Chinese

Journal of
Natural
Chinese Journal of Natural Medicines 2012, 10(4): 02550259 Medicines

Antioxidant and -amylase inhibition activities of

phenolic compounds in the extracts of Indian honey
Subashini Devarajan, Subhashree Venugopal*
School of Biosciences and Technology, VIT University, Vellore - 632014, Tamil Nadu, India
Available online 20 July 2012

[ABSTRACT] AIM: To evaluate the antioxidant and -amylase inhibition potential of phenolic compounds in the extracts of Indian
honey. METHODS: Phenolic compounds were extracted from Indian honey through column chromatography. The antioxidant poten-
tial of extracted phenolic compounds was measured by two different biochemical assays: ferric reducing antioxidant power (FRAP)
assay and scavenging activity on 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Moreover, -amylase inhibition assay of phenolic
compounds of honey was also evaluated. RESULTS: The scavenging inhibition rate varied from 86.8% to 78.6% from the highest (6
mgmL1) to the lowest (1.5 mgmL1) concentration, whereas, reducing power assay varied from 0.89 Abs to 0.19 Abs from the highest
to the lowest concentration. Butylated hydroxytoluene (BHT) was used as reference compound for antioxidant assays. -amylase inhi-
bition assay is reported from the phenolic honey extracts for the first time. The inhibition rate for -amylase varied from 88.8% to
30.5% from the highest (20 gmL1) to the lowest concentration (4 gmL1). CONCLUSION: Honey phenolic extract possessed
antioxidant and -amylase inhibition activity, thus increasing its potential therapeutic property.
[KEY WORDS] Honey; Phenolic compounds; Antioxidant activity; DPPH and FRAP; -Amylase inhibition activity
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2012)04-0255-05

1 Introduction are reported to exhibit anti-carcinogenic, anti-inflammatory,

Honey has been used since the earliest times. It is a nat-
ural substance produced by bees, Apis mellifera, from the
nectar of blossoms or from exudates of trees and plants giv-
ing nectar honey or honey dews, respectively. In most ancient
cultures honey was used for both nutritional and medical
purposes[1]. It is widely appreciated as the concentrated form
of sugar available worldwide[2] and is also used as food pre-
servative[3-4]. Antioxidant activity is the ability of honey to
reduce oxidative reactions within the food system and human
health. These oxidative reactions can cause deleterious reac-
tions in food products and adverse health effects[5-6]. The
factor contributing to honey antioxidant activity are lysozyme,
phenolic acids and flavonoid[7]. Phenolics are among the
most important compounds occurring in plants, comprising at
least 8 000 different known structures[8]. These compounds
[Received on] 11-July-2011
[
Corresponding author] V. Subhashre: Assistant Prof., Tel:
91-9486947377, Fax: 91-416-2243092/2240411, E-mail: vsub-
hashree@vit.ac.in; v_subhashree@hotmail.com
These authors have no any conflict of interest to declare.
Copyright 2012, China Pharmaceutical University.
Published by Elsevier B.V. All rights reserved
anti-thrombotic, immune modulating and analgesic activities
as antioxidants[9].
The honey phenolic acids are divided into two subclasses:
the substituted benzoic acid and cinnamic acids. The flavon-
oids in honey are divided into three classes with similar
structure: flavonols, flavones and flavonones. These are im-
portant due to their contribution to honey color, taste and
flavour and also due to their beneficial effects on health.
Moreover, the composition and consequently antioxidant
capacity of honey phenolic compounds depend on the floral
sources used to collect honey which are dependent on sea-
sonal and environmental factors[10-11]. Since the composition
of active compounds in honey from different locations are
different, difference in properties of honey is expected.
Alpha amylase inhibitory activity has been reported pre-
viously for various plant extracts[12] and microbial extract[13].
Inhibitory activity against amylase by flavonoids and antho-
cyanin has been reported[14-15]. Flavonoids, which are widely
distributed in the plant kingdom and present in considerable
quantities in common food products, spices and beverages,
have been used since ancient times by physicians and laymen
to treat a great variety of human diseases such as diabetes,
coronary heart diseases and cancers[16]. Our study, for the
 Subashini Devarajan, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 255259
first time, revealed the alpha amylase inhibitory potential of
phenolic extracts of honey and this study could be helpful to
develop medicinal preparation against diabetes and related
symptoms. The main approach for treating diabetes is to de-
crease the post-prandial hyperglycemia. This is done by re-
tarding the glucose absorption through the inhibition of car-
bohydrate hydrolyzing enzymes such as -amylase and
-glucosidase in the digestive tract. Inhibitors of this en-
zymes delay carbohydrate digestion time causing a reduction
in the rate of glucose absorption and consequently blunting
the postprandial plasma glucose rise[17].
In this study, phenolic compounds of honey were ex-
tracted. Antioxidant properties of honey extracts were inves-
tigated by ferric reducing antioxidant power (FRAP) assay
and scavenging effect on 1, 1-diphenyl-2-picrylhydrasyl
(DPPH) radicals. Moreover, -amylase inhibition assay of
honey extracts was also studied.
2 Materials and Methods
2.1 Honey sample
Honey samples were collected from Pudur (Vellore dis-
trict, Tamil Nadu, India). This sample was harvested from
honey bee nests of tamarind tree, with the help of profes-
sional honey collectors. Honey samples were stored in the
dark until analyzed.
2.2 Chemicals
Amberlite XAD-2 was purchased from supelco. 1,
1-diphenyl-2-picrylhydrazyl (DPPH), -amylase, methanol,
diethyl ether, potassium ferricyanide, trichloro acetic acid,
ferric chloride, Butylated hydroxyltoluene (BHT), sodium
phosphate buffer, dinitro salicylic acid reagent solution, so-
dium carbonate and all chemical agents used were purchased
from Sigma-Aldrich.
2.3 Extraction of phenolic compounds
Extraction was carried out as described previously[18-21].
Sixty grams of Amberlite XAD-2 resin, pore size 9 nm, parti-
cle size 0.31.2 mm (Supelco, Bellefonte, PA, USA) were
soaked in methanol for 10 min. After that most of methanol
was decanted and replaced by distilled water. The mixture
was stirred, allowed to stand for 510 min and was packed
into the glass column (25 cm 2 cm).
Honey (2550) g was dissolved in 250 mL of distilled
water, and the pH of the solution was adjusted to pH 2.0 by
adding concentrated HCl. The solution was filtered slowly
through the column with Amberlite XAD-2 resin. The col-
umn was washed with 250 mL of acidified water (pH 2 with
HCl) and subsequently rinsed with 300 mL of neutral dis-
tilled water to remove all sugars and other polar compounds
of honey or beebread. The phenolic compounds were eluted
from the sorbent with 250 mL of methanol. The methanol
extracts were concentrated under vacuum at 40 C in a rotary
evaporator (Super fit continental private limited, Mumbai).
The residue was dissolved in 5 mL of distilled water and
extracted three times with 5 mL of diethyl ether. The ether
extracts were combined, and the diethyl ether was removed
by flushing with nitrogen. The dried residue was then
re-dissolved with methanol for antioxidant activities and
-amylase inhibition test.
2.4 Antioxidant activities
2.4.1 Reducing power assay
The reducing power assay was carried out according to
the procedure described by Oyaizu[22]. A volume of 2.5 mL of
solution of honey extracts and standard butylated hydroxy-
toluene with different concentrations (1.5, 3, 4.5 and
6 mgmL1 of methanol) were mixed with 2.5 mL of
200 mmolL1 sodium phosphate buffer (pH 6.6) and 2.5 mL
of 1% potassium ferricyanide. After incubation of this mix-
ture at 50 C for 20 min, a volume of 2.5 mL of 10% tri-
chloroacetic acid (W/V) was added and the mixture was cen-
trifuged at 1 000 rmin1 in a refrigerated centrifuge at 4 C
(Remi Elektrotechnik Ltd, Vasai, India). The homogenized
mixture consists of 5 mL of the upper layer, 5 mL of deion-
ized water and 1 mL of 0.1% of ferric chloride. Then the
solution absorbance was measured spectrophotometrically at
700 nm. All the assays were carried out in triplicate.
2.4.2 Scavenging effect assay
The capacity to scavenge the 2, 2-diphenyl-1-picrylhy-
drazyl (DPPH) free radical was monitored according to a
method reported before[23]. Volumes of 0.3 mL of various
concentrations of honey extracts and standard butylated hy-
droxytoluene (1.5, 3, 4.5 and 6 mgmL1 of methanol) were
mixed with 2.7 mL of methanolic solution containing DPPH
radicals (6 105 molL1). Each mixture was shaken vigor-
ously and left to stand in the dark until a stable absorption
value was obtained at 517 nm. DPPH scavenging effect was
calculated as percentage of DPPH discoloration using the
equation: scavenging effect (%) = [(ADPPH-AS)/ADPPH]
100, where AS is the absorbance of the solution at each level
of the sample extract added and ADPPH is the absorbance of
the DPPH solution. All the assays were carried out in tripli-
cate.
2.5 -Amylase inhibition assay
The -amylase inhibition assay was carried out accord-
ing to the procedure reported by[13]. 500 L of various con-
centrations (4, 8, 12, 16 and 20 gmL1 of methanol) of ho-
ney extract and 500 L of 0.02 molL1 sodium phosphate
buffer (pH 6.9 with 0.006 molL1 NaCl) containing
-amylase solution (0.5 mgmL1) were incubated for 10 min
at 25 C. After pre-incubation, 500 L of 1% starch solution
in 0.02 molL1 sodium phosphate buffer (pH 6.9 with 0.006
molL1 NaCl) was added to each tube. The reaction mixtures
were then incubated at 25 C for 10 min. The reaction was
stopped with 1.0 mL of dinitrosalicylic acid color reagent.
The test tubes were then incubated in a boiling water bath for
5 min and cooled to room temperature. The reaction mixture
was then diluted after adding 10 mL of distilled water and
absorbance was measured at 540 nm; -amylase inhibition
assay was calculated using the formula (%) = [(A540 con-
 Subashini Devarajan, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 255259

trolA540 extract)/A540 control] 100 Phenolic compounds contribute significantly to the anti-
2.6 Statistical analysis oxidant activity of honey, but in spite of this, it seems that
All analyses were carried out in triplicate and the data antioxidant activity appears to be a result of the combined
were expressed as mean standard error (SE). The standard activity of honey flavonoids (chrysin, pinocembrin, pino-
errors were calculated using MS Excel software. banksin, quercetin, kaempferol, luteolin, galangin, apigenin,
hesperetin, and myricetin), phenolic acids (caffeic, coumaric,
3 Results
ferrulic, ellagic, and chlorogenic), ascorbic acid, catalase,
3.1 Antioxidant assays peroxidase, carotenoids and products of the Maillard reac-
Several publications dealing with antioxidant activity of tion[5-6]. The quantity of these components varies widely ac-
raw honey have been reported[24-25] and no such information cording to the floral and geographical origin of honey. Many
about antioxidant properties of the phenolic compounds in studies indicated that the antioxidant activity of honey varies
the extracts of Indian honey was available. So the present widely, depending on the floral source. It was reported that
study dealt with the antioxidant potential of phenolic com- the botanical origin of honey is not only the factor contribut-
pounds in honey extract and it was measured by two different ing to its antioxidant activity, but the phytochemical compo-
biochemical assays: reducing power and scavenging activity sition of the same botanical species depending on plant che-
on DPPH radicals. motype and various environmental factors has the greatest
3.2 Reducing power assay influence on its antioxidant activity[26]. Jose and Sara ana-
The reducing power of phenolic compounds of honey lyzed that the phytochemicals present in honey may augment
extract is shown in Fig. 1. The presence of antioxidants (i.e. defences against oxidative stress and might be able to protect
reducers) caused the reduction of ferricyanide complex to the humans, thus creating potentially protective antioxidant bar-
ferrous form, which can be measured by the formation of rier[27].
Perls Prussian blue at 700 nm. The reducing power of honey By comparing our present findings with those reported
extracts and standard BHT increases with increase in concen- by Leticia et al[28], we noted that the radical scavenging and
tration (Table 1). Different concentrations (1.5, 3, 4.5 and 6 reducing power activities of Indian honey were higher than
mg) of methanolic honey extract and standard BHT were the activities of Northeast Portugal honey, Hawaiian Christ-
examined (Fig. 1). At 6 mg of honey extract and BHT, the mas berry, Tupelo, Sweet clover, Soy bean and Acacia honeys.
reducing power was 0.895 and 0.99 respectively; at 4.5 mg, In our findings, at 6 mg concentration of honey extract, the
the reducing power was 0.535 and 0.65 respectively; at 3 mg, scavenging effect and reducing power was 86.8 % and 0.895
the reducing power was 0.3 and 0.465 respectively; at 1.5 mg, Abs whereas, in Northeast Portugal honey extract, the scav-
the reducing power was 0.195 and 0.34 respectively. All these enging effect and reducing power was 69.2% and 0.79 Abs at
50 mg concentration. Drastic differences in the extract con-
centration were observed. These results showed that honey
extract has both higher scavenging activity and reducing
power. Therefore, further studies of the antioxidant compo-
nents of Indian honey are required, especially identification
and quantification of individual flavonoids and phenolic ac-
ids.
3.3 Radical scavenging effect assay
The radical scavenging effects of honey extracts were
tested using methanolic solution of the stable DPPH free
radical which exhibits a deep purple color with maximum
Fig. 1 Reducing power of methanolic extracts from honey absorption at 517 nm. Antioxidant molecules can quench
DPPH radicals, resulting in discoloration of DPPH because
Table 1 Reducing power of methanolic extracts from honey
of their conversion into a colorless product (i.e. 2,
(mean SE, n = 3)
2-diphenyl-1-hydrazine or a substituted analogous hydrazine).
Reducing power activity, A700 nm The more rapidly the absorbance decreases, the more potent

No. c/(mgmL 1) the antioxidant activity of the honey extract is. Different
Butylated hydroxy- Honey ex-
toluene (BHT) tract concentrations (1.5, 3, 4.5 and 6 mg) of methanolic honey
1 1.5 0.34 0.02 0.19 0.01 extract and standard BHT were examined (Fig. 2). Results are
2 3 0.46 0.02 0.3 0.02
expressed as the percentage (Table 2) of sample absorbance
decrease relatively to the absorbance of DPPH solution in the
3 4.5 0.65 0.02 0.53 0.01
absence of extract at 517 nm. The scavenging effects of
4 6 0.99 0.01 0.89 0.03
 Subashini Devarajan, et al. /Chinese Journal of Natural Medicines 2012, 10(4): 255259

Table 3 -amylase inhibition of methanolic extracts from

honey (mean SE, n = 3)
 -amylase inhibition/%
No. c/(gmL 1)

1 4 30.5 0.92
2 8 64.8 1.85
3 12 70.3 3.70
4 16 87.0 3.70
5 20 88.8 1.85

Fig. 2 Scavenging effect (%) on DPPH radicals of methano- as the percentage of sample absorbance decrease relative to
lic extracts from honey the absorbance of control solution in the absence of extract at
540 nm. The percent inhibition varied from 87% to 30.5 %
Table 2 Scavenging effect (%) on DPPH radicals of metha-
from the highest (20 gmL1) to the lowest concentration (4
nolic extracts from honey (mean SE, n = 3)
gmL1).
DPPH Scavenging activity/% Weston et al revealed that during honey extraction proc-

No. c/(mgmL 1) Butylated hydroxy- Honey ess by column chromatography, sugar and polar compounds
toluene (BHT) extract
were washed with water[29]. In addition by using diethylether,
1 1.5 82.3 0.27 78.6 1.44
the non-flavonoid phenolic compounds were eliminated,
2 3 91.7 0.45 77.8 0.45 which contaminated the flavonoid peaks; thus the main di-
3 4.5 95.3 0.09 82.3 0.27 ethyl ether contents contain only flavonoids. Kanjiro et al
4 6 96.7 0 86.8 0.27 suggested that luteolin, myrcetin and quercetin were potent
inhibitors against porcine pancreatic amylase[30]. All these
honey extract on DPPH radicals increases with increase in flavonoids were present in honey extract, so flavonoids pre-
concentration. At 6 mg concentration of honey extract and sent in honey extract might act as potent inhibitors against
BHT, the scavenging effect was 86.8% and 96.7%, whereas porcine pancreatic amylase. Natural -amylase inhibitors
82.3% and 95.3% at 4.5 mg concentration, 77.8% and 91.7% from herbal sources offer an attractive therapeutic approach
at 3 mg concentration, 78.6 % and 82.3 % at 1.5 mg concen- to the treatment of postprandial hyperglycemia by decreasing
tration. All these assays are averages of the results obtained glucose release from starch and have potential for the treat-
from triplicate assays. ment of diabetes mellitus and obesity[31-32]. -amylase inhibi-
3.4 -amylase inhibition assay tors from honey are potentially safer, and therefore may be a
The inhibitory activity of phenolic honey extracts against preferred alternative for inhibition of carbohydrate break-
porcine pancreatic amylase is shown in Fig 3. The honey down and control of glycemic index of food products.
extract showed a significant -amylase inhibitory activity Therefore, the phenolic compounds namely the flavon-
under in vitro conditions. Concentration dependant inhibitory oids in honey extract, may render a good source of both an-
activity of -amylase was observed at 4, 8, 12, 16 and 20 tioxidant and -amylase inhibition activity besides their ef-
gmL1 of phenolic compounds of honey (Table 3). At 20 g fect as antibacterial thus increasing its potential therapeutic
of honey extract, the inhibitory activity was 88.8 %, whereas, activity.
16, 12, 8 and 4 g of honey extract, exhibited 87 %, 70.3 %,
Acknowledgement
64.8 %, and 30.5 % inhibitory activity. Results are expressed
The authors wish to thank the management of VIT Uni-
versity for providing necessary facilities. We would like to
thank, Prof. LI Zhi-Yong, Shanghai Jiao Tong University for
translating abstract into Chinese language.

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