2013

NPC Natural Product Communications Vol. 8

No. 1
Phenolic Content and DPPH Radical Scavenging Activity 99 - 102
of the Flowers and Leaves of Trifolium repens
Agnieszka Kicel*and Maria Wolbiś

Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Łódź, Muszyńskiego 1,

90-151 Łódź, Poland

agnieszka.kicel@umed.lodz.pl

Received: October 3rd, 2011; Accepted: November 22nd, 2012

In order to facilitate the quality control of Trifolium repens flowers and leaves, a RP-HPLC method with UV detection was developed for the simultaneous
quantitative determination of flavonols and isoflavones. The total flavonoid and phenolic (TPC) contents were determined spectrophotometrically in a visible part
of the light spectrum at 425 and 760 nm, respectively. Additionally, evaluation of the antioxidant properties of the plant materials was performed using the DPPH
in vitro test. The results showed that the flowers are the richest source of phenolics ranging from 28.7 to 38.8 mg GAE/g, and flavonoids, calculated for hyperoside,
up to 20 mg HP/g, which hydrolyzed mainly to flavonols (the quercetin level greater than 6 mg/g). T. repens is poor in isoflavones; similar quantities of ca. 0.2 mg/g
were detected in the flowers and leaves. The flower and leaf extracts showed antioxidant activity towards DPPH˙ with EC50 values ranging from 72.3 to
179.3 µg/mL. Significant linear correlations were found between antioxidant potentials of the studied plant materials and total phenolic and flavonoid contents
determined by HPLC and spectrophotometric methods (R2 in the range of 0.97 – 0.99).

Keywords: Trifolium repens L., Flavonols, Isoflavones, Quantitative determination, Spectroscopic and RP-HPLC analyses, Antioxidant activity.

Trifolium repens L. (white clover), family Fabaceae, originally
native to the south of Europe, has become naturalised in a great
range of habitats and climates [1a]. In Poland, it is known as a
forage crop distributed in meadows and as a weed spreading along
pathways, roadsides and lawns [1b]. Due to its high feed value it is
used as grazing food for cattle and other livestock. Clover is also
known as a medicinal herb. The flowers of white clover relieve
rheumatism and arthritis, and the aerial parts possess anti-diarrheal
and mildly analgesic activities [1a,1c]. Moreover, its aerial shoots
have medicinal values as a deworming remedy [1d].
According to literature, phytochemical interest in the polyphenolic
composition of T. repens has mainly concerned its flavonoids.
Several studies indicate that the flowers and leaves of T. repens
contain flavonols, including kaempferol, quercetin, and myricetin in
glycoside forms, and their 6’’-O-acetyl glycoside derivatives [2a-
2d]. Among isoflavones, 2”-O-acetylated formononetin and
genistein monoglycosides were detected [2e,2f]. Moreover, the
literature reports coumarins [2g,2h], saponin monodesmosides [2i],
phenolic acids [2j] and essential oil [2k,2l]. Although several
studies investigated flavonoids in the genus Trifolium [2m-2q], the
ratio of isoflavones to flavonols in the flowers and leaves of
T. repens has not been reported to date. The objective of this study
was to record the flavonoid profile, including flavonols and
isoflavones of T. repens using a RP-HPLC method. The Folin-
Ciocalteu and colorimetric aluminum chloride methods were used
to determine the total phenolic and flavonoid contents. Additionally,
the antioxidant activity of T. repens extracts was further evaluated
by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical
scavenging assay.
RP-HPLC-UV methods were developed for the determination of the
flavonoid profile of T. repens flowers and leaves. Due to the
initially observed differences in the flavonoid contents, the sample
weight of the studied materials and the conditions of extraction and
hydrolysis had to be optimized separately for the analysis of
flavonols and isoflavones. The sample solutions of the hydrolyzed
T. repens extracts were then separated under the optimized
RP-HPLC conditions. For the quantification of the flavonol
aglycones, a short gradient system was elaborated, which enabled
the good resolution of adjacent peaks in an analysis time of less
than 12 min. A fast and steep linear solvent gradient consisted of
45 – 60 % (v/v) MeOH in an aqueous solution containing 0.5 %
(v/v) H3PO4. The peaks were analyzed at a flow rate 1.6 mL/min
and monitored at 370 nm, where none of the isoflavones absorb.
Moreover, the best isoflavone separation profile was observed
during 31min using variable gradient elution: 13 – 65% (v/v) ACN
– water containing 0.5% (v/v) H3PO4 and 0.4% (v/v) THF, flow
rates between 0.8 and 1.4 mL/min and detection at 260 nm. Table 1
shows the retention parameters, linearity ranges and regression
parameters of the eight flavonoid standards. Good linearity (R2 >
0.9912) was obtained for all the compounds in the concentration
range studied. The R2 values confirmed the linearity of the applied
methods. The high sensitivity of the methods used was
demonstrated with the low LOD (0.002 – 0.17 g/mL) and LOQ
(0.005 – 0.53 g/mL). At this level of LOD and LOQ, the HPLC
methods suggested full capacity for the quantification of each
flavonol and isoflavone studied.
The HPLC determinations summarized in Table 2 show that the
level of flavonol aglycones in the flowers of T. repens (10.7 –
13.4 mg/g) is five times as high as in the leaves (1.8 – 2.6 mg/g).
The flowers were characterized by a significantly high content of
quercetin – greater than 6 mg/g and a low level of myricetin – 2.7 –
3.1 mg/g, kaempferol – ca. 1.2 mg/g and isorhamnetin ca.
0.2 mg/g. The leaves contain exclusively kaempferol and quercetin,
in amounts of ca. 1.3 and 0.9 mg/g, respectively. T. repens is poor
in isoflavones; similar quantities of ca. 0.2 mg/g were detected in
the flowers and leaves. In the flowers, the predominant isoflavones
are genistein and formononetin and their concentrations are similar
– ca. 0.07 and 0.06 mg/g, respectively. The leaves contain more
formononetin, ca. 0.2 mg/g, and less genistein ca. 0.05 mg/g. The
results show that the flowers contain a much higher concentration of
flavonols than isoflavones. They can, therefore, be regarded as an
exclusive flavonol source (Figure 1). Despite the slightly higher
contribution of isoflavones to the overall flavonoid composition
100 Natural Product Communications Vol. 8 (1) 2013 Kicel and Wolbiś

standards was justified because these compounds were found as

9
my ricetin
dominant flavonoids in the studied materials.
quercetin
8
kaempferol Table 3 also shows the contents of total phenols (TPC), which were
isorhamnetin measured by the Folin-Ciocalteu reagent (FCR) in terms of gallic
7 daidzein
genistein acid equivalent (GAE). The TPC varied among plant material type
6 formononetin from 28.7 – 38.8 mg GAE/g in the flowers to 10.7 – 11.9 mg
biochanin A GAE/g in the leaves. Interestingly, the flowers with higher
5
quantities of total phenolics and flavonoids also showed significant
mg/g

4
DPPH scavenging activity (EC50 ≤ 90.0 µg/mL). The major
flavonoid antioxidant in the flowers has been reported to be
3 hyperoside [2a]. The activity of the leaves has been characterized
by higher EC50 values of 179.3 – 168.7 µg/mL, which correspond
2 with a lower antioxidant power. Moreover, all studied plant extracts
showed lower DPPH scavenging activity (EC50 ≥ 72.3 µg/mL)
1
compared with the values obtained for trolox (EC50 = 4.1 µg/mL)
0 and quercetin (EC50 = 1.9 µg/mL).
flowers 2005 flowers 2006 leaves 2005 leaves 2006

Figure 1: Flavonol and isoflavone contents (mg/g), after hydrolysis, of the flowers and leaves As shown in Table 4, significant positive correlations were found
of T. repens. Results indicate averages ± standard deviations for triplicate analyses. between the DPPH assay and the total phenolic content (TPC)
(R2 = 0.968, p < 0.02), flavonoid content determined using the
observed in the leaves, T. repens is not typical of the Fabaceae spectrophotometric method (R2 = 0.987, p < 0.007), and flavonol
family, whose many members are noted to produce significant content quantified by the HPLC method (R2 = 0.979, p < 0.02). A
quantities of isoflavones. significant and linear relationship existing between the antioxidant
activity and phenolic content suggests that the reducing ability of
In view of the fact that for the quantification of plant flavonoids,
polyphenols, including mainly flavonols, seemed to be an important
most pharmacopoeias [3,4] require more accessible and economic
factor dictating free radical scavenging capacity of T. repens
spectrophotometric methods, a comparison was made between the
flowers and leaves.
HPLC total flavonoids assay and the spectrophotometric method
recommended by the Polish Pharmacopoeia VIII [3]. The results
For T. repens flowers and leaves, this is the first report regarding
shown in Table 3 indicate that the highest level of flavonoids was
investigation of total phenols (TPC), including flavonoids, and
detected in the flowers of T. repens and reached 12.4 – 14.1 mg
simultaneous determination of flavonols and isoflavones. Moreover,
QE/g and 17.8 – 20.1 mg HP/g. The leaves were characterized by a
it was demonstrated that the crude methanolic extract (CME) of the
markedly lower content of flavonoids: 5.4 – 7.3 mg QE/g and 7.7 –
flowers and leaves possessed an antioxidant activity which
10.4 mg HP/g. The results from the spectrophotometric analysis
correlated well with the quantitative result. The observed levels of
including the flowers and leaves of T. repens showed good
polyphenols and the antioxidant properties of the studied plant
correlation with the HPLC method (R2 = 0.942, p < 0.03). The high
materials indicate that the flowers of T. repens should be
degree of correlation may be related to the 3-glycoside forms of
particularly desirable, mainly due to the highest level of flavonols,
flavonols, dominating compounds in the flowers and leaves [2a],
which may also significantly influence the pharmacological
which can easily hydrolyze and form aluminum chloride complexes
properties of this material.
[5]. Moreover, the choice of quercetin and hyperoside as calibration

Table 1: Linearity data of standard curves, limits of detection and quantification and amounts for the eight flavonoid aglycones.
Retention timea (min)
Peak Correlation Coefficientc Linear range LOD d LOQ d
Standard Calibration equationb
No. SystemI SystemII (R2) (g/mL) (g/mL) (g/mL)
370 nm 260 nm
1 Myricetin 4.33 12.54 y = 234548 x - 276181 0.9912 1.1-26.9 0.002 0.005
2 Quercetin 6.67 17.17 y = 365915 x -579344 0.9985 1.5-105.2 0.015 0.045
3 Kaempferol 9.50 22.19 y = 373608 x- 226478 0.9983 0.9-50.8 0.174 0.528
4 Isorhamnetin 10.27 22.66 y = 353327 x -105798 0.9986 0.6-15.1 0.060 0.183
5 Daidzein ----- 15.03 y = 974505 x + 135937 0.9999 0.9-9.5 0.025 0.076
6 Genistein ----- 21.33 y = 106 x + 292781 0.9999 1.4-14.3 0.038 0.116
7 Formononetin ----- 24.95 y = 661112 x + 205519 0.9999 1.4-70.7 0.025 0.077
8 Biochanin A ----- 29.25 y = 787467 x + 90072 0.9999 0.5-48.3 0.058 0.175
a
Retention times (tR) are the mean values from three replicates. b For estimation of regression equation parameters (y=ax+b), y = peak area, x = concentration of standards in g/mL.
System I was used for flavonols and System II for isoflavones. c R2 = correlation coefficient for five data points in the calibration curves (n = 3). d LOD: limit of detection, LOQ: limit of
quantification.

Table 2: Flavonol and isoflavone contents (mg/g) in the flowers and leaves of T. repens determined using the RP-HPLC-UV methoda.
Samples myricetin quercetin kaempferol isorhamnetin Flavonols (mg/g)
flowers 07.05 2.67 (2.17) 6.71 (0.54) 1.19 (0.42) 0.15 (0.68) 10.7
flowers 07.06 3.09 (2.85) 8.94 (1.15) 1.21 (0.58) 0.16 (1.22) 13.4
leaves 09.05 nd 1.09 (0.04) 1.48 (1.01) nd 2.6
leaves 09.06 nd 0.71 (1.54) 1.08 (1.21) nd 1.8
daidzein genistein formononetin biochanin A Isoflavones (mg/g)
flowers 07.05 0.016 (3.25) 0.070 (1.00) 0.08 (2.67) 0.008 (1.10) 0.17
flowers 07.06 0.014 (2.29) 0.075 (1.20) 0.05 (3.92) 0.005 (3.00) 0.14
leaves 09.05 0.011 (0.03) 0.060 (3.33) 0.23 (0.88) nd 0.30
leaves 09.06 0.012 (3.33) 0.034 (1.18) 0.12 (0.81) nd 0.17
a
Mean aglycone contents in mg per g of dry weight of the plant materials (n = 3), values in parentheses are relative standard deviations RSD (%), nd: peak not detected.
Phenolics in Trifolium repens Natural Product Communications Vol. 8 (1) 2013 101

Table 3: Flavonoid and phenolic contentsa (mg/g), and antioxidant activity of flowers 36 – 39 % C + 0.4 % D in A, 23 – 31 min: 39 – 65 % C + 0.4 % D in A;
and leaves of T. repens.
flow rate: 0 – 16 min: 0.8 mL/min, 16 – 31 min: 0.9 - 1.4 mL/min;
Total flavonoidsb Total phenols (TPC)c DPPH detection at 260 nm.
Samples Aglycones Glycosides
GAE EC50d TEAAe
QE HP
flowers 07.05 12.4 (2.3) 17.8 28.7 (2.1) 90.0 (1.7) 0.2 Calibration curves: 0.3-1.0 mg of myricetin, quercetin, kaempferol,
flowers 07.06 14.1 (0.4) 20.1 38.8 (0.4) 72.3 (0.5) 0.2 isorhamnetin and, separately, 0.9 - 1.4 mg of daidzein, genistein,
leaves 09.05 5.4 (0.5) 7.7 10.7 (4.0) 179.3 (1.0) 0.09
leaves 09.06 7.3 (0.9) 10.4 11.9 (3.1) 168.7 (0.8) 0.09 formononetin and biochanin A were dissolved in 10 mL of MeOH.
trolox 4.1 (1.1) 1.0 Then, by a successive dilution procedure, 5 different working
quercetin 1.9 (0.7) 2.7
a
solutions were prepared in the concentration ranges of 0.5 –
Results are mean values (n = 3); the values in parentheses are relative standard deviations
RSD (%). b Total flavonoid content expressed in quercetin (mg QE/g dry weight of plant 105 μg/mL and analyzed by triplicate injections (20 μL) into the
material) and hyperoside (mg HP/g dry weight of plant material) equivalents. c Total phenolic optimized HPLC systems. The linearity of analyses for each
content (TPC) expressed in mg gallic acid equivalent (GAE) per g dry weight of the plant
materials. d EC50 values are in µg/mL, amount of dry plant material required for 50% reduction
aglycone was verified by regression analysis and plotted using
of DPPH radical after 60 min. e TEAA trolox equivalent antioxidant activity expressed in linear regression of the peak area versus the concentration.
mmol standard equivalent per 1 g of dry plant material
Limit of detection (LOD) and limit of quantification (LOQ):
Table 4: Correlation coefficients (R2) for relationships between content of phenolic
compounds and antioxidant activity of T. repens flowers and leaves extracts.
Sensitivity was evaluated by determining the limits of detection
(LOD) and quantification (LOQ). The LOD was defined as the
R2 DPPH EC50 (µg/mL)
TPC (GAE, mg/g) 0.968 amount of analyte for which the signal-to-noise ratio was 3
Total flavonoids-UV(QE, mg/g) 0.987 (S/N = 3). The LOQ was defined as the level at which the
Total flavonols-HPLC (aglycones, mg/g) 0.979
measurement precision is satisfactory for the quantitative analysis.
Its value was evaluated as the amount of analyte that produced a
Experimental peak height close to 10 times the baseline noise (S/N = 10). Under
Materials: All samples of the flowers and leaves of Trifolium these chromatographic conditions, LOD and LOQ for each
repens L. were collected from July to September, 2005 - 2006, from 4 compound were determined by analysing a series of diluted
and 5 year-old plants cultivated in Łódź, Poland. The seeds were solutions until S/N was approximately 3 for LOD and 10 for LOQ.
provided by the Plant Breeding and Acclimatization Institute of the
Polish Academy of Science at Radzików (Poland). The plant Sample preparation: For the quantification of flavonols, samples of
materials were dried in natural conditions, then pulverized and sieved 0.2 g and 0.4 g of T. repens flowers and leaves, respectively were
(sieves 0.315 and 0.074 mm). Voucher specimens (Tr-K01, K-02, Tr- refluxed for 1 h with 8 mL of 35 % aqueous HCl (11.2 M, 413 g/L)
L-01, L-02) were deposited at the Department of Pharmacognosy, and 30 mL of MeOH. After filtration, the sample was extracted
Medical University of Łódź. twice with 20 mL of MeOH, each time for 10 min. The combined
hydrolyzates were evaporated and, after dissolution in MeOH, were
The 8 flavonoid standards, myricetin, quercetin, kaempferol, diluted to 25 mL. For the quantification of isoflavones samples of
isorhamnetin, daidzein, genistein, formononetin and biochanin A, 1g of T. repens flowers and leaves were heated under reflux for 2 h
were HPLC–grade purity from Roth (Switzerland) and Fluka with 20 mL of 35% aqueous HCl (11.2 M, 413 g/L) and 50 mL of
(Germany). Gallic acid and (±)-6-hydroxy-2,2,7,8- 70% (v/v) aqueous MeOH. After filtration, the sample was then
tetramethylchroman-2-carboxylic acid (trolox) were purchased from extracted twice with 30 mL of 70 % (v/v) aqueous methanol for
Sigma-Aldrich, (Germany/USA). All reagents: aluminum chloride 15 min. The combined extracts were concentrated under vacuum to
(AlCl3 x 6H2O), hydrochloric acid, metenamin (POCh, Poland) 30 mL (to remove the methanol) and extracted with diethyl ether
2,2 diphenyl-1-picrylhydrazyl (DPPH) (Sigma-Aldrich, Germany/ (4 x 20 mL). Then, the combined ether extracts were evaporated to
USA), Folin-Ciocalteu reagent (FCR) (POCh, Poland), and other dryness and the residue dissolved in MeOH and diluted to 10 mL.
chemicals: acetone, ethyl acetate, glacial acetic acid, hydrochloric For each of the studied plant materials, the extraction and injection
acid and methanol (POCh, Poland) were of analytical purity. procedures were conducted in triplicate. The contents of flavonoid
Methanol, acetonitrile (POCh, Poland) and orthophosphoric acid aglycones were calculated from peak areas from experiments to
(Merck, Germany) were of HPLC-grade purity. assess linearity.

RP-HPLC-UV analysis: RP-HPLC-UV analyses were performed
using a Hewlett-Packard 1100 series chromatograph equipped with
a quaternary pump HP1311A, an HP1322A vacuum degasser, an
HP3395 series integrator, an HP1314A variable wavelength
UV/VIS detector, and a 20 μL manual injector (Rheodyne 7725i,
Cotati, CA, USA). Flavonoids were separated on a steel column
(250 x 4 mm i.d., 5 μm particle), LiChrospher 100 RP - 18 (Merck,
Germany). The mobile phases were a gradient prepared from
solvent A (0.5% aqueous solution of orthophosphoric acid, v/v),
solvent B (MeOH), solvent C (acetonitrile - ACN) and solvent D
(tetrahydrofuran - THF). The identification of flavonoid peaks was
based on the internal and external standard methods using the same
chromatographic conditions. Two chromatographic procedures were
optimized for analyses of flavonols (System I) and isoflavones
(System II). System I: elution profile: 0 – 12 min: 45 – 60 % B in A;
flow rate: 1.6 mL/min; detection at 370 nm. System II: elution profile:
0 – 8 min: 13 – 30 % C + 0.4 % D in A, 8 - 15 min: 30 % C + 0.4 % D
in A (isocratic), 15 – 18 min: 30 – 36 % C + 0.4 % D in A, 18 – 23 min:
Total flavonoid determination: The quantity of flavonoids in the
plant materials was determined by the spectrophotometric method
recommended by the Polish Pharmacopoeia VIII [3]. The applied
procedure involves the hydrolysis of flavonoid glycosides, the
formation of coloured complexes of the aglycones with AlCl3, the
measurement of absorbance at 425 nm on a Carl Zeiss Jena
spectrophotometer (GDR) and the calculation of the total flavonoid
content, expressed as mg of quercetin (mg QE/g dry weight of plant
material) and hyperoside (mg HP/g dry weight of plant material)
equivalents. The analytical procedure for the samples of 0.3 g and
0.5 g of T. repens flowers and leaves, respectively was repeated 3
times.
Total phenols determination: The total phenolic content (TPC) was
determined using the Folin-Ciocalteu reagent (FCR) method [6].
This method depends on the reduction of FCR by phenols to a
mixture of blue oxides, which have a maximal absorption in the
region of 760 nm using a Lambda 25 spectrophotometer (Perkin-
Elmer, USA). Quantities of 0.2 and 0.4 g of T. repens flowers and
102 Natural Product Communications Vol. 8 (1) 2013 Kicel and Wolbiś

leaves, respectively were refluxed for 30 min with 30 mL 70% (v/v)
aqueous MeOH, filtered and twice extracted for 15 min with 20 mL
of 70% MeOH to give the crude methanolic extract (CME). 1 mL of
the CME sample was mixed with 5 mL of FCR (previously pre-
diluted 10 times with water) and allowed to react for 3 min. Then,
the mixture solution was diluted to 10 mL with aqueous sodium
carbonate solution (Na2CO3 x 10 H2O, 202.5 g/L). The reaction was
kept in the dark for 2 h, after which its absorbance was read at
760 nm. The solution without the FCR was used as a blank. The
TPC was expressed in terms of gallic acid equivalent (mg GAE/g
dry weight of plant material), as calculated from the following 6-
point calibration curve of gallic acid (GA):
A760 = 0.0356 [GA (µg/mL)] + 0.0113 (R2 = 0.9980), linearity:
0.99 – 9.86 µg/mL
DPPH free radical scavenging assay: The DPPH test for each
CME sample was determined using the method described by [6]. A
solution of DPPH˙ (36 µg/mL, 91 µM) was prepared in MeOH and
kept in the dark at 4ºC. The initial absorbance of the DPPH˙ in
MeOH (2:1, v/v) was measured at 517 nm to give a value of
0.700 ± 0.030. The analytical samples for DPPH˙ measurements (5-
times) were prepared by mixing 1 mL methanolic solution of CME
at 3 different concentrations (33.33 to 240.00 µg/mL) with 2 mL of
equilibrated DPPH˙ solution. After 60 min of incubation in the dark
at room temperature, the reduction of DPPH˙ was measured by
reading the absorbance at 517 nm. The DPPH˙ control (containing
no sample of DPPH˙) was prepared using the same procedure. The
DPPH˙ concentration in the reaction medium was calculated from
the following 5-point calibration curve:
A517 = 0.0274 [DPPH˙ (µg/mL)] – 0.0071 (R2 = 0.9999), linearity:
2.58 – 77.33 µg/mL
For each CME, its 3 different concentrations were plotted against
the percentage of remaining DPPH˙ to obtain the amount of the
extracts (EC50) necessary to decrease the initial DPPH˙
concentration by 50%. Trolox and quercetin were used as reference
compounds. The activity of the extracts was then expressed in terms
of trolox equivalent antioxidant activity (TEAA).

References
[1] (a) Sabudak T, Guler N. (2009) Trifolium L. – A review on its phytochemical and pharmacological profile. Phytotherapy Research, 23, 439 – 446; (b) Szafer W,
Kulczyński S, Pawłowski B. (1988) Polish Plants. Warszawa, PWN (in Polish), 354-360; (c) Strzelecka H, Kowalski J. (2000) The Encyclopedia of
Herbs and Herbalism. Warszawa PWN (in Polish), 242; (d) Tangpu V, Temjenmongla K, Yadav AK. (2004) Anticestodal activity of Trifolium repens
extract. Pharmaceutical Biology, 42, 656-658.
[2] (a) Kicel A, Wolbiś M. (2012) Study on the phenolic constituents of the flowers and leaves of Trifolium repens L. Natural Product Research, 26, 2050-2054;
(b) Carlsen SCK, Mortensen AG, Oleszek W, Piacente S, Stochmal A, Fomsgaard S. (2008) Variation in flavonoids in leaves, stems and flowers of white
clover cultivars. Natural Product Communications, 3, 1299-1306; (c) Foo LY, Lu Y, Molan AL, Woodfield DR, McNabb WC. (2000) The phenols and
prodelphinidins of white clover flowers. Phytochemistry, 54, 539-548; (d) Hofmann RW, Swinny EE, Bloor SJ, Markham KR, Ryan KG, Campbell BD,
Jordan BR. (2000) Responses of nine Trifolium repens L. populations to ultraviolet-B radiation: differential flavonol glycoside accumulation and
biomass production. Annals of Botany, 86, 527-537; (e) Saxena VK, Jain AK. (1986) Genistein 7-(2''-p-coumaroylglucoside) from Trifolium repens.
Phytochemistry, 25, 2687-2688; (f) Saxena VK, Jain AK. (1989) A new acetylated isoflavone glycoside from Trifolium repens. Fitoterapia, 60, 85;
(g) Zhan Q-F, Xia Z-H, Wang J-L, Lao A-N. (2003) Two new bicoumarins from Trifolium repens L. Journal of Asian Natural Product Research, 5,
303-306; (h) Kicel A, Wolbiś M. (2012) Coumarins from the flowers of Trifolium repens. Chemistry of Natural Compounds, 48, 130-132; (i)
Sakamoto S, Kofuji S, Kuroyanagi M, Ueno A, Sekita S. (1992) Saponins from Trifolium repens. Phytochemistry, 31, 1773–1777; (j) Kicel A,
Wolbiś M. (2006) Phenolic acids in flowers and leaves of Trifolium repens L and Trifolium pratense L. Herba Polonica, 54, 52-60; (k) Tava A,
Ramella D, Grecchi M, Aceto P, Paoletti R, Piano E. (2009) Volatile constituents of Trifolium pratense and T. repens from N.E. Italian Alpine
pastures. Natural Product Communications, 4, 835-838; (l) Kicel A, Wolbiś M. (2010) Identification of volatile constituents in flowers and leaves
of Trifolium repens L. Journal of Essential Oil Research, 22, 624-627; (m) Kagan IA. (2011) Effect of pH, sample size, and solvent partitioning on
recovery of soluble phenolic acids and isoflavonoids in leaves and stems of red clovers (Trifolium pretense cv. Kenland). Natural Product
Communications, 6, 1657-1660; (n) Janda B, Stochmal A, Montoro P, Piacente S, Oleszek W. (2009) Phenolics in aerial parts of Persian clover Trifolium
resupinatum. Natural Product Communications, 4, 1661-1664; (o) Oleszek W, Stochaml A, Janda B. (2007) Concentration of isoflavones and other
phenolics in the aerial parts of Trifolium species. Journal of Agricultural and Food Chemistry, 55, 8095-8100; (p) Wu Q, Wang M, Simon JE. (2003)
Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass
spectrometric detection. Journal of Chromatography A, 1016, 195-209; (q) Vetter J. (1995) Isoflavones in different parts of common Trifolium species.
Journal of Agricultural and Food Chemistry, 43, 106-108.
[3] Farmakopea Polska VIII (2008) Warszawa,Wyd. PTF-arm, 1180.
[4] European Pharmacopoeia, 5th ed., (2004) European Pharmacopoeia Commission, Aubin, Liguge, France, 1104.
[5] Veit M, Beckert C, Hohne C, Bauer K, Geiger H. (1995) Interspecific and intraspecific variation of phenolic in the genus Equisetum subgenus
Equisetum. Phytochemistry, 38, 881-891.
[6] Olszewska MA, Nowak S, Michel P, Banaszczak P, Kicel A. (2010) Assessment of the content of phenolics and antioxidant action of
inflorescences and leaves of selected species from the genus Sorbus sensu strict. Molecules, 15, 8769-8783.