Journal of Saudi Chemical Society (2013) 17, 295–301

King Saud University

Journal of Saudi Chemical Society

www.ksu.edu.sawww.sciencedirect.com

ORIGINAL ARTICLE

Antioxidant activity and inhibition of key enzymes linked

to type-2 diabetes and hypertension by Azadirachtaindica-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

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KEYWORDS

Yogurt;

Azadirachta indica;

Angiotensin-1 converting

enzyme;

a-Glucosidase;

a-Amylase

Corresponding author.

-mail address: shori_7506@

19-6103 ª 2011 King Saud

sevier B.V. All rights reserve

er review under responsibilit

i:10.1016/j.jscs.2011.04.006

Production and h

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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.ª 2011 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

om (A.B. Shori).

y. Production and hosting by

Saud University.

lsevier

1. Introduction

Yogurt is considered a healthy food due to high digestibilityand 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 modification ofallergenic 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,

296 A.B. Shori, A.S. Baba

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 potentialuse 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 havelimited resources and access to modern treatments (Marlesand Farnsworth, 1994). These plants have been investigated

with respect to the suppression of glucose production fromcarbohydrates 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). Thephenolic compounds, for instance, play a role in mediatingamylase inhibition and therefore have potential to contributeto the management of type 2 diabetes (McCue and Shetty,

2004). Phenolic compounds also have lower inhibitory effectagainst a-amylase activity but a stronger inhibition activityagainst 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 toarthritis and rheumatism (Van der Nat et al., 1991), skin dis-eases and inflammation (Clayton et al., 1996). Early studies

showed that aqueous extract of neem leaves decreased bloodadrenaline as well as glucose-induced hyperglycaemia (Shiba-ta, 1955) whereas oral doses of neem leaf extracts significantly

reduced insulin requirements for insulin dependent diabetes(Murthy and Sirsi, 1958). The biologically active componentsof 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, antioxidantcapacity 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 andthen oven dried (50 �C) for 72 h. The dried leaves were thenground 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 occasionalshaking, 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,

Bifidobacterium bifidum Bb-12, Lactobacillus casei LC-01 andStreptococcus thermophilus Th-4 4 in the ratio of 4:4:1:1 anda capsule of probiotic mix containing L. bulgaricus,Lactobacil-

lus rhamnosus,Bifidobacterium infantis andBifidobacterium lon-gum in the ratio of (1:1:1:1). The milk-bacteria mixture wasincubated in a water bath (41 �C) for 12 h and the yogurt

formed was stored at 4 �C and used as starter culture within7 days. Viable bacteria in the starter culture on day 7 of stor-age ranged 2.0–5.0 · 106 cfu/g and 6.0–10.0 · 108 cfu/g forLactobacillus 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 hasbeen 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 wasprepared 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 readusing a digital pH meter (Mettler-Toledo 320, Shanghai).Homogenised yogurt was mixed with phenolphthalein (pH

indicator, 0.1% w/v, 2–3 drops) and the TTA was determinedby titration using NaOH as alkali. Titration with 0.1 N NaOHwas carried out under continuous stirring until the develop-ment of a consistent pink colour. TTA (% lactic acid) pro-

duced after predetermined refrigeration was calculated asfollows:

TTAð%Lactic AcidÞ ¼ Dilution factor� VNaOH � 0:1N

� 0:009� 100%

where V is volume of NaOH required to neutralize the acidand 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 acidified to pH 4.0 with HCl(0.1 M) followed by heating (water bath; 45 �C, 10 min) andcentrifugation (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 andsalts. The supernatants were harvested and kept in the refrig-

erator (4 �C) and used within 12 h of preparation.

Antioxidant activity and inhibition of key enzymes linked to type-2 diabetes and hypertension by Azadirachta 297

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 dH2O (Shetty et al., 1995). Folin–Ciocalteu

reagent (0.5 ml; 50% v/v) was added to each sample and after athorough mixing the solutions were allowed to stand for 5 min.at room temperature. Na2CO3 (1.0 ml, 5% g/100 ml) was then

added and absorbance at 725 nm was read after another60 min. incubation at room temperature. Known concentra-tions of gallic acid (Sigma–Aldrich, Germany; 5–60 lg/ml inethanol) were treated in the same manner as for water extracts

of yogurts and the regression of gallic acid standards was usedto 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). Briefly, DPPH (Sigma–Aldrich, Germany, 3 ml of

60 mmol/L in ethanol) were mixed with 250 ll yogurt extractsor 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 ¼Acontrol

517 � Aextract517

� �

Acontrol517

� � � 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 10–50 ll con-taining 5–100 lg protein) was added directly into 1.0 ml ofOPA reagent. The solution was mixed briefly by inversionand 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 Vermeirssenet al. (2002). ACE reagent (500 ll) was added to 300 ll of

water yogurt extracts in a cuvette and the contents were mixedand then incubated in a water bath (37 �C) for 2 min followedby the addition of 300 ll of rabbit lung extract. Absorbance

was measured at 340 nm and the readings were compared toa 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:

ACEinhibitionð%Þ ¼ ACEcontrol �ACEyogurt water extracts

ACEcontrol

� 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 of0.02 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 eachtube at pre-determined time intervals. The reaction mixtures

were incubated at 25 �C for 10 min. The reaction was stoppedwith 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 addedto each tube after the boiling prior to cooling to room temper-ature. The reaction mixture was then diluted by adding 10 ml

of dH2O and the absorbance was read at 540 nm. The readingswere 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%

¼ 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) wasincubated in water bath at 25 �C for 10 min. After 10 min.,

500 ll of 5 mM p-nitrophenyl-a-D-glucopyranoside solutionin 0.1 M potassium phosphate buffer (pH 6.90) was added toeach tube at predetermined time intervals. The reaction mix-

tures were incubated at 25 �C for 5 min. prior to the readingof absorbance at 405 nm. The readings were compared to acontrol which had 500 ll of buffer solution instead of the ex-

tract. The a-glucosidase inhibitory activity was expressed asinhibition% as follows:

Inhibition% ¼ Absorbancecontrol �AbsorbanceextractsAbsorbancecontrol

� 100

2.13. Sensory analysis

Sensory analyses were carried out by 15 untrained panels agedbetween 20 and 41 years old. Each panel was presented withfour 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 evaluationwas scored on 1–10 point hedonic scale (1–2 = extremely

poor, 3–4 = poor, 5–6 = fair, 7–8 = good, 9–10 = excellent)according to texture (presence of whey), consistency (grainy,lumpy and firmness), 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). Thestatistical analysis was performed using one way analysis ofvariance (ANOVA, SPSS 14.0), followed by Duncan’s posthoc test for mean comparison. The criterion for statistical sig-

nificance was p < 0.05.

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 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

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 daysresulted in further reduction of pH towards f4.25 for plain-yo-gurt whereas A. indica-yogurt increased to 4.22 during the first

week followed by gradual decrease towards 4.05. Post-acidifi-cation of yogurt during storage at 4 �C is a common featureassociated with small but measurable microbial metabolic

activity (Saint-Eve et al., 2008). Fluctuations in pH duringstorage 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 inplain-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 sinceTTA measures organic acids (lactic, citric, formic, acetic andbutyric 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. indicawas 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. HighestDPPH inhibitions were shown by both yogurts on days 7

and 14 with extended storage to 28 days resulted in a loss ofDPPH inhibition to values lower than day 0 by plain-yogurtbut not by A. indica-yogurt. A. indica has been previously re-

ported to show antioxidant activities properties (Sithisarnet al., 2005; Madhi et al., 2003) which may be explained byits high concentration of vitamin C and riboflavin (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-acidificat-

ion during storage. Proteolysis and TTA for instance were alsoshown 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 beneficial in two respects,firstly to delay the oxidation process of lipids in yogurt thatare responsible for the formation of off-flavours and undesir-

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).

able chemical compounds (Berset et al., 1994) and secondly,to increase dietary antioxidants which are crucial in preventingthe progressive impairment of pancreatic beta-cell functiondue 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 inreducing 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 transientreduction in TPC by day 14 (17.5 ± 3.1 and

38.5 ± 7.2 lgGAE/ml, respectively), but increased to highestvalues (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 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 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).

Antioxidant activity and inhibition of key enzymes linked to type-2 diabetes and hypertension by Azadirachta 299

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 withincreased antioxidant activities (Velioglu et al., 1998) despitesome disagreements (Kahkonen et al., 1999). In the present

study A. indica-yogurt showed higher antioxidant activity thanplain-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 beresponsible for the elevated TPC and sustained antioxidantactivities in A. indica-yogurt during refrigerated storage.

3.4. Proteolysis in yogurt

OPA values of yogurt in the presence of A. indica was higherthan plain-yogurt during the 28 days of storage (Fig. 4).OPA values after day 14 of storage tended to reduce forplain-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 caseinto 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 excessamino acids liberated by L. bulgaricus. Therefore, the balancebetween amino acid liberation and utilization was suggested

(Rasic and Kurmann, 1978) to explain the degree ofproteolysis.

3.5. Inhibition of angiotensin-1 converting enzyme (ACE) byyogurts

Fresh plain-yogurt inhibited about 50% ACE activity but thisreduced 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 day14 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 isshown 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-

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).

tion to OPA values with storage time for both plain- and A.indica-yogurts (Figs. 4 and 5) suggest that the relatively less

specific peptides produced during fermentation were furthercleaved to smaller and possibly more bioactive proteins duringthe first 7 days of refrigeration. However, extensive proteolysis

of these proteins during extended storage may yield muchsmaller and less bioactive proteins (Fig. 5). The addition ofA. indica into yogurt may thus change the manner in which

the microbial enzymes affected proteolysis and subsequentlythe formation and deactivation of proteins with anti-ACEactivities.

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 first 14 days to 47% but

this reduced (p< 0.05) to 23–28% between the 3rd and 4thweek 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; Fujitaet al., 2003). This may be explained by the inhibition of carbo-

hydrate digestive enzymes (pancreatic a-amylase) important incausing postprandial hyperglycaemia commonly found intype-2 diabetes (McDougall and Stewart, 2005). A non-sus-

tained increase in a–amylase inhibition with storage may, alsobe related to proteolysis as described earlier for ACE. Ourfindings showed that A. indica did not only enhance but may

also increase the yogurt a-amylase inhibitory capacity withstorage. To our best knowledge, this has not been previously

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).

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 1–10 point hedonic scale (1–2 = extremely poor, 3–4 = poor, 5–6 = fair, 7–8 = good, 9–10 = excellent).

300 A.B. Shori, A.S. Baba

reported and the apparent strong inhibition of a-amylaseactivity even as long as 28 days of storage should be taken to

advantage. This is because plant phenolic compounds whichbind to the reactive sites of enzymes thus altering its catalyticactivity (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 yogurtsshowed similar changes in a-glucosidase inhibition activityduring 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 a–glucosidase as itaffected a–amylase.

3.8. Organoleptic properties

A. indica-yogurt was not different from plain-yogurt for allcharacteristics 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 overallpreference scores (p > 0.05) for A. indica-yogurt compared toplain-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 isconsumed together with fish 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 didnot markedly change yogurt organoleptic properties exceptfor its bitterness. The addition of natural non-caloric sweetener

such as Stevia rebaudiana leaves may increase the acceptabilityof 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 significant decrease (53%) in blood glucose levels,reactivation of the antioxidant enzymes, and recovery of theoriginal body weight. The amount used was 170 times stronger

that the dosage used in the present studies (i.e. 145 mg/100 mlA. indica-yogurt, equivalent to a daily dosage of 2.9 mg/kg

body weight for a 50 kg person). As such, it is anticipated thatthe amount of A. indica water extract used in the preparationof 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 acidificat-ion, total phenolic content and antioxidant activities of yogurt.

A. indica-yogurt also has higher inhibitory effects on in vitroa-amylase and ACE activities. Given the existing medicinalvalues of A. indica and evidence of improved A. indica-yogurt

with respect to acidification, anti-oxidant and inhibition of en-zymes related to diabetes and hypertension in the presentstudy, 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 financial support of University of Malaya

Research Grant (RG023-090BIO).

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