetic acid (5-HIAA, growth hormone) and 5-hydroxytryptophan (5-HTP, growth inhibitor)
from alcoholic extracts [
28
]. 5-HTP is a natural molecule, also used as a drug [
29
], of
particular importance for humans. It is obtained from tryptophan (TP) by enzymatic
reaction, and its decarboxylation produces serotonin (5-hydroxytryptamine, 5-HT), an
important neurotransmitter. Moreover, 5-HT can be transformed into melatonin (N-acetyl-
5-methoxytryptamine), an important hormone.
Tryptophan is one of the essential amino acids that cannot be synthesized by humans
and must be supplied with food. Besides its role of an amino acid in peptide synthesis
and its importance as a precursor of 5-HTP and derived compounds, it is a precursor of
niacin (vitamin B3) and auxins [
30
,
31
]. Tryptophan is present in various vegetals (soy, rice,
corn, cotton, etc.) [
32
], but no paper reports the presence of tryptophan in Agropyron repens
rhyzome (to the best of our knowledge).
The Agropyron repens components were recently reviewed by the European Medicine
Agency [
33
], and the literature data are summarized in Table 1[
33
,
34
], along with the
relevant bibliography [5,6,10,11,16,19,3541].
There is thus a lack of information on the real composition of Agropyron repens herbal
tea, the most used formulation in traditional medicine and herbalism. In the use of the infu-
sion for therapeutic purposes, not only are the active ingredients ingested, but everything
that from the dry rhizome is extracted with water.
For this reason, since the herbal tea sees only water as a solvent, we focused on Agropy-
ron repens rhizome aqueous extracts at three different temperatures: 25 (room temperature),
50 and 100
C (boiling water, herbal tea normal infusion). We decided to investigate 25 and
50 C extracts in order to understand the role of temperature on extract constituents.
Table 1. Chemical constituents of Agropyron repens rhizome extracts (literature data) [33,34].
Class Compounds Quantitation Extraction
Solvent Ref.
Polysaccharides
fructosan (triticin), inulin ~12% water [10,35]
Disaccharides
not identified water [35]
Monosaccharides
fructose, glucose, rhamnose (17.1, 3.4, nd%) water [10,35]
Sugar
alcohols mannitol, inositol nd nr [6,36]
Phenolic
acids,
free (and
bound)
chlorogenic acid nd nr [10]
p-coumaric acid 87 (24) µg/g water [37]
p-hydroxybenzoic acid 10 (55) µg/g water [37]
vanillic acid 27 (11) µg/g water [37]
ferulic acid 38 (12) µg/g water [37]
vanillin 4 (17) µg/g water [37]
p-hydroxybenzaldehyde tr (4) µg/g water [37]
Flavonoids
tricin
nd nr [10,19,38]
rutin
hyperoside
baicalein
quercetin
luteolin
Molecules 2022,27, 4962 3 of 19
Table 1. Cont.
Class Compounds Quantitation Extraction
Solvent Ref.
Tannins 5% water [10]
Antraquinones
emodin 0.06–0.2 µg/g
acetonitrile/
water [39]
chrysophanol 0.05–0.2 µg/g
physcion 0.08–0.3 µg/g
Si silicon species 0.4% water [40]
Main
Volatiles
palmitic acid
0.05% steam dis-
tillation [6,16]
carvacrol
trans-anethole
carvone
thymol
menthol
menthone
Vitamins ascorbic acid nd nr [10]
β-carotene
Organic
acids
acetic acid
nd water [10,41]
propionic acid
butyric acid
hexanoic acid
phenylacetic acid
succinic acid
cinnamic acid
p-hydroxyphenylpropionic acid
p-coumaric acid
3,4-dihydroxyphenylpropionic acid
Total polyphenols contents 257.95 µgGAE/mL
ethanol/water
[11]
Total flavonoid contents 70.89 µgRE/mL
ethanol/water
[11]
Antioxidant activity ~35% (DPPH)
ethanol/water
[11]
~60% (reducing power)
ethanol/water
[11]
nd: not determined; nr: not reported; GAE: gallic acid equivalent; RE: rutin equivalent; DPPH: 2,2-diphenyl-1-
picrylhydrazyl; reducing power: with respect to Fe3+ to Fe2+.
2. Results and Discussion
Three independent commercial samples of Agropyron repens rhizome, named AR1, AR2,
AR3, were used to prepare the herbal tea in Milli-Q water brought to a boil, according to the
dose and procedure recommended by the Official Pharmacopoeia of the Italian Republic,
as reported below in Materials and Methods, with the aim to evaluate the metabolic profile
and the individual phenolic compounds content that consumers may take with a cup of
tisane. The rhizome samples were also extracted with water at room temperature (25
C)
and at 50
C to evaluate the effect of temperature. The extracts were first analyzed by
tandem mass spectrometry (MS/MS) by direct infusion into the ESI source, with the aim to
explore the matrix as a whole.
2.1. Untargeted ESI-MS/MS Profile
The untargeted analysis of the extracts evidenced quite similar profiles, regardless
of type of sample and extraction temperature. The ESI-MS/MS metabolic profile of the
Agropyron repens rhizome herbal tea is shown in Figure 1, in both ESI- (a) and ESI+ (b), in
which the profiles of AR1, AR2 and AR3, from bottom to top, respectively, were reported.
No signals attributable to phenolic compounds were evidenced. However, the phenolic
fraction in natural sources is generally low compared to other fractions such as organic
acids or sugars; therefore, the phenolic signals might be expected to be hidden by the others
in the direct infusion experiments. Conversely, three sets of well-evidenced signals were
ascribed to saccharides, small organic acids and amino acids, based on m/zvalues relative to
deprotonated/protonated ions [M-H]
/[M+H]
+
, sodium, potassium and chloride adducts
[M+Na]
+
, [M+K]
+
and [M+Cl]
, respectively, and fragmentation spectra, obtained in
daughter scans by selecting each precursor ion.
Molecules 2022,27, 4962 4 of 19
Molecules 2022, 27, x FOR PEER REVIEW 4 of 21
2.1. Untargeted ESI-MS/MS Profile
The untargeted analysis of the extracts evidenced quite similar profiles, regardless of
type of sample and extraction temperature. The ESI-MS/MS metabolic profile of the Ag-
ropyron repens rhizome herbal tea is shown in Figure 1, in both ESI- (a) and ESI+ (b), in
which the profiles of AR1, AR2 and AR3, from bottom to top, respectively, were reported.
Figure 1. ESI-MS/MS profile of rhizome Agropyron repens herbal tea, samples AR1, AR2, AR3, in
negative ionization ESI- (a) and positive ionization ESI+ (b), with signals normalized to the highest
one for a direct comparison among samples. (a) Pyr ac (pyruvic acid, [M-H]
= 87 m/z); Lac ac (lactic
acid, [M-H]
= 89 m/z); Pho ac (phosphoric acid, [M-H]
= 97 m/z); Fur ac (2-furoic acid, [M-H]
= 111
m/z); Fum ac (fumaric acid, [M-H]
= 115 m/z); Mal ac (malic acid, [M-H]
= 133 m/z); cis-Aco ac
(cis-aconitic acid, [M-H]
= 173 m/z); hex (hexose, [M-H]
= 179 m/z); Cit ac (citric acid, [M-H]
= 191
m/z); di-hex (di-hexose, [M-H]
= 341 m/z, [M+Cl]
= 377 m/z, [M+Cl+2]
= 379 m/z); di-hex-pen
(di-hexose-pentose, [M-H]
= 473 m/z). (b) GABA (γ-aminobutyric acid, [M+H]
+
= 104 m/z); Pro
(proline, [M+H]
+
= 116 m/z); Val (valine, [M+H]
+
= 118 m/z); Asn (asparagine, [M+H]
+
= 133 m/z); His
(histidine, [M+H]
+
= 156 m/z); Arg (arginine, [M+H]
+
= 175 m/z); hex ([M+Na]
+
= 203 m/z, [M+K]
+
=
219 m/z); di-hex ([M+Na]
+
= 365 m/z, [M+K]
+
= 381 m/z); di-hex-pen ([M+H]
+
= 475 m/z, [M+Na]
+
= 497
m/z, [M+K]
+
= 513 m/z).
No signals attributable to phenolic compounds were evidenced. However, the
phenolic fraction in natural sources is generally low compared to other fractions such as
organic acids or sugars; therefore, the phenolic signals might be expected to be hidden by
the others in the direct infusion experiments. Conversely, three sets of well-evidenced
signals were ascribed to saccharides, small organic acids and amino acids, based on m/z
Figure 1.
ESI-MS/MS profile of rhizome Agropyron repens herbal tea, samples AR1, AR2, AR3,
in negative ionization ESI- (
a
) and positive ionization ESI+ (
b
), with signals normalized to the
highest one for a direct comparison among samples. (
a
) Pyr ac (pyruvic acid,
[M-H]= 87 m/z)
;
Lac ac (lactic acid, [M-H]
= 89 m/z); Pho ac (phosphoric acid, [M-H]
= 97 m/z); Fur ac
(2-furoic acid,
[M-H]= 111 m/z)
; Fum ac (fumaric acid, [M-H]
= 115 m/z); Mal ac (malic
acid,
[M-H]= 133 m/z)
;cis-Aco ac (cis-aconitic acid, [M-H]
= 173 m/z); hex (hexose, [M-
H]
= 179 m/z); Cit ac (citric acid,
[M-H]= 191 m/z)
; di-hex (di-hexose, [M-H]
= 341 m/z,
[M+Cl]
= 377 m/z,
[M+Cl+2]= 379 m/z)
; di-hex-pen (di-hexose-pentose, [M-H]
= 473 m/z).
(
b
) GABA (
γ
-aminobutyric acid,
[M+H]+= 104 m/z)
; Pro (proline, [M+H]
+
= 116 m/z); Val (va-
line,
[M+H]+= 118 m/z)
; Asn (asparagine, [M+H]
+
= 133 m/z); His (histidine,
[M+H]+= 156 m/z)
;
Arg (arginine, [M+H]
+
= 175 m/z); hex ([M+Na]
+
= 203 m/z, [M+K]
+
= 219 m/z); di-hex
([M+Na]+= 365 m/z
, [M+K]
+
= 381 m/z); di-hex-pen
([M+H]+= 475 m/z
, [M+Na]
+
= 497 m/z,
[M+K]+= 513 m/z).
2.1.1. Saccharides
A di-hexose C
12
H
22
O
11
with M = 342 Da was suggested by signals in both ESI- and
ESI+; (a) and (b) in Figure 1, respectively. The assignment was supported by the ESI-
signals at m/z341, 377 and 379, relative to the anions [M-H]
, [M+Cl]
and [M+Cl+2]
,
respectively, and by the ESI+ signals at m/z365 and 381, relative to [M+Na]
+
and [M+K]
+
,
respectively, in good agreement with data reported in the literature for the di-hexoses
maltose [
42
,
43
], and sucrose [
44
]. The ESI- fragmentation spectrum of [M-H]
= 341 m/z
evidenced fragments at m/z179 and 161, deriving from the scission of the glycoside bond
into the hexose (hex) units, and 149, 119, 89 and 71 as hexose fragments, in agreement with
Molecules 2022,27, 4962 5 of 19
those reported for D-fructose [
45
]. The ESI+ fragmentation spectrum of
[M+K]+= 381 m/z
evidenced fragments at m/z219 and 201, corresponding to [M(hex)+K]
+
and [M(hex)+K-
H
2
O]
+
, respectively; analogously, the fragmentation spectrum of [M+Na]
+
= 365 m/z
evidenced the ions [M(hex)+Na]
+
and [M(hex)+Na-H
2
O]
+
at m/z203 and 185, respectively.
These data agree with literature for sucrose [44].
A di-hexose–pentose trisaccharide C
17
H
30
O
15
with M = 474 Da was suggested by
signals in both ESI- and ESI+; (a) and (b) in Figure 1, respectively. The assignment was
supported by signals at m/z473, 475, 497 and 513, relative to the ions [M-H]
, [M+H]
+
,
[M+Na]
+
and [M+K]
+
, respectively. The ESI- fragmentation spectrum of [M-H]
= 473 m/z
evidenced fragments at m/z341 and 131, in agreement with the glycoside bond scission
leading to the disaccharide discussed above and a pentose residual unit (
= 132 Da) [
45
,
46
].
Lastly, the presence of free hexoses with M = 180 Da was suggested by signals in both
ESI- and ESI+; (a) and (b) in Figure 1, respectively. The assignment was supported by
the signal [M-H]
= 179 m/z, giving the same fragments discussed above when it was
selected as a precursor ion, and signals at m/z203 and 219, relative to [M+Na]
+
and [M+K]
+
,
respectively, reported in the literature for glucose [47].
The di-hexose and free hexoses were well evidenced in all samples, with a different
distribution: the hexose/di-hexose signal ratio was in the order AR2 < AR1 < AR3. The di-
hexose–pentose was well characterized, albeit weak, in AR2, while a low signal was evidenced
in AR1 and AR3. Since a similar profile was observed for extracts at room temperature (25
C),
these mono-, di- and tri-saccharides do not derive from any hydrolysis due to temperature,
but they must be present as such in the dry rhizomes. Differences among AR1, AR2 and
AR3 might be due to different geographical origin, collection period, drying process and/or
storage of the rhizomes, information not available to consumers. Slight differences among the
samples were also observed regarding humidity, water activity and microbiological analysis,
important data regarding food safety and quality. As shown in Table 2, all samples were below
the threshold values recommended by the World Health Organization (
TMC < 107CFU/g
and Y/M < 10
5
cfu/g) [
48
50
]. Differences in the TAC values could be attributed to the
microbial contamination that may occur during the product preparation from the harvesting
to packaging, handling, dispensing and storage. The microbiological data were in agreement
with water activity results, shown in Table 2, that were below the limit for all the samples.
Water activity indicates the unbound water available to microorganisms for their growth:
high values of water activity (0.90) inhibit the growth of most microorganisms: for yeasts and
molds and for all microorganisms, the limit values are 0.70 and 0.60, respectively.
Table 2.
Plant material characterization: absolute humidity (Abs. H); active water at 21.5
C (aw);
total aerobic count (TAC); yeast and molds (Y&M); lactic acid bacteria (LAB); Enterobacteriaceae (EB);
CFU: colony-forming unit.
Sample Abs. H
(g/m3)
aw
(21.5 C)
TAC
(CFU/g)
Y&M
(CFU/g)
LAB
(CFU/g)
EB
(CFU/g)
AR1 9.53 0.495 ±0.001 1.41 ±0.15 ×10318.0 ±0.10 <10 <10
AR2 10.60 0.545 ±0.001 3.05 ±0.20 ×10310.0 ±0.20 <10 <10
AR3 8.63 0.485 ±0.001 3.83 ±0.50 ×10322.00 ±0.14 <10 <10
Compared with the literature, the mono-, di- and tri-saccharides described above
might be free fructose and glucose, sucrose and di-hexose-xylose, respectively, taking into
account what is reported in the literature for triticin, the Agropyron repens characteristic
polysaccharide fructosan [
33
]. Triticin is mainly composed of fructose with terminal glucose
residue linked as a sucrose unit, at least according to the hydrolysis products reported in
hot or boiling water [
35
]; furthermore, fructose, glucose and a disaccharide, hydrolizing
mainly to glucose and fructose with xylose, were reported for aqueous alcohol extracts [
35
].
Fructose has the lowest glycemic index compared with some widespread carbohydrate-
containing foods [
51
]. A further dedicated analysis is needed for definitively elucidating
the structures of the di-hexose and the di-hexose-pentose.
Molecules 2022,27, 4962 6 of 19
2.1.2. Small Organic Acids
Among the ESI- signals at low mass values, eight small organic acids were suggested
by the ESI- signals at m/z87, 89, 97, 111, 115, 133, 173 and 191, likely corresponding to
the deprotonated forms [M-H]
of pyruvic, lactic, phosphoric, 2-furoic, fumaric, malic, cis-
aconitic and citric acids, respectively (Figure 1a). The assignments were supported by the
absence of the corresponding [M+H]
+
or adducts in ESI+ (Figure 1b), and by fragmentation
spectra obtained in daughter scans by selecting the precursor ion [M-H]
, besides a good
agreement with the literature [5256]. Data are shown in Table 3.
Table 3.
Fragmentation data obtained by negative ESI-MS/MS infusion experiments in daughter
scans for the precursor ions assigned to pyruvic (Pyr ac), lactic (Lac ac), phosphoric (Pho ac), 2-furoic
(Fur ac), fumaric (Fum ac), malic (Mal ac), cis-aconitic (cis-Aco ac) and citric (Cit ac) acids.
Organic
Acid
Precursor [M-H]
(m/z)
Fragments
(m/z)Ref.
Pyr ac 87 43 [M-H-CO2][52]
Lac ac 89 71 [M-H-H2O]; 43 [M-H-HCO2H][52]
Pho ac 97 79 [M-H2O][53]
Fur ac 111 67 [M-H-CO2]; 41 [M-H-CO2-C2H2][54]
Fum ac 115 71 [M-H-CO2][55]
Mal ac 133 115 [M-H-H2O]; 89 [M-H-CO2];
71 [M-H-CO2-H2O][55]
cis-Aco ac 173 111 [M-CO2-H2O][56]
Cit ac 191 129 [M-H-CO2-H2O]; 111
[M-H-CO2-2H2O][55]
Phosphoric, fumaric and malic acids were prevalent in all samples; pyruvic and lactic
acids were differently distributed in the samples; 2-furoic acid was more abundant in
AR2; cis-aconitic acid was found only in AR2. To the best of our knowledge, among
these, only maleic and citric acids were reported in ethyl acetate extracts from Agropyron
repens shoot and root exudates [
6
], along with trans-aconitic acid, for which a different
fragmentation spectrum with respect to cis-aconitic acid discussed above was reported in
the literature [57].
2.1.3. Amino Acids
Among the ESI+ signals at low mass values, six amino acids were suggested by the ESI+
signals at m/z104, 116, 118, 133, 156 and 175, likely corresponding to the protonated forms
[M+H]
+
of
γ
-aminobutyric acid (GABA), proline, valine, asparagine, histidine and arginine
(Figure 1b). The assignments were supported by the absence of the corresponding anions
[M-H]
in ESI- (Figure 1a) and by fragmentation spectra, obtained in daughter scans by
selecting each precursor ion [M+H]
+
, besides a good agreement with the literature [
58
60
].
Data are shown in Table 4.
Table 4.
Fragmentation data obtained by positive ESI-MS/MS infusion experiments in daughter
scans for the precursor ions assigned to
γ
-aminobutyric acid (GABA), proline (Pro), valine (Val),
asparagine (Asn), histidine (His) and arginine (Arg).
Amino
Acid
Precursor [M+H]+
(m/z)
Fragments
(m/z)Ref.
GABA 104 87 [58]
69 [59]
Pro 116 70 [C4H8N]+[60]
Val 118 72 [M+H-HCO2H]+[60]
Asn 133 87 [M+H-HCO2H]+; 74 [NH2=CHCO2H]+[60]
His 156 110 [M+H-HCO2H]+[60]
Arg 175 116 [M+H-NH2C(NH)=NH2]+; 70 [C4H8N]+[60]
Molecules 2022,27, 4962 7 of 19
GABA and proline were prevalent in all samples. Valine was better evidenced in
AR1 and AR3, while AR2 was in general richer in asparagine, histidine and arginine.
Differences in amino acids composition might be ascribed to different collection periods
and/or storage [60].
All masses discussed above (saccharides, small organic acids, amino acids) were
evidenced as not retained compounds in the full scan chromatogram by HPLC-ESI-MS/MS
analysis, within the first 2–3 min elution, in agreement with the high polarity of compounds
such as sugars, small organic acids and amino acids.
On the whole, a similar behavior was observed for the three samples AR1, AR2
and AR3 with regard to extraction temperature. Extraction time being equal (2 h), slight
differences were observed between 25 and 50
C: a weak increase to no effect was observed
on sugar and amino acid signals at 50
C, and a slight increase was observed for organic
acid signals at 50
C. Since weak differences were observed regarding the sugar signals
in the herbal tea (100
C, 10 min extraction), with respect to 25 and 50
C extracts, the
extraction time seemed to affect the sugar extraction more than temperature. Conversely, a
strong increase was generally observed regarding organic acids signals, and a decrease was
observed for amino acid signals, mainly for GABA. Temperature therefore seemed to affect
the extraction of organic acids and amino acids more than extraction time.
The ESI mass spectral fingerprinting of Agropyron repens rhizome herbal tea is here
provided for the first time, at least to the best of our knowledge, with useful applica-
tion as fast and simple quality control or fraud (the three independent samples showed
similar profiles).
2.2. HPLC-PDA-ESI-MS/MS Targeted Analysis
The HPLC-PDA chromatograms of the aqueous extracts results were quite similar,
regardless of the type of sample and extraction temperature, at least from a qualitative
point of view. A few abundant peaks were evidenced at low elution times, within the
first 18 min, with characteristic absorption at
λ
322–325 nm, and one peak was observed
with characteristic absorption at 279 nm. The PDA chromatogram of AR2 herbal tea, as an
example, is shown in Figure 2a, along with the extracted chromatograms (EC) at 324 nm (b)
and 279 nm (c).
Molecules 2022, 27, x FOR PEER REVIEW 8 of 21
Figure 2. AR2 herbal tea diluted 1:10 with mobile phase: (a) PDA chromatogram; (b) EC at λ = 324
nm; (c) EC at λ = 279 nm. TP: tryptophan; 5-CQA: 5-caffeoylquinic acid; 3-FQA: 3-feruloylquinic
acid; CA: caffeic acid; 4-FQA: 4-feruloylquinic acid; CouA: coumaric acid; FA: ferulic acid.
The absorptions at 322–325 nm are characteristic of hydroxycinnamic acids (HCAs)
and their quinic esters (CQAs) that were confirmed by the targeted analysis of phenolic
compounds (vide infra), while the peak absorbing at 279 nm and retention time tR = 5.51
min were identified as tryptophan (TP) by comparing retention time tR, UV–Vis spec-
trum, ESI+, ESI- and fragmentation spectra with a standard sample. Although TP has
been found in various vegetals [32], no paper reports its presence in Agropyron repens
rhizome, at least to the best of our knowledge. Since it was found in all samples including
extracts at room temperature, the possibility that it might have been generated from any
hydrolytic process seemed to be excluded. TP is the precursor of various bioactive com-
pounds, as growth hormones and allelopathic molecules, i.e., IAA, 5-HIAA, 5-HTP,
TβC-3-COOH and 6-HTβC-3-COOH (see Scheme 1), were reported mainly in alcoholic
extracts of Agropyron repens rhizome [20,25,28]. The presence of these compounds in the
herbal tea was investigated by selecting the corresponding m/z monoisotopic values for
the ions [M-H] in dedicated chromatographic runs: 174 for IAA, 190 for 5-HIAA, 219 for
5-HTP, 215 for TβC-3-COOH, and 231 for 6-HTβC-3-COOH.
No signal was evidenced in the SIR channel 219; therefore, the presence of 5-HTP
was excluded. Conversely, peaks evidenced in the SIR channels 174, 190, 215 and 231
might support the presence of IAA, 5-HIAA, TβC-3-COOH and 6-HTβC-3-COOH, alt-
hough in low amounts, as shown in Figure 3ad, from bottom to top, respectively, in
which AR3 herbal tea SIR (selected ion recording) chromatograms are reported as an
example.
5-HIAA seemed to be further supported by the fragment [M(5-HIAA)-H-16] = 174
m/z evidenced in the SIR channel 174 (Figure 3a). The elution order, 7.70 min (5-HIAA),
11.71 min (IAA), 21.69 min (6-HTβC-3-COOH) and 29.97 min (TβC-3-COOH), was con-
sistent with the structures (see Scheme 1), but it is obvious that further investigation with
comparing standards will be needed to confirm their presence. However, no interference
of these compounds on human health has been reported in the literature, at least to the
best of our knowledge [20].
Considering that TP is an essential amino acid and precursor of key biomolecules
important for humans, such as 5-HTP and serotonin (5-HT), new perspectives of appli-
cations might arise for Agropyron repens rhizome.
Figure 2.
AR2 herbal tea diluted 1:10 with mobile phase: (
a
) PDA chromatogram; (
b
) EC at
λ= 324 nm
;
(
c
) EC at
λ
= 279 nm. TP: tryptophan; 5-CQA: 5-caffeoylquinic acid; 3-FQA: 3-feruloylquinic acid; CA:
caffeic acid; 4-FQA: 4-feruloylquinic acid; CouA: coumaric acid; FA: ferulic acid.
The absorptions at 322–325 nm are characteristic of hydroxycinnamic acids (HCAs)
and their quinic esters (CQAs) that were confirmed by the targeted analysis of phenolic com-
pounds (vide infra), while the peak absorbing at 279 nm and retention time
tR= 5.51 min
were identified as tryptophan (TP) by comparing retention time t
R
, UV–Vis spectrum, ESI+,
Molecules 2022,27, 4962 8 of 19
ESI- and fragmentation spectra with a standard sample. Although TP has been found
in various vegetals [
32
], no paper reports its presence in Agropyron repens rhizome, at
least to the best of our knowledge. Since it was found in all samples including extracts at
room temperature, the possibility that it might have been generated from any hydrolytic
process seemed to be excluded. TP is the precursor of various bioactive compounds, as
growth hormones and allelopathic molecules, i.e., IAA, 5-HIAA, 5-HTP, T
β
C-3-COOH and
6-HT
β
C-3-COOH (see Scheme 1), were reported mainly in alcoholic extracts of Agropyron
repens rhizome [
20
,
25
,
28
]. The presence of these compounds in the herbal tea was inves-
tigated by selecting the corresponding m/zmonoisotopic values for the ions [M-H]
in
dedicated chromatographic runs: 174 for IAA, 190 for 5-HIAA, 219 for 5-HTP, 215 for
TβC-3-COOH, and 231 for 6-HTβC-3-COOH.
No signal was evidenced in the SIR channel 219; therefore, the presence of 5-HTP was
excluded. Conversely, peaks evidenced in the SIR channels 174, 190, 215 and 231 might
support the presence of IAA, 5-HIAA, T
β
C-3-COOH and 6-HT
β
C-3-COOH, although in
low amounts, as shown in Figure 3a–d, from bottom to top, respectively, in which AR3
herbal tea SIR (selected ion recording) chromatograms are reported as an example.
5-HIAA seemed to be further supported by the fragment
[M(5-HIAA)-H-16]= 174 m/z
evidenced in the SIR channel 174 (Figure 3a). The elution order, 7.70 min (5-HIAA),
11.71 min
(IAA), 21.69 min (6-HT
β
C-3-COOH) and 29.97 min (T
β
C-3-COOH), was con-
sistent with the structures (see Scheme 1), but it is obvious that further investigation with
comparing standards will be needed to confirm their presence. However, no interference of
these compounds on human health has been reported in the literature, at least to the best of
our knowledge [20].
Considering that TP is an essential amino acid and precursor of key biomolecules im-
portant for humans, such as 5-HTP and serotonin (5-HT), new perspectives of applications
might arise for Agropyron repens rhizome.
Molecules 2022, 27, x FOR PEER REVIEW 9 of 21
Scheme 1. Biosynthetic pathways of growth hormones and allelopathic molecules starting from
tryptophan (TP).
Some of the other chromatographic peaks evidenced in Figure 2 were assigned, and
quantitated in most cases, by HPLC-ESI-MS/MS SIR mode analysis, carried out with a
method previously developed [43,61,62] and briefly described in Materials and Methods,
for the identification and quantitation of 26 phenolic compounds including ten hy-
droxybenzoic acids (HBAs), four hydroxycinnamic acids (HCAs), two quinic esters of
caffeic acid (CQAs), eight flavonoids and two prenylflavonoids (Fs). In detail, HBAs:
p-hydroxybenzoic acid (p-HBA), m-hydroxybenzoic acid (m-HBA), o-hydroxybenzoic
acid (salicylic acid, SaA), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,4-dihydroxybenzoic
acid (protocatechuic acid, PCA), 2,5-dihydroxybenzoic acid (gentisic acid, GeA),
2,6-dihydroxybenzoic acid (2,6-DHBA), 3,4,5-trihydroxybenzoic acid (gallic acid, GA),
vanillic acid (VA), syringic acid (SyA) (structures in Scheme 2); HCAs: caffeic acid (CA),
p-coumaric acid (CouA), sinapic acid (SiA), ferulic acid (FA) (structures in Scheme 3);
CQAs: 4-caffeoylquinic acid (4-CQA), 5-caffeoylquinic acid (5-CQA) (structures in
Scheme 4); Fs: catechin (Ca), quercetin (Q), kampferol (K), rutin (Ru), myricitrin (My),
quercetin-3-O-glucoside (Q-3-G), kaempferol-3-O-rutinoside (K-3-R), hesperidin (He),
isoxanthohumol (i-XH) (structures in Scheme 5) and xanthohumol (XH) (structure in
Scheme 4).
TP was quantitated with the same chromatographic run by using the calibration
curve y = 34379x 1228.3, R
2
= 0.9995, obtained as described in Materials and Methods,
with LOD = 2.64 μg/mL and LOQ = 8.00 μg/mL.
In addition, ellagic acid (EA) (structure in Scheme 5) was identified by comparison
with standard and was quantitated as Q content by using the quercetin calibration curve
[43].
Lastly, 3-feruloylquinic acid (3-FQA) and 4-feruloylquinic acid (4-FQA) (structures
in Scheme 4) were tentatively assigned, supported by chromatographic data, UV–Vis
spectral data and mass fragmentation spectra, in strong agreement with the literature
[63], and quantitated as FA content by using the ferulic acid calibration curve [43].
Scheme 1.
Biosynthetic pathways of growth hormones and allelopathic molecules starting from
tryptophan (TP).
Molecules 2022,27, 4962 9 of 19
Some of the other chromatographic peaks evidenced in Figure 2were assigned, and
quantitated in most cases, by HPLC-ESI-MS/MS SIR mode analysis, carried out with a
method previously developed [
43
,
61
,
62
] and briefly described in Materials and Methods, for
the identification and quantitation of 26 phenolic compounds including ten hydroxybenzoic
acids (HBAs), four hydroxycinnamic acids (HCAs), two quinic esters of caffeic acid (CQAs),
eight flavonoids and two prenylflavonoids (Fs). In detail, HBAs: p-hydroxybenzoic acid
(p-HBA), m-hydroxybenzoic acid (m-HBA), o-hydroxybenzoic acid (salicylic acid, SaA),
3,5-dihydroxybenzoic acid (3,5-DHBA), 3,4-dihydroxybenzoic acid (protocatechuic acid,
PCA), 2,5-dihydroxybenzoic acid (gentisic acid, GeA), 2,6-dihydroxybenzoic acid (2,6-
DHBA), 3,4,5-trihydroxybenzoic acid (gallic acid, GA), vanillic acid (VA), syringic acid
(SyA) (structures in Scheme 2); HCAs: caffeic acid (CA), p-coumaric acid (CouA), sinapic
acid (SiA), ferulic acid (FA) (structures in Scheme 3); CQAs: 4-caffeoylquinic acid (4-CQA),
5-caffeoylquinic acid (5-CQA) (structures in Scheme 4); Fs: catechin (Ca), quercetin (Q),
kampferol (K), rutin (Ru), myricitrin (My), quercetin-3-O-glucoside (Q-3-G), kaempferol-3-
O-rutinoside (K-3-R), hesperidin (He), isoxanthohumol (i-XH) (structures in Scheme 5) and
xanthohumol (XH) (structure in Scheme 4).
TP was quantitated with the same chromatographic run by using the calibration curve
y = 34379x
1228.3, R
2
= 0.9995, obtained as described in Materials and Methods, with
LOD = 2.64 µg/mL and LOQ = 8.00 µg/mL.
In addition, ellagic acid (EA) (structure in Scheme 5) was identified by comparison with
standard and was quantitated as Q content by using the quercetin calibration curve [
43
].
Lastly, 3-feruloylquinic acid (3-FQA) and 4-feruloylquinic acid (4-FQA) (structures in
Scheme 4) were tentatively assigned, supported by chromatographic data, UV–Vis spectral
data and mass fragmentation spectra, in strong agreement with the literature [
63
], and
quantitated as FA content by using the ferulic acid calibration curve [43].
Molecules 2022, 27, x FOR PEER REVIEW 10 of 21
The presence of CouA quinic derivative (p-CouQA, structure in Scheme 4) was also
investigated by SIR analysis of the ion [M-H]
= 337 m/z, but the acquired mass spectral
and chromatographic data results were insufficient to any conclusion. All data are shown
in Table 5, in which the amounts are reported in μg/g of dry sample of rhizome, as mean
values ± standard deviation.
Figure 3. AR3 herbal tea, diluted 1:10 with mobile phase, ESI-SIR channels at m/z 174 (a), 190 (b),
215 (c) and 231 (d), corresponding to the ion [M-H]
of IAA (a), 5-HIAA (b), TβC-3-COOH (c) and
6-HTβC-3-COOH (d).
Scheme 2. Hydroxybenzoic acids (HBAs) structures.
Figure 3.
AR3 herbal tea, diluted 1:10 with mobile phase, ESI-SIR channels at m/z174 (
a
), 190 (
b
),
215 (
c
) and 231 (
d
), corresponding to the ion [M-H]
of IAA (
a
), 5-HIAA (
b
), T
β
C-3-COOH (
c
) and
6-HTβC-3-COOH (d).
Molecules 2022,27, 4962 10 of 19
Molecules 2022, 27, x FOR PEER REVIEW 10 of 21
The presence of CouA quinic derivative (p-CouQA, structure in Scheme 4) was also
investigated by SIR analysis of the ion [M-H]
= 337 m/z, but the acquired mass spectral
and chromatographic data results were insufficient to any conclusion. All data are shown
in Table 5, in which the amounts are reported in μg/g of dry sample of rhizome, as mean
values ± standard deviation.
Figure 3. AR3 herbal tea, diluted 1:10 with mobile phase, ESI-SIR channels at m/z 174 (a), 190 (b),
215 (c) and 231 (d), corresponding to the ion [M-H]
of IAA (a), 5-HIAA (b), TβC-3-COOH (c) and
6-HTβC-3-COOH (d).
Scheme 2. Hydroxybenzoic acids (HBAs) structures.
Scheme 2. Hydroxybenzoic acids (HBAs) structures.
Molecules 2022, 27, x FOR PEER REVIEW 11 of 21
Scheme 3. Hydroxycinnamic acids (HCAs) structures.
Scheme 4. Hydroxycinnamic acids derivatives structures
Scheme 3. Hydroxycinnamic acids (HCAs) structures.
Molecules 2022, 27, x FOR PEER REVIEW 11 of 21
Scheme 3. Hydroxycinnamic acids (HCAs) structures.
Scheme 4. Hydroxycinnamic acids derivatives structures
Scheme 4. Hydroxycinnamic acids derivatives structures.
The presence of CouA quinic derivative (p-CouQA, structure in Scheme 4) was also
investigated by SIR analysis of the ion [M-H]
= 337 m/z, but the acquired mass spectral
and chromatographic data results were insufficient to any conclusion. All data are shown
Molecules 2022,27, 4962 11 of 19
in Table 5, in which the amounts are reported in
µ
g/g of dry sample of rhizome, as mean
values ±standard deviation.
Scheme 5. Flavonoids (Fs) structures.
Quite similar phenolic profiles were found for the three samples of AR1, AR2 and
AR3 herbal tea. Among the 26 analyzed phenolic compounds, five of them, i.e., 3,5-DHBA,
m-HBA, Ca, My and Ru, were not detected in any samples, and three of them, i.e., EA, Q
and K, were detected in all samples with content under LOD, 70 and 60
µ
g/L of extract,
respectively [
43
]. Furthermore, similar contents of GA, PCA, VA, SyA, SiA, SaA and i-XH
were found for AR1 and AR2, while AR3 was found to be significantly different (p< 0.05)
for the same compounds, except for i-XH, which was not detected.
Conversely, similar contents of CA, FA and 2,6-DHBA were found for AR2 and AR3,
while AR1 was found to be significantly different (p< 0.05) for the same compounds. The
three sample results were significantly different (p< 0.05) for GeA, p-HBA, CouA, 4-CQA,
5-CQA, 3-FQA, 4-FQA and He, except for TP. Q-3-G and K-3-R were identified only in AR2
in amounts under LOD. XH was identified only in AR1 and AR2, in amounts under LOD.
Despite some differences regarding the individual amount of the detected compounds,
likely due to geographical origin, collection period, drying process and/or storage of the
AR1, AR2 and AR3 rhizomes, analogously to what is discussed above, a well-defined
phenolic profile was found for the Agropyron repens herbal tea, mainly composed of HCAs,
in their free forms and as ester derivatives of quinic acid. In particular, 5-CQA was the most
abundant in all samples (57.40–112.22
µ
g/g), followed by 4-CQA (32.43–65.66
µ
g/g), 3-FQA
(9.99–53.99
µ
g/g), 4-FQA (3.97–27.14
µ
g/g), CouA (5.83–8.53
µ
g/g), CA (2.55–6.98
µ
g/g)
and to a lesser extent FA (1.97–4.05
µ
g/g) and SiA (0.40–0.78
µ
g/g) (Table 5; Figure 2a,b).
Lower but still significant amounts were found for HBAs, among which p-HBA was the
most abundant (3.64–9.52
µ
g/g), followed by VA (3.18–5.22
µ
g/g), PCA (1.21–7.16
µ
g/g),
SyA (1.89–3.69
µ
g/g), and to a lesser extent GA (0.67–2.80
µ
g/g), GeA (0.86–2.07
µ
g/g)
and SaA (0.29–1.37
µ
g/g). Among the investigated flavonoids, only He was quantitated
(1.37–4.40
µ
g/g). With regard to the prenylflavonoid i-XH and its precursor XH, they
were detected only in AR1 and AR2, and in both cases, XH was found in lower amounts
(<LOD, 1.13
µ
g/L) than i-XH. Taking into account that i-XH generally generates from XH
by heating [64], these data seemed consistent with some rhizome drying processes.
Molecules 2022,27, 4962 12 of 19
Table 5.
Qualitative and quantitate data of phenolic compounds and tryptophan, identified by comparison with standard, in AR1, AR2, AR3 herbal tea (IN), aqueous
extracts at 50
C (50) and 25
C (25), reported as
µ
g/g of dry rizhome, mean value of analysis in triplicate
±
standard deviation (
σ
). t
R
: retention time; [M-H]
:
monoisotopic mass of the deprotonated ion used for the SIR channel; values with different letters within rows are significantly different (p< 0.05): a, b, c refer to the
same rhizome (AR1, AR2 or AR3) extracts at different temperatures; A, B, C, refer to the three herbal teas AR1, AR2 and AR3. nd: not detected; < LOD: under LOD,
calculated from the corresponding calibration curve. AR1-IN, AR2-IN, AR3-IN: herbal tea; AR1-50, AR2-50, AR3-50: aqueous extracts at 50
C; AR1-25, AR2-25,
AR3-25: aqueous extracts at 25 C. EA quantitated as quercetin equivalent; 3-FQA and 4-FQA tentatively assigned and quantitated as ferulic acid equivalent.
Compound tR
(min)
AR1-IN
(µg/g ±σ)
AR1-50
(µg/g ±σ)
AR1-25
(µg/g ±σ)
AR2-IN
(µg/g ±σ)
AR2-50
(µg/g ±σ)
AR2-25
(µg/g ±σ)
AR3-IN
(µg/g ±σ)
AR3-50
(µg/g ±σ)
AR3-25
(µg/g ±σ)
[M-H]
(m/z)
GA 3.15 0.70 ±0.08 a,A 2.78 ±0.08 b0.63 ±0.04 a0.67 ±0.09 a,A 0.60 ±0.08 a0.61 ±0.07 a2.80 ±0.13 a,B 1.90 ±0.25 a5.10 ±0.00 b169
3,5-DHBA 4.61 nd nd nd nd nd nd nd nd nd 153
PCA 5.15 1.49 ±0.19 a,A 6.64 ±0.25 b2.99 ±0.12 c1.21 ±0.09 a,A 1.85 ±0.08 a2.03 ±0.05 b7.16 ±0.56 a,B 6.19 ±0.19 a7.39 ±0.92 a153
TP 5.51 19.92 ±1.19 a,A 20.52 ±1.92 a12.61 ±0.41 b203.66 ±18.70 a,B
116.20
±
18.64
b196.05 ±3.30 c13.01 ±0.63 a,C 20.76 ±3.89 b20.99 ±2.13 b203
5-CQA 6.90 57.40 ±4.40 a,A 59.02 ±5.08 a20.06 ±0.60 b107.56 ±7.04 a,B 101.23 ±0.59 b
105.10
±
6.02
b112.22 ±5.96 a,C
220.06
±
22.42
b
202.31
±
26.06
b353
GeA 7.14 0.86 ±0.02 a,A 2.38 ±0.03 b0.87 ±0.06 a1.23 ±0.06 a,B 1.10 ±0.09 a1.63 ±0.19 b2.07 ±0.27 a,B 4.19 ±0.34 a3.46 ±0.34 b153
Ca 7.32 nd nd nd nd nd nd nd nd nd 289
4-CQA 7.55 32.43 ±0.65 a,A 35.69 ±0.64 b17.45 ±1.75 c65.66 ±3.92 a,B 47.09 ±1.56 b54.34 ±6.24 b46.39 ±4.75 a,C 85.84 ±2.55 b74.88 ±8.81 c353
p-HBA 7.73 5.32 ±0.47 a,A 3.01 ±0.78 b4.82 ±0.59 a3.64 ±0.47 a,B 6.04 ±0.57 b4.69 ±0.82 a9.52 ±0.94 a,C 8.73 ±0.55 b10.67 ±1.00 b137
3-FQA 7.77 9.99 ±0.83 a,A 53.70 ±3.42 b13.03 ±0.04 c53.99 ±0.18 a,B 33.43 ±0.79 b65.19 ±4.89 c16.08 ±0.25 a,C 25.01 ±3.84 b56.21 ±2.75 c367
VA 9.13 3.18 ±0.24 a,A 6.37 ±0.19 b2.60 ±0.21 c3.36 ±0.24 a,A 4.07 ±0.36 b4.39 ±0.42 b5.22 ±0.54 a,B 6.91 ±0.59 b8.87 ±0.38 c167
CA 9.45 2.55 ±0.14 a,A 34.88 ±0.30 b16.57 ±2.54 c6.98 ±0.34 a,B 25.90 ±1.69 b12.13 ±1.29 c6.91 ±0.59 a,B 26.86 ±2.21 b86.52 ±2.09 c179
SyA 9.56 1.89 ±0.26 a,A 3.25 ±0.40 b1.76 ±0.08 a1.95 ±0.20 a,A 1.77 ±0.06 b2.20 ±0.11 a3.69 ±0.20 a,B 4.59 ±0.29 b4.80 ±0.44 b197
2,6-DHBA 10.48 <LOD 1.60 ±0.06 <LOD 0.85 ±0.11 a,A 1.58 ±0.12 b1.35 ±0.06 b1.02 ±0.08 a,A 2.61 ±0.05 b2.42 ±0.16 b153
m-HBA 10.66 nd nd nd nd nd nd nd nd nd 137
4-FQA 12.12 3.97 ±0.35 a,A 26.50 ±1.37 b5.14 ±0.38 c27.14 ±2.82 a,B 17.59 ±0.06 b30.30 ±2.97 a7.97 ±0.18 a,C 23.25 ±3.52 b28.84 ±3.23 c367
CouA 14.19 5.83 ±0.56 a,A 10.21 ±1.41 b4.62 ±0.03 c8.53 ±0.27 a,B 14.58 ±0.40 b10.24 ±0.67 c7.60 ±0.08 a,C 11.92 ±1.38 b20.74 ±1.94 c163
EA 15.74 <LOD <LOD <LOD <LOD 0.18 ±0.02 a1.24 ±0.08 b<LOD nd nd 301
SiA 15.92 0.40 ±0.06 a,A 0.73 ±0.11 b0.41 ±0.11 a0.50 ±0.03 a,A 0.54 ±0.04 a0.69 ±0.10 a0.78 ±0.03 a,B 1.18 ±0.10 b0.93 ±0.09 b223
FA 16.13 1.97 ±0.08 a,A 7.55 ±0.56 b2.36 ±0.99 c3.58 ±0.47 a,B 11.13 ±0.01 b6.29 ±0.18 c4.05 ±0.20 a,B 8.28 ±0.87 b13.98 ±0.79 c193
My 16.74 nd nd nd nd nd 0.73 ±0.07 nd nd nd 463
Molecules 2022,27, 4962 13 of 19
Table 5. Cont.
Compound tR
(min)
AR1-IN
(µg/g ±σ)
AR1-50
(µg/g ±σ)
AR1-25
(µg/g ±σ)
AR2-IN
(µg/g ±σ)
AR2-50
(µg/g ±σ)
AR2-25
(µg/g ±σ)
AR3-IN
(µg/g ±σ)
AR3-50
(µg/g ±σ)
AR3-25
(µg/g ±σ)
[M-H]
(m/z)
Ru 16.83 nd nd nd nd nd nd nd nd nd 609
Q-3-G 17.75 nd <LOD <LOD <LOD 0.59 ±0.03 a0.74 ±0.06 bnd nd 0.73 ±0.03 463
K-3-R 20.07 nd nd nd <LOD <LOD 0.16 ±0.02 nd <LOD nd 593
SaA 20.33 0.30 ±0.04 a,A 0.43 ±0.03 a0.34 ±0.05 a0.29 ±0.52 a,A 0.55 ±0.08 b1.08 ±0.11 c1.37 ±0.15 a,B 1.94 ±0.19 b3.44 ±0.17 c137
He 23.26 1.37 ±0.22 a,A 1.01 ±0.02 a1.05 ±0.04 a1.71 ±0.28 a,B 1.30 ±0.07 b1.17 ±0.12 b4.40 ±0.58 a,C 1.11 ±0.11 b6.91 ±0.58 c609
Q 27.76 <LOD 0.19 ±0.01 <LOD <LOD 0.16 ±0.06 a1.66 ±0.15 b<LOD nd <LOD 301
K 30.27 <LOD <LOD <LOD <LOD <LOD 0.42 ±0.12 <LOD <LOD <LOD 285
i-XH 32.70 0.22 ±0.005 a,A 0.23 ±0.002 a0.25 ±0.01 a0.24 ±0.005 a,A 0.28 ±0.004 a0.49 ±0.12 bnd nd nd 353
XH 38.04 <LOD <LOD <LOD <LOD 0.24 ±0.02 a4.14 ±0.21 bnd nd nd 353
Molecules 2022,27, 4962 14 of 19
TP was found in the herbal tea among the most abundant compounds, with quite
similar content in AR1 (19.92
µ
g/g) and AR3 (13.01
µ
g/g) and in high levels in AR2
(203.66 µg/g)
, a trend confirmed in the extracts at different temperatures. Such a difference
might be ascribed to different origin and/or treatment of the three samples of rhizome;
however, all data confirm the presence of TP as a free amino acid, which was unexpected
data since it was never reported before in Agropyron repens.
The statistical analysis carried out for each compound quantitated in the three different
extracts (herbal tea, 2 h stirring at 25
C and 2 h stirring at 50
C), evidenced different
behaviors for different compounds: weak effects were evidenced for some molecules, tem-
perature/extraction time combined effects were observed for others. In general, the herbal
tea resulted in a good compromise of temperature (100
C) and extraction time
(10 min)
,
providing a total of extracted phenolic antioxidants in the range of
126.89–289.09 µg/g
of
rhizome, with TP as an added value.
Conversely, the presence of some phytotoxins in the herbal tea cannot be excluded. The
presence of DIBOA, DIMBOA and HBOA (structures in Scheme 6), reported as allelopathic
constituents of ethyl acetate extracts from some parts of the plant [
6
], was investigated in
the herbal tea as well in the aqueous extracts at different temperatures by selecting the
corresponding m/zmonoisotopic values for the ion [M-H]
in dedicated chromatographic
runs: 180 for DIBOA, 210 for DIMBOA and 164 for HBOA.
Molecules 2022, 27, x FOR PEER REVIEW 15 of 21
Scheme 6. Allelopathic molecules DIMBOA, DIBOA and HBOA structures.
Three chromatographic peaks, each one in the corresponding SIR channel, were
observed at 9.21, 9.77 and 13.76 min, as shown in Figure 4a–c, respectively, in which data
for AR3 herbal tea are reported as an example. Besides the molecular mass, chromato-
graphic data as t
R
and elution order seemed in good agreement with the structures of
DIBOA, DIMBOA and HBOA (see Scheme 6).
Figure 4. ESI- SIR channels at (a) 180, (b) 210 and (c) 164 m/z, corresponding to the ion [M-H]
of (a)
DIBOA, (b) DIMBOA and (c) HBOA, respectively. AR3 herbal tea as an example.
Lastly, the presence of the flavonoids luteolin, as free form (L) and as glucoside de-
rivative (L-3-G), baicalein (B) and tricin (Tr) (structures in Scheme 5), and the hexadecyl
coumaric acid ester (CouAHDE) (structure in Scheme 4), reported in the literature as
constituents of Agropyron repens rhizome extracts [10,19,21,22,38], were investigated by
the SIR technique, selecting in independent channels the monoisotopic ion [M-H]
m/z
values 285 for L, 447 for L-3-G, 269 for B, 329 for Tr and 387 for CouAHDE. On the basis
of the molecular mass values and chromatographic data, the presence of luteolin was
excluded, in the free form as well as for the glucoside derivative. Analogously, the
presence of CouAHDE was excluded, as expected due to its low polarity. Ambiguous
data were found for baicalein, whose presence cannot be supported. Conversely, a
well-defined peak with [M-H]
= 329 m/z at t
R
= 31.07 min suggested the presence of Tr,
the elution time being consistent with the structure.
If the absence of a peak in the SIR channel may exclude the presence of any isobaric
compounds with that of the m/z value, including the searched one, the presence of a peak
in the SIR channel needs further investigation to definitively assign it to a target mole-
cule, which cannot be excluded until then.
3. Materials and Methods
3.1. Chemicals and Solvents
All chemicals were of analytical grade, purchased from Sigma-Aldrich (Milano, It-
aly) and used as received. HPLC-grade acetonitrile and methanol were purchased from
Carlo Erba (Milano, Italy); HPLC-grade water was freshly prepared with the Milli-Q pu-
rification system (Millipore, Vimodrone, Italy).
Scheme 6. Allelopathic molecules DIMBOA, DIBOA and HBOA structures.
Three chromatographic peaks, each one in the corresponding SIR channel, were
observed at 9.21, 9.77 and 13.76 min, as shown in Figure 4a–c, respectively, in which data for
AR3 herbal tea are reported as an example. Besides the molecular mass, chromatographic
data as t
R
and elution order seemed in good agreement with the structures of DIBOA,
DIMBOA and HBOA (see Scheme 6).
Molecules 2022, 27, x FOR PEER REVIEW 15 of 21
Scheme 6. Allelopathic molecules DIMBOA, DIBOA and HBOA structures.
Three chromatographic peaks, each one in the corresponding SIR channel, were
observed at 9.21, 9.77 and 13.76 min, as shown in Figure 4a–c, respectively, in which data
for AR3 herbal tea are reported as an example. Besides the molecular mass, chromato-
graphic data as t
R
and elution order seemed in good agreement with the structures of
DIBOA, DIMBOA and HBOA (see Scheme 6).
Figure 4. ESI- SIR channels at (a) 180, (b) 210 and (c) 164 m/z, corresponding to the ion [M-H]
of (a)
DIBOA, (b) DIMBOA and (c) HBOA, respectively. AR3 herbal tea as an example.
Lastly, the presence of the flavonoids luteolin, as free form (L) and as glucoside de-
rivative (L-3-G), baicalein (B) and tricin (Tr) (structures in Scheme 5), and the hexadecyl
coumaric acid ester (CouAHDE) (structure in Scheme 4), reported in the literature as
constituents of Agropyron repens rhizome extracts [10,19,21,22,38], were investigated by
the SIR technique, selecting in independent channels the monoisotopic ion [M-H]
m/z
values 285 for L, 447 for L-3-G, 269 for B, 329 for Tr and 387 for CouAHDE. On the basis
of the molecular mass values and chromatographic data, the presence of luteolin was
excluded, in the free form as well as for the glucoside derivative. Analogously, the
presence of CouAHDE was excluded, as expected due to its low polarity. Ambiguous
data were found for baicalein, whose presence cannot be supported. Conversely, a
well-defined peak with [M-H]
= 329 m/z at t
R
= 31.07 min suggested the presence of Tr,
the elution time being consistent with the structure.
If the absence of a peak in the SIR channel may exclude the presence of any isobaric
compounds with that of the m/z value, including the searched one, the presence of a peak
in the SIR channel needs further investigation to definitively assign it to a target mole-
cule, which cannot be excluded until then.
3. Materials and Methods
3.1. Chemicals and Solvents
All chemicals were of analytical grade, purchased from Sigma-Aldrich (Milano, It-
aly) and used as received. HPLC-grade acetonitrile and methanol were purchased from
Carlo Erba (Milano, Italy); HPLC-grade water was freshly prepared with the Milli-Q pu-
rification system (Millipore, Vimodrone, Italy).
Figure 4.
ESI- SIR channels at (
a
) 180, (
b
) 210 and (
c
) 164 m/z, corresponding to the ion [M-H]
of
(a) DIBOA, (b) DIMBOA and (c) HBOA, respectively. AR3 herbal tea as an example.
Lastly, the presence of the flavonoids luteolin, as free form (L) and as glucoside
derivative (L-3-G), baicalein (B) and tricin (Tr) (structures in Scheme 5), and the hexadecyl
coumaric acid ester (CouAHDE) (structure in Scheme 4), reported in the literature as
constituents of Agropyron repens rhizome extracts [
10
,
19
,
21
,
22
,
38
], were investigated by
Molecules 2022,27, 4962 15 of 19
the SIR technique, selecting in independent channels the monoisotopic ion [M-H]
m/z
values 285 for L, 447 for L-3-G, 269 for B, 329 for Tr and 387 for CouAHDE. On the basis
of the molecular mass values and chromatographic data, the presence of luteolin was
excluded, in the free form as well as for the glucoside derivative. Analogously, the presence
of CouAHDE was excluded, as expected due to its low polarity. Ambiguous data were
found for baicalein, whose presence cannot be supported. Conversely, a well-defined peak
with [M-H]
= 329 m/zat t
R
= 31.07 min suggested the presence of Tr, the elution time
being consistent with the structure.
If the absence of a peak in the SIR channel may exclude the presence of any isobaric
compounds with that of the m/zvalue, including the searched one, the presence of a peak
in the SIR channel needs further investigation to definitively assign it to a target molecule,
which cannot be excluded until then.
3. Materials and Methods
3.1. Chemicals and Solvents
All chemicals were of analytical grade, purchased from Sigma-Aldrich (Milano, Italy)
and used as received. HPLC-grade acetonitrile and methanol were purchased from Carlo
Erba (Milano, Italy); HPLC-grade water was freshly prepared with the Milli-Q purification
system (Millipore, Vimodrone, Italy).
3.2. Plant Material Characterization
Three commercial samples of rhizomes of Agropyron repens (L.) P. Beauv. (Quack-
grass), marked above as AR1, AR2 and AR3, were purchased from three different local
herbal medicine shops; each sample was characterized by dealer name, batch number
and expiration date. The samples, stored under vacuum, are kept at our laboratory for
further reference. Water activity and absolute humidity of each sample were measured
(in duplicate) before use, by a Schaller Humimeter Rh2. The microbiological analysis was
performed on 5.00 g of each sample aseptically removed from the shop package, placed in a
stomacher bag, diluted with 0.9% NaCl solution and homogenized with a Stomacher LAB
Blender 400 (Pbi International); the total aerobic count was determined on 3M
®
Petrifilm
aerobic total count, after incubation at 30
C for 24–48 h; yeasts and molds were detected on
3M
®
Petrifilm yeast and molds at 30
C for 24–72 h; lactic acid bacteria were enumerated
on 3M
®
Petrifilm lactic acid bacteria incubated at 37
C for 24–72 h; Enterobacteriaceae
were counted on 3M
®
Petrifilm Enterobacteriaceae at 37
C for 24
±
2 h. All measurements
were performed in duplicate.
3.3. Agropyron repens (L.) P. Beauv. Rhizome Extraction Procedure
The rhizome herbal tea was obtained according to dose and procedure recommended
by the Official Pharmacopoeia of the Italian Republic, by adding 1.00 g di AR1, AR2 and
AR3, in turn, to 10 mL of Milli-Q purified water previously brought to boil. After 10 min,
the mixture was filtered at 0.22
µ
m, brought back to room temperature, appropriately
diluted (1:10, 1:100, 1:200) with the mobile phase (A:B, 95:5, v/v, vide infra) and injected
(20 µL) in triplicate for the analysis by HPLC-PDA-ESI-MS/MS.
Aqueous extracts (25 or 50
C) were obtained by adding 10 mL of Milli-Q-purified
water to 1.00 g of AR1, AR2 and AR3, in turn, and keeping the mixtures at room temperature
or at 50
C in thermostated bath under magnetic stirring for 2 h. The supernatant was then
filtered at 0.22
µ
m, appropriately diluted (1:10, 1:100, 1:200) with the mobile phase (A:B,
95:5, v/v, vide infra) and injected (20
µ
L) in triplicate for the analysis by HPLC-PDA-ESI-
MS/MS.
3.4. HPLC-PDA-ESI-MS/MS Analysis
The aqueous extracts were analyzed with a Waters 1525
µ
HPLC (Milford, MA, USA)
equipped with a Waters 996 PDA detector and a Quattro Micro Tandem MS/MS with a Wa-
ters ESI source (Micromass, Manchester, UK), by using a Waters XBridge C18 (150
×
2.1 mm
Molecules 2022,27, 4962 16 of 19
i.d.) 5
µ
m analytical column. Milli-Q water/formic acid 5 mM (A) and acetonitrile/formic
acid 5 mM (B) were used as mobile phase, flowing at 0.20 mL/min. A chromatographic
separation method previously developed was applied for the analysis [
43
,
61
,
62
]. Briefly:
0
1 min, 5% B; 1–20 min, 16.5% B; 20–30 min, 40% B; 30–35 min, 60% B; 35–36 min, 80%
B; 36–40 min, 80% B; 40–41 min, 5% B; 41–61 min, 5% B. The PDA detector recorded one
UV–Vis spectrum per second in the range of 200–800 nm, resolution 1.2 nm. Mass spectral
data were acquired in: (a) full scan in negative (ESI-) and positive (ESI+) ionization; (b)
selected ion recording (SIR) mode in ESI-, by using separate acquisition channels for each
different m/zmonoisotopic value of the deprotonated forms [M-H]
, under the following
source conditions, previously optimized [
43
]: capillary voltage 2.7 kV, cone voltage 27 V,
source temperature 120
C, desolvation temperature 350
C, cone gas flow 40 L/h, desol-
vation gas flow 500 L/h, dwell cell value of 0.200 s. Data acquisition, data handling, and
instrument control were performed by MassLynx Software 4.1 v (Data Handling System
for Windows, Micromass, UK).
3.5. Tryptophan Calibration Curve by HPLC-ESI-MS/MS
The methanol stock solution containing 1.0 mg/mL tryptophan was diluted with the
mobile phase (A:B, 95:5, v/v) until the final concentrations of 0.1, 0.2, 0.5, 1.0 and 5.0 mg/L.
Each final solution was analyzed in triplicate (20
µ
L injected) by HPLC-ESI-MS/MS-SIR
mode, by selecting 203 m/zfor the tryptophan monoisotopic anion [M-H]
. The calibration
curve was calculated with equal-weighted least-squares linear regression analysis of the
SIR peak area against the standard nominal concentration; limit of detection (LOD) and
quantitation (LOQ) were obtained as LOD = 3Sa/b and LOQ = 10Sa/b, respectively, where
Sa and b are the estimated standard deviation and the slope of the analytical calibration
function with a 95% confidence level, respectively [65].
3.6. Untargeted ESI-MS/MS Profile
The untargeted analysis of the aqueous extracts, degassed, filtered and diluted 1:10
with the mobile phase (A:B, 95:5, v/v) was carried out by direct infusion into the ESI source
with an external syringe flowing at 5
µ
L/min. Full scan spectral data were acquired for
2 min in the mass range 80–800 Da, in both ESI- and ESI+, with cone voltage 27 and 24 V,
respectively, ionization source temperature 100
C, desolvation gas temperature 150
C,
cone gas flow 30 L/h, and desolvation gas flow 400 L/h. The signals were normalized to
the highest one for a direct comparison among samples. Fragmentation data were acquired
in daughter mode in ESI- and/or ESI+, using argon as collision gas, by selecting each m/z
value evidenced in the full scan as precursor ion, in turn, and using collision energy (CE) in
the range of 10–20 eV.
3.7. Statistical Analysis
All samples were analyzed in triplicate for quantitation, and results were reported
as mean values
±
standard deviation (SD). Data were analyzed by using the one-way
analysis of variance (ANOVA). The significance of differences (p< 0.05) among samples
was determined by the Tukey test.
4. Conclusions
In conclusion, a comprehensive study of the Agropyron repens (L.) P. Beauv. rhizome
herbal tea was carried out by ESI tandem–mass spectrometry, analyzing three independent
commercial samples. ESI- and ESI+ mass spectra, acquired by direct infusion experiments,
provided characteristic “rhizome” ions, ascribed to fructose/glucose, a di-hexose, and a di-
hexose–pentose as typical saccharides, pyruvic, lactic, phosphoric, 2-furoic, fumaric, malic,
cis-aconitic and citric acids as typical small organic acids, and
γ
-aminobutyric acid, proline,
valine, asparagine, histidine and arginine as typical amino acids, with useful application for
fast and simple quality control analysis. The HPLC-PDA-ESI-MS/MS analysis evidenced
the unexpected presence of tryptophan, never reported previously, with the amounts
Molecules 2022,27, 4962 17 of 19
lying in the range of 13.01–203.66
µ
g/g of dry rhizome, with possible new perspectives of
applications for Agropyron repens rhizome. In addition, the targeted analysis evidenced at
least twenty phenolic compounds representative of the Agropyron repens rhizome, among
which caffeoyl and feruloyl quinic esters and caffeic and coumaric acids were the most
abundant. From a comparison with aqueous extracts at different temperatures, the herbal
tea resulted in a good compromise of temperature and extraction time, with a total of
extracted phenolic antioxidants in the range of 126.89–289.09 µg/g of rhizome.
Author Contributions:
Conceptualization, writing—original draft preparation, supervision, project
administration and funding acquisition, M.F. and R.P.; methodology, formal analysis, data evaluation
and statistical analysis, M.B. and P.D.M.; writing—revision: D.R. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was financially supported by Sapienza University of Rome (project no.
RP11715C63A2EC44).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank the Ministero dell’Universitàe della Ricerca for
financial support.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Sample Availability: Samples of the analyzed compounds are available from the authors.
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