Diabetes & Metabolic Syndrome: Clinical Research & Reviews 5 (2011) 148–152

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Diabetes & Metabolic Syndrome: Clinical Research &

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Original article

Shaddock peels (Citrus maxima) phenolic extracts inhibit a-amylase,

a-glucosidase and angiotensin I-converting enzyme activities:
A nutraceutical approach to diabetes management
Ganiyu Oboh *, Ayokunle O. Ademosun
Department of Biochemistry, Federal University of Technology, Akure, Ondo-State, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, the interactions of free and bound phenolic-rich extracts from shaddock peels (popular in
Diabetes folklore for the management of diabetes and hypertension) with a-amylase and a-glucosidase (key
Shaddock enzymes linked to type-2 diabetes) and angiotensin I-converting enzyme (ACE) (key enzyme linked to
a-Glucosidase hypertension) were assessed. The free phenolics of shaddock (Citrus maxima) peels were extracted with
a-Amylase 80% acetone, while the bound phenolics were extracted from the alkaline and acid hydrolyzed residue
Phenolics
with ethyl acetate; and their interaction with the enzymes were assessed. The phenolic extracts inhibited
a-amylase, a-glucosidase and ACE enzyme activities in a dose-dependent manner; however, bound
phenolics had significantly higher (P < 0.05) a-amylase inhibitory activities, than free phenolics, which
had significantly higher (P < 0.05) ACE inhibitory activities. There was no significant difference (P > 0.05)
in their a-glucosidase inhibitory activities. The stronger inhibition of a-glucosidase when compared to
a-amylase is of great pharmaceutical importance. The phenolic inhibited sodium nitroprusside induced
lipid peroxidation in pancreas in a dose dependent manner. Therefore, free and bound phenolic extracts
from shaddock peels could be used as nutraceutical for the management of hypertension and type-2
diabetes.
ß 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.

1. Introduction Natural inhibitors of these enzymes have been reported to be

The digestion of starch, which is the major source of
carbohydrates in the diet for most humans begins in the mouth,
where salivary a-amylase hydrolyzes the internal glycosidic
linkages of starch, producing short polysaccharide fragments or
oligosaccharides. However, salivary a-amylase is inactivated by
the low pH of the stomach, but pancreatic a-amylase continues the
breakdown process [1]. In the intestine, a-glucosidases break
down disaccharides into monosaccharides which is readily
absorbed through the epithelia cells of the small intestine into
blood stream.
Many commercially available drugs used in the management of
type-2 diabetes are inhibitors of pancreatic a-amylase and a-
glucosidase, as the inhibition of these enzymes as served as an
effective strategy for type-2 diabetes management [2]. a-Amylase
and a-glucosidase inhibition delays the absorption of glucose
following starch and sucrose conversion, thereby moderating the
postprandial blood glucose elevation [3,4].
* Corresponding author.
E-mail address: goboh2001@yahoo.com (G. Oboh).
effective in decreasing postprandial hyperglycaemia with minimal
side effects [4]. Usual side effects observed with synthetic a-
amylase and a-glucosidase inhibitors such as abdominal disten-
sion, bloating, flatulence and diarrhoea [5,6], are linked to
excessive inhibition of the pancreatic a-amylase [3]. Natural a-
amylase and a-glucosidase inhibitors therefore offer an attractive
therapeutic approach to the treatment of postprandial hypergly-
caemia by ultimately slowing glucose release from starch [7].
Hypertension and type-2 diabetes are interrelated metabolic
disorders [8,9] and persistent hypertension is one of the risk factors
for strokes, heart attacks, heart failure and is a leading cause of
chronic renal failure [10]. The rennin–angiotensin system (RAS)
plays a pivotal role in blood pressure regulation, and in the
pathophysiology of cardiovascular diseases such as congestive heart
failure and hypertension [11]. Renin produces angiotensin I from
angiotensinogen, after which it is cleaved by angiotensin I-
converting enzyme (ACE) to release angiotensin II, a potent
vasoconstrictor. ACE also inactivates bradykinin, which has
depressor action. As such, inhibition of ACE activity may also yield
major anti-hypertension benefits. Inhibition of ACE is considered a
useful therapeutic approach in the treatment of high blood pressure
in both diabetic and non-diabetic patients [12], and dietary phenolic

1871-4021/$ – see front matter ß 2012 Diabetes India. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dsx.2012.02.008
 G. Oboh, A.O. Ademosun / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 5 (2011) 148–152
phytochemicals have promising potential. Previous in vitro and in
vivo animal and clinical studies have also indicated the potential of
specific phenolic phytochemicals in hypertension management
with direct absorption into the blood [4].
Shaddock, like other citrus fruits has a small edible portion and
large amounts of waste materials such as peels and seeds. Belitz
and Grosch [13] reported that peels contain more total phenol
content than the peeled citrus fruits. Citrus fruits’ peels contain
significant amount of phenolic compounds, especially phenolic
acids and flavonoids [14]. Natural polyphenols are capable of
removing free radicals, chelate metal catalysts, activate antioxi-
dant enzymes, reduce a-tocopherol radicals and inhibit oxidases
[15]. Citrus peels are used in folk medicine, in the management of
diabetes and hypertension, though there is very limited informa-
tion on the mode of action of these peels in the management of
diabetes and hypertension. It is therefore expedient to investigate
the possible mechanism of action of the phenolic constituents of
shaddock peels in the management/prevention of type-2 diabetes
and hypertension.
2. Methods
2.1. Sample collection
Shaddock (Citrus maxima) peels were collected from the Akure
main market, Akure, Nigeria. The peels were sun dried and ground
to fine powder.
2.2. Extraction of free soluble phenolics
The extraction of free and bound phenolics was carried out
according to the method reported by Chu et al. [16]. 50 g of the
ground peels was extracted with 80% acetone and was filtered
(Whatman no. 2) under vacuum. The filtrate was then evaporated
using a rotary evaporator under vacuum at 45 8C until about 90% of
the filtrate had been evaporated. The phenolic extracts were
frozen, while the residues were kept for the extractions of bound
phenolics [16].
2.3. Extraction of bound phenolics
The residue from free soluble extraction above was flushed with
nitrogen and hydrolyzed with about 20 ml of 4 M NaOH solution at
room temperature for 1 h with shaking. Then, the pH of the
mixture adjusted to pH 2 with concentrated HCl and the bound 5
phytochemicals were extracted with ethylacetate (6 times). The
ethyl acetate fractions were then evaporated at 45 8C [16].
2.4. Determination of total phenol content
The total phenol content was determined according to the
method of Singleton et al. [17]. Briefly, appropriate dilutions of the
extracts were oxidized with 2.5 ml 10% Folin–Ciocalteau’s reagent
(v/v) and neutralized by 2.0 ml of 7.5% sodium carbonate. The
reaction mixture was incubated for 40 min at 45 8C and the
absorbance was measured at 765 nm in the spectrophotometer.
The total phenol content was subsequently calculated as gallic acid
equivalent.
2.5. Lipid peroxidation assay
2.5.1. Preparation of tissue homogenates
The rats were decapitated under mild diethyl ether anaesthesia
and the pancreas was rapidly isolated and placed on ice and
weighed. This tissue was subsequently homogenized in cold saline
(1/10, w/v) with about 10-up-and-down strokes at approximately
149
1200 rev/min in a Teflon glass homogenizer. The homogenate was
centrifuged for 10 min at 3000  g to yield a pellet that was
discarded, and a low-speed supernatant (S1) was kept for lipid
peroxidation assay [18].
2.5.2. Lipid peroxidation and thiobarbibutric acid reactions
The lipid peroxidation assay was carried out using the modified
method of Ohkawa et al. [19]. 100 ml S1 fraction was mixed with a
reaction mixture containing 30 ml of 0.1 M pH 7.4 Tris–HCl buffer,
extract (0–100 ml) and 30 ml of 70 mM freshly prepared sodium
nitroprusside. The volume was made up to 300 ml by water before
incubation at 37 8C for 1 h. The colour reaction was developed by
adding 300 ml 8.1% SDS (sodium dodecyl sulphate) to the reaction
mixture containing S1, this was subsequently followed by the
addition of 600 ml of acetic acid/HCl (pH 3.4) mixture and 600 ml
0.8% TBA (thiobarbituric acid). This mixture was incubated at
100 8C for 1 h. TBARS (thiobarbituric acid reactive species)
produced were measured at 532 nm and the absorbance was
compared with that of standard curve using MDA (malondialde-
hyde).
2.6. a-Amylase inhibition assay
Appropriate dilution of the phenolic extracts (500 ml) and
500 ml of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M
NaCl) containing Hog pancreatic a-amylase (EC 3.2.1.1) (0.5 mg/
ml) were incubated at 25 8C for 10 min. Then, 500 ml of 1% starch
solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M
NaCl) was added to each tube. The reaction mixtures was
incubated at 25 8C for 10 min and stopped with 1.0 ml of
dinitrosalicylic acid colour reagent. Thereafter, the mixture was
incubated in a boiling water bath for 5 min, and cooled to room
temperature. The reaction mixture was then diluted by adding
10 ml of distilled water, and absorbance measured at 540 nm [20].
2.7. a-Glucosidase inhibition assay
Appropriate dilution of the phenolic extracts (50 ml) and 100 ml
of a-glucosidase solution (1.0 U/ml) in 0.1 M phosphate buffer (pH
6.9) was incubated at 25 8C for 10 min. Then, 50 ml of 5 mM p-
nitrophenyl-a-D-glucopyranoside solution in 0.1 M phosphate
buffer (pH 6.9) was added. The mixtures were incubated at
25 8C for 5 min, before reading the absorbance at 405 nm in the
spectrophotometer. The a-glucosidase inhibitory activity was
expressed as percentage inhibition [21].
2.8. Angiotensin I-converting enzyme (ACE) inhibition assay
Appropriate dilution of the phenolic extracts (50 ml) and ACE
solution (50 ml, 4 mU) was incubated at 37 8C for 15 min. The
enzymatic reaction was initiated by adding 150 ml of 8.33 mM of
the substrate Bz–Gly–His–Leu in 125 mM Tris–HCl buffer (pH 8.3)
to the mixture. After incubation for 30 min at 37 8C, the reaction
was arrested by adding 250 ml of 1 M HCl. The Gly–His bond was
then cleaved and the Bz–Gly produced by the reaction was
extracted with 1.5 ml ethyl acetate. Thereafter the mixture was
centrifuged to separate the ethyl acetate layer; then 1 ml of the
ethyl acetate layer was transferred to a clean test tube and
evaporated. The residue was re-dissolved in distilled water and its
absorbance was measured at 228 nm. The ACE inhibitory activity
was expressed as percentage inhibition [22].
2.9. Data analysis
The results of three replicates were pooled and expressed as
mean  standard error (S.E.). Student’s t-test, one-way analysis of
150 G. Oboh, A.O. Ademosun / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 5 (2011) 148–152

Table 1 Shaddock-free Shaddock-bound

Phenolic distribution in Shaddock peels (mg/g).

Sample Total Phenol Content (mg/g) 80

α-amylase inhibition (%)

Free 6.5  0.8a 70
Bound 3.4  0.3b 60
The total phenol content was calculated as gallic acid equivalent.
50
Values represent means of triplicate readings. 40
Values with the same abet in a column are not significantly different (P > 0.05). 30
20
10
variance (ANOVA) and least significance difference (LSD) were carried 0
out [23]. Significance was accepted at P  0.05. -10 0 50 100 150 200 250 300 350
Concentration of extracts (μg/ml)

3. Results Fig. 2. a-Amylase inhibitory activity of shaddock peels’ phenolic extract. The citrus
peel extracts concentration for the plot is 0, 80.0, 160.0, 240.0, and 320.0 mg/ml.
3.1. Phenolic distribution

The phenolic distribution in the peels as shown in Table 1 shows
that the free soluble phenolics content (6.5  0.8 mg/g) of the
shaddock peels was significantly (P < 0.05) higher than the bound
phenolics extract (3.4  0.3 mg/g).
3.2. Effect of the free and bound phenolics extracts against sodium
nitroprusside induced lipid peroxidation in cultured rats’ pancreas
The protective ability of the free and bound phenolics extracts
against sodium nitroprusside induced lipid peroxidation in
cultured rats’ pancreas is presented in Fig. 1. Incubation of the
pancreas tissues in the presence of sodium nitroprusside caused a
significant (P < 0.05) increase in the MDA content (189.0%).
However, the free phenolics percentage inhibition of
90.00  2.4% to 50.92  2.0% and bound phenolics (91.74  2.3% to
61.46  8.2%) showed that the extracts inhibited MDA production in a
dose-dependent manner.
3.3. Effect of the free and bound phenolics extracts on a-amylase
activity
Fig. 2 shows the inhibition of a-amylase activities by the
phenolic extracts. 80.0–320.0 mg/ml of extracts were tested, and
the extracts caused a dose dependent inhibition of a-amylase
activities [free phenolics (2.08  3.4% to 25.22  3.1%), bound
phenolics (12.91  2.3% to 64.24  3.2%)].
Shaddock-free Shaddock-bound
200
MDA Produced (% control)
3.4. Effect of the free and bound phenolics extracts on a-glucosidase
activity
The a-glucosidase inhibitory effect of the peels as shown in
Fig. 3 revealed that the free phenolics caused a 74.45  1.3% to
89.05  4.2% inhibition, while the bound phenolics caused a
45.99  2.3% to 95.99  2.1% inhibition of the enzyme activity at
the concentrations (80.0–320.0 mg/ml) of extracts tested.
3.5. Effect of the free and bound phenolics extracts on angiotensin I-
converting enzyme (ACE) activity
The ACE inhibitory activity of the phenolic extracts reveals that
free phenolic extracts showed stronger inhibition of ACE (75.21–
89.43%) than the bound phenolics (8.93–49.74%) at the concen-
trations (31.58–105.26 mg/ml) tested.
4. Discussion
Earlier reports on the phenolic distribution in some commonly
consumed vegetables [16] and fruits [24] agree with the findings in
Table 1, where they have more free soluble phenolic content than
the bound phenolic extracts.
The soluble free phenolic contents of the shaddock peels was
significantly higher (P < 0.05) than the soluble free phenolic
content of red pepper, potato, lettuce, cucumber, carrot, onion,
spinach, cranberry, apple, red grape and broccoli [16,24,25]; but
lower than that of green and sour teas [26]. However, the bound
phenolics compound of the pepper was generally higher than that
of broccoli, cucumber, grape, onion, apple, and red pepper
[16,24,25].

180 Shaddock-free Shaddock-bound

160
140 120
α-glucosidase inhibition (%)

120
100
100
80 80
60
40 60
20
40
0
0 50 100 150 200 250 300 20
Concentration of ex tracts (μg/ml)
0
Fig. 1. Inhibition of sodium nitroprusside induced lipid peroxidation in pancreas by 0 50 100 150 200 250 300 350
shaddock peels’ phenolic extracts. TBARS (thiobarbituric acid reactive species) Concentration of ex tracts (μg/ml)
produced were measured at 532 nm and the absorbance was compared with that of
standard curve using MDA (malondialdehyde). The citrus peel extracts Fig. 3. a-Glucosidase inhibitory activity of shaddock peels’ phenolic extracts. The
concentration for the plot is 0, 62.5, 125.0, 187.5, and 250.0 mg/ml. citrus peel extracts concentration for the plot is 0, 80.0, 160.0, 240.0, and 320.0 mg/ml.
 G. Oboh, A.O. Ademosun / Diabetes & Metabolic Syndrome: Clinical Research & Reviews 5 (2011) 148–152
Free phenolics are more readily absorbed and thus, exert
beneficial bioactivities in early digestion. The significance of bound
phytochemicals to human health is not clear [16,24]. However, it is
possible that different plant foods with different amounts of bound
phytochemicals can be digested and absorbed at different sites of
the gastrointestinal tract and play their unique health benefits.
Bound phytochemicals, mainly in b-glycosides, cannot be digested
by human enzymes and could survive stomach and small intestine
digestion to reach the colon and be digested by bacteria flora to
release phytochemicals locally to have health benefits [16,24].
Oxygen radicals are involved in the cause of diabetes and they
also play a role in some of the complications seen in long-tern
treatment of diabetes. Drugs such as alloxan and streptozotocin are
used to induce diabetes in animals by different mechanisms of
action, but both result in the production of reactive oxygen species
[27]. In humans, the white cell production of reactive oxygen
species mediates the immune destruction of the beta cells [27].
Sodium nitroprusside, a component of antihypertensive drugs
causes cytotoxicity through the release of cyanide and/or nitric
oxide (NO). NO is a universal neuronal messenger in the central
nervous system and acts independently, it may also cause neuronal
damage in cooperation with other reactive oxygen species (ROS)
[28]. The protective ability of the free and bound phenolic extracts
against sodium nitroprusside induced lipid peroxidation in
cultured rats’ pancreas is presented in Fig. 1.
The phenolic extracts caused a dose dependent inhibition of the
sodium nitroprusside induced oxidative stress in the pancreas, this
findings agree with earlier report, where phenolics had been
reported to be a potent inhibitor of lipid peroxidation in several
animal tissues [25]. Nevertheless, there was no significant
difference (P > 0.05) in the inhibition of oxidative stress between
the free phenolic extracts and the bound phenolic extracts, at the
concentrations (62.5–250.0 mg/ml) tested.
Inhibition of enzymes involved in the metabolism of carbohy-
drates such as a-amylase and a-glucosidase is one of the
therapeutic approaches for reducing postprandial hyperglycaemia
[29]. The inhibitions of a-amylase by both the free and bound
phenolic extracts could have contributed to the use of these peels
in folklore in the management of diabetes, thereby slowing down
the breakdown of starch.
The a-glucosidase inhibitory activities of the free and bound
phenolic extracts are shown in Fig. 3. Both phenolic extracts
inhibited a-glucosidase in a dose dependent manner. This
inhibition of the enzyme slows down the breakdown of disaccha-
ride to simple glucose, by so doing reduces the amount of glucose
absorbed in the blood [4,30]. However, at a lower concentration of
80.0 mg/ml free Phenolic extracts had significantly (P < 0.05)
higher inhibition than the bound phenolic extracts, but there was
no significant difference at higher concentrations (160.0–
320.0 mg/ml). On comparing the inhibition of a-amylase and a-
glucosidase; both phenolic extracts were stronger inhibitors of a-
glucosidase than a-amylase; this agrees with earlier findings
where orange and grape peels had stronger inhibition of a-
glucosidase than of a-amylase [31,32]. Pharmaceutically, a weaker
inhibition of a-amylase when compared to a-glucosidase is of
great importance in addressing some of the side effects associated
with drugs such as acarbose and voglibose presently used for the
management of diabetes.
Angiotensin I-converting enzyme (ACE) cleaves angiotensin I to
produce angiotensin II, a powerful vasoconstrictor that has been
identified as a major factor in hypertension [33]. Therefore, ACE
inhibitors have been widely developed to prevent angiotensin II
production in cardiovascular diseases, and utilized in clinical
applications since the discovery of ACE inhibitors in snake venom
[34]. As shown in Fig. 4, the phenolic extracts from the peels
inhibited ACE activity in a dose-dependant manner. The results of
151
Shaddock-free Shaddock-bound
100
90
80
ACE inhibition (%)
70
60
50
40
30
20
10
0
0 20 40 60 80 100 120
Concentration of extracts (μg/ml)
Fig. 4. ACE inhibitory activity of shaddock peels’ phenolic extracts. The citrus peel
extracts concentration for the plot is 0, 31.58, 52.63, 73.68, and 105.26 mg/ml.
the ACE inhibition assay clearly shows that the extracts from the
shaddock peels inhibited ACE and this could explain the possible
mechanism for their use in the treatment of hypertension.
5. Conclusion
The inhibition of key enzymes linked to type-2 diabetes (a-
amylase and a-glucosidase) and hypertension (angiotensin I-
converting enzymes) and lipid peroxidation by the Phenolic
extracts could be part of the mechanism through which the peels
manage/prevents diabetes and hypertension. The strong a-
glucosidase inhibition and mild inhibition of a-amylase makes
the extracts good nutraceuticals for the management of type-2
diabetes with minimum side effects currently observed with some
of the drugs currently in use for the management of non-insulin
dependent diabetes mellitus.
Conflict of interest
The authors declare that they have no conflict of interest.
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