ORIGINAL ARTICLE

Antioxidant activity and inhibition of key enzymes linked

to type-2 diabetes and hypertension by Azadirachta
indica-yogurt
A.B. Shori
*
, A.S. Baba
Biomolecular Research Group, Division of Biochemistry, Institute of Biological Sciences, Faculty of Science,
University of Malaya, 50603 Kuala Lumpur, Malaysia
Received 22 January 2011; accepted 13 April 2011
Available online 3 May 2011
KEYWORDS
Yogurt;
Azadirachta indica;
Angiotensin-1 converting
enzyme;
a-Glucosidase;
a-Amylase
Abstract Azadirachta indica is widely used in traditional medicine to treat diabetes and hyperten-
sion. In the present study A. indica-yogurt was prepared and refrigerated up to 28 days. pH of A.
indica-yogurt was lower whereas total titratable acid (TTA) was higher than plain-yogurt during
storage. The total phenolic content (TPC) and antioxidant capacity increased during storage. A.
indica-yogurt had highest TPC (74.9 5.1 lgGAE/ml; p < 0.05) on day 28 and DPPH inhibition
(53.1 5.0%; p < 0.05) on day 14 compared to plain-yogurt (29.6 1.1 lgGAE/ml and
35.9 5.2%, respectively). The OPA values increased between day 7 and 21 of storage but reduced
on the 4th week of storage with values for A. indica-yogurt being higher (p < 0.05) than plain-
yogurts. Maximum inhibition of a-amylase (47.4 5.8%), a-glucosidase (15.2 2.5%) and angio-
tensin-1 converting enzyme (ACE, 48.4 7.2%) by plain-yogurt water extract occurred on day 7,
14 and 0, respectively. A. indica-yogurt water extract increased the inhibition to maximal values for
a-glucosidase and ACE on day 14 of storage (15.9 10.1% and 79.70 11.2%, respectively) and
for a-amylase on day 21 of storage (54.8 3.2%). A. indica-yogurt has higher TPC, antioxidant
activities and enzymes inhibitory effects than plain-yogurt. Thus A. indica-yogurt may have the
potential to serve as enhanced functional yogurt with anti-diabetic and anti-hypertension activities.
2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
Yogurt is considered a healthy food due to high digestibility
and bioavailability of its protein, energy and calcium. In addi-
tion, the yogurt microbial fermentative activities (e.g. proteol-
ysis) during the making of yogurt resulted in modication of
allergenic properties of milk (Viljoen et al., 2001) and the re-
lease of a range of bioactive peptides encrypted within the
sequence of the native proteins (Gobbetti et al., 2004).
Amongst the bioactive components detected in dairy products,
*
Corresponding author.
E-mail address: shori_7506@hotmail.com (A.B. Shori).
1319-6103 2011 King Saud University. Production and hosting by
Elsevier B.V. All rights reserved.
Peer review under responsibility of King Saud University.
doi:10.1016/j.jscs.2011.04.006
Production and hosting by Elsevier
Journal of Saudi Chemical Society (2013) 17, 295301
King Saud University
Journal of Saudi Chemical Society
www.ksu.edu.sa
www.sciencedirect.com
inhibitors of angiotensin I-converting enzyme (ACE), which
has a central role in the regulation of blood pressure in mam-
mals, have been studied extensively because of their potential
use in the treatment of elevated blood pressure (Meisel, 1998).
In recent years, there has been increasing interest in the use
of natural food additives and incorporation of health-promot-
ing substances into the diet. Medicinal plants play an impor-
tant role in the treatment of diabetes and hypertension,
particularly in developing countries where most people have
limited resources and access to modern treatments (Marles
and Farnsworth, 1994). These plants have been investigated
with respect to the suppression of glucose production from
carbohydrates in the gut or glucose absorption from the intes-
tine (Matsui et al., 1987), as well as the inhibition of ACE-I
activities (Actis-Goreta et al., 2003; Kwon et al., 2005). The
phenolic compounds, for instance, play a role in mediating
amylase inhibition and therefore have potential to contribute
to the management of type 2 diabetes (McCue and Shetty,
2004). Phenolic compounds also have lower inhibitory effect
against a-amylase activity but a stronger inhibition activity
against a-glucosidase and therefore, can potentially be used
as effective therapy for postprandial hyperglycaemia with min-
imal side effects (Kwon et al., 2006).
Neem (Azadirachta indica) has long been used to treat some
pathological conditions linked to oxidative disorders which in-
clude treatment of fever, diabetes, and disorders that relate to
arthritis and rheumatism (Van der Nat et al., 1991), skin dis-
eases and inammation (Clayton et al., 1996). Early studies
showed that aqueous extract of neem leaves decreased blood
adrenaline as well as glucose-induced hyperglycaemia (Shiba-
ta, 1955) whereas oral doses of neem leaf extracts signicantly
reduced insulin requirements for insulin dependent diabetes
(Murthy and Sirsi, 1958). The biologically active components
of dried A. indica leaves were found to be b-sitosterol, gluco-
sides, nimbin, azadirones, azadirachtin and alkaloids (Schmut-
terer, 1995).
The purpose of this present study was to assess the inhibi-
tory potentials of A. indica-yogurt extract as anti-diabetic
(i.e. against a-amylase and a-glucosidase) and anti-hyperten-
sive (i.e. against angiotensin-I converting enzyme) agents.
Quantitative measurements were also carried out on the A. in-
dica-yogurt water extracts total phenolic content, antioxidant
capacity and OPA values.
2. Materials and methods
2.1. Plant material
A. indica leaves were collected from an eighteen-year old tree in
Petaling Jaya, Selangor, Malaysia. The leaves were washed and
then oven dried (50 C) for 72 h. The dried leaves were then
ground to powder form, placed in an airtight container and
stored at room temperature away from direct sunlight.
2.2. Water extraction of herb
Powdered A. indica leaves (10 g) were soaked in 100 ml of dis-
tilled water for 12 h in a water bath (70 C) with occasional
shaking, followed by centrifugation (2000 rpm, 15 min at
4 C) and the supernatant was harvested. The clear solution
obtained was refrigerated (4 C) and used within 3 days as
water herbal extract in the making of yogurt.
2.3. Preparation of starter culture
Pasteurized full cream milk (4% fat, 1 l) inoculated with a sa-
chet of bacteria mixture with the following composition: Lac-
tobacillus acidophilus LA-5, Lactobacillus bulgaricus,
Bidobacterium bidum Bb-12, Lactobacillus casei LC-01 and
Streptococcus thermophilus Th-4 4 in the ratio of 4:4:1:1 and
a capsule of probiotic mix containing L. bulgaricus, Lactobacil-
lus rhamnosus, Bidobacterium infantis andBidobacterium lon-
gum in the ratio of (1:1:1:1). The milk-bacteria mixture was
incubated in a water bath (41 C) for 12 h and the yogurt
formed was stored at 4 C and used as starter culture within
7 days. Viable bacteria in the starter culture on day 7 of stor-
age ranged 2.05.0 10
6
cfu/g and 6.010.0 10
8
cfu/g for
Lactobacillus spp. and S. thermophilus, respectively.
2.4. Preparation of yogurt
A. indica water extract (10 ml) was added into pre-warmed
(41 C) pasteurized full cream (4%) milk (85 ml) which has
been previously mixed with starter culture (5 ml). The milk so-
lid content was corrected to 15% g/ml by adding 2 g milk pow-
der (4% fat). The mixture was thoroughly mixed and aliquoted
(50 ml) into disposable plastic containers. Plain yogurt was
prepared as described for herbal-yogurt but distilled water
(10 ml) was used instead of herbal extract. Yogurts were incu-
bated in water bath (41 C) for 6 h and stored in a refrigerator
(4 C) for 2 h (fresh yogurt or 0-day storage) up to 28 days.
2.5. pH and total titratable acid (TTA) determination
Yogurt was homogenized in water (1:9) and the pH was read
using a digital pH meter (Mettler-Toledo 320, Shanghai).
Homogenised yogurt was mixed with phenolphthalein (pH
indicator, 0.1% w/v, 23 drops) and the TTA was determined
by titration using NaOH as alkali. Titration with 0.1 N NaOH
was carried out under continuous stirring until the develop-
ment of a consistent pink colour. TTA (% lactic acid) pro-
duced after predetermined refrigeration was calculated as
follows:
TTA%Lactic Acid Dilution factor V
NaOH
0:1N
0:009 100%
where V is volume of NaOH required to neutralize the acid
and dilution factor = 10.
2.6. Preparation of yogurt water extracts
Yogurts (10 g) were homogenized with 2.5 ml of sterile distilled
water and the homogenates were acidied to pH 4.0 with HCl
(0.1 M) followed by heating (water bath; 45 C, 10 min) and
centrifugation (5000g, 10 min, 4 C). The pH of supernatants
was brought to 7.0 using NaOH (0.1 M) and re-centrifuged
(5000g, 10 min 4 C) for further precipitation of proteins and
salts. The supernatants were harvested and kept in the refrig-
erator (4 C) and used within 12 h of preparation.
296 A.B. Shori, A.S. Baba
2.7. Total phenolic content assay
Yogurt water extract (1.0 ml) was mixed with ethanol (1.0 ml,
95% v/v) and 5 ml dH
2
O (Shetty et al., 1995). FolinCiocalteu
reagent (0.5 ml; 50% v/v) was added to each sample and after a
thorough mixing the solutions were allowed to stand for 5 min.
at room temperature. Na
2
CO
3
(1.0 ml, 5% g/100 ml) was then
added and absorbance at 725 nm was read after another
60 min. incubation at room temperature. Known concentra-
tions of gallic acid (SigmaAldrich, Germany; 560 lg/ml in
ethanol) were treated in the same manner as for water extracts
of yogurts and the regression of gallic acid standards was used
to convert unknown samples to total phenolic content (lg gal-
lic acid equivalent, (lgGAE)/ml).
2.8. Antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH) inhibition assay
DPPH inhibition was determined as described by Shetty et al.
(1995). Briey, DPPH (SigmaAldrich, Germany, 3 ml of
60 mmol/L in ethanol) were mixed with 250 ll yogurt extracts
or 250 ll of water which serve as control. The mixture was sha-
ken thoroughly and allowed to stand at room temperature.
The constant absorbance readings at 517 nm were recorded
after 5 min and the inhibition of DPPH oxidation (%) was cal-
culated as follows (Apostolidis et al., 2007):
%inhibition
A
control
517
A
extract
517

A
control
517
100
2.9. The o-pthaldialdehyde (OPA) assay
This assay was used to evaluate proteolysis of milk proteins.
The OPA reagent was prepared as described by Church et al.
(1983). A small aliquot of yogurt extract (usually 1050 ll con-
taining 5100 lg protein) was added directly into 1.0 ml of
OPA reagent. The solution was mixed briey by inversion
and incubated for 2 min. at room temperature. The absor-
bance readings were, taken at 340 nm. The peptide concentra-
tion was estimated against the tryptone standard curve.
2.10. ACE inhibition assay
The ACE reagent was prepared as described by Vermeirssen
et al. (2002). ACE reagent (500 ll) was added to 300 ll of
water yogurt extracts in a cuvette and the contents were mixed
and then incubated in a water bath (37 C) for 2 min followed
by the addition of 300 ll of rabbit lung extract. Absorbance
was measured at 340 nm and the readings were compared to
a control which had 300 ll of buffer solution instead of the ex-
tract. The rate of the reduction in ACE inhibition activity was
calculated as follows:
ACEinhibition%
ACE
control
ACE
yogurt water extracts
ACE
control
100
2.11. a-Amylase inhibition assay
The a-amylase inhibition assay was adapted from Apostolidis
et al. (2006). Yogurt water extracts (500 ll) and 500 ll of
0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M sodium
chloride containing 0.5 mg/ml a-amylase solution were pre-
incubated at 25 C for 10 min. This was followed by the addi-
tion of 500 ll of a 1% starch solution in 0.02 M sodium phos-
phate buffer, pH 6.9 with 0.006 M sodium chloride to each
tube at pre-determined time intervals. The reaction mixtures
were incubated at 25 C for 10 min. The reaction was stopped
with 1.0 ml of dinitrosalicyclic acid (DNSA) colour reagent.
The test tubes were then incubated in a boiling water bath
for 7 min. Then, 1.0 ml of 18.2% tartrate solution was added
to each tube after the boiling prior to cooling to room temper-
ature. The reaction mixture was then diluted by adding 10 ml
of dH
2
O and the absorbance was read at 540 nm. The readings
were compared to a control which had 500 ll of buffer solu-
tion instead of the extract. The enzyme inhibition was calcu-
lated as below:
Inhibition%

Absorbance of control Absorbance of extracts

Absorbance of control
100
2.12. a-Glucosidase inhibition assay
The a-glucosidase inhibition assay was performed in reference
to the method of Apostolidis et al. (2006). Yogurt water ex-
tract (500 ll) and 1000 ll of 0.1 M potassium phosphate buffer
(pH 6.90) containing a-glucosidase solution (1.0 U/ml) was
incubated in water bath at 25 C for 10 min. After 10 min.,
500 ll of 5 mM p-nitrophenyl-a-D-glucopyranoside solution
in 0.1 M potassium phosphate buffer (pH 6.90) was added to
each tube at predetermined time intervals. The reaction mix-
tures were incubated at 25 C for 5 min. prior to the reading
of absorbance at 405 nm. The readings were compared to a
control which had 500 ll of buffer solution instead of the ex-
tract. The a-glucosidase inhibitory activity was expressed as
inhibition% as follows:
Inhibition%
Absorbance
control
Absorbance
extracts
Absorbance
control
100
2.13. Sensory analysis
Sensory analyses were carried out by 15 untrained panels aged
between 20 and 41 years old. Each panel was presented with
four coded yogurt samples (10 ml) of the following treatments:
A. indica-yogurt, A. indica-yogurt + sugar (10 g/100 g yogurt),
plain-yogurt and commercially available organic strawberry-
yogurt containing sugar (10 g/100 g yogurt). The evaluation
was scored on 110 point hedonic scale (12 = extremely
poor, 34 = poor, 56 = fair, 78 = good, 910 = excellent)
according to texture (presence of whey), consistency (grainy,
lumpy and rmness), taste (sour, sweet and bitter), aroma
and overall preference.
2.14. Statistical analysis
Three separate experiments were carried out and triplicate as-
says from the same experiment were performed. Data were ex-
pressed as mean SEM. (standard error of the mean). The
statistical analysis was performed using one way analysis of
variance (ANOVA, SPSS 14.0), followed by Duncans post
hoc test for mean comparison. The criterion for statistical sig-
nicance was p < 0.05.
Antioxidant activity and inhibition of key enzymes linked to type-2 diabetes and hypertension by Azadirachta 297
3. Results and discussions
3.1. pH and TTA in yogurt
The pH at the end of fermentation was lower for A. indica-yo-
gurt than that for plain-yogurt (4.11 0.03 and 4.50 0.04,
respectively, p < 0.05, Fig. 1). Refrigerated storage for 28 days
resulted in further reduction of pH towards f4.25 for plain-yo-
gurt whereas A. indica-yogurt increased to 4.22 during the rst
week followed by gradual decrease towards 4.05. Post-acidi-
cation of yogurt during storage at 4 C is a common feature
associated with small but measurable microbial metabolic
activity (Saint-Eve et al., 2008). Fluctuations in pH during
storage may be explained by the relative changes in the forma-
tion of organic acids and alkaline nature of milk protein break-
down products (Sefa-Dedeh et al., 2001; Papadimitriou et al.,
2007). The consistently higher TTA in A. indica-yogurt than in
plain-yogurt, both at the end of fermentation and during stor-
age may indicate differential microbial population during fer-
mentation and possibly storage. This is not surprising since
TTA measures organic acids (lactic, citric, formic, acetic and
butyric acids), with the exception of those bound to alkaline
ions, accumulated in the yogurt (Ostlie et al., 2003).
3.2. Antioxidant activity
DPPH inhibition of day 0 yogurt in the presence of A. indica
was higher than plain-yogurt (30.1 5.1% and 23.5
5.0%, respectively, Fig. 2). Refrigerated storage increased
DPPH inhibition in both yogurts with A. indica-yogurt show-
ing consistently stronger effect than plain-yogurt. Highest
DPPH inhibitions were shown by both yogurts on days 7
and 14 with extended storage to 28 days resulted in a loss of
DPPH inhibition to values lower than day 0 by plain-yogurt
but not by A. indica-yogurt. A. indica has been previously re-
ported to show antioxidant activities properties (Sithisarn
et al., 2005; Madhi et al., 2003) which may be explained by
its high concentration of vitamin C and riboavin (Atangwho
et al., 2009). Other possible sources of DPPH inhibitors attrib-
uted to yogurt may be derived from milk protein proteolysis
(Lourens-Hattingh and Viljoen, 2001) and organic acids (Cor-
reia et al., 2004) as a result of fermentation and post-acidicat-
ion during storage. Proteolysis and TTA for instance were also
shown to increase during storage in the presence of A. indica
(Figs. 4 and 1b, respectively). Higher yogurt antioxidant activ-
ity in the presence of A. indica is benecial in two respects,
rstly to delay the oxidation process of lipids in yogurt that
are responsible for the formation of off-avours and undesir-
able chemical compounds (Berset et al., 1994) and secondly,
to increase dietary antioxidants which are crucial in preventing
the progressive impairment of pancreatic beta-cell function
due to oxidative stress (Liu et al., 2005). The presence of A. in-
dica in yogurts may thus be viewed advantageous in prolong-
ing the shelf life of yogurt and the consumption of which in
reducing the occurrence of type 2 diabetes.
3.3. Total phenolic content
The TPC in A. indica-yogurt was higher (p < 0.05) than plain-
yogurt both after fermentation and during storage (Fig. 3).
Refrigerated plain- and A. indica-yogurts showed transient
reduction in TPC by day 14 (17.5 3.1 and
38.5 7.2 lgGAE/ml, respectively), but increased to highest
values (29.6 2.5 and 74.9 6.2 lgGAE/ml, respectively;
p < 0.05) by day 28. The transient decrease and increase in
TPC in both yogurts may be explained by the action of yogurt
Figure 1 Changes in (a) pH and (b) TTA during refrigerated
(4 C) storage of yogurts. Plain-yogurt (control), A. indica-
yogurt. Values are presented as mean SEM (n = 3).
Figure 2 Changes in antioxidant activity (inhibition % of
DPPH) in yogurt during refrigerated (4 C) storage. Plain-
yogurt (control), A. indica-yogurt. Values are presented as
mean SEM (n = 3).
Figure 4 Changes in OPA values (mg/g) in yogurt during
refrigerated (4 C) storage. Plain-yogurt (control), A. indica-
yogurt. Values are presented as mean SEM (n = 3).
Figure 3 Changes in total phenolic content (TPC; lgGAE/ml) in
yogurt during refrigerated (4 C) storage. Plain-yogurt (control),
A. indica-yogurt. Values are presented as mean SEM (n = 3).
298 A.B. Shori, A.S. Baba
bacteria during refrigerated storage to further break down the
polymeric phenolics in the presence of A. indica (Dalling,
1986). Elevated TPC in food was commonly associated with
increased antioxidant activities (Velioglu et al., 1998) despite
some disagreements (Kahkonen et al., 1999). In the present
study A. indica-yogurt showed higher antioxidant activity than
plain-yogurt, although not all of these activities can be attrib-
uted to A. indica (Fig. 2). Further studies are required to char-
acterize the phytochemicals in A. indica which may be
responsible for the elevated TPC and sustained antioxidant
activities in A. indica-yogurt during refrigerated storage.
3.4. Proteolysis in yogurt
OPA values of yogurt in the presence of A. indica was higher
than plain-yogurt during the 28 days of storage (Fig. 4).
OPA values after day 14 of storage tended to reduce for
plain-yogurt but the opposite was true for A. indica-yogurt.
OPA values were highest for A. indica-yogurt (30 0.02 mg/
g) on day 21 of storage but lowest for plain-yogurt
(10.2 0.03 mg/g) on day 28. Proteolysis in fermented milks
is related to the activity of yogurt bacteria (Wood, 1981).
The proteinase of L. bulgaricus for instance hydrolyses casein
to yield polypeptides (Tamine and Robinson, 1985) which
may be broken down further by the peptidases of S. therrno-
philus. On the other hand, S. thermophilus metabolizes excess
amino acids liberated by L. bulgaricus. Therefore, the balance
between amino acid liberation and utilization was suggested
(Rasic and Kurmann, 1978) to explain the degree of
proteolysis.
3.5. Inhibition of angiotensin-1 converting enzyme (ACE) by
yogurts
Fresh plain-yogurt inhibited about 50% ACE activity but this
reduced to about 20% by the 3rd week of storage (Fig. 5). A.
indica-yogurt had higher ACE inhibition than plain-yogurt
with maximal inhibition of 79.7 11.1% occurring on day
14 of storage. Both plain- and A. indica-yogurts recorded sim-
ilar values on day 28 of storage (23.1 0.3%). Pripp et al.
(2005) showed that a high ACE-inhibitory potential is accom-
panied by a high degree of proteolysis (OPA values), which is
shown as higher OPA values in A. indica-yogurt than in plain-
yogurt in the present studies (Fig. 5). The exopeptidase activ-
ities on milk proteins are regarded responsible for the forma-
tion of many fragmented peptides with anti-ACE activity
(FitzGerald et al., 2004). The changes in ACE activity in rela-
tion to OPA values with storage time for both plain- and A.
indica-yogurts (Figs. 4 and 5) suggest that the relatively less
specic peptides produced during fermentation were further
cleaved to smaller and possibly more bioactive proteins during
the rst 7 days of refrigeration. However, extensive proteolysis
of these proteins during extended storage may yield much
smaller and less bioactive proteins (Fig. 5). The addition of
A. indica into yogurt may thus change the manner in which
the microbial enzymes affected proteolysis and subsequently
the formation and deactivation of proteins with anti-ACE
activities.
3.6. a-Amylase inhibitory potentials in yogurt
Fresh A. indica-yogurt (44.4 3.0%, day 0) had higher
(p < 0.05) inhibitory effects on a-amylase than plain-yogurt
(29.8 5.3%, Fig. 6). Refrigerated storage increased plain-yo-
gurt a-amylase inhibition during the rst 14 days to 47% but
this reduced (p < 0.05) to 2328% between the 3rd and 4th
week of storage. In contrast, a-amylase inhibition of A. indi-
ca-yogurt increased to 55.0 2.5% by day 21 of storage with
small reduction to 42.0 3.0% by day 28 of storage. The con-
sumption of yogurt was reported effective to check rapid post-
prandial increase in blood glucose (Djomeni et al., 2006; Fujita
et al., 2003). This may be explained by the inhibition of carbo-
hydrate digestive enzymes (pancreatic a-amylase) important in
causing postprandial hyperglycaemia commonly found in
type-2 diabetes (McDougall and Stewart, 2005). A non-sus-
tained increase in aamylase inhibition with storage may, also
be related to proteolysis as described earlier for ACE. Our
ndings showed that A. indica did not only enhance but may
also increase the yogurt a-amylase inhibitory capacity with
storage. To our best knowledge, this has not been previously
Figure 6 Changes in a-amylase inhibitory activity (%) in yogurt
during refrigerated (4 C) storage. Plain-yogurt (control), A.
indica-yogurt. Values are presented as mean SEM (n = 3).
Figure 7 Changes in a-glucosidase inhibitory activity (%) in
yogurt during refrigerated (4 C) storage. Plain-yogurt (control),
A. indica-yogurt. Values are presented as mean SEM (n = 3).
Figure 5 Changes in inhibition (%) of angiotensin-1 converting
enzyme (ACE) by yogurt refrigerated (4 C) up to 28 days.
Plain-yogurt (control), A. indica-yogurt. Values are presented as
mean SEM (n = 3).
Antioxidant activity and inhibition of key enzymes linked to type-2 diabetes and hypertension by Azadirachta 299
reported and the apparent strong inhibition of a-amylase
activity even as long as 28 days of storage should be taken to
advantage. This is because plant phenolic compounds which
bind to the reactive sites of enzymes thus altering its catalytic
activity (McCue and Shetty, 2004) in general are considered
potent and safe inhibitors of carbohydrate hydrolysing en-
zymes (Lee et al., 2008).
3.7. a-Glucosidase inhibitory potentials in yogurt
A.indica-yogurt has higher mean a-glucosidase inhibition
activity than plain-yogurt (p > 0.05; Fig. 7) but both yogurts
showed similar changes in a-glucosidase inhibition activity
during refrigerated storage with highest inhibition (15%) re-
corded on day 14. Therefore, refrigeration of yogurts for two
weeks resulted in maximum increase of a-glucosidase inhibi-
tion but A. indica per se was not affecting aglucosidase as it
affected aamylase.
3.8. Organoleptic properties
A. indica-yogurt was not different from plain-yogurt for all
characteristics except for higher score for bitterness
(4.3 0.7 and 1.9 0.3, respectively, p < 0.05). The addition
of sugar increased the sweetness (p < 0.05), aroma and overall
preference scores (p > 0.05) for A. indica-yogurt compared to
plain-yogurt. However, when compared with strawberry-yo-
gurt, sweetened A. indica-yogurt had lower sourness but higher
sweetness scores (p < 0.05). Despite its bitterness, A. indica is
consumed together with sh and sweet sauce in order to pro-
mote good health (Clayton et al., 1996). Yogurt may become
a good carrier for A. indica extract because its presence did
not markedly change yogurt organoleptic properties except
for its bitterness. The addition of natural non-caloric sweetener
such as Stevia rebaudiana leaves may increase the acceptability
of A. indica-yogurt without causing concern to diabetic con-
sumers (Table 1).
Toxicology studies was not attempted, but published re-
ports indicate the application of A. indica water extract rang-
ing from 10 to 500 mg/kg body weight (Boeke et al., 2004)
did not produce toxic effects. An example of extreme adminis-
tration of A. indica was reported by Chandra et al. (2007)
whereby diabetic rats survived after a 30 days oral administra-
tion of water extracts of dried A. indica leaves (500 mg/kg/ani-
mal) with signicant decrease (53%) in blood glucose levels,
reactivation of the antioxidant enzymes, and recovery of the
original body weight. The amount used was 170 times stronger
that the dosage used in the present studies (i.e. 145 mg/100 ml
A. indica-yogurt, equivalent to a daily dosage of 2.9 mg/kg
body weight for a 50 kg person). As such, it is anticipated that
the amount of A. indica water extract used in the preparation
of A. indica-yogurt was in the range of safety dose and it is as-
sumed to be safe for human consumption.
4. Conclusions
The addition of A. indica into yogurt enhanced the acidicat-
ion, total phenolic content and antioxidant activities of yogurt.
A. indica-yogurt also has higher inhibitory effects on in vitro
a-amylase and ACE activities. Given the existing medicinal
values of A. indica and evidence of improved A. indica-yogurt
with respect to acidication, anti-oxidant and inhibition of en-
zymes related to diabetes and hypertension in the present
study, A. indica-yogurt has the potential to be further devel-
oped as a functional yogurt for consumers with diabetes and
hypertension.
Acknowledgement
We acknowledge the nancial support of University of Malaya
Research Grant (RG023-090BIO).
References
Actis-Goreta, L., Ottaviani, J.I., Keen, C.L., Fraga, G., 2003.
Inihbition of angiotensin converting enzyme (ACE) activity by
avan 3-ols and procyanidins. FEBS Lett. 555, 597600.
Apostolidis, E., Kwon, Y.I., Shetty, K., 2007. Inhibitory potential of
herb, fruit,and fungal-enriched cheese against key enzymes linked
to type 2 diabetes and hypertension. Innov. Food Sci. Emerg.
Technol. 8, 4654.
Apostolidis, E., Kwon, Y.I.I., Shetty, K., 2006. Potential of cranberry-
based herbal synergies for diabetes and hypertension management.
Asia Pac J Clin Nutr (15), 433441.
Atangwho, I.J., Ebong, P.E., Eyong, E.U., Williams, I.O., Eteng,
M.U., Egbung, G.E., 2009. Comparative chemical composition of
leaves of some antidiabetic medicinal plants: Azadirachta indica,
Vernonia amygdalina and Gongronema latifolium. Afr. J. Biotech-
nol. 8 (18), 46854689.
Berset, C., Brand-Williams, W., Cuvelier, M.E., 1994. Use of a free
radical method to evaluate antioxidant activity. Lebensm. Wiss.
Technol. 28 (11), 2530.
Boeke, S.J., Boersma, M.G., Alink, G.M., van Loon, J.J.A., van Huis,
A., Dicke, M., Rietjens, I.M.C.M., 2004. Safety evaluation of neem
Table 1 Mean taste panel scores for samples of yogurt.
Criteria Plain-yogurt A. indica-yogurt A. indica-yogurt + sugar Strawberry-yogurt
Texture 5.9 0.6 5.8 0.6 5.7 1.1 5.9 1.2
Consistency 6.3 0.7 6.1 0.4 6.8 0.9 7.5 0.4
Sourness 4.4 0.9 4.2 0.5 3.1 1.1 6.2 1.0
Sweetness 2.5 0.7 2.9 0.5 7.7 0.5 5.3 0.6
Bitterness 1.9 0.3 4.3 0.7 2.9 0.4 1.9 0.1
Aroma 5.1 0.6 5.5 0.9 6.5 0.8 7.8 0.3
Overall preference 5.6 0.1 5.4 0.7 6.5 0.7 7.4 0.3
*
Values are presented as mean SEM, n = 12.
*
A 110 point hedonic scale (12 = extremely poor, 34 = poor, 56 = fair, 78 = good, 910 = excellent).
300 A.B. Shori, A.S. Baba
(Azadirachta indica) derived pesticides. J. Ethnopharmacol. (94),
2541.
Chandra, A., Mahdi, A.A., Ahmad, S., Singh, R.K., 2007. Indian
herbs result in hypoglycemic responses in streptozotocin-induced
diabetic rats. Nutr. Res. (27), 161168.
Church, F.C., Swaisgood, H.E., Porter, D.H., Catignani, G.L., 1983.
Pectrophotometric assay using o-phthaldialdehyde for determina-
tion of proteolysis in milk and isolated milk proteins. J. Dairy Sci.
(66), 12191227.
Clayton, T., Soralump, P., Chaukul, W., Temsiririrkkul, R., 1996.
Medicinal Plants in Thailand, vol. 1. Amarin Printing, Bangkok.
Correia, I., Nunes, A., Duarte, I.F., Barros, A., elgadillo, I., 2004.
Sorghum fermentation followed by spectroscopic techniques. Food
Chem., 853859.
Dalling, M.J., 1986. Plant Proteolytic Enzymes, vols. I and II. CRC
Press, Boca Raton, FL.
Djomeni, P.D.D., Te dong, L., Asongalem, E.A., Dimo, T., Sokeng,
S.D., Kamtchouing, P., 2006. Hypoglycaemic and antidiabetic
effect of root extracts of Ceiba Pentadra in normal and diabetic
rats. Afr. J. Tradit. Complement. Altern. Med. 3, 129136.
FitzGerald, R.J., Murray, B.A., Walsh, D.J., 2004. Hypotensive
peptides from milk proteins. J. Nutr. 134, 980S988S.
Fujita, H., Yamagami, T., Ohshima, K., 2003. Long-term ingestion of
touchi-extract, an a-glucosidase inhibitor, by borderline and mild
type-2 diabetic subjects is safe and signicantly reduces blood
glucose levels. Nutr. Res. 23, 713722.
Gobbetti, M., Minervini, F., Rizzello, C.G., 2004. Angiotensin I-
converting-enzyme-inhibitory and antimicrobial bioactive peptide.
Int. J. Dairy Technol. 57, 172188.
Kahkonen, M.J., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K.,
Kujala, T.S., 1999. Antioxidant activity of plant extracts containing
phenolic compounds. J. Agric. Food Chem. 47, 39543962.
Kwon, Y.I., Apostolidis, E., Shetty, K., 2005. Traditional diet of
Americas for management of diabetes and hypertension. J. Med.
Food 10, 266275.
Kwon, Y.I., Vattem, D.A., Shetty, K., 2006. Clonal herbs of
Lamiaceae species against diabetes and hypertension. Asia Pac. J.
Clin. Nutr. 15, 107118.
Marles, R.J., Farnsworth, N., 1994. Antidiabetic plants and their
active constituents. Phytomedicine 2 (2), 137189.
Lee, Y.A., Cho, E.J., Yokozawa, T., 2008. Effects of proanthocyanidin
preparations on hyperlipidemia and other biomarkers in mouse
model of type 2 diabetes. J. Agric. Food Chem. 56 (17), 77817789.
Liu, D.-Z., Lin, Y.-S., Hou, W.-C., 2005. Monohydroxamates of
aspartic acid and glutamic acid exhibit antioxidant and angiotensin
converting enzyme inhibitory activities. J. Agric. Food. Chem. (52),
23862390.
Lourens-Hattingh, A., Viljoen, B.C., 2001. Yogurt as probiotic carrier
food. Int. Dairy J. (11), 117.
Madhi, A.A., Chandra, A., Singh, R.K., Shukia, S., Mishra, L.C.,
Ahmad, S., 2003. Effect of herbal hypoglycemic agents on oxidative
stress and antioxidant status in diabetic rats. Ind. J. Clin. Biochem.
18 (2), 815.
Matsui, T., Ueda, T., Oki, T., Sugita, K., Terahara, N., Matsumoto,
K., 1987. -glucosidase inhibitory action of natural acylated
anthocyanins 1. Survey of natural pigment with potent inhibitory
activity. J. Agric. Food. Chem. 49, 19481951.
McCue, P., Shetty, K., 2004. Inhibitory effects of rosmarinic acid
extracts on porcine pancreatic amylase in vitro. Asia Pac. J. Clin.
Nutr. 13, 101106.
McDougall, G.J., Stewart, D., 2005. The inhibitory effects of berry
extracts on digestive enzymes. BioFactors (23), 189195.
Meisel, H., 1998. Overview on milk proteins-derived peptides. Int.
Dairy J. (8), 363373.
Murthy, P.S., Sirsi, M., 1958. Pharmacological studies on Melia
azadirachta. Indian J. Psychol. 2 (2), 387396.
Ostlie, H.M., Treimo, J., Narvhus, J.A., 2003. Effect of temperature on
growth metabolism of probiotic bacteria in milk. Int. Dairy J. 15,
989997.
Papadimitriou, C.G., Mastrojiannaki, A.V., Silva, A.V., Gomes,
A.M., Malcata, F.X., Alichanidis, E., 2007. Identication of
peptides in traditional and probiotic sheep milk yoghurt with
angiotensin I-converting enzyme (ACE)-inhibitory activity. Food
Chem. 105, 647656.
Pripp, A.H., Sorensen, R., Stepaniak, L., Sorhaug, T., 2005. Rela-
tionship between proteolysis and angiotensinand-I-converting
enzyme inhibition in different cheeses. LWTFood Sci. Technol.
(39), 677683.
Rasic, J.L., Kurmann, J.A., 1978. In: Yogurt: Scientic Grounds,
Technology, Manufacture, and Preparations. Tech. Dairy Publ.
House, Copenhagen, Denmark.
Saint-Eve, A., Levy, C., Le Moigne, M., Ducruet, V., Souchon, I.,
2008. Quality changes in yogurt during storage in different
packaging materials. Food Chem. 110, 285293.
Sefa-Dedeh, S., Kluvitse, Y., Afoakwa, O.E., 2001. Inuence of
fermentation and cowpea steaming on some quality characteristics
of maize-cowpea blends. Afr. J. Sci. Technol. 2 (2), 7180.
Schmutterer, H., 1995. The neem tree, source of unique natural
products for integrated pest management, medicine, industry and
other purposes, vol. 7. VCH Publishers Inc., New York, USA, pp.
3589.
Shetty, K., Curtis, O.F., Levin, R.E., Witkowsky, R., Ang, W., 1995.
Prevention of verication associated with in vitro shoot culture of
oregano (Origanum vulgare) by Pseudomonas spp. J. Plant Physiol.
147, 447451.
Shibata, K., 1955. Basic Statistics for Biologist. Tokyo Shobun
Publishing Co., pp. 6478.
Sithisarn, P., Supabphol, R., Gritsanapan, W., 2005. Antioxidant
activity of Siamese neem tree (VP1209). J. Ethnopharmacol. 99 (1),
109112.
Tamine, A.Y., Robinson, R.K., 1985. In: Yogurt science and
technology. Oxford, Pergamon Press, pp. 365373.
Van der Nat, J.M., Van der Sluis, W.G., Silva, K.T.D., Labadie, R.P.,
1991. Ethnopharmacognostical survey of Azadirachta indica A.
Juss (Meliaceae). J. Ethnopharmacol. (35), 124.
Velioglu, Y.S., Mazza, G., Gao, L., Oomah, B.D., 1998. Antioxidant
activity and total phenolics in selected fruits, vegetables, and grain
products. J. Agric. Food. Chem. 46 (10), 41134117.
Vermeirssen, V., Camp, J.V., Verstraete, W., 2002. Optimization and
validation of an angiotensin-converting enzyme inhibition assay for
the screening of bioactive peptides. J. Biochem. Biophys. Methods
51, 7587.
Viljoen, C., Lourens-Hattingh, A., Bennie, 2001. Yogurt as probiotic
carrier food. Int. Dairy J. (11), 117.
Wood, B.J.B., 1981. The yeast/lactobacillus interaction; a study in
stability. In: Bushell, M.E., Slater, J.H.S. (Eds.), . In: Mixed
Culture Fermentation. Academic Press, London, pp. 138R150R
(Chapter 7).
Antioxidant activity and inhibition of key enzymes linked to type-2 diabetes and hypertension by Azadirachta 301