Physicochemical and radical scavenging activities of honey samples from Malaysia

doi:10.4236/as.2013.45B009

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Vol.4, No.5B, 46-51 (2013) Agricultural Sciences

doi:10.4236/as.2013.45B009

Physicochemical and radical scavenging activities of

honey samples from Malaysia

Norul Liza A-Rahaman, Lee Suan Chua*, Mohamad Roji Sarmidi, Ramlan Aziz

Metabolites Profiling Laboratory, Institute of Bioproduct Development, Universiti Teknologi Malaysia, Johor Bahru, Johor, Malaysia;

*Corresponding Author: [email protected]

Received 2013

ABSTRACT

The physical properties, total phenols, total fla-

vonoid content and free radical scavenging ac-

tivities of honey samples from Malaysia were

investigated. The physical properties of Tualang,

Gelam and Acacia honey samples, in terms of pH,

color, moisture, electrical conductivity and total

soluble solid were significantly different (p =

0.000). Gelam honey was reported to have the

highest total phenols (606.17 mg GAE/kg honey)

and flavonoid content (183.43 mg RE/kg honey).

Tualang honey was reported to have the highest

free radical scavenging activity with the IC50,

72.75 g/L compared to Gelam (77.41 g/L) and

Acacia (90.83 g/L). There is no significant dif-

ference has been revealed among honey samples

for radical scavenging activity (p = 0.827). Nev-

ertheless, strong correlation was obtained be-

tween pH, color, electrical conductivity and total

soluble solid with the scavenging activity of all

honey samples with the correlative coefficient, r

= 0.979, 0.902, 0.917 and 0.957, respectively. The

establishment of the statistical correlation could

be useful for honey related industry.

Keywords: Total Phenolic Content; Total Flavonoid

Content; Radical Scavenging Activity

1. INTRODUCTION

Honey is a natural food source that widely used as a

traditional medicine. To the best of our knowledge, more

than 200 substances including complex sugar and small

amount of other substances have been found in honey.

The common types of honey found in lowland rain forest

of Peninsular Malaysia are Tualang and Gelam honey,

originates from Tualang (Koompassia excels) and Gelam

(Melaleuca cajuputi) trees. Another common source of

honey in Malaysia is Acacia honey and this honey is

easily available all over the world with different physical

and chemical characteristics.

Honey is widely used as antioxidant to reduce the risk

of heart disorder, cancer and to increase the immune

system [1]. The antioxidant activity of honey is believed

mainly contributed by the presence of phenolic acids,

flavonoids, catalases, peroxides, carotenoids and non-

peroxidal components. According to Gheldof and Enge-

seth [2] and Turkmen et al. [3], the composition of honey

is highly dependent on the botanical and geographical

origin, as well as the handling process during harvesting

and storage. There are also studies reported that the bo-

tanical origin is the critical factor influencing the anti-

oxidant activity of honey [4,5].

There are several methods commonly used for the

measurement of antioxidant activity of honey based on

different principles and experimental condition. Accord-

ing to Bertoncelj et al. [6], the methods include FRAP

assay (ferric reducing antioxidant power), TEAC (Trolox

equivalent antioxidant activity), DPPH (1, 1-diphenyl-

2-picrylhydrazyl) and ORAC (oxygen radical absorbance

capacity). DPPH method is likely to be the most reliable

technique to determine the antioxidant activity of honey,

since DPPH produces stable free radicals for testing [7].

Since honey varies according to their origin, the physical

properties of honey samples including pH, color, mois-

ture, electrical conductivity and total soluble solid were

studied and correlated statistically with their total phe-

nols, total flavanoids and antioxidant activity. The estab-

lishment of the physicochemical correlation enhances the

understanding of honey characteristics from tropical

country which is beneficial to honey related industry.

The findings from this study could be compared to the

data reported previously which found the physical prop-

erties were highly correlated with the antioxidant activity

of honey [6,8,9]. Therefore, this study was investigated

the physical properties, phenolic and flavonoid contents,

as well as the antioxidant activity of honey samples from

Malaysia.

2. MATERIALS AND METHODS

2.1. Honey Samples

All honey samples were collected from the West Coast

N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51 47

of Peninsular Malaysia, which consist of Tualang, Gelam

and Acacia honey. Tualang and Gelam honey was ob-

tained from the Federal Agricultural Marketing Author-

ity (FAMA), Kedah, whereas MB An-Nur Apiary, Johor,

supplied the Acacia honey. Honey samples were stored

in glass containers at room temperature (25℃ - 30℃) in

the dark place without any processing or treatment prior

to analysis. The analyses were carried out within six

months after harvesting.

2.2. Reagents and Standard

All reagents are of analytical grade otherwise stated.

HPLC-grade of methanol from Merck (Darmstadt, Ger-

many). Hydrochloric acid, sodium hydroxide and alu-

minium trichloride were purchased from Fisher Scien-

tific (Pittsburg, USA). Deionized water was produced by

Barnstead NANOpure Diamond water purification sys-

tem at 18.2 MΩ-cm resistivity (State of Illinois, USA).

The standard reagents such as Folin-Ciocalture reagent,

2-2-diphenyl-1-picryhydrazyl and gallic acid were pur-

chased from Sigma-Aldrich (Missouri, USA). Ascobic

acid (98 %) and rutin (97 %) were obtained from Across

Organics (Pittsburg, USA).

2.3. Physical Analysis

Determination of pH: The pH value of honey was

determined based on the method described by Saxena et

al. [9] using a pH meter (Mettler Toledo Delta 320) for the

measurement. A 10% w/v of honey solution was prepared

in aqueous medium.

Determination of color: The color of honey was meas-

ured by using Lovibond Honey Photometer based on the

AOAC Official Method 985.25 (2000) with minor modi-

fication. Honey samples were placed in a 10 mm path-

length plastic cuvette. The color values was determined

in triplicate and expressed in millimeter Pfund scale.

Determination of moisture content: The moisture con-

tent was measured based on the AOAC Method 969.38

(2000). A handheld Refractometer (Atago, Japan) was

used to determine the water content in honey based on

the refractive index. The readings were corrected for a

standard temperature of 20℃ by adding the correction

factor of 0.00023/℃. The moisture content was calcu-

lated by using Wedmore’s table referring to the refractive

index. All measurements were carried out in triplicate

and the moisture content is expressed in percentage (%).

Determination of total soluble solid: The total soluble

solid was measured by using a refractometer (Atago

Pocket Refractometer) at ambient temperature. The re-

sults were expressed in % Brix.

Determination of electrical conductivity: The electrical

conductivity was determined based on the electrical re-

sistance by using a conductivity meter (EcoScan COND

6+ Conductivity Meter from Eutech Instrument) [9]. All

honey samples were analyzed in triplicate and the results

are expressed in mS/cm.

2.4. Analysis of Chemical and Scavenging

Activity

Total phenolic content in honey samples was determined

by using the Folin-Ciocalteu method described by Sin-

gleton et al. [10] with minor modification. A 0.5 mL of

honey solution (0.1 g/mL) was mixed with 2.5 mL of 0.2

N Folin-Ciocalteu reagent and incubated for five minutes.

Then, 2 mL of sodium carbonate (75 g/L) was added and

incubated for two hours. After incubation, the absorbance

was measured against methanol as blank at 760 nm using

a UV-Vis spectrophotometer (Perkin Elmer, Lamda 25).

The calibration curve was constructed using gallic acid

(0 - 100 mg/L). The measurement was performed in trip-

licate and the total phenolic content was expressed in mg

of gallic acid equivalents (GAE) per kg of honey.

Total flavonoid content was determined by using the

method described by Singleton et al. [10] with minor

modification. A 5 mL of honey solution (0.1 g/mL) was

mixed with 5 mL of 2% aluminium trichloride (AlCl3).

The complex formation (flavonoid-aluminium) was meas-

ured at 415 nm by using a UV-Vis spectrophotometer

(Perkin Elmer, Lambda 25) after ten minutes incubation.

The standard curve for total flavonoids content was plot-

ted using rutin (0 - 100 mg/L) as a standard chemical.

The measurement was performed in triplicate and the

total flavonoids content was expressed in mg rutin equi-

valents (RE) per kg of honey.

The radical scavenging activity of honey was meas-

ured according to the 2, 2-diphenyl-1-picryhydrazyl (DPPH)

method as described by Velazquez et al. [11] with minor

modification. This method measures the reduction of

DPPH purple color to yellow color. A 0.75 mL of honey

solution (0.02 - 0.04 g/mL methanol) was mixed with 1.5

mL of 0.02 mg/mL DPPH solution. The complex mixture

against DPPH was measured by using a UV-Vis spectro-

photometer (Perkin Elmer, Lambda 25) at 517 nm after

15 minutes incubation at 25℃. The sample blank con-

sisted of 0.75 mL methanol and 1.5 mL of methanolic

DPPH. The ascobic acid standard curve (0 - 10 mg/L)

was plotted for calibration. The free radical scavenging

activity was expressed as IC50.

2.5. Statistical Analysis

The data were analyzed by the analysis of variance

(ANOVA) using SPSS software (SPSS 16.0). Correla-

tions were carried out by using the Pearson’s correlation

coefficient (r) in bivariate linear correlations.

N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51

3. RESULTS AND DISCUSSION

3.1. Physical Properties of Honey

From Table 1, the pH values of honey samples; Tualang,

Gelam and Acacia, were ranging from 3.14 to 3.52, where

Tualang honey showed the most acidic value in pH. The

pH values of all honey samples were significantly dif-

ferent (p = 0.000) due to the difference in botanical and

geographical origin of honey. The parameter of pH is

crucial as it is strongly related to the stability, life

expectancy of honey product and fermentation process

due to storage [8,12]. The pH values for Malaysian honey

reported by Khalil et al. [13]] was slightly higher, which

ranged from 3.44 to 3.89 compared to the pH values

reported by Alqarni et al. [14]. However, honey samples

from Serbian, Brazil and Uruguayan showed the com-

parable findings with this study, the pH were ranging from

3.1 - 4.14, 2.9 - 3.7 and 3.00 - 4.30, respectively[8,15,16].

Gelam honey was reported to have darker color

compared to Acacia and Tualang honey with Pfund scale,

139.00, 97.00 and 74.00 mm, respectively. All honey

samples were statistically different (p = 0.000) in color

depending on the type of floral fed by bees. Based on the

National Honey Board [1], the color of Tualang, Gelam

and Acacia honey was categorized as light amber to dark

amber. The color of honey might be contributed by

pigments such as chlorophylls, carotenoids, flavonoids

and derivatives of tannis and polyphenols [17]. The total

soluble solid are primarily consisted of sugars; fructose,

glucose, and sucrose. Moreover, organic compounds

such as acids and minerals also contributed to the total

soluble solid in honey. Acacia honey showed the highest

soluble solid (74.80%), followed by Gelam (74.07%) and

Tualang (72.93%). The difference of soluble solid might

due to the difference in chemical composition of honey.

Acacia honey was reported to have the lowest mois-

ture content compared to Tualang and Gelam honey;

19.53, 24.07, 25.20%, respectively. Tualang and Gelam

honey contained high moisture content, which exceeding

20%, while the moisture content of Acacia honey was

within the permitted ranged [18]. The moisture content

of honey is a crucial parameter to determine the quality

and shelf-life of honey product. The factor of storage

combined with the high moisture content in honey could

increase the fermentation rate, and leading to the forma-

tion of acidified honey because of acetic acid formation.

Somehow, the moisture content of honey is highly de-

pending on the climatic change, maturity of honey and

harvesting season [19]. Based on Kayacier and Karaman

[20], the moisture content might indicate the origin of

honey. The tropical country of Malaysia with rainy sea-

son all over the year leads to high moisture content in

honey. Therefore, Malaysian honey is always treated by

evaporation to reduce the water content, simultaneously

increase the quality of honey.

The electrical conductivity was the highest in Acacia

honey, followed by Gelam and Tualang honey. All honey

samples were significantly different in electrical conductive-

ity (p = 0.045). The electrical conductivity measures the

ability of honey solution to conduct an electric current.

This electric current was carried by ions and the chemi-

cal changes in the solution. The factors that contributed

to the high value of electrical conductivity might be storage,

time, temperature, water content and concentration of

ions and minerals [14,21,22]. The electrical conductivity

affects the pH of honey, which indicated in the acidity

value, depending on the dissolved chemical compound

and biochemical process [23].

3.2. Total Phenols, flavonoids and

Antioxidant Properties

The amount of total phenolic contents was predicted

by using the Folin-Ciocalteu reagent from methanolic

honey solution. The total phenolic contents in Malaysian

honey were expressed in milligram gallic acid equivalent

to kilogram of honey. The result obtained (Table 1) showed

that the total phenolic contents for honey samples were

ranged from 383.79 to 606.17 mg GAE/kg. Floral honey,

Gelam was reported having the highest phenolic contents,

followed by Acacia (honeydew honey) and Tualang (floral

honey) with 606.17, 550.00 and 383.79 mg GAE/kg,

Table 1.

Honey (Mean ± SD)

Parameters (Unit) Tualang Gelam Acacia

pH 3.14 ± 0.06c 3.52 ± 0.07a 3.33 ± 0.08b

Color (mm Pfund) 74.00 ± 0.00c 139.00 ± 0.00a 97.00 ± 0.00b

Moisture (%) 25.20 ± 0.17a 24.07 ± 0.12b 19.53 ± 0.12c

Electrical Conductivity, (mS/cm) 0.91 ± 0.0012c 1.02 ± 0.0089b 1.09 ± 0.0030a

Total soluble solid, (%) 72.93 ± 0.06c 74.07 ± 0.15b 74.80 ± 0.10a

Total phenolics content, (mggallic acid/kg) 383.79 ± 13.57a 606.17 ± 20.36a 550.00 ± 21.93a

Total flavonoids content, (mgrutin/kg) 49.04 ± 0.53c 183.43 ± 1.70a,b 115.96 ± 4.69a,b

DPPH Radical Scavenging Activity (IC50, g/L) 72.75 ± 4.52a 77.41 ± 62.29a 90.83 ± 12.31a

a,b,cSuperscript in the same row was compared based on LSD’s test (p < 0.05). Same superscript means no significant difference.

N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51 49

respectively. However, there is no significant difference

(p = 0.471) between floral and honeydew honey for total

phenolic contents. The similar finding was reported by

Beretta et al. [24] and Bertoncelj et al. [6] using floral

and honeydew honey for their studies.

The total flavonoid contents of all honey samples were

expressed in mg rutin equivalent/kg by using rutin stan-

dard curve (r2 = 0.995). Once again, Gelam honey was

reported to have the highest flavonoid contents (183.43

mg RE/kg), followed by Acacia (115.96 mg RE/kg) and

Tualang (49.04 mg RE/kg). There is no significant dif-

ference between Gelam and Acacia honey (p = 0.650) for

total flavonoid content. The amount of flavonoid con-

tents was lower than the phenolic contents in all honey

samples. The values of phenolic and flavonoid contents

in honey samples were in close agreement with the re-

sults reported by Meda et al. [25] and Yao et al. [26]

The free radical scavenging activity was determined

based on the DPPH method using the stable radical 2,2-

diphenyl-1-picryhydrazyl. The radicals are scavenged by

delocalization of the spare electron over the molecule.

This delocalization would give the purple color in metha-

nol solution and turned to pale yellow after reacting with

the substance by donating hydrogen atom. The free radi-

cal scavenging activity typically expressed as IC50, to

represent the quantity of antioxidant required to reduce

the initial concentration of DPPH by 50 %. By using IC50,

the antioxidant activity increase with the de- creasing of

IC50 value [27]. From the Ta b l e 1 , the IC50 values were

ranged from 72.75 to 90.83 g/L, where Acacia honey

reported to have the highest value (90.83 g/L), followed

by Gelam (77.41 g/L) and Tualang (72.75 g/L). Acacia

honey was the least reactive honey to trap the free radical,

compared to Gelam and Tualang honey. However, there

is no statistically different between all honey samples (p

= 0.827) for the radical scavenging activity. Somehow, it

is important to note that, the presence of peptide, organic

acids, enzymes and the product of Maillard reaction

would contribute to the antioxidant activity in honey [6].

3.3. Correlation between Physical,

Chemical and Antioxidant

Properties

The physical properties in honey closely related to the

presence of antioxidant properties. Indeed, the physical

properties of honey depend on the geographical and

botanical origin of the nectar. This present study reported

that the antioxidant properties have strong correlation

with pH, color, electrical conductivity and total soluble solid.

Nevertheless, the moisture and free radical scavenging

activity of honey samples showed a negative correlation.

From Ta bl e 2, the total phenolic contents showed the

strong correlation between pH, color and total soluble

solid with correlation coefficient, r = 0.979, 0.902 and

0.957, correspondingly. Besides, the total flavonoid con-

tent also exhibited similar correlation with the pH (r =

0.997), color (r = 0.986) and total soluble solid (r =

0.839). The presence of phenolic acids and flavonoids in

honey samples might act as a proton donator leading to the

for- mation of acidified honey with low pH value. The pig-

ments of polyphenolics and flavonoids that exist in honey

might contribute to the variable color of honey [17]. The

dark honey contributed by the higher content of phenolic

compounds [28]. These organic compounds and acids

also explained to the strong correlation with the total

soluble solid. The free radical scavenging activity exhib-

ited strong correlation with electrical conductivity and

total soluble solid with correlation coefficient, r = 0.917

and 0.921, respectively. This electric current was carried

by ions and controlled by dissolved chemical compound

and biochemical process [23]. Strong negative correla-

tion was reported between free radical scavenging active-

ity and moisture content (r = -0.998). The high moisture

content could lead to the fermentation process that could

decrease the antioxidant capacity.

Table 2.

pH Color Moisture EC TSS TPC TFC DPPH-IC50

pH 1

Color 0.971 1

Moisture -0.188 -0.016

EC 0.710 0.474 -0.881

TSS 0.878 0.738 -0.675 0.944 1

TPC 0.979 0.902 -0.432 0.807 0.957 1

TFC 0.997 0.986 -0.165 0.612 0.839 0.961 1

DPPH-IC50 0.246 0.083 -0.998 0.917 0.921 0.624 0.246 1

Correlations were carried out by using Pearson’s correlation coefficient (r); EC: Electrical conductivity, TSS: Total soluble solid, TPC: Total phenolic content,

TFC: Total flavonoid content.

N. L. A-Raham an et al. / Agricultura l Sciences 4 (2013) 46-51

4. CONCLUSIONS

The physical properties of honey are closely related

with the chemical composition of honey. The composi-

tion is dependent on the botanical and geographical ori-

gins, in addition to the shelf life of honey. Gelam was

reported to have high phenolic and flavonoid contents.

The high scavenging activity was also reported in Tu-

alang honey. The physical properties could be used to

predict the antioxidant activity in honey based on the

strong correlation for pH, color, electrical conductivity

and total soluble solid. The establishment of the correla-

tion could be the easiest way to determine the quality of

honey.

5. ACKNOWLEDGEMENTS

The authors would like to thank Ms. Nur Ardawati Adnan and Ms.

Norfahana Abd-talib for their support and guidance. This research work

was granted by GUP (Tier 1) Fund under the Research Alliance of

Biotechnology (7125.00H05), UTM.

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