Antioxidant activity of water extract of propolis from different regions in kafrelsheikh governorate



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ANTIOXIDANT ACTIVITY OF WATER EXTRACT OF PROPOLIS FROM DIFFERENT REGIONS IN KAFRELSHEIKH GOVERNORATE
aOSMAN, M. F. and bTAHA, E. A.

a Food Technology Dept., b Economic Entomol. Dept., Fac. Agric., Kafrelsheikh Univ., Egypt.
ABSTRACT

A water extract of propolis collected from three geographic regions (Motobes, Kafrelsheikh and Desouk) in Kafrelsheikh Governorate, Egypt was prepared. The extracts were analyzed for the determination of total polyphenol (from 5.70 to 8.79 g/100 g sample and from 22.80 to 34.30 g/100 g powder) and total flavonoid (from 3.05 to 4.85 g/100 g sample) contents. Water extract of propolis were evaluated for antioxidant activities using the β- carotene bleaching and 1,1-diphenyl- 2- picrylhydrazyl (DPPH) free radical- scavenging assay systems. It was observed that all propolis had strong antioxidant activities and also, related to the total polyphenol and flavonoid contents. The highest activities were found for samples from Desouk followed by from Kafr elsheikh, then from Motobes. Freeze-dried extracts of propolis can be used as natural antioxidants in sunflower oil compared to BHT and TBHQ. Propolis from Desouk and Kafrelsheikh at 200 and 300 ppm were similar in reducing peroxide values and both of them at 300 ppm were better than BHT and lower than TBHQ at 200 ppm concentration in reducing peroxides and hydroperoxides production in sunflower oil at 63 0C for 4 days.



Keywords: Propolis; antioxidants; water extracts; DPPH; free radical scavenging.

INTRODUCTION

Propolis (bee glue) is a resinous substance of complex mixture of several compounds collected by honeybees from trees and leaf buds. The sources of propolis were poplar (Populus spp.), birch (Betula alba), beech (Fagus stylvatica), horse chestnut (Aesculus hippocastanum), alder (Alnus glutinosa) and various conifers (Ghisalberti, 1979; Amoros et al., 1992; Bankova et al., 2000). Park et al. (2002) reported that the botanical origin of propolis was resinous coatings from young leaves of Hyptis divaricata (Lamiaceae) and Baccharis dracunculifolia (Asteracea). Also, Baccharis dracunculifolia, is an established source of propolis (Santos et al., 2003).

Propolis used mainly to cover the hive interior and the breeding cells and also to repair cracks and fissures. These uses are interest, because propolis avoids hive colonization with diseases (Walker and Crane, 1987).

Propolis contains a variety of chemical compounds such as polyphenols (flavonoid aglycons, phenolic acids and their esters, phenolic aldehydes, alcohols and ketones), sesquiterpene quinines, coumarins, steroids, amino acids, and inorganic compounds (Bankova et al., 2000). Propolis samples contain more than 160 constituents and differ greatly due to the variation in their geographical and botanical origins (Kujumgiev et al., 1999; Moreno et al., 2000 and Kumazawa et al., 2004). It has been used in folk medicine to maintain health. Pharmacological activities such as anticancer (Marcucci, 1995) anti-inflammatory (Wang et al., 1993), antibiotic (Koo et al., 2000), antioxidative (Moreno et al., 2000), antiviral, antifungal (Kujumgiev et al., 1999), anaesthetic and cytostatic effects (Ghisalberti, 1979) have been ascribed to ethanolic extract of propolis.

Most propolis components are of phenolic nature, mainly flavonoids. It is known that simple phenols, phenolic acids and polyphenols are active antimicrobial agents (Cowan, 1999). Flavonoids are synthesized by plants as a response to microbial infections and are recognized to have effective antimicrobial effects against a wide range of microorganisms (Recio et al., 1989).

Although ethanol extract of propolis is the most common, it is known that this extract poses immunological properties in animals and patients (Scheller et al., 1988). Studies concerning water extract of propolis are increasing (Basnet et al., 1996).



Nagai et al. (2003) reported that the quantity of phenolic compounds in fresh propolis from Brazil was about 168 µg/mg powder of lyophilized water extract. They suggested that water extract of propolis contains a mixture of natural substances, such as amino acids, phenolic acids, phenolic acid esters, flavonoids, cinnamic acid, and caffeic acid.

The purpose of the present study was to examine the antioxidative effects of propolis collected from three different regions in Kafrelsheikh Governorate.



MATERIALS AND METHODS

Materials

Fresh propolis was obtained from three apiaries in Kafrelsheikh Governorate, at divergent regions (Kafrelsheikh, Desouk and Motobes) at 2007 season. Survey of propolis sources in the previous regions was done. Sunflower oil (free of antioxidants) were purchased from Tanta Company for oils and soaps, Tanta, Egypt.



Chemicals

Linoleic acid, ascorbic acid and butylated hydroxy toluene (BHT) were purchased from Al Gomhoria Company for Chemical and Drugs in Cairo. 1,1-diphenyl- 2- picrylhydrazyl (DPPH), β- carotene, quercetin were purchased from Sigma. Tert- butyl hydroquinone (TBHQ) was obtained from Tanta Company for oils and soaps in Tanta City.



Preparation of water extract of propolis

Water extract of propolis was obtained as described by Suzuki, (1990) with slight modification by Nagai et al. (2003) as follows: 20.0 g of propolis were suspended and extracted with 5 volumes of distilled water with shaking at laboratory temperature for 24 hrs. The extracts were centrifuged at 3000 g for 20 min., and the supernatants were pooled. The residue was re-extracted under the same conditions. The extracts were centrifuged under the same conditions and the supernatants were pooled. Supernatants obtained were combined and dialyzed against distilled water, and then the dialysate was lyophilized. Each solution (10, 50, 100 mg/ml H2O) was used as the sample solution for the following tests.



Determination of total polyphenol and flavonoid contents

Total polyphenol contents in extracts were determined by the Folin- Ciocalteau Colorimeteric Method (Singleton et al., 1999). Extract solution (0.5 ml) was mixed with 0.5 ml of the Folin- Ciocalteau reagent and 0.5 ml of 100 mg/ml Na2Co3, and the absorbance was measured at 760 nm after 1 h of incubation at room temperature. Extract samples were evaluated at the final concentration of 20 μg/ml. Total polyphenol contents were expressed as mg/g (tannic acid equivalents).

Total flavonoid contents in extract were determined by the method of Woisky and Salatino (1998) with minor modifications by Nagai et al. (2003). To 0.5 ml of the extract solution, 0.05 ml of 20 mg/ml AlCl3 ethanol solution was added. After 1 h at room temperature, the absorbance was measured at 420 nm. Extract samples were evaluated at the final concentration of 20 μg/ml. Total flavonoid contents were calculated as quercetin (mg/g) from a calibration curve.

Antioxidant assay to determine DPPH scavenging activity

The scavenging effect of propolis samples as well of ascorbic and chlorogenic acid (as positive control samples) corresponded to the quenching intensity of 1,1- diphenyl -2– picrylhydrazyl (DPPH) as carried out by Yamaguchi et al. (1998) as follows: Dilutions of propolis extracts (10, 50 and 100 μg/ml) were added to 0.5 ml of 300 m mol/l DPPH in ethanol. The mixtures were shaken vigorously and left to stand at room temperature for 20 min in the dark. Absorbance at 514 nm was measured using ethanol as a blank. The degradation of DPPH was evaluated by comparison with control (0.5 ml of DPPH solution and 1.5 ml of ethanol). Results were expressed by the proportion of DPPH degradation compared with the control.



Antioxidant activity on linoleic acid oxidation

This experiment was carried out according to the method of Emmons et al. (1999) with some modification. ß- Carotene (3 mg) was dissolved in 30 ml of chloroform, and 3 ml were added to 40 mg of linoleic acid and 400 mg of Tween 40. Chloroform was removed under a stream of nitrogen gas. Then distilled water (100 ml) was added, and the solution was well mixed. Aliquots (3 ml) of the ß- carotene/linoleic acid emulsion were mixed with 50 µl of sample solution and incubated in a water bath at 50 0C. Oxidation of the emulsion was monitored spectrometrically by measuring absorbance at 470 nm over a 60 min period. The control sample contained 50 µl of solvent in place of the extract. The antioxidant activity was expressed as percent inhibition relative to the control after a 60 min incubation using the equation:

AA = (DRc – DRs)/ DRc x 100

Where: AA is the antioxidant activity, DRc is the degradation rate of the control (=1n (a/b)/60), DRs is the degradation in the presence of the sample (=1n (a/b)/60), a is the initial absorbance at time 0, and b is the absorbance at 60 min. Propolis extract samples were evaluated at a final concentration of 10 µg/ ml, and ascorbic acid and BHT at 1 µg/ml were used as a reference samples.



Assay of propolis freeze -dried extracts as antioxidants in sunflower oil

Propolis phenolic extracts (as powder) were assayed as natural antioxidants for sunflower oil (free of antioxidants). These extracts were added by concentrations 200 and 300 ppm (as phenolic compounds in powder) compared to 200 ppm for BHT and TBHQ as synthetic antioxidants. Triplicate portions of each solution (50g) were put in an open 100- ml beaker. The solutions were incubated in an oven, thermostatically controlled at 63 0C, for 4 days. Peroxide values in the stored oil samples were determined every 12 hrs (Rodriguez de Sotillo et al., 1994).



Peroxide value (PV)

Peroxide value (PV) was determined as described by Leonard et al. (1987) by dissolving 1 g of oil samples in a 30 ml glacial acetic acid- chloroform solution (60:40, v/v), adding of 1 ml potassium iodide (15%) and titrating the iodine librated with 0.1 n sodium thiosulphate solution. The peroxide value was expressed as milliequivalents of peroxide per 1000 g of sample.



Statistical analysis

The obtained data were statistically analyzed using General Linear Models Procedure Adapted by Statistical Package for the Social Sciences (SPSS, 1997).



RESULTS AND DISCUSSIONS

Total polyphenol and flavonoid contents

Table (1) shows the total polyphenol and flavonoid contents of propolis samples. The results indicated that propolis samples collected from Desouk had significantly the highest amounts of total polyphenols in crude samples and freeze-dried extract. It recorded 8.57 and 34.38 g/100g ; respectively. The samples collected from Kafrelsheikh were significantly, the second, and collected from Motobes was the third.

Table (1): Total polyphenol and total flavonoid (g/100 g) contents of propolis from three regions in Kafrelsheikh Governorate.


Sample region

Total polyphenols (g/100g)

Total flavonoids

Propolis sample

Lyophilized extract

Motobes

c5.70 ± 0.123

c22.80 ± 0.564

b3.05 ± 0.233

Kafr elsheikh

b7.32 ± 0.233

b29.30 ± 0.322

a4.11 ± 0.122

Desouk

a8.57 ± 0.242

a34.38 ± 0.227

a4.85 ± 0.253

Values are Means ± S. D. Means of treatments having the same case letter(s) within a column are not significantly different (p > 0.05).
No significant differences in total flavonoid contents were found between the samples collected from Desouk and Kafrelsheikh and both of them were the best followed by collected from Motobes.

Phenolic compounds are commonly found in both edible and non-edible plants, and they have been reported to have multiple biological effects, including antioxidant activity. Propolis contains a wide variety of phenolic compounds, mainly flavonoids. Variation in the flavonoid content of propolis is mainly attributable to the difference in the preferred regional plants collected by honeybees (Kahkonen et al., 1999).



DPPH radical scavenging activity

DPPH is a free radical compound and has been widely used to test the free radical scavenging ability of various samples (Hatano et al., 1997). It is accepted that the DPPH free radical scavenging by antioxidants is due to their hydrogen- donating ability (Tang et al., 2002). To evaluate the scavenging effect of DPPH on water extract of propolis, DPPH inhibition was investigated and these results are shown as relative activities against control.



As shown in Table (2) and Fig. (1), the activities of propolis samples and synthetic antioxidants as free radical scavenging increased as a function of concentration increment.
Table (2): The DPPH radical scavenging activities (%) of water propolis extracts collected from three regions in Kafrelsheikh Governorate.

Treatments
Region

Concentration µg powder/ ml water

10 µg

50 µg

100 µg

Absorbance

Activity

Absorbance

Activity

Absorbance

Activity

Control

0.173±0.0002

0.00

0.173±0.0002

0.00

0.173±0.0002

0.00

Ascorbic

0.117±0.0004

32.37 d

0.054±0.0003

68.79 b

0.033±0.0004

80.92 b

BHT

0.102±0.0004

41.04 a

0.045±0.0004

73.99 a

0.020±0.0002

88.44 a

Motobes

0.125±0.0003

27.75 e

0.085±0.0003

50.87 e

0.057±0.0003

67.05 e

Kafr elsheikh

0.115±0.0003

33.53 c

0.073±0.0002

57.80 d

0.041±0.0002

76.30 d

Desouk

0.107±0.0002

38.15 b

0.067±0.0002

61.27 c

0.038±0.0004

78.03 c

Values are means ± S. D.


Figure (1): The DPPH radical scavenging activities (%) of water propolis extracts

collected from three regions in Kafrelsheikh Governorate.


All propolis samples showed free radical scavenging activity but less than synthetic antioxidants. The samples collected from Desouk had the highest free radical scavenging at all used concentrations compared to others propolis samples. It may be related to its contents from total polyphenol and flavonoid contents.

Generally, the abilities of synthetic and natural extracts as free radical scavenging at all used concentrations in order: BHT > ascorbic acid > Desouk propolis > Kafrelsheikh propolis > Motobes propolis.



Nagai et al. (2003) reported that the activities of water extract of fresh propolis from Brazil (at concentrations 1, 5, 10, 50 and 100 mg/ml of extract) as DPPH radical scavenger were between those of 0.1 and 1.0 mM ascorbic acid. Hegazi and Abd El Hady, (2002) found that caffeic and vitamin C at concentrations 1, 10 and 100 µg showed the highest activity as free radical scavenger compared to the same concentrations of propolis samples collected from reclaimed land in Egypt. Ahn et al. (2007) observed that propolis samples collected in various area of China showed free radical scavenging activity and there were positive correlation between the activities and total polyphenol contents.

Effect of various propolis samples on linoleic acid oxidation

The antioxidant assay, using the discoloration of β- carotene is extremely susceptible to free radical- mediated oxidation. β- Carotene is discolorized easily by the oxidation of linoleic acid, due to its double bonds being sensitive to oxidation (Unten et al., 1997 and Singh et al., 2002).

Water extracts of propolis samples were evaluated at the final concentration of 10 µg powder/ml for the assay, and ascorbic acid and BHT were compared at 1 µg/ml under the same conditions. As shown in Table (3) and Fig. (2), antioxidant activities of synthetic antioxidants were higher than those of natural freeze dried extracts, and BHT was the highest. Freeze dried extract of propolis from Desouk had a strong antioxidant activity than those from others regions followed by collected from Kafr elsheikh, then collected from Motobes. It may be related to its high contents of both total polyphenol and flavonoid contents compared with others samples. Flavonoids have been reported to be the most abundant and most effective antioxidant in propolis from Argentine (Bonvehi and Coll, 1994 and Isla et al., 2001). Kumazawa et al. (2004) reported that the antioxidant activity correlated with total flavonoid contents of ethanolic extracts of propolis.

Table (3): Antioxidant activities (%) of water propolis extracts collected from different regions in Kafrelsheikh Governorate.



Sample region

cAntioxidant activities (%)

aAscorbic acid

b79.58 ± 0.132

aBHT

a85.13 ± 0.437

bMetobes

e65.49 ± 0.257

bKafr elsheikh

d71.68 ± 0.193

bDesouk

c77.13 ± 0.325

a= Synthetic antioxidants at 1 µg/ml solvent. b= Propolis extracts at 10 µg powder/ ml water.

c= Values are Means ± S. D. Means of treatments having the same case letter(s) within a column are not significantly different (p > 0.05).


Figure (2): Antioxidant activities (%) of water propolis extracts collected from different regions in Kafrelsheikh Governorate.

The DPPH free radical- scavenging activity seemed to relate with the antioxidant activity also, has DPPH free radical –scavenging activity. However, more detailed qualitative and quantitative analyses of the compounds with antioxidant activity are necessary to elucidate the antioxidant activity of propolis (Choi et al., 2006).

Assay of Freeze-dried extracts of propolis as antioxidant in sunflower oil

Freeze-dried extracts of propolis samples were applied on sunflower oil (free of antioxidants) as natural antioxidants. The used concentrations were 200 and 300 ppm (total polyphenols in freeze dried) compared to the recommended concentration of synthetic antioxidants (200 ppm).

The results in Table (4) shows peroxide values (PV) of untreated and treated sunflower oil with natural and synthetic antioxidants which incubated at 63 0C for 4 days. The results indicated that there were significant differences (at p > 0.05) between PV of untreated and treated samples. PV of samples treated with natural and synthetic antioxidants were lower than those of untreated samples with storage time for 4 days. Peroxide values of oil treated with TBHQ were significantly the lowest among all treated samples. So that TBHQ had strong antioxidant activity higher than those of others additives. No significant difference was found in PVs for oil treated with Kafrelsheikh propolis and Desouk propolis at 300 ppm concentration, and both of them were the second. BHT was significantly the third in reducing peroxides and hydroperoxides production. Also, Kafrelsheikh and Desouk propolis at 200 ppm concentration were similar in reducing peroxide values and both of them were the fourth. Propolis collected from Motobes at 300 ppm concentration was the fifth, then at 200 ppm concentration.

Generally, these findings demonstrate that the antioxidant activity was correlated with total polyphenol contents in propolis samples. Also, these activities increased depending on the concentration of the added total polyphenol content.



Conclusion

In this study, the in vitro antioxidant activity of propolis collected from three regions in Kafrelsheikh was investigated. Differences were observed in total polyphenol and flavonoid contents. Propolis samples had strong antioxidant activities, and the highest activities were found in Desouk propolis. Also, freeze-dried extracts of propolis can be used as natural antioxidants in edible oils and fatty foods against oxidative deterioration instead of synthetic antioxidants.



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النشاط المضاد للأكسدة في المستخلص المائي لصمغ النحل في أماكن مختلفة من محافظة كفر الشيخ

محمد فوزي عثمان1 ، القذافى عبده طه2

قسم تكنولوجيا الأغذية1، قسم الحشرات الاقتصادية2كلية الزراعة – جامعة كفرالشيخ – مصر

الملخص العربي

تم إعداد المستخلص المائي لصمغ النحل من أماكن مختلفة في محافظة كفرالشيخ وهى مطوبس – كفرالشيخ – دسوق0 تم تقدير الفينولات العديدة الكلية في المستخلص المائي منسوبة للعينة الأصلية وكذلك في المستخلص المائي بعد تجفيدة حيث تراوحت النسبة من 5.7 إلى 8.87 جم/100 جم عينة أصلية، ومن 22.80 إلى 34.30 جم/ 100 جم مستخلص مجفد0

أيضا" تم تقدير الفلافونويدات الكلية وتراوحت من 3.05 – 5.85 جم/ 100 جم عينة اصلية0 أيضا" تم اختبار النشاط المضاد للأكسدة للمستخلص المائي لهذه العينات باستخدام اختبار تبييض البيتا كاروتين وكذلك اختبار إيقاف نشاط الأصل الحر DPPH 0



حيث أظهرت النتائج أن كل المستخلصات المستخدمة لها نشاط مضاد للأكسدة قوى وكان ذلك مرتبط بمحتوى المستخلصات المائية من المركبات الفينولية والفلافونويدات0 وكان النشاط المضاد للأكسدة للعينات المأخوذة من دسوق الأعلى يليها المأخوذة من كفرالشيخ ثم المأخوذة من مطوبس0 كما تم استخدام المستخلص المائي المجفد للعينات المختلفة بتركيزات 200 ، 300 جزء/ مليون (الفينولات الموجودة بها) كمضاد أكسدة طبيعي لزيت عباد الشمس مقارنة بـ BHT ، TBHQ بتركيزات 200 جزء/ مليون كمضادات أكسدة صناعية حيث أظهرت النتائج أن العينات المأخوذة من دسوق وكفرالشيخ بتركيزات 300 جزء/ مليون لها تأثير مشابه (لا يوجد فروق معنوية بينهما) في خفض رقم البيروكسيد لزيت عباد الشمس وكانتا أفضل من BHT ولكنهما أقل من TBHQ0



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