Isolation of two new bioactive proanthocyanidins from Cistus salvifolius herb extract

Article abstract of DOI:10.1691/ph.2011.0839

Two new proanthocyanidins, epigallocatechin-3-O-p-hydroxybenzoate- (4β→8)-epigallocatechin (1) and epigallocatechin-3-O-p- hydroxybenzoate-(4β→8)-epigallocatechin-3-O-gallate (2) in addition to the known compound epigallocatechin-(4β→6)-epigallocatechin-3-O- gallate (3), were isolated from the air-dried herb of Cistus salvifolius. The chemical structures were determined on the basis of 1D-and 2D-NMR-spectra (HSQC, HMBC) of their peracetylated derivatives, MALDI-TOF-mass spectra, and by acid-catalysed degradation with phloroglucinol. The isolated compounds 1-3 and the water extract of C. salvifolius herb were tested for their inhibitory activities against COX-1 and COX-2. Compound 2 showed the strongest inhibitory effect on COX-2 followed by compound 3, compound 1 and the water extract, while compounds 1-3 exhibited moderate in vitro inhibition against COX-1.

Full text of DOI:10.1691/ph.2011.0839

ORIGINAL ARTICLES
Faculty of Pharmacy1, The University of Petra, Jordan, Institute of Pharmaceutical Biology and Phytochemistry2,
University of Münster, HerbResearch3, Tussenhausen-Mattsies, Germany
Isolation of two new bioactive proanthocyanidins from Cistus salvifolius
herb extract
F. Qa’dan1, A. Nahrstedt2, M. Schmidt3
Received November 8, 2010, accepted December 10, 2010
Dr. Fadi Qa’dan, Petra University, Faculty of Pharmacy PO Box: 961343, Airport street, Amman-11196 Jordan
fqadan@gmail.com
Pharmazie 66: 454–457 (2011)
doi: 10.1691/ph.2011.0839
Two new proanthocyanidins, epigallocatechin-3-O-p-hydroxybenzoate-(4␤→8)-epigallocatechin (1) and
epigallocatechin-3-O-p-hydroxybenzoate-(4␤→8)-epigallocatechin-3-O-gallate (2) in addition to the known
compound epigallocatechin-(4␤→6)-epigallocatechin-3-O-gallate (3), were isolated from the air-dried herb
of Cistus salvifolius. The chemical structures were determined on the basis of 1D-and 2D-NMR-spectra
(HSQC, HMBC) of their peracetylated derivatives, MALDI-TOF-mass spectra, and by acid-catalysed degra-
dation with phloroglucinol. The isolated compounds 1–3 and the water extract of C. salvifolius herb were
tested for their inhibitory activities against COX-1 and COX-2. Compound 2 showed the strongest inhibitory
effect on COX-2 followed by compound 3, compound 1 and the water extract, while compounds 1–3
exhibited moderate in vitro inhibition against COX-1.
1. Introduction
acetate (1a), which suggests a B-type dimeric prodelpinidin
with a p-hydroxybenzoic acid acylation. The HNMR of 1a
1
The genus Cistus (Cistaceae) comprises about 20 shrub species
found in wide areas throughout the whole Mediterranean region
(Comandini et al. 2006). Cistus species are used as anti-
diarrheic, as general remedies in folk medicine for the treatment
of various skin diseases, and as anti inflammatory agents
(Attaguile et al. 2000; Petereit 1992). Furthermore, a specific
polyphenol containing Cistus incanus extract (CYSTUS052)
demonstrated antiviral activity against influenza A virus infec-
tions (Droebner et al. 2007; Ehrhardt et al. 2007). In Jordan,
a tea prepared from Cistus salvifolius L. herb has traditionally
been used for the treatment of gout (Al-Khalil 1995). Pharmaco-
logical investigations on traditional Turkish plants have shown
that C. salvifolius is highly effective for the treatment of ulcer
(Yesilada et al. 1999). The ethyl acetate-soluble fraction from
the aqueous acetone extract was found to contain the following
flavan-3-ols and dimeric prodelphinidins: catechin, gallocat-
echin, epicatechin, epigallocatechin, epicatechin-3-O-gallate,
epigallocatechin-3-O-gallate, epigallocatechin-3-O-p-hydroxy-
benzoate, epigallocatechin-(4␤→8)-epigallocatechin, epigallo-
catechin-3-O-gallate-(4␤→8)-epigallocatechin, and epigallo-
catechin-(4␤→6)-epigallocatechin-3-O-gallate (Danne et al.
1994). In a recent study, a higher oligomeric proanthocyani-
din containing fraction isolated from the title plant exhibited the
strongest antioxidant activity of all isolated fractions (Qa’dan
etal. 2006). Incontinuationofourphytochemicalandpharmaco-
logical studies on Cistus species, we report here on the isolation,
structure elucidation and testing of new polyphenolic com-
pounds for their inhibitory activities against COX-1 and COX-2.
in CDCl3 (600 MHz) was similar to that of the analogous
proanthocyanidin epigallocatechin-(4␤→8)-epigallocatechin-
3-O-p-hydroxybenzoate peracetate (De Mello et al. 1996).
The 1H-NMR of 1a gave two two-proton singlets at δ
6.90 and 7.22 ppm typical for pyrogallol-type-B rings of
the constituent flavan-3-ol units. The heterocyclic coupling
constants (J2,3 < 2 MHz) confirmed the relative 2,3-cis con-
figuration of the “upper” and “lower” constituent units
(Fletcher et al. 1977). The acyl substituent was identified
from the AA’BB’ type aromatic signal set at ␦ 7.11 and
7.88 ppm, which indicate the equivalent protons of a p-
hydroxybenzoyl group. The 3J cross peak of H-3 (C) to
the carboxyl group was the direct proof for the point of
attachment of the acyl substituent on C-3 position of the
upper flavan-3-ol unit. The location of the interflavanoid
linkage was recognized for 1a by long-range correlation
(HMBC) of the H-4 (C) with the C-8a (D) (Balas and
Vercauteren 1994). This key correlation indicates that the
flavan-3-ol units are C-4/C-8 linked. The correlation between
H-8 (A) and H-4 (C) to the C-8a (A) confirmed the “upper”
flavan-3-ol unit as epigallocatechin-3-O-p-hydroxybenzoate.
Compound 1 gave epigallocatechin-3-O-p-hydroxybenzoate-
(4␤→2)-phloroglucinol as the main degradation product, and
epigallocatechin as releasing terminal flavan-3-ol (Foo and
Karchesy 1989). These degradation products were identified by
NMR-spectroscopy and compared with authentic compounds.
The intense positive Cotton effect at 210–240 nm in the CD
spectrum of 1a is in line with the 4␤-linkage, and thus a 4R
absolute configuration (Barrett et al. 1979; Botha et al. 1981).
In conjunction with the optical rotation [α]2D0 = –47.3◦ (C 0.18,
MeOH), compound 1 was identified as epigallocatechin-3-O-p-
hydroxybenzoate-(4␤→8)-epigallocatechin.
2. Investigations, results and discussion
Compound 1 showed a prominent quasi-molecular ion peak
at m/z 1257 (M + Na(+ in the MALDI-TOF-MS of its per-
454
Pharmazie 66 (2011)

ORIGINAL ARTICLES
Table: IC50 values of isolated compounds as inhibitors of COX-
1 and COX-2a
OR
OR
6'
1
8
B
COX-1 [␮M]
COX-2 [␮M]
8a
4a
RO
O
C
OR
G
2'
2
A
1''
4''
WE
1
2
3
ASA
63 1.0
44.7 0.8
37.6 3.2
12.3 0.3
29.3 1.1
17.5 0.3
1a
6
O
OR
3
78.9 1.7
65.2 0.9
73.5 2.1
21.3 0.4
4
OR
O
OR
E
OR
OR
a
RO
O
F
Values are means data obtained from three different assays. WE water extract
D
OR
of the constituent flavan-3-ol units. The heterocyclic cou-
pling constants (J2,3 < 2 MHz) confirmed the relative 2,3-cis
configuration of the “upper” and “lower” constituent units
(Fletcher et al. 1977). The identity of the acyl substituents
was obvious from an AA’BB’ type aromatic signal set at
␦ 7.18 and 7.90 ppm and a sharp low-field two-proton sin-
glet (δ 7.72 ppm), which indicate the equivalent protons of
a p-hydroxybenzoyl group and a galloyl moiety, respectively.
The long-range correlation signals between the H-3 (C) and
H-3 (F) with the corresponding carboxyl functions of the
attached acyl moieties were the direct proof for the point of
attachment at C-3 position of both units. The location of the
interflavanoid linkage was recognized for 2a by long-range
correlation (HMBC) of the H-4 (C) with the C-8a (D) (Balas
and Vercauteren 1994). This key correlation indicates that the
flavan-3-ol units are C-4/C-8 linked. The correlation between
H-8 (A) and H-4 (C) to the C-8a (A) confirmed the “upper”
flavan-3-ol unit as epigallocatechin-3-O-p-hydroxybenzoate.
Compound 2 gave epigallocatechin-3-O-p-hydroxybenzoate-
(4␤→2)-phloroglucinol as the main degradation product, and
epigallocatechin-3-O-gallate as releasing terminal flavan-3-
ol (Foo and Karchesy 1989). These degradation products
were identified by NMR-spectroscopy and in comparison
with authentic compounds. The intense positive Cotton effect
at 210–240 nm in the CD spectrum of 2a is in line with
the 4␤-linkage, and thus a 4R absolute configuration (Bar-
rett et al. 1979; Botha et al. 1981). In conjunction with the
optical rotation [α]2D0 = –33.6◦ (C 0.15, MeOH), compound
2 was identified as epigallocatechin-3-O-p-hydroxybenzoate-
(4␤→8)-epigallocatechin-3-O-gallate.
OR
1: R = H
1a: R = Acetyl
OR
OR
6'
1
8
B
8a
RO
O
C
OR
G
2'
1a
2
A
1''
4''
6
O
OR
3
4a
OR
4
O
OR
E
OR
OR
RO
O
F
OR
D
1a
O
O
OR
H
1''
4''
OR
OR
2: R = H
2a: R = Acetyl
OR
OR
OR
RO
O
OR
OR
The structure of compound 3 was identified on the basis of 1D-
and 2D-NMR (HSQC, HMBC) experiments of its peracetylated
derivatives. Comparison of the data with authentic samples from
earlier work and published values, identified this compound
as epigallocatechin-(4␤→6)-epigallocatechin-3-O-gallate (De
Mello et al. 1996). To the best of our knowledge, compounds 1
and 2 are described here for the first time.
RO
OR
RO
RO
O
RO
O
O
The Cistus water extract (WE) and the isolated compounds
were tested for their in vitro inhibitory activities against
cyclooxygenase (COX-I and COX-2, Table 1). The WE and
the pure compounds 1–3 were found to have potent COX-
2 inhibitory effects, with IC50 values of 44.7 ␮M, 37.6 ␮M,
12.3 ␮M and 29.3 ␮M, respectively; the WE and all the iso-
lated compounds (1–3) exhibited moderate inhibition against
COX-1 (Table). The new compound epigallocatechin-3-O-
p-hydroxybenzoate-(4␤→8)-epigallocatechin-3-O-gallate (1)
showed higher COX-2 inhibitory activity (IC50 = 12.3 ␮M) than
the positive control acetyl salicylic acid (ASA, IC50 = 17.5 ␮M).
The presence of a p-hydroxybenzoyl moiety in addition to the
galloylated moiety, and two trihydroxylated B-rings in the iso-
lated new compound 2, and its relative higher molecular weight
might be responsible for the potent inhibitory activity against
COX-2 (Seeram et al. 2003). The water extract showed rela-
tively high inhibition (Table), possibly because it contains in
RO
OR
OR
3: R = H
3a: R = Acetyl
Compound 2 showed a prominent quasi-molecular ion peak at
m/z 1493 (M + Na(+ in the MALDI-TOF-MS of its peracetate
(2a), indicating an acylated prodelphinidin dimer. The1H-
NMR of 2a in CDCl3 (600 MHz) was similar to that of
the analogous proanthocyanidin epigallocatechin-3-O-gallate-
(4␤→8)-epigallocatechin-3-O-gallate-peracetate (De Mello
et al. 1996). The 1H NMR of 2a gave two two-proton sin-
glets at (6.82 and 7.07 ppm typical for pyrogallol-type-B rings
Pharmazie 66 (2011)
455

ORIGINAL ARTICLES
addition to the known tannins (Danne et al. 1994; Qa’dan et al.
2006) also other compounds like flavonols and phloroglucinol
glucosides (Danne et al. 1994) which were reported to have
anti-inflammatory effects (Clavin et al. 2007). Further studies
are necessary to explore in more depth the anti-inflammatory
potency of compound 2.
In conclusion, the two new isolated proanthocyanidins (Fig. 1)
showed great similarity in the chemical structure to the flavan-3-
ols and oligomeric proanthocyanidins isolated previously from
the title plant (Danne et al. 1994; Qa’dan et al. 2006) in the
predominance of 2,3-cis-configuration, 3’,4’,5’-trihydroxylated
B-rings and the presence of galloylated units.
H-2 (F)], ␦ 5.53 [brs, H-2 (C)], ␦ 5.59 [m, H-3 (C)], ␦ 5.75 [m, H-3 (F)], ␦
6.30 [d, J = 2.3 Hz, H-8 (A)], ␦ 6.33 [d, J = 2.3 Hz, H-6 (A)], ␦ 6.71 [s, H-6
(D)], ␦ 6.90 [s, H-2’/H-6’ (E)], ␦ 7.11 [d, J = 8.7 Hz, H-3”/H-5” (G)], ␦ 7.22
[s, H-2’/H-6’ (H)], 7.88 [d, J = 8.7 Hz, H-2”/H-6” (G)]. 13C-NMR (CDCl3,
150 MHz): ␦ 29.3 [C-4 (F)], ␦ 36.5 [C-4 (C)], ␦ 67.7 [C-3 (F)], ␦ 72.4 [C-3
(C)], ␦ 74.7 [C2 (C)], ␦ 76.6 [C-2 (F)], ␦ 108.1 [C-6 or C-8 (A)], ␦ 108.5
[C-6 or C-8 (A)], ␦ 111.3 [C-8 (D)], ␦ 121.6 [C-2”/C-6” (G)], ␦ 127.1 [C-1”
(G)], ␦ 138.8 [C-4” (G)], ␦ 142.3 [C-3”/C5” (G)], ␦ 163.6 [Carboxyl (G)].
G represents the 1H/13C signals of the attached hydroxybenzoyl moiety.
3.6. Epigallocatechin-3-O-p-hydroxybenzoate-(4␤→8)
epigallocatechin-3-O-gallate (2)
Fraction
8 (17500 - 18200 ml, 560 mg) achieved from Sephadex
LH-20 column was subjected to chromatography on MCI gel CHP
20P (25 × 450 mm) with a 10 - 60% (3 L) MeOH linear gradient
(16 ml/subfraction) to afford an amorphous white powder (compound 2,
subfractions 43–47, 33 mg); Acetylation yielded epigallocatechin-3-O-p-
3. Experimental
hydroxybenzoate-(4␤→ 8)-epigallocatechin-3-O-gallate -peracetate (1a).
3.1. General experimental procedures
[α]2D0 = + 46,65◦ (c 0.19, MeOH); MALDI-TOF-MS: [M + Na]
m/z
+
NMR spectra were recorded in CDCl3 with a a Bruker AM 600 instrument.
Chemical shifts were recorded relative to CHCl3. CD spectra were measured
in MeOH on a CD spectrometer AVIV 62A DS. Acetylation was performed
in Ac2O pyridine (1.2:1) at ambient temperature for 24 h. MALDI-TOF-
mass spectrometer: LAZARUS II (home-built), N2-laser (LSI VSL337ND)
337 nm; 3 ns puls width, focus diameter, 0.1 mm; acceleration voltage 16 kV;
1 m drift length; data logging with LeCroy9450A; sampling time 2.5 ns,
expected mass accuracy, +/– 0.1%; compounds were deposited from a solu-
tion in CHCl3 on a thin layer of 2,5-dihydroxybenzoic acid (DHB) crystals.
Analytical TLC was done on silica gel GF254 plates (Merck) with the mobile
phase EtOAc/HCOOH/H2O (18:1:1). Compounds were visualized as red
spots by spraying with vanillin/HCl-reagent. Optical rotation ([␣]) was
measured using a Perkin-Elmer polarimeter 241.
1377. CD [␪]295 + 3029, [␪]278 –10337, [␪]234 + 172395, [␪]216–16155,
[θ]210 + 91. Degradation of 18 mg 1 with 15 mg phloroglucinol in 2.5 mL
1% ethanolic HCl yielded 7 mg epigallocatechin-3-O-p-hydroxybenzoate-
(4␤→2)-phloroglucinol and 5.5 mg of the intact epigallocatechin-3-O-
gallate 5.5 mg, which were purified using a Sephadex LH-20 column
(60 × 25 mm) with first 500 ml EtOH-MeOH (1:1), then 400 mL MeOH as
eluent and identified as peracetates with NMR and MALDI-TOF-MS exper-
iments. 1HNMR (CDCl3, 600 MHz, HR = assignment of rotameric signals):
␦ 2.07–2.31 [m, aliphatic and aromatic OAc], ␦ 2.78 [d, J = 16.9 Hz, HR-4a
(F)], ␦ 2.93 [d, J = 16.9 Hz, H-4a (F)], ␦ 3.09 [s, H-4b (F)], ␦ 4.34 [s, HR-4
(C)], ␦ 4.51 [s, H-4 (C)], ␦ 5.29 [s, H-2 (F)], ␦ 5.43 [s, H-2 (C)], ␦ 5.53 [s,
H-3 (C)], ␦ 5.65 [m, HR-3 (F)], ␦ 5.70 [m, H-3 (F)], ␦ 6.70 [d, J = 2.2 Hz, H-6
or H-8 (A)], ␦ 6.79 [d, J = 2.2 Hz, HR-6 or HR-8 (A)], ␦ 6.82 [d, J = 2.2 Hz,
H-6 or H-8 (A)], ␦ 6.76 [s, H-8 (D)], ␦ 6.87 [s, HR-8 (D)], ␦ 7.09–7.41
[proton signals of the rings B and E], ␦ 7.10 [d, J = 8.8 Hz, HR-3” HR-5”
(H)], ␦ 7.14 [d, J = 8.8 Hz, H-3” H-5” (H)], ␦ 7.51 [s, H-2” H-6” (G)], ␦ 7.54
[s, HR-2” HR-6” (G)], ␦ 7.86 [d, J = 8.8 Hz, HR-2” HR-6” (H)], ␦ 7.92 [d,
J = 8.8 Hz, H-2” H-6” (H)]. 13C-NMR (CDCl3, 150 MHz, CR = assignment
of rotameric signals): ␦ 26.52 [C-4 (F)], ␦ 35.15 [C-4 (C)], ␦ 67.45 [C-3
(F)], ␦ 67.53 [CR-3 (F)], ␦ 72.35 [C-3 (C)], ␦ 74.97 [CR-2 (C)], ␦ 74.02 [C-2
(C)], ␦ 76.88 [C-2 (F)], ␦ 107.33 [C-6 or C-8 (A)], ␦ 107.35 [CR-6 or CR-8
(A)], ␦ 108.88 [C-6 or C-8 (A)], ␦ 109.80 [CR-8 (D)], ␦ 110.67 [C-8 (D)],
␦ 121.60 [C-3” C-5” (H)], ␦ 122.24 [C-2” C-6” (G)], ␦ 127.48 [C-1” (G)],
␦ 131.50 [C-2” C-6” (H)], ␦ 139.00 [C-4” (G)], ␦ 143.52 [C-3” C5” (G)],
␦ 154.14 [C-4” (H)], ␦ 162.93 [Carboxyl (G)], ␦ 165.44 [Carboxyl (H)]. G
represents the 1H/13C signals of the attached galloyl moiety, H represents
the 1H/13C signals of the attached hydroxybenzoyl moiety.
3.2. Chemicals and reagents
COX-1 and COX-2 were purchased from Sigma (Munich, Germany). 1-14C-
labeled arachidonic acid (>200 ␮Ci (370 kBq), NEN) was purchased from
New England Nuclear Co. (Boston, USA). Other chemicals and reagents
were purchased from Roth Chemicals (Dubai, UAE).
3.3. Plant material
Cistus salvifolius L. was collected in El Majdal (Jordan; 04/2006) and iden-
tified in comparison with authentic Cistus salvifolius obtained from the
Botanical Garden, University of Jordan (Amman). A voucher specimen is
deposited at the Petra University, Amman-Jordan under PUAM19.
3.4. Extraction and isolation
3.7. Effect on cyclooxygenase –1 and –2
Air-dried material (4 kg) was exhaustively extracted with 30 L hot water
(40 ◦C) and the combined extracts were evaporated in vacuo to dryness
(534 g). A portion (100 g) of the water-extract was applied to CC on
Sephadex LH-20 (55 × 900 mm) and successively eluted with 5 L EtOH-
H2O and 20 L MeOH-H2O (1:1) to afford 10 fractions. Fraction 8 (17500
- 18200 ml, 560 mg) obtained from Sephadex LH-20 column was subjected
to chromatography on MCI gel CHP 20P (25 × 450 mm) with a 10–60%
MeOH (3 L) linear gradient (16 ml/subfraction) to afford an amorphous
whitepowder(compound 3, subfractions16-23, 15.5 mg). Compound 3was
identified after acetylation by its physical data (NMR, MS, CD) and by com-
parisonwithanauthenticsampleandpublishedvalues(DeMelloetal. 1996).
The effect on cyclooxygenase-1 and –2 (COX-1 and COX-2) was deter-
mined by measuring PGE2 production. The reaction mixtures were prepared
in tris(hydroxymethyl)aminomethane-HCl buffer (pH 8.0), containing glu-
tathione (350 ␮M), epinephrine (350 ␮M), hematin (1.5 ␮M), enzyme
(COX-1 or COX-2, 50 ␮l) and various concentrations of the water extract
(WE) or isolated compounds. 1–14C arachidonic acid (10 ␮l) was added
to start the reaction. The mixture was first incubated for 30 min at 37 ◦C,
then the reaction was terminated by adding the reaction mixture (20 ␮l)
to 30 ␮M indomethacin (200 ␮l). Arachidonic acid and its radio-labeled
metabolites were separated and determined by reversed-phase HPLC using
aBertholdradioactivitymonitor(PharmaTechR&D, Amman, Jordan). Inhi-
bition refers to the reduction of PGE2 formation, in comparison to a blank
run without inhibitor. ASA was used as a positive control. The results are
means of three independent experiments.
3.5. Epigallocatechin-3-O-p-hydroxybenzoate-(4␤→ 8)-
epigallocatechin
(1): Fraction 7 (16700–17500 ml, 760 mg) obtained from Sephadex LH-
20 column was subjected to chromatography on MCI gel CHP 20P
(25 × 450 mm) with a 10 - 60% MeOH (3 L) linear gradient (compound
1, 16 ml/subfraction) to afford an amorphous white powder (compound
subfractions 33 - 38, 41 mg); Acetylation yielded epigallocatechin-3-
O-p-hydroxybenzoate-(4␤ → 8)-epigallocatechin-peracetate (1a). [α]D20
Acknowledgements: We acknowledge the University of Petra (Jordan) for
a grant and the help of Prof. Dr. R. Peters (Institut fuer Organische Chemie,
Stuttgart, Germany) for the NMR spectra, Dr. H. Luftmann (Institut fuer
Organische Chemie, Muenster, Germany) for the MALDI-MS spectra, M.
Odwan (Pharma Tech R&D, Amman, Jordan) for the HPLC-determinaion
of radio-labeled metabolites and Prof. Dr. V. Buß (Theoretische Chemie,
Duisburg, Germany) for the CD spectra.
= + 47,3◦ (c 0.18, MeOH); MALDI-TOF-MS: [M + Na] m/z 1257. CD
+
[␪]293 + 2879, [␪]274 –12134, [␪]237 + 131455, [␪]216 – 15685, [θ]210 + 171.
Degradation of 12 mg 1 with 15 mg phloroglucinol in 2.5 mL 1% ethano-
lic HCl yielded 5 mg epigallocatechin-3-O-p-hydroxybenzoate-(4␤→2)-
phloroglucinol and 3.7 mg of the intact epigallocatechin-3-O-gallate 5.5 mg,
which were purified using a Sephadex LH-20 column (60 × 25 mm) with
first 500 ml EtOH-MeOH (1:1), then 400 mL MeOH as eluent and identi-
fied as peracetates with NMR and MALDI-TOF-MS experiments. 1H-NMR
(CDCl3, 600 MHz): ␦ 1.88–2.28 [m, aliphatic and aromatic OAc], ␦ 2.91
[m, H-4ax (F) and H-4eq (F)], ␦ 4.53 [d, J = 1.9 Hz, H-4 (C)], ␦ 4.66 [brs,
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