Beta vulgaris subspecies cicla var. flavescens (Swiss chard): Flavonoids, hepatoprotective and hypolipidemic activities

Article abstract of DOI:10.1691/ph.2016.5821

The novel flavonoids, 2″,2″′-di-O-α-rhamnopyranosyl-vicenin II, a di-C-glycosyl flavone, and herbacetin 3-O-β-xylopyranosyl- (1″′→2″)-O-β-glucopyranoside, were isolated from the leaves of Beta vulgaris subspecies cicla L. var. flavescens, an edible plant which is consumed in the Mediterranean areas, additional to the known flavonoids, 6-C-glucosyl isoscutellarein, vitexin-(1″′→2″)-O-β-xylopyranosyl, vitexin-(1″′→2″)-O-α-rhamnopyranosyl and vitexin. All metabolites were established by conventional methods of analysis and their structures were confirmed by spectroscopic analysis, including 1D and 2D-NMR and by HR-ESIMS, as well. The extract of the plant leaves shows hepatoprotective effects in rats intoxicated by administration of acetaminophen and exhibits hypolipidemic activity in rats with high-fat-diet induced hypercholesterolemia. The evaluation was done through measuring the liver function enzymes (aspartate and alanine aminotransferases and alkaline phosphatase, the lipid profile (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and triglycerides) and histopathological analysis of liver slides.

Full text of DOI:10.1691/ph.2016.5821

ORIGINAL ARTICLES
Department of Phytochemistry and Plant Systematics1; Therapeutic Chemistry Department2, National Research Centre,
Dokki, Cairo, Egypt; Microbiology & Pharmacognosy Department3, Faculty of Pharmacy, The British University in Egypt
(BUE); Institute of Pharmacy Pharmaceutical Biology4, Ernst-Moritz-Arndt-Universität, Greifswald, Germany
Beta vulgaris subspecies cicla var. flavescens (Swiss chard): flavonoids,
hepatoprotective and hypolipidemic activities
1
2
2
3
4
1
A. N. HASHEM , M. S. SOLIMAN , M. A. HAMED , N. F. SWILAM , U. LINDEQUIST , M. A. NAWWAR
Received October 17, 2015, accepted November 20, 2105
Prof. Mahmoud Nawwar, Department of Phytochemistry and Plant Systematics, National Research Centre, 14,
El Tahrier Str., Dokki, Cairo, Egypt
mahmoudnawwarhesham@yahoo.com
Pharmazie 71: 227–232 (2016)
doi: 10.1691/ph.2016.5821
The novel flavonoids,2’’,2’’’-di-O-α-rhamnopyranosyl-vicenin II, a di-C-glycosyl flavone, and herbacetin 3-O-β-xy-
lopyranosyl- (1”’→2”)-O-β-glucopyranoside, were isolated from the leaves of Beta vulgaris subspecies cicla L.
var. flavescens, an edible plant which is consumed in the Mediterranean areas, additional to the known flavo-
noids, 6-C-glucosyl isoscutellarein, vitexin-(1”’→2”)-O-β-xylopyranosyl, vitexin-(1”’→2”)-O-α-rhamnopyranosyl
and vitexin. All metabolites were established by conventional methods of analysis and their structures were
confirmed by spectroscopic analysis, including 1D and 2D-NMR and by HR-ESIMS, as well. The extract of the
plant leaves shows hepatoprotective effects in rats intoxicated by administration of acetaminophen and exhibits
hypolipidemic activity in rats with high-fat-diet induced hypercholesterolemia. The evaluation was done through
measuring the liver function enzymes (aspartate and alanine aminotransferases and alkaline phosphatase,
the lipid profile (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol and
triglycerides) and histopathological analysis of liver slides.
disease and a causative factor in the development of atheroscle-
rosis and coronary heart disease (Rizvi et al. 2003).
Besides we report here the isolation and structure elucidation of
two new flavonoids and four known compounds from the plant
Beta vulgaris subspecies cicla L. var. flavescens.
1. Introduction
In the frame of our continuing investigations of Egyptian edible and
medicinal plants we investigated the aqueous methanol leaf extract
of Beta vulgaris subspecies cicla L. var. flavescens (Amarantha-
ceae) for its flavonoids as well as for possible hepatoprotective
and hypocholesteremic activities. The plant is known in English as
Swiss chard, sea beet, foliage beet or leaf beet and is the ancestor
of cultivated beet. It can grow as a biennial or a perennial, under
favorable conditions, although many Mediterranean populations
are annuals. It has long wedge shaped leaves in a rosette arrange-
ment and is without a swollen root. The glom rules occur in groups
of around three and are crowded together on the inflorescence.
It has a chromosome number of 18. Swiss chard grows wild in
Egypt. It also occurs along the Atlantic coasts up into Scandinavia,
throughout the Near and Middle East, and into India. It thrives on
stony and sandy beaches, rocky cliff sides, coastal grass lands, and
salt marshes. It is also found in some inland sites in the Mediterra-
nean region, and in Iran and Azerbaijan (Bramwell and Bramwell
1974). The plant is known in folk medicine as a hypoglycemic
(Bolkent et al. 2000, Yanardag et al. 2002), anti-inflammatory and
hemostatic herb (Kim et al. 2003).
In the present work we evaluated Beta vulgaris aqueous metha-
nolic extract for hepatoprotective and hypolipidemic activities.
The evaluation was done through measuring the liver function
enzymes, the lipid profile and the histopathological picture of
the liver of rats. Hepatotoxicity was induced by acetaminophen
(paracetamol). Overdoses of this analgetic drug lead to acute liver
failure and hepatic cytolysis (Michaut et al. 2014). The mode of
action of acetaminophen on the liver is by covalent binding of its
toxic metabolite, n-acetyl-p-benzoquinone-amine to the sulfhydryl
group of proteins resulting in cell necrosis and lipid peroxidation
(Kapur et al. 1994). Acetaminophen induced hepatotoxicity has
been used successfully as an experimental model to evaluate the
efficacy of hepatoprotective agents (Sreedevi et al. 2009). On the
other hand, hypercholesterolemia is one of the main causes of liver
2. Investigations, results and discussion
2.1. Isolation and structure elucidation of flavonoids
The phenolics of the aqueous methanol leaf extract of Beta vulgaris
subspecies cicla were isolated by standard procedures (CC and
prep. PC). Two of the separated compounds (1 and 2) are new. The
1
remaining isolates exhibited mass spectrometric, 1-D and 2-D H
and 13C NMR analytical data identical with those of vitexin-
(1”’→2”)-O-α-rhamnosyl (3) (Ninfli et al. 2007), vitexin-
(1”’→2”)-O-β-xylosyl (4) (Wen et al. 2007), 6-C-glucosyl isoscut-
telarin (5) (Rigano et al. 2007) and vitexin (6) (Nawwar et al.
2014).
Compound 1 was obtained as a light yellow amorphous powder. It
possessed chromatographic properties and color reactions similar
to those of polyglycosylated apigenin derivatives. It showed an [M
-1]- ion at m/z 885.4 in the negative ESI-mass spectrum, corre-
sponding to a Mr of 886. Fragments ions at m/z 739.1 and 593
were also observed. The molecular formula was concluded to be
C39H50O23 from its negative HRESI-MS, which showed an [M –
–
1] ion at m/z = 885.7688 (calculated for C H50 O : 886.8005).
The compound exhibited the following UV 3s9pectral23data (λmax, in
MeOH (nm): 270, 334, bassochromic shift with NaOAc; no shift
with H3BO3; stable shift with NaOMe) reminiscent of apigenin
with unsubstituted OH groups. Complete acid hydrolysis (reflux
with methanolic 2 M HCl, for 7 h) 1 gave 6,8-di-C-glucosylapi-
genin, or vicenin II (CoPC, UV and 1H NMR spectral analysis) and
rhamnose (CoPC). This finding together with the above obtained
analytical data proved that 1 is a di-rhamnosyl vicenine II. The 1H
NMR spectrum revealed a vicenin II pattern of resonances at δ 6.72
Pharmazie 71 (2016)
227

ORIGINAL ARTICLES
Table 1: 13C NMR chemical shifts (ppm) of the isolated flavonoids of Beta vulgaris subspecies cicla L.
2’,2”’-Di-O-rhamnosyl vicenin II
Herbacetin 3-O-xylosylglucoside
Herbacetin
Aglycone moiety
Aglycone moiety
2
3
4
5
6
7
8
9
10
1’
2’
3’
4’
5’
6’
164.31
102.82
182.49
158.75
107.60
161.37
105.43
155.26
104.09
121.67
129.17
116.09
160.96
116.09
129.17
2
3
4
5
6
7
8
9
10
1’
2’
3’
4’
5’
6’
157.6
132.9
176.3
153.1
100.9
153.6
125.5
151.3
98.9
121.5
130.0
115.5
159.8
115.5
129.5
146.6
135.4
177.8
152.3
98.01
152.8
124.7
144.9
102.5
121.5
129.7
115.4
159.2
115.4
129.5
C-glucosyl moietie s
Glucoside moiety
1”&1”’
2”&2”’
3”&3”’
4”&4”’
5”&5”’
6”&6”’
71.22 & 72.12
1”
2”
3”
4”
5”
6”
98.23 & 98.46
82.07 & 81.98
79.10 & 79.03
70.54 & 70.6
81.44 & 80.96
61.47& 61.58
77.11& 77.20
77.35 & 77.32
72.32 & 72.41
76.97 & 76.92
61.40 &61.46
O-rhamnosyl moieties
Xylosyl moiety
1””&1””’
2””&2””’
3””&3””’
4””&4””’
5””&5””’
OMe
100.17 & 100.09
1”’
2”’
3”’
4”’
5”’
103.90 & 104.o1
73.78 & 73.82
77.45 & 77.49
68.12 & 68.20
65.44 & 65.43
70.30 & 70.34
71.40 & 71.38
70.34 & 70.30
68.10 & 68.16
17.88 & 17.90
(s, H-3); 6.9 (d, J = 8 Hz, H-3’ & H-5’) and 8.0 (8 J = 8 Hz, H-2’
& H-6’). In addition, the spectrum showed two broad singlets, each
of Δν1/2 = 4 Hz, at δ 5.05 and 5.1 ppm , assignable to the two α-ano-
meric rhamnosyl protons, together with a multiplet, integrated to
two protons at δ 4.83 ppm , attributable to the two glucosyl β-ano-
meric protons. Other glucosyl and rhamnosyl protons together
with the hydroxyl and H2O protons showed resonances as a
complex pattern localized in the region from δ 3.3 to 4.15 ppm.
The spectrum revealed also two doublets at δ 0.88 and 89 ppm,
each with J = 6 Hz, and integrated to three protons assignable to
the two methyl rhamnosyl protons. The 13C NMR spectral analysis
of 1 exhibited, in addition to the characteristic 6,8-di-C-substituted
apigenin pattern of carbon resonances (Table 1), a group of 12
glucose and 12 rhamnose carbon resonances, each possessing a
distinct chemical shift value. Di-O-rhamnosylation followed from
the anomeric carbon signals at δ 100.74 and 100.93, while di-C-glu-
cosylation was proved by the anomeric glucosyl C- l resonances at
δ 71.15 and 71.11 ppm. The two carbon resonances localized in
this spectrum at δ 60.84, 60.85 were assigned to the C-6 carbons in
both of the C-glucosyl moieties and reflect no substitution of the
geminal hydroxyl groups of the C-6 in the C-glucosyl moieties.
That O-rhamnosylation in 1 is present at each of the C-2 carbons
of the C-glucosyl moieties was then confirmed by the downfield
location of the C-2 carbon resonances in both the 6- and 8-C-glu-
cosyl moieties at δ 73.88 and 74.21 ppm (α-effect) as well as by the
upfield shift of the signals of the vicinal C- l and C- 3 carbons in
each of these two moieties to δ 71.33, 72.01, 79.19 and 79.06 ppm
, respectively (all in comparison with the resonances of the corre-
sponding carbons in 6- and 8-C-glucosylapigenin (Agrawal 1989).
The assignment of the 13C NMR signals followed directly from
the HMQC and HMBC spectra. That the glucose moieties are C-C
linked to the apigenin moiety at its C-6 and C-8 atoms in 1, which
were previously deduced from the lowfield shifts of their anomeric
proton signals, is unambiguously confirmed by long-range correla-
tion between these protons and the flavone carbons C-6 and C-8
atoms at δ 107:51 and 105.25 ppm , respectively in the HMBC
spectrum. This latter spectrum also allowed the unambiguous
determination of the interconnectivity of the two rhamnosyl units
in 1. In this spectrum the rhamnosyl anomeric protons at δ 5.05 and
5.10 showed correlations with the glucosyl C-2 carbon signals at δ
73.88 and 74.21 ppm . Hence, the structure of 1 was determined to
be 2’’,2’’’-di-O-α-rhamnopyranosyl vicenin II, which represents, to
the best of our knowledge, a new natural product.
Fig. 1: 2”, 2”’-Di-O-α-rhamnopyranosyl vicenin II (1)
Compound 2 was found to possess chromatographic proper-
ties (light yellow color in UV and dull black color, unchanged
by ammonia vapor on PC under UV light) and UV spectral data
resembling those reported for 3-O-substituted herbacetin deriv-
atives (bassochromic shift with NaOAc, negligible shift with
NaOAc + H3BO3 and stable shift with NaOMe) (Harborne 1969).
Negative ESI-mass spectrometric analysis gave a spectrum which
exhibited a [M-H]- molecular ion at m/z 595, corresponding to a
Mr of 596. The molecular formula was concluded to be C26H28O16
from its negative HRESI-MS, which showed an [M – 1] – ion at m/z
= 595.4909 (calculated for C26H28O16: 596.4909). On acid hydro-
228
Pharmazie 71 (2016)

ORIGINAL ARTICLES
lysis, 2 yielded herbacetin (CoPC, UV and 1H NMR spectral data)
together with glucose and xylose (CoPC). The H spectrum of 2
2.2.2. Hepatoprotective effect of Beta vulgaris extract
against acetaminophen induced liver toxicity in rats
1
is closely similar to that of the aglycone herbacetin (Saleh et al.
1988). In this spectrum, the glucose anomeric proton resonance
was located at δ 5.35 ppm as a doublet of J = 8 Hz, thus proving
the attachment of this moiety to a flavonol phenolic OH group,
most probably OH-3 in herbacetin and confirms the β-configura-
tion of that proton. On the other hand, the anomeric xylose proton
was found resonating as a doublet (J = 7.1 Hz) at 4.77 ppm to
prove the attachment of this moiety to an alcoholic sugar moiety
and confirms the β-configuration at the anomeric xylose carbon
C-1. Consequently, 2 is suggested to be a herbacetin 3-O-β-xylo-
syl-β-glucoside. Confirmation of the structure of 2 was achieved
Administration of acetaminophen caused liver damage as
evidenced by the altered serum biochemical parameters. The
results revealed significant increase in alanine aminotransferase
(ALT), aspartate aminotransferase (AST) and alkaline phospha-
tase (ALP) levels by 128.73, 150.91 and 200.12 %, respectively
as compared with the control group. Due to the liver injury caused
by acetaminophen overdose, the transport function of the hepato-
cytes gets disturbed resulting in leakage of the enzymes through
the plasma membrane (Zimmerman and Seeff 1970) to the circu-
lation. However, administration of the extract of Beta vulgaris at
a dose of 200 mg/kg attenuated the increased level of ALT, AST
and ALP by 37.44, 42.07 and 48.01 %, respectively, as compared
with the acetaminophen treated rats. Treatment with 500 mg/kg
significantly decreased ALT, AST and ALP levels by 47.50, 94.61
and 49.32 %, respectively. Treatment with silymarin as reference
compound attenuated the levels of ALT, AST and ALP by 44.85,
57.74 and 62.56 %, respectively as compared with the acetamino-
phen group (Table 2). The observed effects may be due to the pres-
ence of the flavonoid glycosides in the investigated Beta vulgaris
extract (Jain and Singhai 2012).
by 2D- H and 13C NMR spectroscopy, including H-1H COSY,
HMQC and HMBC analysis, which allowed the full assignment
of all carbon and proton resonances. The IH and 13C NMR spectra
(Table 1) unambiguously identified 2 as a herbacetin derivative
with a 3-O-substituent (Markham and Chari 1982). The number
and characteristic shifts of the 13C glycosidic signals indicated
the presence of one hexose and one pentose system in the pyra-
1
1
1
nose form. The assignment of their H and 13C signals followed
directly from the COSY and HMQC spectra. The 13C shifts of the
signals of the hexose C-6 and C-4 indicated that these positions
were unsubstituted. Both sugars had β-glycosidic linkages from
the magnitude of the vicinal proton couplings of the anomeric
protons in the 1H spectrum. That the hexose linked to the aglycone
hydroxyl group at C-3 was readily identified from the low field
shift of its anomeric proton (H-l” at δ ppm 5.35) and unambigu-
ously confirmed by a long-range correlation between this proton
and the flavonol C-3 in the HMBC spectrum. This latter spectrum
also allowed the interconnectivity of the two sugar units to be
unambiguously determined. In this spectrum, the xylosyl anomeric
proton (δ 4.90 ppm) showed a correlation with C-2 of the glucoside
moiety. Hence, the structure of 2 was determined as herbacetin
3-O-β-xylopyranosyl-(1”’→2”)-O-β-glucopyranoside, a flavonoid
structure which has not been reported in nature before.
Table 2: Effect of treatment with plant extracts on liver function
enzymes against acetaminophen induced hepatotoxicity
Groups
ALT
AST
ALP
Control
18.48a 1.40 33.72a 3.01 120.52a 4.23
--- --- ---
Acetaminophen
42.27b 3.43 84.61b 3.98 361.71b 16.36
(+128.73) (+150.91) (+200.12)
Acetaminophen+extract
(200mg/kg)
26.44d 2.05 49.01d 4.06 188.03d 10.23
[-37.44] [-42.07] [-48.01]
Acetaminophen+extract
(500mg/kg)
22.19c 2.08 38.62c 1.62 147.11c 8.80
[-47.50] [-94.61] [-49.32]
Acetaminophen+silymarin 23.31c 1.80 36.60ac 1.97 135.39c 6.08
(100mg/kg) [-44.85] [-57.74] [-62.56]
Data are mean SD of six rats in each group. Values are expressed as U/L. Statis-
tical analysis are done using one way analysis of variance (ANOVA) using Co
Stat Computer program accompanied with least significance level (LSD) between
groups at p<0.05. Unshared superscript letters are significant values between groups
at p<0.0001. Values between brackets are % changes over control group = [(mean
control- mean treated)/mean of control] x 100. Values between parenthesis are %
changes over acetaminophen group= [(mean of acetaminophen - mean treated)/mean
of acetaminophen] x 100.
The histology of the liver section of control animals exhibited
normal liver architecture with well-preserved cytoplasm, prom-
inent nucleus and hepatocytes radially arranged around the
central vein (Fig. 3a). In rats treated with acetaminophen, dilated
congested portal tract, necrosis of the hepatocytes, inflammatory
and infiltration in portal area and many nuclear pyknosi were seen
(Fig. 3b). Rats treated with 200 mg/kg b.wt. of the plant extract
showed mild sinusoid congestion, central vein dilation, scattered
pyknotic nuclei and focal areas of centrilobular necrosis (Fig. 3c).
Rats treated with 500 mg/kg. b.wt. of Beta vulgaris or silymarin
showed amelioration of the histopathological picture by marked
decrease of necrosis in the hepatocytes and normal morpholog-
ical appearance of hepatocytes and central vein (Fig. 3d, e). The
maximum protection against hepatic damage was achieved with
the extract dose of 500 mg/kg.
Fig. 2: Herbacetin3-O-β-xylopyranosyl-(1”’→2”’)-O-β-glucopyranoside (2)
2.2. Biological activities of methanolic extract of Beta
vulgaris
2.2.3. Hypolipidemic effect of Beta vulgaris extract in
hypercholesterolemic rats
With respect to the lipid profile in hypercholesterolemic rats, the
results revealed significant elevation in total cholesterol (TC,
41.71 %), high density lipoprotein-cholesterol (HDL-C, 47.19 %),
2.2.1. Acute toxicity
Beta vulgaris aqueous methanolic extract did not cause any
mortality up to 2000 mg/kg. Hence, we selected the doses 200 and
500 mg/kg b.wt. for the hepatotoxicity study and 500 mg/kg b.wt.
for the evaluation of hypocholesterolemic activities.
Pharmazie 71 (2016)
229

ORIGINAL ARTICLES
Data are mean SD of six rats in each group. Values are expressed
as mg/dL. Statistical analysis are done using one way analysis of
variance (ANOVA) using Co Stat Computer program accompanied
with least significance level (LSD) between groups at p<0.05.
Unshared superscript letters are significant values between groups
at p<0.0001. Values between brackets are % changes over control
group = [(mean control- mean treated)/mean of control] x 100.
Values between parenthesis are % changes over hypercholesterol-
emic group= [(mean of hypercholesterolemia -mean treated)/mean
of hypercholesterolemia] x 100.
2.2.4. Effects of Beta vulgaris extract on liver function
indices in hypercholesterolemic rats
Regarding to the liver function indices in hypercholesterolemic
rats, the results revealed significant increase in AST, ALT and ALP
levels by 52.22, 142.60 and 88.01 %, respectively as compared
with the control group. Treatment of hypercholesterolemic rats
with the plant extract attenuated the level of AST, ALT and ALP
by 26.76, 31.89 and 19.62 %, respectively as compared with the
hyperlipidemic rats. The positive control lipanthyl decreased
the levels of AST, ALT and ALP by 27.73, 40.50 and 27.56 %
(Table 4). This improvement may be due to the attenuation of the
hypercholesterolemic state and the elaborated free radicals, which
in turn ameliorate the liver function.
Fig. 3: (a) Liver section of control group showed hepatic tissue with preserved
(intact) lobular hepatic architecture, normal hepatocytes (black arrow) and
normal morphological appearance with central vein (red arrow) and blood
sinusoid (yellow arrow). (b) Liver section treated with acetaminophen showed
moderate dilated congested portal tract (black arrow), moderate periportal
necrosis of the hepatocytes that surrounded the portal area, inflammatory
infiltration (red arrow), moderate dilated central vein (yellow arrow), binu-
cleated nuclei (green arrow) and many nuclear pyknosis (blue arrows). (c)
Liver section treated with plant extract (low dose) showed mild sinusoid con-
gestion (red arrow), central vein dilation (black arrow), scattered pyknotic
nuclei (green arrows) and focal areas of centrilobular necrosis (yellow arrow).
(d) Liver section treated with plant extract (high dose) showed hepatic tissue
with preserved (intact) lobular hepatic architecture, normal hepatocytes (black
arrow), normal morphological appearance with central vein (red arrow) and
congested dilated blood sinusoid (yellow arrow). (e) Liver section treated with
silymarin showed hepatic tissue with preserved (intact) lobular hepatic archi-
tecture with normal (red arrow) and vacuolated hepatocytes (black arrow) and
normal morphological appearance, congested dilated blood sinusoid (yellow
arrow) (H&E, x200, x400).
Table 4: Effect of treatment with Beta vulgaris extract on liver func-
tion indices in hypercholesterolemic rats
Parameters Control
Cholesterol
Plant extract
Lipanthyl
AST
ALT
ALP
2.70b 0.05
---
1.15c 0.04
---
4.11a 0.25
(+52.22)
2.79a 0.04
(+142.60)
3.01b 0.25
[-26.76]
1.90b 0.07
[-31.89]
2.97b 0.41
[-27.73]
1.66b 0.04
[-40.50]
120.55d 15.37 226.65a 16.80 182.18b 6.55 164.17c 8.11
--- (+88.01) [-19.62] [-27.56]
low density lipoprotein cholesterol (LDL-C, 127.19 %) and
triglycerid (TG, 79.71 %) levels as compared with the normal
control group. These results were in accordance with the results of
Awad et al. (2011, 2012) and Hamed (2011) who found significant
increase in lipid profile in rats after administration of cholesterol.
Treatment of hypercholesterolemic rats with plant extract resulted
in a significant decrease in TC, HDL-C, LDL-C and TG levels by
23.72, 13.32, 26.15 and 12.30 %, respectively as compared with the
untreated hypercholesterolemic rats. Lipanthyl (fenofibrate) as refer-
ence compound attenuated the TC, HDL-C, LDL-C and TG level
by 25.74, 18.71, 32.73 and 19.49 %, respectively as compared with
the hypercholesterolemic rats (Table 3). Adaramoye et al. (2008)
attributed the effect of treatment to reduced hepatic triglyceride
biosynthesis and favoring the redistribution of cholesterol among
the lipoprotein molecules. The reduction of total cholesterol by plant
extract was associated with a decrease of its LDL fraction, which is
the target of several hypolipidemic drugs. This result suggests that
cholesterol-lowering activity of the Beta vulgaris extract does not
result from the rapid catabolism of LDL-cholesterol through its
hepatic receptors for final elimination in the form of bile acids. The
results show promising potential of the plant extract for protection
against diseases caused by elevated lipid levels.
Data are mean SD of six rats in each group. Values are expressed as U/L. Statis-
tical analysis are done using one way analysis of variance (ANOVA) using Co
Stat Computer program accompanied with least significance level (LSD) between
groups at p<0.05. Unshared superscript letters are significant values between groups
at p<0.0001. Values between brackets are % changes over control group = [(mean
control- mean treated)/mean of control] x 100. Values between parenthesis are %
changes over hypercholesterolemic group= [(mean of hypercholesterolemia -mean
treated)/mean of hypercholesterolemia] x 100.
With respect to the histopathological finding of normal rat livers,
the results showed normal lobular hepatic architecture and insignif-
icant pathological changes (Fig. 4 a). Hypercholesterolemic rats’
liver showed lost of lobular hepatic architecture with moderate
hydropic degeneration (black arrows), sever micro and macroste-
atotic changes (red arrow), sever interlobular inflammation (yellow
arrow), congested blood vessels (green arrow) (Fig. 4 b). This was
in agreement with Zhen et al. (2008), Awad et al. (2011, 2012)
and Hamed (2011) who observed the same structure of liver after
high fat diet. This was attributed to the presence of fatty liver as a
result of hypercholesterolemia and accumulation of large vacuoles
of fat. This microscopic examination also confirmed the biochem-
ical determinations, where cholesterol and triglycerides were
increased.
Treatment of hypercholesterolemic rats with plant extract led to
intact (preserved) lobular hepatic architecture, normal hepatocytes
with mild sinusoidal congestion and dilatation (red arrow) and
mild interlobular inflammation (black arrow) (Fig. 4 c). Hypercho-
lesterolemic rats treated with lipanthyl also showed intact lobular
hepatic architecture and normal hepatocytes with fibrotic improve-
ment. Mild sinusoidal and blood vessels congestion and dilatation
were also seen (Fig. 4 d).
Table 3: Effect of treatment with Beta vulgaris extract on lipid profile
in hypercholesterolemic rats
Parameters
Control
Cholesterol
Plant extract
Lipanthyl
(500mg/kg b.wt.)
TC
69.55c 6.51 98.56a 12.14 75.18b 7.66 73.19b 13.56
--- (+41.71) [-23.72] [-25.74]
HDL-C
LDL-C
TG
63.33c 2.53 93.22a 4.70 71.771b 7.21 75.77b 5.53
-- (+47.19) [-13.32] [-18.71]
13.45c 2.70 30.55a 4.16 22.56b 3.11 20.55b 3.26
--- (+127.13) [-26.15] [-32.73]
100.35d 5.10 180.34a 6.37 158.15b 6.22 145.17c 6.88
--- (+79.71) [-12.30] [-19.49]
2.3. Conclusion
In conclusion, the aqueous methanolic extract of Beta vulgaris
subsp. cicla var. flavescens had no toxicity even at a higher dose of
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3.3.2. Herbacetin3O-β-xylopyranosyl-(1”’→2”)-β-O-
glucopyranoside (2)
UV data: λmax (nm): in MeOH: 273, 325, 360; +NaOAc: 283, 313, 390; +NaOAc-
H3BO3: 279, 315, 367; +AlCl3: 283, 361, 418; +NaOMe: 280, 410. Mr: 596, ESI-MS:
negative ion: m/z 595 [M-H]-, 462.9 [M - xylosyl moiety]-, 301 [herbacetin – 1]-.
Acid hydrolysis (2 N aq. HCl, 100o C, 2 hr) of 2 gave glucose, xylose (coPC) and
herbacetin. UV data of herbacetin: λmax (nm): in MeOH: 276, 335 shoulder, 380;
_ NaOAc: 278,320 shoulder, 375; NaOAc-H3BO3: 285,320 shoulder, 375; AlCl3:
280,370,448; NaOMe: decomposition; 1H NMR of herbacetin: δ 6.28 (s, H-6), 7.0 (d.
J = 8 Hz, H-3’ and H-5’), 8.2 (d, J = 8 Hz, H-2’ and H-6’). 1H NMR of 2: herbacetin
moiety: δ ppm 6.25 (s, H-6), 6.87 (d, J = 8 Hz, H-3’ and H-5’), 8.08 (d, J = 8 Hz, H-2’
and H-6’); sugar moieties: δ ppm: 535 d, J = 8 Hz, anomeric glucose proton), 4.77
(d, J = 7 Hz, anomeric xylose proton), 3.27-3.95 (m, 9sugar protons hidden by OH
protons). 13C NMR of 2: Table 1.
3.4. Biological assays
3.4.1 Animals
Fig. 4: Hematoxylin and eosin (H&E) stained section (200×) of normal rat liver (a),
liver of hypercholesterolemic rats (b), liver of hypercholesterolemic rats treat-
ed with plant extract (c), and liver of hypercholesterolemic rats treated with
lipanthyl (d).
Male Wistar albino rats (120 – 150 g) and Swiss albino mice (25-30 g) were selected
and obtained from the animal house, National Research Center, Egypt. All animals
were kept in standard environmental conditions (23-25 C and 12h light/dark cycle)
0
and were fed on standard pellet and water ad libitum. The animals were housed in
groups for a minimum of 7 days prior to experiments.
3.4.2. Ethics
2000 mg/kg and exhibits in vivo hepatoprotective and hypolipid-
emic activities. The extract was rich in flavonoids which probably
contribute to the observed pharmacological properties. Further
experiments are underway to identify the responsible compounds
and to discern their molecular mechanisms.
Anesthetic procedures and handling with animals complied with the ethical guide-
lines of Medical Ethical Committee of the National Research Centre in Egypt and
performed for being sure that the animals do not suffer at any stage of the experiment.
3.4.3. Acute toxicity study
Twenty four Swiss albino mice were divided into four groups (n=6). Animals were
starved overnight and the Beta vulgaris methanolic extract was administered orally at
a dose level of 200, 500, 1000 and 2000 mg/kg b.wt. Animals were observed for 24 h
and observed for signs of toxicity and mortality for 7 days (Silva et al. 2007). As we
observed no died animals, we selected the 200 and 500 mg/kg b.wt. for the hepato-
toxicity study and the 500 mg/kg b.wt. for the evaluation of hypolipidemic activities.
3. Experimental
3.1. General experimental procedures
1H NMR spectra were measured by a Jeol ECA 500MHz NMR spectrometer, at
500 MHz. 1H chemical shifts (δ) were measured in ppm, relative to TMS and 13C
NMR chemical shifts to DMSO-d6 and converted to TMS scale by adding 39.5.
ESIMS spectra were measured on a Finnigan LTQ-FTMS (Thermo Electron, Bremen,
Germany) (Department of Chemistry, Humboldt-Universität zu Berlin). UV record-
ings were made on a Shimadzu UV–Visible-1601 spectrophotometer. Paper chro-
matographic analysis was carried out on Whatman No. 1 paper, using solvent systems:
(1) H2O; (2) 6 % HOAc; (3) BAW (n-BuOH–HOAc–H2O, 4:1:5, upper layer).
3.4.4. Study for hepatoprotective effect
Hepatotoxicity was induced by acetaminophen (paracetamol). Acetaminophen was
suspended in 0.5 % Tween-80 and administered orally at a dose of 3 g/kg (Abou
El-Kassem et al. 2012). Rats were divided into 5 groups of 6 animals each. Group
1 served as control. Group 2 were treated with acetaminophen (3 g/kg, p.o.). Group
3 received a single dose of acetaminophen and 200 mg/kg of Beta vulgaris extract.
Group 4 received a single dose of acetaminophen and 500 mg/kg of Beta vulgaris
extract. Group 5 received a single dose of acetaminophen and 100 mg/kg of stan-
dard silymarin (Motawi et al. 2011). The administration of plant extract and silymarin
started three hours after the administration of acetaminophen and continued one time
per day for seven days.
3.2. Plant material
Leaves of Beta vulgaris subsp. cicla var. flavescens were collected from plants culti-
vated in the Nile Delta near Cairo, in February 2014. The plant was identified by Dr.
Mohamed El Gebali, National Research Centre (NRC), Cairo. A voucher specimen (B
201) is deposited at the Herbarium of the NRC.
3.3. Extraction and isolation
3.4.5. Study for hypocholesterolemic effect
The fresh leaves (3 kg) were exhaustively extracted with 75 % MeOH in water (v/v).
Dryness in vacuum afforded a sticky dark brown extract (350 g) from which 70 g
were dissolved in 100 ml MeOH and applied to a polyamide column (Macherey and
Nagel). Separation was initiated with H2O-MeOH (9: 1) and the MeOH content grad-
ually increased in 10 % steps. Compounds 1 and 2 were eluted as fraction I, with
H2O-MeOH (3:7). Further separation was performed by CC on Sephadex LH-20 with
MeOH (1:1) of 1.02 g of the material of fraction I to afford individually pure samples
of 1 (99 mg) and 2 (104 mg). Compounds 3 (88 mg) and 4 (102 mg) were desorbed
by H2O-MeOH (5:5) as fraction II (2.76 g). Each was separated pure from a Sephadex
LH-20 column of II (898 mg, eluted with 70 % MeOH-H2O). Further purification was
performed by applying prep. PC, using BAD as solvent, thus yielding samples of pure
3 (122 mg) and 4 (59 mg). Compounds 5 (44 mg) and 6 (101 mg) were obtained from
308 mg of fraction III (eluted with MeOH –H2O 80:20) through prep. PC using 6 %
AcOH as solvent.
Administration of test samples was done five times per week for nine consecutive
weeks (Adaramoye et al. 2008). The dose was selected according to the toxicity study,
500 mg/kg body weight. Cholesterol was orally given at a dose 30 mg/animal (Adar-
amoye et al. 2008). Lipanthyl (fenofibrate, Mina Pharm., Egypt) was orally given at a
dose of 50 mg/kg b.wt. (Petit et al. 1988).
A total of 24 male rats was divided into five groups (six rats each) as follows: Group
1: normal healthy control rats. Group 2: cholesterol-treated rats. Group 3: rats forced
with cholesterol and plant extract at the same time and for the same duration. Group 4:
rats forced with cholesterol and lipanthyl. Control groups were fed with standard diet
(El- Kahira Co. for Oil and Soap), while hypercholesterolemic groups were fed with
standard diet containing 150g lard/kg diet (Auger et al. 2002). The modified diet was
taken along with oral administration of cholesterol to get a condition of high fat and
cholesterol level (Kim et al. 2008) and to ensure triglycerides elevation (Gershkovich
and Hoffman 2007).
3.3.1. 2”,2”’-Di- O-α-rhamnopyranosyl vicenin II (1)
3.4.6. Samples preparation
UV data: λmax (nm): in MeOH: 272, 330; +NaOAc: 278, 390; +NaOAc-H3BO3: 281,
389; +AlCl3: 280, 305, 344, 385; +NaOMe: 274, 357, 410
Blood was collected from each animal by puncture of the sub-lingual vein, left for
10 min to clot and centrifuged at 3000 rpm for serum separation. The separated serum
was stored at -80 oC for further determinations of liver function enzymes (AST, ALT,
ALP), lipid profile (TC, HDL-C, LDL-C, TG) and serum total protein content.
Mr: ESI-MS: negative ion: 886, fragments ions at m/z 739.1[mono-O-rhamnosy
vicenin-II-1]- and 593 [vicenin II – 1]-. Compound 1 was hydrolysed with 2 N aq.
methanolic HCl (1: 1) at 100’ for 7 h to give vicenin II and rhamnose. Vicenin II
was precipitated from the cold aq. hydrolysate after evaporating the MeOH. Vicenin
II: UV data: λmax in MeOH (nm): 272, 333; +NaOAc: 282, 293; +NaOAc-H3BO3:
283, 390; + AlCl3: 280, 305, 345, 385; +NaOMe: 275, 361, 402. 1H NMR: aglycone
moiety: δ ppm, 6.74 (s, H-3), 6.92 (d, J = 8 Hz, H-3’ & H-5’), 8.0 (d, J = 8 Hz, H-2’
and H-6’); sugar moieties: 4.84 (m, two anomeric glucosyl protons), 3.08-3.88 (m, 10
sugar protons overlapped by OH protons). 1H NMR of 1: δppm 6.74 (s, H-3), 6.95 (d,
J = 8 Hz, H-3’ and H-5’), 8.1 (d, J = 8Hz, H-2’ and H-6’), 5.10 (broad s, Δν1/2 = 4 Hz,
rhamnosyl anomeric proton), 5.05 (broad s, Δν1/2 = 4 Hz, rhamnosyl anomeric proton),
4.84 (m, two anomeric glucosyl protons), 3.12-3.92 (m, sugar protons hidden by OH
protons). 13C NMR of 1: Table 1.
3.4.7. Biochemical determinations
Aspartate and alanine aminotransferases (AST and ALT) were estimated by the
method of Reitman and Frankel (1957), alkaline phosphatase (ALP) by the method
of Belfield and Goldberg (1971). Total cholesterol (TC) was determined by the
method of Meiattini et al. (1978), high density lipoprotein-cholesterol (HDL-C) by
the method of Bustein et al. (1980), low density lipoprotein-cholesterol (LDL-C) by
the method of Assmann et al. (1984) and triglycerides (TG) by the method of Fossati
and Prencipe (1982).
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