Bioactivity of diosmetin glycosides isolated from the epicarp of date fruits, Phoenix dactylifera, on the biochemical profile of alloxan diabetic male rats

Article abstract of DOI:10.1002/ptr.4777

The new natural flavonoid compounds - diosmetin 7-O-β-L- arabinofuranosyl (1 → 2) β-D-apiofuranoside (1) and diosmetin 7-O-β-D-apiofuranoside (2) - were isolated from the acetone extract of date fruits epicarp belonging to family Arecaceae (Palmae). Elucidation of their chemical structures was determined by different spectroscopic methods in addition to the chemical and physical methods of analysis. These compounds were assessed for their biological activity on alloxan diabetic rats. A dose of 1.5 ml of (1) and (2) suspensions/100 gm b. wt were orally administrated to alloxan diabetic rats for 30 days. The treatment of diabetic rats with these compounds resulted in marked improvement of the different biochemical results, i.e. The serum glucose level (highly significant, from 330 + 5.5 mg/dL to 140 + 1.2 mg/dL) treated with (1); liver functions markedly developed both by AST and ALT levels, (reduced significantly from 68.3 + 4.8 μ/L to 54 + 5.5 μ/L and from 61.0 + 3.6 μ/L to 40.1 + 3.6 μ/L, respectively) treated with (2), accompanying with mild decrease in both cholesterol and triglycerides levels with (1) or (2). Decrease of TBARS level was observed in whole blood when treated with (1) or (2), while levels of glutathione peroxidase and superoxide dismutase were increased in liver. Serum testosterone level was highly significantly increased (from 705.1 + 3.6 mg/100 ml to 720 + 4.7 mg/100 ml), total acid phosphatase and prostate acid phosphatase activities were highly significantly decreased (from 16.9 + 0.28 μ/L to 10.7 + 1.2 μ/L and from 9.7 + 0.7 μ/L to 6.5 + 1 μ/L, respectively) for compound (1). Copyright

Full text of DOI:10.1002/ptr.4777

PHYTOTHERAPY RESEARCH
Phytother. Res. (2012)
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/ptr.4777
Bioactivity of Diosmetin Glycosides Isolated from
the Epicarp of Date Fruits, Phoenix dactylifera,
on the Biochemical Profile of Alloxan Diabetic
Male Rats
1
1
2
*
Helana Naguib Michael, Josline Yehia Salib and Emad Fawzi Eskander
1Chemistry of Tanning Materials Department, National Research Centre, Dokki, Cairo, Egypt
2Hormones Department, National Research Centre, Dokki, Cairo, Egypt
The new natural flavonoid compounds – diosmetin 7-O-b-L-arabinofuranosyl (1 ! 2) b-D-apiofuranoside (1) and
diosmetin 7-O-b-D-apiofuranoside (2) – were isolated from the acetone extract of date fruits epicarp belonging to
family Arecaceae (Palmae). Elucidation of their chemical structures was determined by different spectroscopic
methods in addition to the chemical and physical methods of analysis. These compounds were assessed for their
biological activity on alloxan diabetic rats. A dose of 1.5 ml of (1) and (2) suspensions/100 gm b. wt were orally
administrated to alloxan diabetic rats for 30 days. The treatment of diabetic rats with these compounds resulted
in marked improvement of the different biochemical results, i.e. the serum glucose level (highly significant, from
330 + 5.5 mg/dL to 140 + 1.2 mg/dL) treated with (1); liver functions markedly developed both by AST and ALT
levels, (reduced significantly from 68.3 + 4.8 m/L to 54 + 5.5 m/L and from 61.0 + 3.6 m/L to 40.1 + 3.6 m/L,
respectively) treated with (2), accompanying with mild decrease in both cholesterol and triglycerides levels with
(1) or (2). Decrease of TBARS level was observed in whole blood when treated with (1) or (2), while levels of
glutathione peroxidase and superoxide dismutase were increased in liver. Serum testosterone level was highly
significantly increased (from 705.1 + 3.6 mg/100 ml to 720 + 4.7 mg/100 ml), total acid phosphatase and prostate
acid phosphatase activities were highly significantly decreased (from 16.9 + 0.28 m/L to 10.7 + 1.2 m/L and from
9.7 + 0.7 m/L to 6.5 + 1 m/L, respectively) for compound (1). Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: Phoenix dactylifera L. epicarp; Arecaceae; new flavones; antidiabetic activity.
natural antioxidants which have been partially attributed
INTRODUCTION
to their polyphenols contents, in particular flavonoids,
and could potentially be considered as a functional food
ingredient (Hong et al., 2006; Biglari et al., 2008). It could
be used for generations to come due to its remarkable
nutritional, health and economic value in addition to its
aesthetic and environmental benefits as every part of
the date palm is useful (Al-Shahib and Marshall, 2003;
Zangiabadi et al., 2011).
Palms are among the best known and extensively
cultivated plant families, and emphasis is now being
placed on the biological and hormonal activity of new
compounds isolated from the Arecaceae (Palmae). They
have been important to humans throughout much of
history. They have been used as antinociceptive, anti-
inflammatory, antioxidant, molluscicidal, anti-diabetes,
antimicrobial and nitric oxide production inhibition
(Ross, 2001).
Alloxan causes diabetes in experimental animals
through neurosis of the insulin-producing B cells of the
pancreas (Lenzen et al., 1996).
Date palm (Phoenix dactylifera L.) of the Arecaceae
family is an important plantation crop for many countries
extending from North Africa to the Middle East including
many states of the Arabian Gulf. Many Middle Easterners
believe that consumption of date fruits, particularly in the
morning on an empty stomach, can reverse the actions of
any toxic material that the subject may have been exposed
to (Zohget and Elsheikh, 2000). Different parts of
this plant are traditionally claimed to be used for the treat-
ment of a broad spectrum of ailments including memory
disturbances, fever, loss of consciousness and nervous
disorders. Date fruits may serve as a good source of
In non-insulin-dependent diabetes mellitus type II,
insulin resistance is a major patho-physiologic factor
influencing glucose homeostasis and is generally associated
with morbidity and mortality. Type II patients are usually
characterized by an elevated basal metabolic rate
(BMR). It was reported that BMR of hepatic glucose
production and lipid peroxidation are interrelated in type
II diabetic patients (Kallunki and Group, 1992). It has
been suggested that an increased rate of lipid oxidation
could contribute to the increased rate of hepatic glucose
production since beta oxidation of non-esterified fatty
acids provides the abundance of adenosine triphosphate
(ATP) required for gluconeogenesis.
The aim of the present study was to reveal the
degenerative action of oxidative stress played by free
radicals induced by alloxan and to assess the therapeutic
action of the two new natural compounds; diosmetin
* Correspondence to: Prof. Dr. Helana Naguib Michael, Chemistry of
Tanning Materials Department, National Research Centre, Dokki,
Cairo, Egypt.
E-mail: dr_hnmichael@yahoo.com
Received 08 February 2012
Revised 03 June 2012
Copyright © 2012 John Wiley & Sons, Ltd.
Accepted 09 June 2012

H. N. MICHAEL ET AL.
7-O-b-L-arabinofuranosyl (1 ! 2) b-D-apiofuranoside
Extraction, fractionation and isolation
(1) and diosmetin 7-O-b-D-apiofuranoside (2) isolated
from the acetone extract of date fruits epicarp; on the
biological functions of each of the pancreas, the liver
and the testicular tissue of alloxan induced diabetes in
male rats.
Phoenix dactylifera epicarps (1 kg) of fruits were exhaus-
tively extracted with acetone several times (five extractions
each with 3 L) at room temperature. The combined
acetone extracts were concentrated under pressure to give
a dry residue (200 g). The resulting extract was fractio-
nated on a Sephadex LH-20 column (500 g, 40 ꢀ 1000 mm)
using an eluting system of ethanol followed by different
percentage of ethanol/water giving rise to five fractions.
The new natural compound (1) (200 mg) was separated
in a pure form from the second fraction (10 g, 20%
ethanol) by applying on the Sephadex LH-20 column
which eluted by 50% EtOH. Compound (2) (400 mg)
was isolated in a pure form from the third fraction
(40% EtOH, 15 g) by applying the elution technique,
using Whatmann 3MM paper and the solvent system
BAW for irrigation.
MATERIALS AND METHODS
Plant material
Fruits of Phoenix dactylifera were collected from South
Sinai, Egypt. The fruits were then peeled off to collect
its epicarp. Authentication was performed by Dr. M.
El-Gebali, former researcher of botany at the National
Research Centre. A voucher specimen is deposited in
the National Research Centre Herbarium for future
references.
Characterisation of the new natural compounds
Diosmetin 7-O-b-L-arabinofuranosyl (1!2)b-D-
apiofuranoside (1). Rf-values x 100: 31(1), 20(2), 25(3),
17(4); UV (lmax nm) in MeOH: 249, 266, 342; +NaOMe:
264, 278sh, 306sh, 384; + NaOAc: 250, 266, 346, 418;
+NaOAc/H3BO3: 250, 266, 342; +AlCl3: 256, 272, 294sh,
344sh, 375;+AlCl3/HCl: 256, 272, 294sh, 344sh, 375;
(sh = shoulder). 1H-NMR (DMSO-d6): aglycone moiety: d
(ppm) 7.48 (dd, J=8.0 and 2.0 Hz, H-6’); 7.38 (d, J=2.0 Hz,
H-2’); 7.07(d, J=8.0 Hz, H-5’); 6.70 (d, J=2.1 Hz, H-8);
6.63 (s, H-3); 6.47 (d, J =2.1 Hz, H-6), 3.73 (s, OCH3).
Sugar moieties: d (ppm) 5.8 (d, J = 3.2 Hz, H-1" of b-D-
apiofuranose); 5.18 (d, J = 4 Hz, H-1"’ of b-L-arabinofura-
nose); 4.3 (d, J = 3.2 Hz, H-2"); 4.205 (d, J = 3.2 Hz, H-2’");
4.1 (d, J = 9.48 Hz, H-4"a); 4.06 (d, J = 9.48 Hz, H-4"b); 3.9
(d, J = 10 Hz, H-4"’a); 3.8 (d, J = 10Hz, H-4’"b); 3.5-3.7
(m, rest of sugar protons); 13C-NMR (DMSO-d6):
aglycone moiety: d (ppm)166.57 (C-2); 104.85 (C-3);
184.0 (C-4); 163.08 (C-5); 101.04 (C-6); 164.39 (C-7);
96.04 (C-8); 158.95 (C-9); 107.16 (C-10); 124.83 (C-1’);
114.08 (C-2’); 148.29 (C-3’); 152.84 (C-4’); 112.79 (C-5’);
120.28 (C-6’); 56.51 (OCH3). Sugar moieties: b-D-
apiofuranose moiety: d(ppm) 109.1 (C-1"); 84.18
(C-2"); 83.01 (C-3"); 75.4 (C-4"); 64.51 (C-5"); b-L-
arabinofuranose moiety: 108.4 (C-1’"); 82.1 (C-2’");
77.3 (C-3’"); 86.2 (C-4’"); 61.4 (C-5’").
General
1H and 13C (500, 125MHz) NMR: Joel spectrometer in
DMSO-d6; UV: Shimadzu spectrophotometer model
UV-240; CC: Polyamide 6S and Sephadex LH-20; PC:
was carried out on Whatman No.1 and 3MM paper using
solvent systems (1) BAW (n-BuOH: HOAc: H2O, 4:1:5);
(2) H2O; (3) 15% AcOH (AcOH:H2O, 15:85) and (4)
Forestal (AcOH: conc. HCl: H2O; 30:3:10).
Animals
Forty adult Sprague–Dawely male rats, of the same age
(4 months) and weight (120–150 gm), obtained from the
animal house colony of the National Research Centre,
were used in this study. The animals were divided into:
Group I: Ten normal healthy adult male rats served as
control group.
Group II: Thirty adult male rats were rendered
diabetic by intraperitoneal injection of freshly prepared
alloxan monohydrate solution in a dose of 120 mg/kg
body weight (Lazarow and Palay, 1964).
Group III: Ten alloxan diabetic rats were given orally
a solution of compound (1) (20 mg/kg body weight)
using an orogastric tube daily for a period of 30 days.
Group IV: Ten alloxan diabetic rats were given orally
a solution of compound (2) (20 mg/kg body weight)
daily for a period of 30 days.
Diosmetin 7-O-b-D-apiofuranoside (2). Rf-values x 100:
42(1), 17(2), 21(3), 65(4); UV (lmax nm) in MeOH: 250,
265, 340; +NaOMe: 267, 384 dec.; +NaOAc: 250, 266,
342; +NaOAc/H3BO3: 250, 265, 340; +AlCl3: 258, 271,
295sh, 345sh, 375; +AlCl3/HCl: 261, 271, 295sh, 345sh,
375; (sh = shoulder). 1H-NMR (DMSO-d6): aglycone
moiety: d (ppm) 7.57 (dd, J = 8.5 and 2.0 Hz, H-6’); 7.45
(d, J = 2.0 Hz, H-2’); 7.13(d, J = 8.5 Hz, H-5’); 6.77
(d, J = 2.1 Hz, H-8); 6.81 (s, H-3); 6.47 (d, J = 2.1 Hz,
H-6), 3.73 (s, OCH3). Sugar moieties: d (ppm) 5.58
(d, J = 3.16 Hz, H-1" of b-D-apiofuranose); 4.13
(d, J = 3.2 Hz, H-2"); 4.0 (d, J = 9.6 Hz, H-4a"); 3.7
(d, J = 9.6 Hz, H-4b"); 3.4-3.6 (m, H-3" and H-5");
13C-NMR (DMSO-d6): aglycone moiety: d (ppm)
166.57 (C-2); 104.85 (C-3); 184.0 (C-4); 163.08 (C-5);
101.04 (C-6); 164.39 (C-7); 96.04 (C-8); 158.95 (C-9);
107.16 (C-10); 124.83 (C-1’); 114.08 (C-2’); 148.29 (C-3’);
152.84 (C-4’); 112.79 (C-5’); 120.28 (C-6’); 56.2 (OCH3).
Blood samples were collected, using ocular vein
puncture from the fasting control group, alloxan-diabetic
group, the groups treated with compounds (1) and (2),
respectively. Small portions of blood samples were placed
in heparinized plastic tubes and assayed in the same day
of collection to prevent the conversion of glutathione into
its reduced form, for the determination of glutathione
peroxidase (GPX) and superoxide dismutase (SOD)
activities as well as lipid peroxidation. Other portions of
the blood samples were left to clot, and then centrifuged
at 5000 r.p.m. under cooling for 10 min to separate the sera
for the other biochemical analysis.
Copyright © 2012 John Wiley & Sons, Ltd.
Phytother. Res. (2012)

ANTIDIABETIC ACTIVITY OF DIOSMETIN GLYCOSIDES ISOLATED FROM PHOENIX DACTYLIFERA
OH
Sugar moiety: d (ppm) 109.1 (C-1"); 76.5(C-2"); 79.28
(C-3"); 75.0 (C-4"); 64.04 (C-5")
OCH3
HO
O
O
O
O
Biochemical study
OH
O
Determination of serum glucose concentration by
Trinder (1969), serum amino transferase enzyme activities
by Tietz (1976), serum protein concentration by
Weichselbaum (1946), serum albumin concentration by
Doumas et al. (1971), serum cholesterol and triglycerides
by Bucolo and David (1973), determination of lipid
peroxidation by Mihara and Uchiyamo (1978), GPX activ-
ity and SOD were determined by Paglia and Valentine
(1967), glutathione content in liver tissue by Beutler
(1975), serum testosterone concentration by Hill et al.
(1985) and the determination of serum acid phosphatase
activity was determined by Moss (1984).
OH
O
HO
OH
OH
Diosmetin 7-O- -L-arabinofuranosyl (1 2)- -D-apiofuranoside (1)
OH
OCH3
HO
O
O
O
O
RESULTS AND DISCUSSION
OH OH
OH
The use of some plants as medicinal plant is due to
the presence of natural products mainly polyphenols
(flavonoids), hence the use of Dates (which is rich in
flavonoids) in folk medicine is not surprising as polyphenols
have been shown to reduce the development of athero-
sclerosis in animal models (Stocker and O’Halloran
2004; Waddington et al., 2004).
Diosmetin 7-O- -D-apiofuranoside (2)
Figure 1. Chemical structure of the new isolated compounds (1)
and (2).
Our results showed that these compounds from Date
caused a significant marked improvement of the different
biochemical measurements of the serum glucose level,
liver functions markedly developed both by AST and
ALT levels, with mild decrease in both cholesterol and
triglycerides levels. Also, each of GPX and SOD levels
was increased; besides, albumin and total protein levels
were reduced. For these reasons, bioassay-guided phyto-
chemical investigation of the Egyptian plant Phoenix
dactylifera with proven activity was carried out to isolate
secondary metabolites. Two compounds were isolated
from its epicarps: diosmetin 7-O-b-L-arabinofuranosyl
(1! 2) b-D-apiofuranoside (1) and diosmetin 7-O-b-D-
apiofuranoside (2). Their structures (Fig. 1) were
elucidated on the basis of spectroscopic analysis as follow.
Compound (1) was obtained as a yellow amorphous
powder; the UV spectral data in MeOH and by using
diagnostic reagents suggested the 7,4’-disubstituted
flavone structure (Markham, 1982), whereby the addition
of NaOAc produced no shift in band (II) indicating the
substitution of the 7-position; also, the addition of
NaOMe led to a bathochromic shift in band (I) with a
decrease in intensity as well as the addition of NaOAc/
H3BO3 produced no shift in band (I) indicating absence
of an o-dihydroxyl group in ring B and the substitution
of 4’-position and not the 3’.On complete acid hydrolysis
with 2N HCl (1½ h 100 ꢁC) yielded the aglycone
diosmetin identified by Co-PC, UV spectral data and
1H-NMR (Mabry et al., 1985) together with the two sugar
moieties arabinose and apiose (Co-PC) proving the
occupation of a methoxyl group at the 4’-position and
the sugar moieties at 7-position. 1H-NMR spectrum
showed two doublet signals at d 6.70 and d 6.47 ppm
(each 1H, J = 2.1 Hz) assignable of H-8 and H-6, respec-
tively, which were more downfield than that of diosmetin
due to the glycosylation at the 7-position. The singlet
signal at d 6.63 (1H, s, H-3) confirmed its flavone type.
The presence of three aromatic protons was confirmed
through an ABX-type coupling at d 7.48 (1H, dd, J = 2
and 8 Hz, H-6’); 7.38 (1H, d, J = 2 Hz, H-2’); 7.07 (d, J = 8
Hz, H-5’). The glycosidic nature of (1) was confirmed
from the appearance of two anomeric protons; the first
anomeric doublet signals at d5.8 with J = 3.2 Hz identical
of an apiosyl moiety where the J1,2 ~ 3–4 Hz confirmed
the more stable b-D-erythrofuranoside form of the
apiose (Ranganathan et al., 1980; Silva et al., 2000) and
the second upfield anomeric proton doublet signal at dH
5.18 (J = 4 Hz) typical for O-b-L-terminal arabinofuranosyl
unit. The attachment of the apiosyl moiety directly to C-7
with the linkage of the arabinosyl moiety at its H-2" was
suggested by the appearance of its H-2" signal at d 4.205
(d, J = 3.2 Hz) more downfield than the unsubstituted
one at (d 4.13 ppm).
Data of 13C-NMR spectrum were in agreement with
the reported values for 7-substituted diosmetin with
the known substitution effects (Markham, 1982;
Agrawal, 1989). The two anomeric carbon signals
appeared at d (ppm) 109.1 (C-1") and 108.4 (C-1’")
are in accordance with the corresponding data for the
apiofuranoside and arabino-furanosyl moieties, respect-
ively. The shift of the apiosyl carbon C-2" which
appeared at d 84.18 ppm more downfield than the
unsubstituted one at (d 76.5 ppm) confirmed that the
interglycosidic linkage to be arabinofuranose (1 ! 2)
apiofuranose. Thus, the structure of compound (1) was
established as diosmetin 7-O-b-L-arabinofuranosyl
(1!2) b-D-apiofuranoside, a new natural compound.
The molecular formula of (2), a yellow amorphous
powder, was established based on CHO. Its UV spectral
data in MeOH and by using diagnostic reagents suggested
the 7, 4’-disubstituted flavone (Markham, 1982). Acid
Copyright © 2012 John Wiley & Sons, Ltd.
Phytother. Res. (2012)

H. N. MICHAEL ET AL.
hydrolysis of (2) afforded diosmetin as the aglycone and
Table 2 depicts the serious de-arrangement in liver
functions. Each of the serum level of aspartate amino
transferase (AST), alanine amino transferase (ALT),
triglycerides and cholesterol significantly raised to
68.3 + 4.8 m/L, 61.0+ 3.6 m/L, 153.2 + 5.2 mg/dl and
133 + 1.2 mg/dl, respectively, while serum albumin and
total protein levels markedly dropped to 2.4 + 0.4 mg/dl
and 5.1 + 0.3 mg/dl, respectively, in alloxan-diabetic rats
as compared to the control group. The high level of
ALT is indicative of the serious hepatocellular damage,
since it is more liver specific than AST. Triglycerides
are synthesized in the liver from fatty acids and glycerol,
and as such they are transported as very low density
lipoproteins (V-LDL) to adipose tissue stores (Smith
et al., 1998). The protein content is unchanged or
decreased in liver of diabetic animals, but skeletal and
cardiac muscle is most likely the major site of net
protein less during diabetes (Hay and Waterlow, 1967).
This has been observed in alloxan-diabetic rats. Table 2
shows a lowering in the level of total protein in diabetic
rats mechanisms for intracellular protein break down
involve cytosolic ATP - dependent. Meanwhile, treatment
of diabetic rats with either (1) or (2) has raised the level
of total protein. In alloxan-diabetic rats, albumin secre-
tion is reduced in perfuse livers, and the synthesis of
albumin is more markedly depressed than that of other
secreted proteins (Marsh, 1961). Table 2 depicts a highly
significant drop in serum albumin level in diabetic rats
as compared to the control group. Mild increase in
serum albumin level was observed after treatment of
the diabetic rats with (1) and (2).
These compounds have shown protective effect
against the oxidative peroxidation of cells and could
restore the disordered lipid metabolism. In diabetes,
oxidative stress has been found to be mainly due to an
increased production of free radicals attached with a
sharp reduction of antioxidant defenses (Baynes, 1992).
However, thiobarbituric acid reactive substance (TBARS)
were measured as an index of malondialdehyde produc-
tion and hence lipid peroxidation. Compared with
control group, diabetic liver and pancreas showed
significant increase in TBARS levels at all time intervals
(Kakkar et al., 1998). The data of lipid peroxidation
as TBARS, GPX and SOD diabetic rats and diabetic
rats treated with compounds (1) and (2) are shown in
(Table 3). Highly significant increase in lipid peroxidation
was observed in alloxan-diabetic rats as compared to
control ones, while highly significant decrease in lipid
peroxidation was detected after treatment with either
compound (1) or (2). In contrast, highly significant
decrease in both (GPX) and (SOD) activities were
detected in alloxan-diabetic rats as compared to control
apiose as the sugar moiety which was identified by Co-
PC, indicating a methoxyl group in 4’-position, while the
sugar moiety in 7-position. The occurrence of diosmetin
was clearly determined from the 1H 500 MHz NMR
spectrum which displayed six aromatic protons at d
7.57 ppm (dd, J = 8.5 and 2.0 Hz, H-6’); 7.45 (d, J = 2.0 Hz,
H-2’); 7.13(d, J = 8.5 Hz, H-5’); 6.81 (s, H-3), 6.77
(d, J = 2.1 Hz, H-8); 6.47 (d, J = 2.1 Hz, H-6) and one
1
methyl proton singlet at d 3.73 ppm. The remaining H
resonances indicated one anomeric doublet signal at
d5.58 (J = 3.16 Hz) identical with a b-D-apiofuranosyl
moiety (Ranganathan et al., 1980; Silva et al., 2000). These
data was confirmed by 13C NMR spectrum, whereby it
revealed the 7-substituted diosmetin skeleton signals
together with the five additional carbon signals of b-D-
apiofuranosyl moiety.
By studying the treatment of diabetic rats with these
two new natural compounds, marked improvement of
the different biochemical symptoms resulted as follows:
Table 1 shows a very high level of serum glucose of
330 + 5.5 mg/dl in alloxan diabetic rats as compared to
80.2 + 1.2 mg/dl in the control group. It has been
suggested that alloxan initiates an increased rate of lipid
oxidation which contributes to the increased rate of
hepatic glucose production. It is therefore likely that
the necessary energy was peroxide by the increased rate
of lipid oxidation (Kallunki and Group, 1992). Reduc-
tion of serum glucose to 140 + 1.2 mg/dl, 158 + 1.3 and
167 + 1.1 after treatment of alloxan-diabetic rats with
compounds (1) and (2), respectively, indicates that these
compounds could bring about blood glucose homeosta-
sis through regeneration of endocrine pancreas and
increasing insulin secretion and stimulating the enzyme
glycogen synthetase.
Table 1. Serum glucose level of control rats, alloxan-diabetic rats
and diabetic rats treated with compounds (1) and (2) at the end of
30 days
Subject
Serum glucose mg/dl
Control group n = 10
80.2 + 1.2
330 + 5.5**
140 + 1.2**
158 + 1.3**
Alloxan-diabetic rats n =30
Treatment with compound 1 n = 10
Treatment with compound 2 n = 10
Values are mean + S.E. of 10 animals.
**Highly significant change (P < 0.01).
n = number of rats.
Table 2. Liver profile for control group, alloxan-diabetic rats and diabetic rats treated with (1) and (2) at the end of 30 days
Subject
AST m/L
ALT m/L
Total protein mg/dl Albumin mg/dl Cholesterol mg/dl Tri-glycerides mg/dl
Control group n = 10
30.7 + 2.0
30.1 + 1.9
6.1 + 0.2
3.8 + 0.2
82.2 + 5.8
91.1 + 3.3
Alloxan-diabetic rats n = 10 68.3 + 4.8** 61.0 + 3.6**
5.1 + 0.3*
5.4 + 0.1 (N.S)
5.7 + 0.4 (N.S)
2.4 + 0.4**
3.1 + 0.2*
3.2 + 0.3*
133 + 1.2**
130 + 1.6 (N.S)
130 + 2.7 (N.S)
153.2 + 5.2
147.1 + 1.3 (N.S)
145.2 + 2.1 (N.S)
Treatment with (1) n = 10
Treatment with (2) n = 10
55 + 3.9** 42.2 + 1.3**
54 + 5.5** 40.1 + 3.6**
Values are mean + S.E. of 10 animals.
*Significant change (P < 0.05).
**Highly significant change (P < 0.01).
Copyright © 2012 John Wiley & Sons, Ltd.
Phytother. Res. (2012)

ANTIDIABETIC ACTIVITY OF DIOSMETIN GLYCOSIDES ISOLATED FROM PHOENIX DACTYLIFERA
Table 3. Lipid peroxidation as TBARS, glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities in whole blood of
control group, alloxan-diabetic rats and diabetic rats after treatment with (1) and (2) (20 mg/kg) at the end of 30 days
Control group
(n = 10)
Alloxan-diabetic rats
Treatment with
(1) (n = 10)
Treatment with (2)
(n = 10)
Item
(n = 10)
TBARS n mol/g HB
Glutathione peroxidase m/g HB
Superoxide dismutase m/g HB
49.4 + 1.4
58.2 + 1.2
4.9 + 0.12
92.2 + 5.6**
41.1 + 1.3**
2.1 + 0.14**
64.3 + 2.9**
50.4 + 1.1*
3.11 + 0.13**
70.1 + 1.3**
47.2 + 2.1**
2.82 + 0.09**
Values are mean + S.E. of 10 animals.
*Significant change (P < 0.05).
**Highly significant change (P < 0.01).
rats, while the activity of (GPX) and (SOD) showed highly
significant increase after treatment with either compound
(1) or (2). The level of TBARS in the liver of alloxan-
diabetic rats was highly significantly increased, while
glutathione content and (SOD) activity were highly signifi-
cantly decreased as compared to the control group. How-
ever, after treatment of alloxan-diabetic rats with either
compounds (1) or (2), the level of TBARS in the liver
was significantly decreased, while the glutathione content
and (SOD) activity were highly significant increased as
shown in (Table 4).
Tables 3 and 4 show significant increase in the levels of
TBARS in the whole blood (92.2 + 5.6 n mol/g HB) and in
the liver (79.1 + 1.2 n mol/g fresh tissue) of alloxan-
diabetic male rats as compared with the corresponding
control groups. However, treatment of diabetic male rats
with either (1) or (2) resulted in significant regression of
the levels of TBARS, both in the whole blood and in
the liver tissue (Tables 3 and 4). Therefore, low GSH
content implies low (GPX) activity which may produce
increased oxidative stress propensity (Kakkar et al.,
1998). Also, it was shown that (GPX) is more potent on a
molar basis than (SOD) and catalase to protect endothelial
cells from oxidative stress (Michiels et al., 1990). A sharp
drop in levels of both antioxidant enzymes (GPX) and
(SOD) in the whole blood of alloxan-diabetic rats was
detected. Soto et al. (1998) have observed that 6 h after
alloxan administration, there was a decrease in plasma
(GSH) of diabetic rats. Tagami et al. (1992) reported that
the activity of (SOD) was decreased in endothelial cells in
alloxan-diabetic rabbits after 17 days of induction of
diabetes. This may reflect a reaction between alloxan
and plasma glutathione or perhaps through a free radical
detoxicant enzyme that requires glutathione for its forma-
tion (Soto et al., 1998). The decreasing trend of (SOD)
activity with progression of diabetes could be due to
glycation of the enzyme which occurs in the diabetic state
(Taniguchi, 1992) and thus contributes to oxidative
damage through formation of O-2 and H2O2 (Hunt et al.,
1990). Hypo-insulinaemia increases the activity of
Table 4. Level of TBARS, glutathione (GSH) and superoxide dismutase (SOD) in liver of control group, alloxan-diabetic rats and
diabetic rats treated with (1) and (2) (20 mg/Kg) at the end of 30 days
Control group
(n = 10)
Alloxan-diabetic rats
Treatment with (1)
(n = 10)
Treatment with (2)
(n = 10)
Item
(n = 10)
TBARS n mol/g fresh tissue
Glutathione peroxidase mg/g fresh tissue
Superoxide dismutase m/g HB
48.3 + 3.1
7.1 + 0.2
5400 + 110
79.1 + 1.2**
4.8 + 0.3**
2900 + 140**
63.42 + 4.1*
6.2 + 0.2**
4400 + 160**
67.2 +1.1*
5.4 +0.2**
3920 + 180**
Values are mean + S.E. of 10 animals.
*Significant change (P < 0.05).
**Highly significant change (P < 0.01).
Table 5. Testosterone, total and prostatic acid phosphatase levels in serum of control group, alloxan-diabetic rats and diabetic rats
treated with (1) and (2) at the end of 30 days
Control group
(n = 10)
Alloxan-diabetic rats
Treatment with compound 1
(n = 10)
Treatment with compound 2
(n = 10)
Item
(n = 10)
Testosterone mg/100 ml
Total acid phosphatase m/L
Prostatic acid phosphatase
m/L
712 + 3.3
8.2 + 0.33
9.1 + 0.4
705.1 + 3.6**
16.9 + 0.28**
9.7 + 0.7**
720 + 4.7**
10.7 + 1.2**
4.8 + 0.3**
733 +1.1*
14.1 + 0.2**
5.4 + 1.1**
Values are mean + S.E. of 10 animals.
*Significant change (P < 0.05).
**Highly significant change (P < 0.01).
Copyright © 2012 John Wiley & Sons, Ltd.
Phytother. Res. (2012)

H. N. MICHAEL ET AL.
enzyme fatty acyl-COA oxidase that initiates B-oxidation
been reported that diabetes mellitus is associated with
significant reduction in serum testosterone level and
accessory sex gland weight (Balasubramanian et al.,
1991). These results come in consistence with our data
(Table 5) where we found highly significant decrease in
serum testosterone level in alloxan-diabetic rats in
concomitant with highly significant rise in total acid
phosphatase and prostatic acid phosphatase activities.
However, treatment of alloxan-diabetic rats with
either (1) or (2) resulted in highly significant increase
in serum testosterone level and marked modulation in
the level of both total acid phosphatase and prostatic
acid phosphatase activities.
of fatty acids; resulting in increased production of H2O2
(Horie et al., 1981). Treatment of alloxan-diabeteic
male rats with either (1) or (2) resulted in highly signifi-
cant increase in the level of both antioxidant enzymes
(GPX) and (SOD) in the whole blood and of (GSH)
in the liver tissue.
The levels of serum testosterone, total and prostatic
acid phosphatase of control rats, alloxan-diabetic rats
and diabetic rats treated with compounds (1) and (2)
are illustrated in (Table 5). Highly significant decrease
in serum testosterone level with concomitant increase
in total and prostatic acid phosphates activities were
observed in alloxan-diabetic rats as compared to control
rats. After treatment with compounds (1) and (2), highly
significant increase in serum testosterone level accom-
panied with highly significant decrease in total and pros-
tatic acid phosphatase activities were achieved. It has
Conflict of Interest
The authors have declared that there is no conflict of interest.
REFERENCES
Agrawal PK. 1989. Carbon-13 NMR of Flavonoids, Vol. 39.
Elsevier: Amsterdam – Oxford – New York – Tokyo.
Al-Shahib W, Marshall RJ. 2003. The fruit of the date palm: its possible
use as the best food for the future? Int J Food Sci Nutr 54: 247–259.
Balasubramanian KS, Thameemdheen S, Govindarajulu P. 1991.
Effect of diabetes mellitus on epidymal enzymes of adult rats.
Indian J Exp Boil 29(10): 907–909.
Mihara M, Uchiyamo M. 1978. Determination of malonaldehyde
precursor in tissues by thiobarbituric acid test. Anal Biochem
86: 271–278.
Moss DW. 1984. Acid phosphatases. In Methods of enzymatic ana-
lysis, Bergmeyer HU (ed). Verlag-Chemie, (3rd edn). Weinheim:
Florida; 4: 92–106.
Paglia DE, Valentine WN. 1967. Studies on the quantitative and
qualitative characterization of erythrocyte glutathione peroxidase.
J Lab Clin Med 70: 158–169.
Baynes JW. 1992. Role of oxidative stress in development of
complications in diabetes. Diabetes 40: 405–412.
Beutler E. 1975. Red cell metabolism. A manual of Biochemical
methods (2nd edn). Grune and Stration: New York; 69–70.
Biglari F, Alkarkhi AFM, Azhar ME. 2008. Antioxidant activity and
phenolic content of various date palm (Phoenix dactylifera)
fruits from Iran. Food Chem 107: 1636–1641.
Bucolo G, David M. 1973. Quantitative determination of serum
triglycerides by the use of enzymes. Clin Chem 19: 476–482.
Doumas BT, Watson WA, Biggs HG. 1971. Albumin standards and
the measurement of serum albumin with bromocresol green.
Clin Chem Acta 31: 87–96.
Hay AM, Waterlow JC. 1967. The effect of alloxan protein synthesis
in the rat measured by constant infusion of L-14C lysine. J
Physiol (Lond) 191: 111–112.
Hill P, Garbaczewski L, Kasumi F. 1985. Plasma testosterone and
breast cancer. Clin Oncol 21: 1265–1266.
Ranganathan R, Nagarajan S, Mabry TJ, Yong-Long L, Neuman P.
1980. 6-Hydroxy-luteolin 7-O-apioside from Lepidagathis
cristata. Phytochemistry 19: 2505–2506.
Ross IA. 2001. Medicinal Plants of the World, Volume 2, Chemical
Constituents, Traditional and Modern Medicinal Uses. Humana
Press Inc: Totowa, New Jersey.
Silva BP, Velozos LSM, Parente JP. 2000. Biochanin A triglycoside
from Andira inermis. Fitoterapia 71: 663–667.
Smith AF, Beckett GJ, Walker SW, Rae PWH. 1998. (a) Chap. I 1,
Disorders of Carbohydrate Metabolism, p 149 and (b) Chap. 4,
Renal Disease, p 53, 54. In Lecture Notes on Clinical Biochemistry
(6th edn). Blackewell Sci. Publ.: Oxford.
Soto CP, Perez BL, Favari LP, Reyes JL. 1998. Prevention of
alloxan-induced diabetes mellitus in the rat by silymarin. Comp
Biochem Physiol 119c(2): 125–129.
Hong YJ, Tomas-Barberan FA, Kader AA, Mitchell AE. 2006. The
flavonoid glycosides and procyanidin composition of Deglet Noor
dates (Phoenix dactylifera). J Agric Food Chem 54: 2405–11.
Horie S, Ishii H, Suga T. 1981. Changes in peroxisomal fatty
acid oxidation in diabetic rat liver. J Biochem 90: 1691–1696.
Hunt JV, Smith CT, Wolff SP. 1990. Autoxidative glycosylation
and possible involvement of peroxides and free radicals in
LDL modification by glucose. Diabetes 39: 1420–1424.
Kakkar R, Mantha SV, Radhi J, Prasad K, Kalra J. 1998. Increased
oxidative stress in rat liver and pancreas during progression of
streptozotocin-induced diabetes, Clin Sci 94: 623–632.
Kallunki AF, Group L. 1992. Factors associated with basal
metabolic rate in patients with type 2 (non-insulin-dependent)
diabetes mellitus. Diabetologia 35: 962–966.
Lazarow A, Palay SL. 1964. The production and course of alloxan-
diabetes in the rats. J Lab Clin Med 31: 1004–1015.
Lenzen S, Tiedge M, JÖrns A, Munday R. 1996. Alloxan derivatives
as a tool for the elucidation of the mechanism of the diabetogenic
action of alloxan. In lessons from animal diabetes VI, Shafrir E
(ed), Brikhäuser, Boston; 8: 113–122.
Stocker R, O’Halloran RA. 2004. Dealcoholized red wine decreases
atherosclerosis in apolipoprotein
E gene-deficient mice
independently of inhibition of lipid peroxidation in the artery
wall. Am J Clin Nutr 79: 123–130.
Tagami S, Kondo T, Yoshida K.1992. Effect of insulin on impaired
antioxidant activities in aortic endothelial cells from diabetic
rabbits. Metabol Clin Exp 41(10): 1053–1058.
Taniguchi N. 1992. Clinical significance of superoxide dismutase
changes in aging, diabetes, ischemia and cancer. Adv Clin
Chem 29: 1–59.
Tietz NW. 1976. Fundamentals of Clinical Chemistry (2nd Edn).
WB Saunders Co.: Philadelphia; 657–680.
Trinder P. 1969. Determination of blood glucose using an oxidase/
peroxidase system with a non-carcinogenic chromogen. J Clin
Pathol 22(2): 158–161.
Waddington E, Puddey IB, Croft KD. 2004. Red wine polyphenolic
compounds inhibit atherosclerosis in apolipoprotein E–deficient
mice independently of effects on lipid peroxidation. Am J Clin
Nutr 79: 54–61.
Weichselbaum CT. 1946. An accurate and rapid method for the
determination of proteins in small amounts of blood serum
and plasma. Am J Clin Path 7: 40–49
Zangiabadi N, Asadi-Shekaari M, Sheibani V, et al. 2011.
Date fruit extract is a neuroprotective agent in diabetic
peripheral neuropathy in streptozotocin-induced diabetic
Mabry TJ, Markham KR, Chari, VM. 1985. C-13 NMR Spectra
of Flavonoids. In The Flavonoids, Advances in Research,
Harborne JB, Mabry TJ (eds). Chapman and Hall: London, pp. 51.
Markham KR. 1982. Techniques of Flavonoid Identification.
Academic Press: London.
Marsh JB. 1961. Effect of fasting and alloxan diabetes on albumin
synthesis by perfused rat liver. Am J Physiol 201: 55–57.
Michiels C, Toussaint O, Remacle J. 1990. Comparative study of
oxygen toxicity on human fibroblasts and endothelial cells. J
Cell Physiol 144: 295–302.
rats:
976948.
a multimodal analysis. Oxid Med Cell Longev
Zohget M, Elsheikh A. 2000. Wild Plants in the Region of
Riyadh, Riyadh, Saudi Arabia. King Saud University Press:
Riyadh; 185–186.
Copyright © 2012 John Wiley & Sons, Ltd.
Phytother. Res. (2012)