Antihepatotoxic, nephroprotective, and antioxidant activities of phenolic compounds from Satureja macrostema leaves against carbon tetrachloride-induced hepatic damage in mice

Article abstract of DOI:10.1007/s00044-012-0176-x

Satureja macrostema (SM) is used with culinary and medicinal purposes. Methanol extract from SM was investigated for its phenolic content, antioxidant, hepatoprotective, and kidney protective activities. Liver and kidney damage were induced in rats with CCl₄. Hepatoprotective efficacy was measured by the activity of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total bilirubin, cholesterol, high density lipoprotein and total protein, and lipid peroxidation. Kidney function was evaluated by measuring plasma urea and creatinine. Antioxidant activity was evaluated by measuring blood glutathione content, superoxide dismutase and catalase activities, and malondialdehyde equivalent; their activity was comparable to that of silymarin, a well-known hepatoprotective agent. Methanol extracts of S. macrostema showed potent antioxidant, kidney protective, and hepatoprotective activities; in-depth chromatographic investigation resulted in the identification of six new flavonoid glycosides: 5-hydroxy-3,6,4′- trimethoxyflavonol-7-C-α-l-rhamnopyranosyl-(1 → 3)-β-d- glucopyranoside (2), 4′-methoxy-5,7,3′,5′-tetra- hydroxyflavanone-3-O-β-d-rhamnopyranosyl-(1 → 2)-β-d- rhamnopyranoside (3), 5,4′-dimethoxy-7,3′,5′- trihydroxyflavanone-3-O-β-d-rhamnopyranoside (4), 5,3′,4′, 5′-tetrahydroxyflavanone-7-O-β-d-rhamnopyranoside (5), 5,3′,4′,5′-tetramethoxyflavanone-7-O-β-d- rhamnopyranoside (6), and 5,4′-dimethoxy-3′-hydroxyflavone-7-β- d-rhamnopyranoside (8) along with three known compounds: 5-hydroxy-7,4′- dimethoxyflavone (1), prunin (7), and diosmin (9) that were isolated. Structural elucidation of the new compounds was established based on the spectral data. The present study revealed that S. macrostema leaves have a significant radical scavenging and hepatoprotective activity.

Full text of DOI:10.1007/s00044-012-0176-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1846–1855
DOI 10.1007/s00044-012-0176-x
ORIGINAL RESEARCH
Antihepatotoxic, nephroprotective, and antioxidant activities
of phenolic compounds from Satureja macrostema leaves against
carbon tetrachloride-induced hepatic damage in mice
Rosa Martha Perez Gutierrez
Received: 15 February 2011 / Accepted: 7 July 2012 / Published online: 15 August 2012
ꢀ Springer Science+Business Media, LLC 2012
Abstract Satureja macrostema (SM) is used with culinary
and medicinal purposes. Methanol extract from SM was
investigated for its phenolic content, antioxidant, hepato-
protective, and kidney protective activities. Liver and kidney
damage were induced in rats with CCl4. Hepatoprotective
efficacy was measured by the activity of aspartate amino-
transferase, alanine aminotransferase, alkaline phosphatase,
total bilirubin, cholesterol, high density lipoprotein and total
protein, and lipid peroxidation. Kidney function was evalu-
ated by measuring plasma urea and creatinine. Antioxidant
activity was evaluated by measuring blood glutathione con-
tent, superoxide dismutase and catalase activities, and mal-
ondialdehyde equivalent; their activity was comparable to
that of silymarin, a well-known hepatoprotective agent.
Methanol extracts of S. macrostema showed potent antioxi-
dant, kidney protective, and hepatoprotective activities;
in-depth chromatographic investigation resulted in the iden-
tification of six new flavonoid glycosides: 5-hydroxy-3,
6,40-trimethoxyflavonol-7-C-a-L-rhamnopyranosyl-(1 ? 3)
-b-D-glucopyranoside (2), 40-methoxy-5,7,30,50-tetra-hydroxy
flavanone-3-O-b-D-rhamnopyranosyl-(1 ? 2)-b-D-rhamnopyr-
anoside (3), 5,40-dimethoxy-7,30,50-trihydroxyflavanone
-3-O-b-D-rhamnopyranoside (4), 5,30,40,50-tetrahydroxy-
flavanone-7-O-b-D-rhamnopyranoside (5), 5,30,40,50-tetra-
methoxyflavanone-7-O-b-D-rhamnopyranoside (6), and 5,
40-dimethoxy-30-hydroxyflavone-7-b-D-rhamnopyranoside
(8) along with three known compounds: 5-hydroxy-7,40-
dimethoxyflavone (1), prunin (7), and diosmin (9) that were
isolated. Structural elucidation of the new compounds was
established based on the spectral data. The present study
revealed that S. macrostema leaves have a significant rad-
ical scavenging and hepatoprotective activity.
Keywords Satureja macrostema ꢀ
Hepatoprotective activity ꢀ Kidney protective ꢀ
Flavonoids glycosides
Introduction
Oxidative stress resulting from the toxic effects of free
radicals on the tissue plays an important role in the patho-
genesis of various pathological conditions such as ischemia,
anemia, asthma, arthritis, inflammation, neurodegeneration,
Parkinson’s diseases, Alzheimer disease, Down syndrome,
aging process, and perhaps some dementias. Antioxidants
are radical scavengers, which protect the human body
against free radicals (Mahakunakorn et al., 2004). Free
radicals cause one of the most important mechanisms of
liver damage. Metabolism of certain agents like carbon
tetrachloride (CCl4), produce free radicals, which cause
liver damage. Antioxidants may offer resistance against
oxidative stress by scavenging free radicals, inhibiting lipid
peroxidation, and by other mechanisms, and thus prevent
disease (Miller and Rice-Evans, 1997).
CCl4 is one of the most commonly used hepatotoxin in
the experimental study of liver diseases. Various studies
have demonstrated that the liver is not the only target organ
of CCl4; it can also induce free radical generation in other
tissues such as kidneys, heart, lung, testis, brain, and blood
(Ozturk et al., 2003; Perez et al., 1987). Several plants
have been tested for their efficacy in controlling the CCl4-
induced liver damage. Furthermore, it has been evident that
R. M. P. Gutierrez (&)
Laboratorio de Investigacion de Productos Naturales, Escuela
´
´
´
Superior de Ingenierıa Quımica e Industrias Extractivas IPN,
Punto Fijo 16, Col. Torres Lindavista, cp 07708 Mexico, D.F.,
Mexico
e-mail: julio_tellez@yahoo.com; rmpg@prodigy.net.mx
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Med Chem Res (2013) 22:1846–1855
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Chromatographic purification and isolation
several phytoconstituents have the ability to induce
microsomal enzymes either by accelerating the excretion of
CCl4 or by inhibition of lipid peroxidation induced by
CCl4. Phytoconstituents like flavonoids and triterpenoids
are known to possess hepatoprotective activity (Baek et al.,
1996). Damage of liver cells is reflected by an increase in
the levels of hepatospecific cytoplasmic enzymes that are
released into circulation after cellular damage (Sallie et al.,
1991).
The dried leaves of S. macrostema (9 kg) were powdered
and extracted three times with methanol under reflux for
3 h, and concentrated in a rotatory evaporator.
The resulting extract (918 g residue) was loaded onto a
silica gel chromatography column and eluted with CHCl3–
EtOAc 2:1 and 7 fractions (F1 to F7) were obtained. These
fractions were then tested for hepatoprotective and anti-
oxidant activities. Active fractions were pooled together
according to their similarities provided by thin-layer
chromatography analysis. The fraction F4 was the fraction
that showed hepatoprotective and antioxidant properties.
F4 was subjected to silica gel column chromatography
eluted with CHCl3-ethyl ether (2:1) to produce six fractions
(F4-1 to F4-6). The active fraction F4-1 was subjected to
chromatography over the silica gel column using CHCl3–
EtOAc 2:1 to yield five subfractions (F41-1 to F41-5). The
F41-2 fraction was further purified by a preparative plate
using CHCl3-ethyl ether-acetone 7:1:0.5 and visualized
with UV at 254 nm to yield 1 (155 mg), 2 (170 mg), and 3
(190 mg). Fraction F41-4 was separated by Sephadex LH-
20 using a gradient of CHCl3–MeOH (from 10:1 to 5:1) to
yield 4 (1670 mg) and 5 (178 mg). F4-5 was subjected to
silica gel CC using ethyl ether-EtOAc 7:1 to yield eight
fractions (F45-1 to F45-8). Fraction F45-6 was separated
by Sephadex LH-20 using a gradient of CHCl3–MeOH
(from 10:1 to 5:1) afforded 6 (169 mg), 7 (189 mg), 8
(150 mg), and 9 (138 mg).
Satureja macrostema (Benth.) Bricq. (Aguilar, 2001)
original from Mexico is commonly known as ‘‘Nurite.’’
The dark green leaves are not only eaten as spices in tra-
ditional Mexican cuisine, but also used to cure many dis-
eases in the forms of decoctions, infusion, reducing
stomach pain and liver diseases, and improving gastroin-
testinal conditions and liver stones (Espejo and Lopez-
Ferrari, 1998). In the course of a preliminary screening test
for antioxidant and hepatoprotective activities, the metha-
nolic extract of S. Macrostema leaves markedly showed
protective effects in liver damage caused by CCl4 in mice
(Perez and Gallardo, 2010). These results led to the sepa-
ration of the biologically active materials of this plant. This
paper describes the isolation and structure elucidation of
flavonoids as well as their hepatoprotective activity.
Materials and methods
General experimental procedures
Characterization of the flavonoids glycosides
Melting point was measured without correction on an
electrothermal model 9100. IR spectra were obtained on a
Perkin–Elmer FTRI 1720X. A Bruker DRX-300 NMR
spectrometer, operating at 599.19 MHz for 1H and
150.86 MHz for l3C, using the UXNMR software package,
was used for NMR experiments; chemical shifts are
expressed in d (ppm) using TMS as an internal standard.
5-Hydroxy-3,6,40-trimethoxy flavonol-7-C-a-L-
rhamnopyranosyl-(1 ? 3)-b-D-glucopyranoside (2)
Yellow amorphous powder; mp 180 ꢁC; High resolution
HREIMS, m/z ([M ? H])? 652.5994 calcd for C30H36O16,
652.5971; IR (KBr): mmax 3430 (OH), 2940, 1629 (C=O),
2700 (phenolic OH), 1460 (arom); 1H NMR (DMSO,
300 MHz) d: 3.35 (9H, s, 39 OMe), 5.91 (1H, s, H-8), 6.72
(2H, d, J = 6.4 Hz, H-30, H-50), 7.15 (2H, d, J = 6.4 Hz,
H-20, H-60), 12.10 (OH-5); 5.4 (1H, d, J = 7.5 Hz, glc
H-100), 4.68 (1H, d, J = 1.5 Hz, Rha, H-1000), 3.16-3.75
(9H, m, sugar protons); 1.21 (3H, d, J = 6.4, H-6000), 13C
NMR (DMSO-d6, 100 MHz) d: 158.44 (C-2), 146.24 (C-
3), 188.01 (C-4),158.31 (C-5), 132.43 (C-6), 162.54 (C-7),
96.06 (C-8), 152.10 (C-9), 103.21 (C-10), 129.34 (C-10),
128.36 (C-20, C-60), 163.05 (C-40), 116.47 (C-30, C-50),
52.76 (OMe), 53.79 (OMe), 56.98 (OMe), Glc: 75.98 (C-
100), 70.44 (C-200), 88.75 (C-300), 69.39 (C-400), 82.61 (C-500),
64.20 (C-600), Rha: 101.76 (C-1000), 72.2 (C-2000), 72.4 (C-
3000), 74.0 (C-4000), 70.6 (C-5000), 17.50 (C-6000).
l
DEPT l3C, ID TOCSY, H-lH DQF-COSY, and HMBC
NMR experiments were carried out using the conventional
pulse sequences as described in the literature. Mass Spec-
trometry was measured on a JEOL HX 110 mass
spectrometer.
Plant collection
The aerial part of the plant S. macrostema was collected in
the month of December 2007 from the Mexican state of
Oaxaca. The plant material was taxonomically identified
and the voucher specimen was deposited in Herbarium of
Escuela Nacional de Ciencias Biologicas, IPN for further
reference (No. 6456).
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Med Chem Res (2013) 22:1846–1855
40-Methoxy-5,7,30,50-tetrahydroxyflavanone-3-O-b-D-
rhamnopyranosyl-(1 ? 2)-b-D-rhamnopyranoside (3)
H-3ax), 6.20 (1H, d, J = 2.1 Hz, H-6), 6.80 (1H, d,
J = 2.1 Hz, H-8), 7.18 (1H, d, J = 9.1 Hz, H-20, 60), 12.10
(s, C-5-OH), Rha: 4.75 (1H, d, J = 1.5 Hz, H-100), 3.72
(1H, dd, J = 3.0, 1.5 Hz, H-200), 3.71 (1H, dd, J = 9.5,
3.0 Hz, H-300), 3.49 (1H, t, J = 9.5 Hz, H-400), 3.92 (1H, m,
H-500), 1.84 (3H, d, J = 6.0 Hz, H-600); 13C NMR (DMSO-
d6, 100 MHz) d: 157.91 (C-2), 42.3 (C-3), 196.03 (C-4),
165.53 (C-5), 96.12 (C-6), 162.8 (C-7), 96.20 (C-8), 154.49
(C-9), 103.54 (C-10), 122.54 (C-10), 110.9 (C-20,60), 167.53
(C-30,50), 162.32 (C-40), Rha: 101.76 (C-100), 72.2 (C-200),
72.4 (C-300), 74.0 (C-400), 29.69 (C-500), 21.50 (C-600).
Yellow needles, mp 174 ꢁC, High resolution HREIMS,
m/z ([M ? H])? 626.5635 calcd for C28H34O16, 626.5606;
IR (KBr): mmax 3530 (OH), 3360–2700 (phenolic OH),
1630 (C=O), 1458 (arom); 1H NMR (DMSO-d6, 300 MHz)
d: 5.55 (1H, dd, J = 13, 2.6 Hz, H-2), 2.57 (1H, dd,
J = 17, 2.6 Hz, H-3eq), 6.20 (1H, d, J = 2.1 Hz, H-6),
6.80 (1H, d, J = 2.1 Hz, H-8), 7.23 (H, d, J = 9.0 Hz,
H-20,H-60), 12.15 (1H, s, OH-5), 3.35 (s, OMe), 4.58 (1H,
d, J = 7.5 Hz, Rha, H-100), 4.68 (1H, d, J = 7.5 Hz, Rha,
H-1000), 3.16-3.75 (9H, m, sugar protons); 1.21 (C-600), 0.92
(C-6000), 13C NMR (DMSO-d6, 100 MHz) d: 80.36 (C-2),
44.07 (C-3), 198.26 (C-4), 127.99 (C-5), 95.82 (C-6),
165.66 (C-7), 99.90 (C-8), 154.49 (C-9), 103.74 (C-10),
126.02 (C-10), 128.8 (C-20,60), 163.3 (C-30), 157.91 (C-40),
160.5 (C-50), Rha 1: 101.4 (C-100), 74.2 (C-200), 76.9 (C-
300),70.4 (C-400), 76.1 (C-500), 17.5 (C-600), Rha 2: 100.7 (C-
1000), 70.5 (C-2000), 70.8 (C-3000), 72.3 (C-4000), 68.2 (C-5000),
17.8 (C-6000), OMe (61.02).
5,30,40,50-Tetramethoxyflavanone-7-O-b-D-
rhamnopyranoside (6)
Yellow needles, mp 163 ꢁC, High resolution HREIMS,
m/z ([M ? H])? 506.4974 calcd for C25H30O11, 506.4993;
IR (CHCl3): mmax 3541 (OH), 3360–2700 (phenolic OH),
1669 (C=O), 1455 (arom) cm-1 1H NMR (DMSO-d6,
;
300 MHz) d: 5.55 (1H, dd, J = 13, 2.6 Hz, H-2), 2.57 (1H,
dd, J = 17, 2.6 Hz, H-3 eq), 3.11 (1H, dd, J = 17, 13 Hz,
H-3ax), 6.20 (1H, d, J = 2.1 Hz, H-6), 6.80 (1H, d,
J = 2.1 Hz, H-8), 7.23 (1H, d, J = 9.0 Hz, H-20, 60), Rha:
4.85 (1H, d, J = 1.5 Hz, H-100), 3.72 (1H, dd, J = 3.0,
1.5 Hz, H-200), 3.71 (1H, dd, J = 9.5, 3.0 Hz, H-300), 3.49
(1H, t, J = 9.5 Hz, H-400), 3.92 (1H, m, H-500), 1.84 (3H, d,
J = 6.0 Hz, H-600), 3.66 (s, 29 OMe), 3.62 (s, OMe),
3.59 (s, OMe); 13C NMR (DMSO-d6, 100 MHz) d: 157.91
(C-2), 41.8 (C-3), 198.11 (C-4), 165.42 (C-5), 96.45 (C-6),
162.8 (C-7), 96.20 (C-8), 154.49 (C-9), 103.54 (C-10),
147.0 (29 OMe), 144.5 (OMe-40), 153.6 (OMe-5), Rha:
101.76 (C-100), 72.2 (C-200), 72.4 (C-300), 74.0 (C-400), 29.69
(C-500), 21.50 (C-600), 61.02 (29 OMe), OMe (60.54), OMe
(58.40).
5,40-Dimethoxy-7,30,500-trihydroxyflavanone-3-O-b-D-
rhamnopyranoside (4)
Yellow needles, mp 160 ꢁC, High resolution HREIMS,
m/z ([M ? H])? 492.4315 calcd for C23H24O12, 492.4298;
IR (CHCl3): mmax 3530 (OH), 3360-2700 (phenolic OH),
1606 (C=O), 1458 (arom) cm-1 1H NMR (DMSO-d6,
;
300 MHz) d: 6.20 (1H, d, J = 2.1 Hz, H-6), 6.80 (1H, d,
J = 2.1 Hz, H-8), 7.23 (1H, d, J = 9.0 Hz, H-20, 60), 5.55
(1H, dd, J = 13, 2.6 Hz, H-2), 2.57 (1H, dd, J = 17,
2.6 Hz, H-3 eq), 4.86 (1H, d, J = 7.0 Hz, Rha, H-100), 3.96
(1H, dd, J = 1.7, 3.4 Hz, Rha, H-200), 3.70 (1H, dd,
J = 3.4, 9.4 Hz, Rha, H-300), 3.45 (1H, t, J = 9.3 Hz, Rha,
H-400), 3.55 (1H, m, Rha, H-500), 1.26 (3H, d, J = 6.0 Hz,
Rha, Me-600), OMe (s, 3.29); 13C NMR (DMSO-d6,
100 MHz) d: 13C NMR (DMSO-d6, 100 MHz) d: 80.36 (C-
2), 73.07 (C-3), 198.26 (C-4), 127.99 (C-5), 95.82 (C-6),
165.66 (C-7), 99.90 (C-8), 154.49 (C-9), 103.74 (C-10),
126.02 (C-10), 128.8 (C-20,60), 163.3 (C-30), 157.91 (C-40),
160.5 (C-50), Rha: 101.4 (C-100), 74.2 (C-200),76.9 (C-
300),70.4 (C-400), 76.1 (C-500), 17.5 (C-600), OMe (61.02).
5,40-Dimethoxy-30-hydroxyflavone-7-b-D-
rhamnopyranoside (8)
Mp 122 ꢁC, High resolution HREIMS, m/z ([M ? H])?
432.4344 calcd for C23H24O10, 460.4315; IR (CHCl3): mmax
3539 (OH), 3360–2700 (phenolic OH), 1660 (C=O), 1450
(arom) cm-1; 1H NMR (DMSO-d6, 300 MHz) d: 6.72 (1H,
s H-3), 6.17 (1H, d, J = 2.2 Hz, H-6), 6.46 (1H, d,
J = 2.2 Hz, H-8), 7.31 (1H, d, J = 1.8 Hz, H-20), 6.90
(1H, d, J = 8.3 Hz, H-50), 7.25 (1H, dd, J = 8.3, 1.9 Hz,
H-60), OMe (s, 3.42), 3.79 (6H, s, 29 OMe); Rha: 4.85
(1H, d, J = 1.5 Hz, H-100), 3.72 (1H, dd, J = 3.0, 1.5 Hz,
H-200), 3.71 (1H, dd, J = 9.5, 3.0 Hz, H-300), 3.49 (1H, t,
J = 9.5 Hz, H-400), 3.92 (1H, m, H-500), 1.84 (3H, d,
J = 6.0 Hz, H-600); 13C NMR (DMSO-d6, 100 MHz) d:
158.44 (C-2), 146.24 (C-3), 175.1 (C-4), 160.9 (C-5), 95.79
(C-6), 162.54 (C-7), 96.45 (C-8), 152.67 (C-9), 103.20 (C-
5,30,40,50-Tetrahydroxyflavanone-7-O-b-D-
rhamnopyranoside (5)
Yellow needles, mp 115 ꢁC, High resolution HREIMS, m/
z ([M ? H])? 450.3971 calcd for C21H22O11, 450.3932; IR
(CHCl3): mmax 3541 (OH), 3360–2700 (phenolic OH), 1669
(C=O), 1455 (arom) cm-1
;
1H NMR (DMSO-d6,
300 MHz) d: 5.55 (1H, dd, J = 13, 2.6 Hz, H-2), 2.57 (1H,
dd, J = 17, 2.6 Hz, H-3 eq), 3.11 (1H, dd, J = 17, 13 Hz,
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10), 131.9 (C-10), 118.2 (C-20), 146,7 (C-30), 147.4 (C-40),
112.8 (C-50), 108.9 (C-60), 56.16 (OMe), 56.22 (OMe).
dose of 25 lmol/kg by gavage 30 min before and 3 h after
the CCl4 poisoning. At the end of the experimental period,
after 48 h, following CCl4 administration animals were
starved overnight before sacrifice to minimize variations in
hepatic metabolism. Blood was collected by direct cardiac
puncture, and liver and kidneys were immediately excised.
Portions of the liver and kidneys were homogenized (1:5
w/v) in phosphate-buffered saline (pH 7.4). The activities of
alanine aminotransferase (ALT), aspartate aminotransferase
(AST), and alkaline phosphatase (ALP) activities were
determined in serum according to the procedures given in
the respective assay kits. The contents of glutathione (GSH)
and TBARS in serum, liver, and kidneys were determined
by the methods of Ohkawa et al. (1979), respectively. Total
bilirubin (TB), total protein, albumin, urea, and creatinine
contents in serum were also determined using assay kits.
Lipid peroxidation in the liver was measured by malondi-
aldehyde equivalent (MDA) by colorimetric Assay Kit
(Oxis International, Inc).
Hydrolysis
Compounds 2–6 and 8 (8 mg each) were refluxed with
15 % HCl/MeOH (10 ml) at 70 ꢁC. The presence of car-
bohydrates in the mixture was established by comparison
with authentic samples on a TLC plate (silica gel 60,
Merck) in the solvent system MeCOEt/iso-PrOH/Me2CO/
H2O (20:10:7:6). Spots were detected with the aniline-
diphenylamine reagent.
Free radical-scavenging activity assay
Free radical-scavenging activity of flavonoids on the stable
radical DPPH (1,1-diphenyl-2-picryl hydrazyl) was esti-
mated by the method of Pajero et al. (2000).
The hydroxyl radical (.OH) scavenging assay was car-
ried out by measuring the clearance of test compounds for
hydroxyl radicals generated from the H2O2/Fe2? systems
(Sekiya et al., 2005).
Statistical analysis
The determination of superoxide anion scavenging
activity of flavonoids was made according to method of
Nishikimi et al. (1972).
For each set of experiments where two or more groups
were compared, an analysis of variance (ANOVA) test
was used to determine the significance of the differences.
Differences between the control and CCl4-treated group
were compared for significance using student’s t test for
non-paired samples. All the values shown are the
mean standard error (SE) The difference between the
test group and control was determined by least significant
difference method at p \ 0.05 confidence levels.
Experimental animals
The study was conducted in CD1 male mice, weighing
about 25–30 g. They were obtained from the animal house
of the National School of Biological Sciences, IPN. The
animals were grouped and housed in microlon boxes in a
controlled environment (temperature 25 2 ꢁC) with
standard laboratory diet and water ad libitum. Litter in
cages was renewed thrice a week to ensure hygiene and
maximum comfort for animals. All procedures described
were reviewed and approved by the University Animals
Ethical Committee. Ethical clearance for handling animals
(NIH publication No. 85-23 revised 1985) was observed.
Results and discussion
The known compound 1 was characterized as 5-hydroxy-
7,40-dimethoxyflavone, which was previously isolated from
Combretum erythrophyllum, with the poorest antioxidant
activity (Martini et al., 2004). Prunin (7) has been identi-
fied in peach (Erez and Lavee, 1969). Compound 9 was
identified as diosmin isolated from Diosma crenulata
(Rashid et al., 1995). The prophylactic hepatoprotective
properties of diosmine (Dorkina, 2004) and naringenin
(Yen et al., 2009) were studied in a model of acute CCl4
hepatitis. Therefore, 1, 7, and 9 were not tested on hepa-
totoxicity (Structure 1).
Experimental design
Rats were fasted for 24 h prior to experiments, but were
allowed free access to water. Animals were divided into
nine groups—six animals each. Group I remained untreated,
they received only corn oil (1 mg/ml) and were kept as a
control group. Study mice received 0.125 ml of CCl4 in
corn oil (50 %, v/v) per 100 g body weight intraperitoneally
(Upadhay et al., 2010). The mice given CCl4 were ran-
domly divided into five groups. One group remained
untreated, and the other groups were treated with flavonoids
glycosides 2–6, 8, and silymarin. Each compound, dis-
solved in physiological saline, was administered orally at a
The compound 2 was obtained as pale-yellow crystal
needles. Its molecular formula was determined as
C30H36O16 by high resolution HREIMS. The 13C NMR
spectrum indicated the presence of 15 aromatic carbon res-
onances for the aglycon, a three OMe signal, and 12 sugar
signals ascribable to one hexose unit and one deoxyhexose
unit. Acid hydrolysis yielded glucose and rhamnose
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Med Chem Res (2013) 22:1846–1855
3´
6´
CH2OH
OH
OMe
OMe
HO
O
2´
4´
O
O
MeO
2
O
MeO
O
O
7
2
H3C
O
3
HO
OMe
5
OH
HO
OH
O
OH
1
OH
OH
OH
OMe
OH
O
HO
O
O
HO
OMe
O
O
O
HO
O
O
OH
OH
O
OH
O
OH
OH
OH
OH
HO
HO
O
O
OH
HO
OH
OMe
HO
O
HO
O
OH
OH
3
4
5
OMe
OH
CH2OH
OMe
HO
HO
O
O
O
O
O
O
O
OH
OH
OH
OMe
OMe
O
OH
6
7
HO
OH
O
O
HO HO
OMe
HO
O
HO
OH
O
O
O
O
OMe
O
O
O
OH
HO
OH
OMe
OH
8
9
Structure 1 Chemical structures of compounds 1–9 isolated from Satureja macrostema
identified by direct TLC comparison with authentic samples.
1
The H NMR spectrum showed a singlet at dH 5.91 indi-
presence of three methoxyl groups in the molecule was
1
indicated by a peak in the H NMR spectrum at dH 3.35
cating a proton for ring A establishing at C-8. The 13C NMR
spectrum showed signals at dc 128.36 (C-20, C-60) and dc
116.47 (C-30, C-50) assigned to two equivalent aromatic
carbons of the symmetrical B ring, which was confirmed by
the signals at dH 6.72 (J = 6.4 Hz, H-30, H-50) and dH 7.15
(J = 6.4 Hz, H-20, H-60) in 1H NMR spectrum. The
appearing as a singlet and integrating for nine protons and in
the 13C NMR spectrum as three signals at 52.76, 53.79, and
56.98. These three methoxyl group were placed on carbons 3
(dc 146.24), 6 (dc 132.43), and 40 (dc 163.05) based on
HMBC correlations of the methoxyl group with the above-
1
mentioned carbons (Fig. 1). H NMR spectrum indicating
123

Med Chem Res (2013) 22:1846–1855
1851
the presence of an intramolecular hydrogen bond between
OH-5 and C=O groups of flavonols at dH 12.10. The sugar
residue was determined to be a-L-rhamnopyranosyl-
(1 ? 3)-b-D-glucopyranoside, linked to C-7 of the glycon
using 1D TOCSY, 2D DQF-COSY, and HSQC NMR
experiments. Two anomeric signals at dH 5.12 and 4.85,
readily identified in the 1H NMR spectrum of 2, correlated to
carbons at dc 75.98 and dc 101.76, respectively, in the
HSQC spectrum. In the 1D TOCSY spectrum, the H-100
signal at dH 5.12 showed connectivities to four methines and
a methylene. The COSY spectrum established the proton
sequence within this monosaccharide as H-100 to H2-600, of
which the multiplicity and coupling constants were typical
of a glucopyranosyl substituent (D’Agostino et al., 1989).
The b-configuration for the D-glucopyranosyl unit
(JH1–H2 = 7.5 Hz) and the a-configuration for the L-rhamno-
pyranoside (JH1–H2 = 1.5 Hz) were deduced by the 1H
NMR data as well as 13C NMR data of key carbons (C-2,
C-3, and C-5). The absence of any 13C NMR glycosylation
shift for the a-L-rhamnopyranosyl moiety suggested that this
sugar was the terminal unit. Glycosylation shifts were
observed for C-300 (dc 88.75 by b-effect) of the b-D-gluco-
pyranosyl unit, demonstrating the (1 ? 3) linkage between
rhamnosyl and glucosyl units. The disaccharide chain was
confirmed as being at C-7 on ring A on the basis of corre-
lations due to long-range couplings observed between H-100
(dH 5.12) of the glucosyl unit and C-7 (109.2), C-8 (96.06),
and C-6 (132.43) of the aglycon. A cross peak observed
between H-1000 (dH 5.22) and C-300 (88.75) confirmed that
the rhamnopyranosyl substituent was attached to C-3 of
the glucopyranosyl unit. Accordingly, compound 2 was
determined as 5-Hydroxy-3,6,40-trimethoxyflavonol-7-C-
a-L-rhamnopyranosyl-(1 ? 3)-b-D-glucopyranoside.
spectrum of an ABX system with signals at dH 5.55 (1H,
dd, J = 13, 2.6 Hz, H-2), 2.57 (1H, dd, J = 17, 2.6 Hz,
H-3 eq) and 3.25 (1H, dd, J = 17, 2.6 Hz, H-3ax) corre-
sponding to three aliphatic protons at C-2 and C-3. Fur-
thermore, the chemical shifts and the coupling constants of
H-6 and H-8 clearly indicated the A ring to be 5,7
di-substituted. Two protons in the ring B show a meta spin–
spin splitting (J = 2.1) in full agreement with the sugges-
tion that the substituents occur only in the 5 and 7
positions. The carbonyl at C-4 was hydrogen-bonded with
the C-5 OH group, as evidenced by a singlet at dH 12.15 in
the 1H NMR spectrum. In addition, the signals of C-30 and
C-50 observed at dC 163.3 and 160.5 in the 13C NMR
spectrum indicated the position of two hydroxyl groups.
Regarding the sugar moiety, an additional signal was a
three proton singlet at dH 3.35 correlated with the signal at
dC 62.3 in the 13C NMR spectrum indicating the presence
of an aromatic methoxyl group. The assignment of signal
to H-20 and H-60 suggested that three groups were attached
to the B ring. The position of the OMe on ring B was
indicated by the cross peaks between the OMe signal (dH
3.85), C-40 (157.91), and H20/H-60 (dH 7.23). 1H NMR
spectra shows a doublet at dH 4.58 which is attributed to the
proton attached to the anomeric proton carbon C-100 of the
sugar moiety. The doublet coupling constant unambigu-
ously indicates a b configuration (J = 7.5). One more
doublet is present at dH 4.68, corresponding to the proton
attached to the anomeric carbon 1000 of second sugar, and
whose coupling constant (J = 7.5) also indicated a b
configuration. All the chemical shifts exclude any position
for the sugar moiety other than 3. Signals for the sugar pro-
tons are situated between 3.0 and 3.9 ppm. 13C NMR spec-
trum indicated the presence of two molecules of rhamnose.
All these results are in accordance with the data taken from
the literature (Kokkalou and Kapetanidis, 1988). All protons
and carbon signals of 3 were completely assigned by the aid
of two-dimensional NMR spectra 1H-1H COSY and 13C-1H
HMBC (Fig. 2). The compound 3 is therefore identified as
40-Methoxy-5,7,30,50-tetrahydroxyflavanone-3-O-b-D-
Compound 3 was obtained as a pale-yellow amorphous
powder, with the molecular formula C28H34O15. Acid
hydrolysis yielded rhamnose identified by direct TLC. IR
spectrum showed strong absorption peaks at mmax
3400–3100 cm-1 (bonded OH) and 1630 cm-1 (chelated
C=O) suggested that 3 was a flavanone. The flavanone
1
nucleus was confirmed by the presence in the H NMR
rhamnopyranosyl-(1 ? 2)-b-D-rhamnopyranoside.
Compound 4 was obtained as a pale-yellow amorphous
powder, with the molecular formula C23H24O12. IR spec-
trum showed strong absorption peaks at mmax 3400–
3100 cm-1 (bonded OH) and 1606 cm-1 (C=O) suggested
that 4 was a flavanone. The flavanone nucleus was con-
firmed by the presence in the 1H NMR spectrum with
signals at dH 5.55 (1H, dd, J = 13, 2.6 Hz, H-2) and 2.57
(1H, dd, J = 17, 2.6 Hz, H-3eq), corresponding to two
aliphatic protons at C-2 and C-3. In 1H NMR spectra, three
aromatic protons appeared at dH 6.20 (1H, d), 6.80 (1H, d),
and 7.23 (1H, d), and two methoxyl groups at dH 3.29
(3H,s) and 3.85 (3H, s) which were assigned to the 5- and
40-positions. The assignment of signal to H-20 and H-60
H
CH2OH
OH
H
OMe
H
HO
O
H
O
O
O
O
H3C
O
H
H
HO
MeO
OMe
H
OH
HO
OH
H
C
Fig. 1 Major HMBC in 2
123

1852
Med Chem Res (2013) 22:1846–1855
OH
OH
H
H
H
H
OMe
OMe
HO
H
O
H
HO
H
O
H
H
H
O
OH
OH
O
O
O
OH
OH
O
OMe
O
OH
H
H
HO
HO
HO
O
H
O
H
C
OH C
OH
H
Fig. 3 Major HMBC in 4
Fig. 2 Major HMBC in 3
the concerted use of 2D NMR experiments including
COSY and HMBC (Fig. 4). From these results, 5 was
identified as 5,30,40,50-tetrahydroxyflavanone-7-O-b-D-
rhamnoside.
suggested that three groups were attached to the B ring.
Furthermore, the chemical shifts and the coupling constants
of H-6 and H-8 clearly indicated the A ring to be 5,7 di-
substituted (Yoshizaki et al., 1987). From the HMBC
spectrum the proton at dH 6.80 showed correlations with
C-7 and 9 and was therefore assigned to H-8. The position
of the OMe on ring B was indicated by the cross peaks
between the OMe signal (dH 3.85), C-40 (157.91), and H20/
H-60 (dH 7.23). 13C NMR spectrum showed a carbonyl
group at C-4 (dC 179.91) which resonated at its normal
position and indicated that group at C-5 is methylated, this
was also confirmed by the lack of low-field signals in the
1H NMR spectra (dH 10–13). NMR spectrum of 4 indicated
the presence of 15 aromatic carbon resonances for the
aglycon, a two OMe signals, and 6 sugar signals ascribable
to one hexose units. Also, a characteristic glycosylation
shift was observed for C-3, suggesting the location of the
rhamnose moiety to be the C-3 (Brenini et al. (1988). In
addition, show a doublet at dH 5.30 which is attributed to
the proton attached to the anomeric proton carbon C-100 of
the sugar moiety (Fig. 3), the doublet coupling constant
unambiguously indicates a b configuration (J = 7.4).
Furthermore, all the chemical shifts exclude any position
for the sugar moiety other than 3. Thus, 4 was assigned as
5,40-Dimethoxy-7,30,500-trihydroxyflavanone-3-O-b-D-
rhamnopyranoside.
Compound 6 exhibited similar NMR spectra to 5 except
the OH groups of C-5, C-30, C-40, and C-50 which were
replaced by OMe groups (Fig. 5). Thus, on the basis of
above spectroscopic techniques the structure of 6 was
established as 5,30,40,50-Tetramethoxyflavanone-7-O-b-D-
rhamnopyranoside.
Compound 8 presented the same carbohydrate moiety
signal patterns of compounds 5 and 6. The hexose was
determined to be rhamnose unit. The other oxygenated
quaternary at dc 146.7 should, therefore, be recognized at 30
2
3
since it showed J and J connectivity by H-20 and H-50,
respectively; thus methoxy group should be placed at C-40.
The size of the coupling constants (1.9 and 8.3 Hz) is
characteristic of meta and ortho coupling, as found in 30,4-
oxygenated flavonoids. In addition, signals in the 13C NMR
spectrum at dC 146.7 and dC 147.4 indicated that the
oxygenated carbons are adjacent due to the shielding effect
([d 10) each oxygen exerts on its neighboring ortho car-
bons (Matsuda et al., 2002). The presence of two methoxyl
1
groups in the molecule was indicated by a peak in the H
NMR spectrum at dH 3.79 appearing as a singlet and
OH
Compound 5 was obtained as yellow-colored solid. IR
1
absorption bands and H spectrum confirm the presence of
H
H
OH
O
a flavanone nucleus. The anomeric proton of the sugar
HO
O
H
O
H
appeared at dH 4.75 (d, J = 6.2 Hz; dC 101.76). An addi-
tional signal in the H spectrum was a two proton doublet
1
OH
H
OH
at dH 7.18 indicating the presence of three hydroxyl groups
in B ring. The B-ring substitution pattern was determined
on the basis of 2D NMR experiments. The coupling con-
stant of the anomeric proton (J = 6.2 Hz) confirmed the
H
OH
OH
O
1
b-linkage of the sugar (Brenini et al., 1988). The H and
H
C
13C chemical shift assignments, and the structure of the
aglycone and the sugar linkage of 5 were established from
Fig. 4 Major HMBC in 5
123

Med Chem Res (2013) 22:1846–1855
1853
integrating for 6 protons and in 13C NMR spectrum as two
signals at dC 56.16 and 56.22. These two methoxyl groups
were placed on carbons 5 and 30 (160.9 and 147.4) based on
HMBC correlations of the methoxyl group with the above-
mentioned carbons (Fig. 6). In addition, from long-range
COSY connectivities the position of the methoxyl group on
ring A and ring B were confirmed since cross peaks from
H-6 to OMe were found as well as from H-50 to OMe.
Thus, it was assigned as 5,40-Dimethoxy-30-hydroxyflav-
one-7-b-D-rhamnopyranoside (8).
OH
H
H
OMe
HO
O
O
H
O
O
OH
H
H
HO
H
H
OMe
H
C
The absolute configuration at C-2 of flavanones could not
be determined from the coupling constants of H-2 and H-3 in
the lH-NMR spectrum, since natural flavanones exist in the
thermodynamically favored conformation with the C-2 aryl
substituent in the equatorial orientation and it follows that
J2,3axial for both S and R isomers are large. Finally, the
absolute configuration of 3, 4, 5, 6, and 7 was determined on
the basis of circular dicronic (CD) spectroscopic analysis.
Thus, the CD spectra of 3, 4, 5, 6, and 7 showed a negative
Cotton effect at 330 nm and positive maximum in the long
wavelength region at 285 nm, which indicated the absolute
configuration of the 2 and 3 positions to be 2R and 3R ori-
entations, respectively (Gaffield et al., 1975) (Fig. 6).
The free radical scavenging of flavonoids was deter-
mined using a stable DPPH free radical (Table 1). Flavo-
noids were shown to have significant DPPH scavenger
capacity and IC50 values obtained for 5 scavenging activity
were very close to those of quercetine, followed in activity
by 3 [ 4 [ 8 [ 2. In contrast, 6 has the poorest antioxi-
dant activity.
Fig. 6 Major HMBC in 8
Table 1 Hydroxyl, DPPH radical, and superoxide anion scavenging
activities of flavonoids 2–5 and 8
Treatment
(IC50 lg/ml)
DPPH
Hydroxyl radical
Superoxide anion
2
24.8 3.89
17.5 1.32
11.6 1.25
9.3 2.32
21.4 3.45
8.2 0.87
36.42 4.16
22.4 3.42
26.7 3.14
18.2 2.86
32.7 4.38
14.6 1.75
43.12 3.98
30.4 1.23
35.2 2.49
25.8 3.28
39.76 4.79
20.1 1.99
3
4
5
8
Quercetin
Values are IC50 SD of different flavonoids. Each IC50 value was
obtained by testing five doses (5, 10, 25, 50, 100 lg/ml) in a triplicate
experiment
increased biochemical markers like AST, ALT, ALP
activities, TB, cholesterol, high density lipoprotein (HDL),
and lipid peroxidation, and reduced the total protein levels.
The results are shown in Tables 2 and 3. Pretreatment with
flavanoids 2–5 and 8 (25 lmol/kg) reduced elevated levels
of all the above-mentioned biochemical indicators and
increased the total protein levels. As shown in Table 3, the
MDA content in CCl4 group was higher than that of the
control group, flavonoids, and provided good protection.
These data indicate that flavonoids treatment inhibited lipid
peroxidation in the CCl4-induced liver damage.
Superoxide anion radicals, and hydroxyl radical-scav-
enging activity of those compounds followed the order
5 [ 3 [ 4 [ 8 [ 2. These results (Table 1) were statisti-
cally significant (p \ 0.05).
The hepatoprotective properties of flavonoids were
studied in mice with a model of acute CCl4-induced liver
toxicity. The effect was evaluated by the ability of tested
substances to recovering the biochemical characteristics of
the functional state of the liver, in comparison to the ref-
erence drug silymarin. Administration of CCl4 significantly
Kidney function tests are represented by measuring
plasma urea and creatinine in mice as shown in Table 3 and
revealed that the CCl4 receiving group developed signifi-
cant kidney damage as evident from a significant elevation
in plasma urea and creatinine as compared to the control
group. Administration of flavonoids markedly reversed the
kidney damage and exhibited a significant reduction in the
CCl4-induced elevation of plasma urea and creatinine.
The structural aspects of the antioxidant activity of
flavonoids were reviewed in the literature (Moure et al.,
2001); the authors reported that B ring of flavonoids was
the crucial site and 40-OH was the main base, and they
almost determined the strength of antioxidant activity, 30-
OH, 50-OH, 7-OH, and 5-OH. In the present study, 5 have
OMe
H
H
OMe
HO
O
O
H
O
H
OH
OMe
H
HO
H
H
OMe
O
H
C
Fig. 5 Major HMBC in 6
123

1854
Med Chem Res (2013) 22:1846–1855
Table 2 Effect of 2–5 and 8 from S. Macrostema on AST, ALT, ALP, TB, and cholesterol in mice intoxicated with CCl4
Groups (20 mg/kg)
AST (IU/l)
ALT (IU/l)
ALP (IU/l)
Total cholesterol (md/dl)
Control (saline)
CCl4 only
95.89 5.74c
296 7.93
152.4 2.56b
(48)
61.3 4.12c
487.3 6.21
219.4 7.12a
(55)
102.7 6.57c
239.1 5.98
156.5 6.51a
(34)
106.12 2.78c
172.57 4.36
143.28 3.67a
(17)
2 1 CCl4
3 1 CCl4
115.1 6.73b
161.6 7.84a
128.3 4.42b
129.31 3.67a
(61)
124.2 6.16b
(58)
(67)
189.4 3.94b
(61)
(46)
137.5 5.51a
(42)
(25)
136.80 4.36a
(21)
4 1 CCl4
5 1 CCl4
103.4 5.87b
145.7 5.61a
118.1 7.42b
124.34 3.78b
(65)
143.6 2.87b
(51)
(70)
200.6 7.84a
(59)
(50)
149.6 5.78a
(37)
(28)
140.13 3.67a
(19)
8 1 CCl4
Sylimarin 1 CCl4
87.6 2.38c
130.1 2.75a
112.9 7.46c
120.34 4.21c
(70)
(73)
(53)
(30)
a
b
c
Each value represents the mean SD, n = 6; p \ 0.05, p \ 0.01, p \ 0.001, compared with the CCl4 control group. () percentage
reduction
Table 3 Effect of flavonoids 2–5 and 8 from S. Macrostema on TB, LDH, total protein and lipid peroxidation (MDA), and plasma urea and
creatinine in mice intoxicated with CCl4
Group
(20 mg/kg)
MDA
(lmol/mgprotein)
LDH
(lmol/mgprotein)
TB (mg/dl)
Total protein
(g/l)
Plasma urea
(mg/dl)
Creatinine
(mg/dl)
Control
0.22 0.006c
0.39 0.005
0.30 0.007a
(23)
684.73 12.54c
2548.49 32.42
1735.53 24.32b
(32)
0.71 0.08c
4.16 0.075
1.99 0.056b
(52)
82.57 3.32c
61.80 5.48
68.23 4.93a
49.12 1.34c
61.56 3.46
44.78 5.21a
0.82 0.005c
1.2 0.006b
0.67 0.009b
CCl4 only
2 1 CCl4
3 1 CCl4
0.27 0.004a
1466.50 9.49a
1. 44 0.095b
74.76 2.86a
71.57 3.83a
76.10 4.56a
70.24 3.84a
79.62 4.21a
49.43 4.87a
47.26 4.63a
51.10 5.90a
46.62 3.72a
52.13 5.34a
0.74 0.001b
0.71 0.007b
0.76 0.004b
0.69 0.003b
0.81 0.002b
(31)
0.28 0.007a
(28)
42)
1539.29 10.65b
(39)
(65)
1. 79 0.078b
(57)
4 1 CCl4
5 1 CCl4
0.25 0.008b
1365.17 13.38a
1.36 0.032b
(36)
0.29 0.007a
(25)
(46)
1646.12 14.65b
(35)
(67)
1.91 0.061b
(54)
8 1 CCl4
Sylimarin 1 CCl4
0.24 0.004b
1326.14 21.59a
1.27 0.072b
(38)
(48)
(69)
a
b
c
Each value represents the mean SD, n = 6; p \ 0.05, p \ 0.01, p \ 0.001, compared with the CCl4 control group. () percentage
reduction
OH groups in positions 30,40,50, and 5. Compounds 2 and 8
have 5-OH and 30-OH, respectively. In contrast, 3 and 4
have 30-OH, 50-OH, 5-OH and 7-OH, and 30-OH, 50-OH,
and 7-OH, respectively, so the antioxidant activity is con-
sistent with the reported literature.
compounds, since flavonoids compounds have hepatopro-
tective activity, they may delay oxidant injury and cell
death by scavenging oxygen radicals; protecting against
lipid peroxidation; and thereby terminating the chain-rad-
ical reaction and chelating metal ions, to form inert
complexes (Soicke and Leng-Peschlow, 1977). Hepato-
protective effect of the flavonoids from S. macrostema
The hepatoprotective effect of Satureja macrostema
may be attributed to the presence of the isolated
123

Med Chem Res (2013) 22:1846–1855
1855
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Phcog Mag 6:125–131
leaves, against CCl4-induced liver damage, may be due to
the ability of its flavonol glycoside content to act as strong
free radical scavengers intercepting those radicals involved
in CCl4 metabolism by microsomal enzymes. Conse-
quently, by trapping oxygen-related free radicals, the fla-
vonol glycosides of SM leaf could hinder the free radical
interaction with polyunsaturated fatty acids and would
abolish the enhancement of the lipid peroxidative process
leading to hepatic cell damage. The significant ameliora-
tion in the antioxidant parameters might result in the
hepatoprotective action of flavonoids glycosides against
the CCl4-induced oxidative stress induced by CCl•4. Con-
sequently, the flavonoids glycosides content of SM leaves
are responsible for the abolishment of CCl4-induced
hepatic and renal damage through their strong antioxidant
activity.
The results obtained revealed that flavonoids from S.
macrostema leaf had the most potent antioxidant activity;
these antioxidant properties are attributable to the ability of
its phenolic constituents to quench reactive oxygen species.
Also, the present study confirmed that the kidney and liver
protective action of flavonoids against CCl4-induced
damage in mice was comparable to that of the standard
hepatoprotective drug, silymarin.
Perez AL, Courel M, Sobrado J, Gonzalez L (1987) Acute renal
failure after topical application of carbon tetrachloride. Lancet
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