Comparison of antioxidant activity of ethanolic extracts ofpropolis obtained by different extraction methods.

The Particular antioxidant actions in the ethanolic extracts regarding propolis obtained simply bydifferent extraction techniques (high hydrostatic pressure

extraction, leaching with room temperature and heat reflux extraction)

were investigated in relationship for their total polyphenol and

flavonoid contents by a pair of various assays, namely, the particular [beta]-carotene

bleaching and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free of charge radical

scavenging assay systems. Your outcomes showed that your ethanolic extracts

of propolis obtained by substantial hydrostatic pressure extraction and

leaching with space temperature had relatively strong antioxidant

activities, which might become correlated with almost all the total polyphenol and

flavonoid contents. Antioxidant activities of ethanolic extracts of

propolis obtained simply by higher hydrostatic strain extraction were the actual same

as those regarding ethanolic extracts regarding propolis obtained simply by leaching in room

temperature. Leaching with room temperature generally wants a few days, and

can take much more than 7 d, whilst higher hydrostatic pressure extraction

needs only one min. These kind of findings further illustrate that the high

hydrostatic pressure extraction has a bright prospect regarding extracting

flavonoids from propolis.

Les activites antioxydantes des extraits d'ethanol obtenus par

differentes methodes d'extraction (extraction par pression

hydrostatique elevee, lessivage the temperature ambiante et extraction par

reflux de chaleur) ont ete etudiees en relation avec leur teneur totale

en polyphenol et flavonoide par deux essais differents, a new savoir les

methodes d'essai par blanchiment du [beta]-carotene et par

entrainement des radicaux libres par le diphenyl-1, one picrylhydrazyl-2

(DPPH). Les resultats montrent que les extraits d'ethanol de

propolis obtenus au moyen de l'extraction par pression

hydrostatique elevee et du lessivage a new temperature ambiante ont des

activites antioxydantes relativement fortes, ce qui peut etre correle

aux teneurs totales en polyphenol et flavonoide. Les activites

antioxydantes des extraits d'ethanol de propolis obtenues au moyen

de l'extraction par pression hydrostatique elevee sont les memes

que celles des extraits d'ethanol de propolis obtenus par le

lessivage any temperature ambiante. Le lessivage a new temperature ambiante

necessite generalement quelques jours, et peut meme prendre in addition de 7

jours, tandis que l'extraction par pression hydrostatique elevee ne

necessite que one min. Ces decouvertes illustrent davantage que

l'extraction par pression hydrostatique any un bel avenir pour

l'extraction des flavonoides a partir du propolis.

Keywords: propolis, extraction methods, antioxidant activity

INTRODUCTION

Propolis, an just about all natural substance collected simply by honeybees through buds and

exudates involving particular trees as well as plants, continues to be utilized in folk medicines in

many parts of the entire world along with may be reported to have various

biological actions for example antioxidant ability, antibacterial,

antiviral, anti-inflammatory and anticancer properties (Kimoto et al.,

2001). for this reason, propolis will be extensively found in food and

beverages to improve health insurance stop diseases for example inflammation,

heart disease, diabetes as well as cancer (Banskota et al., 2001).

Pharmacological activities, like anticancer, anti-inflammatory,

antibiotic, antioxidative, antiviral, antifungal, anaesthetic and

cytostatic, have been ascribed for you to ethanolic extracts associated with propolis (EEP)

(Nieva et al., 2000). Analysis of EEP confirmed the actual existence of

antibacterial and also totally free radical-scavenging activities (Nieva et al.,

2000). EEP usually contains a assortment of chemical compounds, such as

polyphenols (flavonoids, phenolic acids in inclusion in order to their esters), terpenoids,

and thus on. the antioxidant exercise involving EEP may end up being associated for you topolyphenol

and flavonoid contents since it has been reported in which these phenolic

compounds can break the particular chain response of lipid, inhibit

chemiluminescence reactions, scavenge several ROS (Krol et al., 1996),

etc.

There are usually papers in literature in which offer using the methods of

extraction associated with flavonoids from propolis, such as leaching with room

temperature (LRT) (Murad et al., 2002), and also heat reflux extraction (HRE)

(Gu et al., 2001). LRT may always be the nearly all presently applied technique, however it

usually requirements a few days, as well as will take much more when compared with 7 days. HRE

generally wants an increased temperature (85[degrees]C) in the particular program of the

extracting, which usually can easily lead some heat-sensitive ingredients involving propolis

to shed their particular biological activity. high hydrostatic strain extraction

(HHPE) can always be a novel technique with present, which in turn has been successfully employedin

extracting flavonoids from propolis (Jun, 2005). Experiment outcomes have

shown HHPE has many advantages, like shorter occasion (only one min),

higher extraction yield therefore in (Jun, 2005). Propolis obviously

possesses antioxidant activity, as reported simply by many researchers.

However, you will find couple of studies around the comparison of the antioxidant

activity of EEP obtained through HHPE, LRT as well as HRE. Therefore, the actual present

study is actually directed at the investigation in the comparison in the antioxidant

activity involving EEP obtained simply by different extraction techniques within relationship

to his or her total polyphenol and also flavonoid contents.

MATERIALS AND METHODS

Materials and Instrumentation

Crude propolis that had been collected in Nongan County associated with Jilin

Province (China), which was mainly from the Japan pagoda tree bud or

unexpanded leaves visited by the bees, has been provided from the Jilin

Provincial Institute with regard to Drug Control. Rutin and gallic acid,

pharmaceutical grade standard, was purchased from the National Institute

for Manage regarding Pharmaceutical along with Biological Items (China). Tween 80,

ethanol, chloroform, aluminum chloride and also potassium acetate (Beijing

Chemical Reagents Company; analytical grade) had been used. Tert-butylated

hydroxyquinone (TBHQ), [beta]-carotene, linoleic acid as well as 1,

1-diphenyl-2-picrylhydrazyl (DPPH) were bought in Sigma Aldrich Co.

(St. Louis, U.S.A.). Your spectrophotometer (751-GW) had been through Shanghai

Analytical Instrument overall Factory.

Ultrahigh stress isostatic apparatus (DL700-0.55 x 1.5) was

purchased from Shanghai Dalong Ultrahigh Stress Device Co., Ltd.

(China) (Effective volume associated with vessel: 0.35 L, maximal operating pressure:

700 MPa, inner diameter: 55 mm, pressure transmitting media: mixture of

transformer oil and also kerosene).

Preparation regarding EEP through HHPE

Crude propolis has been frozen from -20[degrees]C and ground in a chilled

disintegrator. Then, ten g associated with crude propolis has been blended with 350 mL involving75%

ethanol as well as positioned into a sterile polyethylene bag. the bag had been sealed

after air inside has been eliminated. The Particular bag had been placed into a

hydrostatic strain vessel inside an ultrahigh stress isostatic

apparatus. after becoming processed (high strain levels: 500 MPa) regarding 1

min at space temperature, the mixture has been filtered through filter paper.

The extracts were centrifuged from 4000 x g with regard to ten min, and the

supernatants were pooled. the residue ended up being re-extracted beneath your same

conditions. The Particular extracts had been centrifuged under the identical circumstances and

the supernatants were pooled. Supernatants obtained were mixed and

concentrated in a rotary evaporator beneath reduced pressure at

40[degrees]C, and then the actual supernatant was lyophilized. Thus, the particular EEP by

HHPE had been ready (Jun, 2005). A New solution (10 [micro]g/mL ethanol) was

used since the sample answer for that next tests.

Preparation regarding EEP simply by LRT

The EEP by simply LRT has been obtained as described through Murad et al. (2002). In

brief, ten g of propolis were suspended along with extracted with 30 mL associated with 70%

ethanol as well as shaken with room temperature to get a week. The Actual mixture was

filtered via filter paper; extracts had been centrifuged in 4000 x g for

10 min, and additionally the supernatants were pooled. The Actual residue had been re-extracted

under exactly the particular same conditions. The Particular extracts were centrifuged beneath theparticular same

conditions and the http://en.wikipedia.org/wiki/Honey_bee supernatants were pooled. Supernatantsobtained were

combined as well as concentrated in the rotary evaporator below reduced pressure

at 40[degrees]C, after which the supernatant was lyophilized. Thus, the particular EEP

by LRT were prepared. Any solution (10 [micro]g/mL ethanol) was utilized as

the taste remedy for the following tests.

Preparation regarding EEP by HRE

The EEP simply by HRE had been obtained as described simply by Gu et al. (2001). In

brief, propolis ethanol extracts had been boiled (10 g regarding propolis, mixed

with 40 mL of 95% ethanol within water) from boiling point, about

85[degrees]C, with regard to 4 h (super boiling of the solution didn't occur). The

mixture had been filtered via filter paper, the extracts had been centrifuged

at 4000 x g for 10 min, as well as the supernatants were pooled. The Particular residue

was re-extracted under the same conditions. Your extracts were

centrifuged below exactly the actual same circumstances as well as the supernatants were pooled.

Supernatants obtained were mixed and concentrated inside a rotary

evaporator beneath reduced stress from 40[degrees]C, then the

supernatant has been lyophilized. Thus, your EEP by HRE were prepared. A

solution (10 [micro]g/mL ethanol) was utilized since the test remedy for

the next tests.

Total Polyphenol and Flavonoid Contents

Phenolic compounds are generally found in plants, plus they have got been

reported to have multiple biological effects, including antioxidant

activity (Kahkonen et al., 1999). Propolis contains a wide selection of

phenolic compounds, mainly flavonoids. Contents regarding flavonoid and other

phenolic substance have been suggested to try out any preventive role in the

development associated with cancer and heart problems (Kahkonen et al., 1999). The

Folin-Ciocalteau approach and the Al[Cl.sub.3] colouration are currently

used to appear with regard to the total polyphenol and also flavonoid contents,

respectively (Liu et al., 2002; Luximon et al., 2002). Inside your present

study, we applied these solutions to decide the particular total polyphenol and

flavonoid contents associated with EEP samples.

Total polyphenol content inside EEP had been decided through the

Folin-Ciocalteau colourimetric technique (Kumazawa et al., 2002). EEP

solution (0.5 mL) had been mixed with 0.5 mL in the Folin-Ciocalteau reagent

and 0.5 mL regarding 10% [Na.sub.2]C[O.sub.3], and the absorbance ended up being measured

at 760 nm after 1 h incubation with room temperature. EEP biological materials were

evaluated with the final concentration regarding ten [micro]g/ mL. Total

polyphenol content material had been expressed as mg/g (gallic acid equivalents).

Total flavonoids content in EEP had been determined by the approach of

Woisky as well as Salatino (1998). 0.5 mL of EEP solution, 0.5 mL regarding 2%

Al[Cl.sub.3] ethanol remedy ended up being added. Right After 40 min from room

temperature, the actual absorbance ended up being measured in 415 nm. EEP samples were

evaluated in the final concentration involving 10 [micro]g/mL. Total flavonoids

content had been calculated as rutin from the calibration curve.

[beta]-Carotene Bleaching Method

This experiment was carried out through the technique involving Emmons et al.

(1999). [beta]-Carotene (3 mg) ended up being dissolved throughout 30 mL of chloroform, and

3 mL were added to 40 mg associated with linoleic acid and 400 mg of Tween 80.

Chloroform had been removed below a stream involving nitrogen gas. Distilled water

(100 mL) had been added along with mixed well. Aliquots (3 mL) regarding the

[beta]-carotene/linoleic acid emulsion had been blended with 50 [micro]l of

EEP answer and also incubated inside a h2o bath with 50 [micro]l. Oxidation of

the emulsion has been monitored spectrometrically by measuring absorbance at

470 nm over a new 60 min period. The Actual manage sample contained 50 [micro]l of

solvent in place in the extract. the antioxidant activity will be expressed

as percent inhibition relative for the manage after a 60 min incubation

using the actual subsequent equation:

AA=100 (D[R.sub.C]-D[R.sub.S])/D[R.sub.C] (1)

where AA may be the antioxidant activity, DRC is the degradation rate

of your control (=ln(a/b)/60), DRS will end up being the degradation price throughout the

presence with the trial (=ln(a/b)/60), any will be the first absorbance at

time 0, and also b is the absorbance at 60 min. EEP trials were evaluated at

the final concentration associated with ten [micro]g/mL.

Free Radical Scavenging Exercise about DPPH

The scavenging activity of the EEP upon DPPH radicals was measured

according for the technique involving Chu et al. (2000) with a new few modifications. An

aliquot associated with two mL involving 1 x [10.sup.4]mol/L DPPH radical throughout ethanol hadbeen added

to an exam tube with two mL EEP taste answer (10 [micro]g/mL ethanol).

Ethanol was used as in opposition to the EEP taste remedy like a control. The

reaction mixture was incubated pertaining to one h with room temperature along with the

absorbance (Abs) had been established right away after mixing by measuring propolis extract sprayat

517 nm with a spectrophotometer. the scavenging exercise (%) (SA) on

DPPH radicals had been calculated by simply Equation (2):

SA =100 (1-Abs within the presence involving sample/ Abs inside the absence of

sample) (2)

RESULTS AND DISCUSSION

Total Polyphenol along with Flavonoids Contents associated with EEP

Propolis is actually commercially accessible as tinctures or perhaps tablets made

from ethanol extracts throughout many countries. The Actual total polyphenol and

flavonoid contents tend to be reported to become one with the most abundant and most

effective antioxidant inside propolis (Scheller et al., 1990). Therefore, we

firstly investigated the actual total polyphenol as well as flavonoid contents regarding EEP

obtained simply by distinct extraction methods.

Table 1 exhibits the total polyphenol as well as flavonoid contents of

different EEP samples. the EEP by simply HHPE and also LRT had no significant

difference (P0.05), as well as the EEP by HRE showed the best value for

polyphenol and also flavonoid contents. Impurities, like beeswax, which

could certainly not or even we had not been fully dissolved in ethanol solution at room

temperature, had been mostly dissolved in higher temperature (Cao along with Wei,

2002). Thus, the actual impurity content regarding EEP would increase, which usually would

lead to a lower of total polyphenol as well as flavonoid contents.

Therefore, the particular total polyphenol along with flavonoids contents associated with EEPobtained

by HRE had been low.

Effects regarding Numerous EEP samples about [beta]-Carotene Bleaching Method

Figure one exhibits your antioxidant exercise of EEP biological materials obtained by

different extraction strategies determined by [beta]-carotene bleaching

method. the antioxidant assay, using the particular discolouration of

[beta]-carotene can be widely used, simply because [beta]-carotene is actually extremely

susceptible to be able to free involving charge radical-mediated oxidation. [beta]-carotene is

discoloured simply from the oxidation of linoleic acid, due for you to its double

bonds becoming sensitive to oxidation (Singh et al., 2002). EEP samples

were evaluated with the last concentration associated with 10 [micro]g/ mL regarding the

assay.

[FIGURE one OMITTED]

As shown within Figure 1, EEP by 3 extraction methods had strong

antioxidant activity-over 60%. EEP examples obtained by simply HHPE as well as LRT had

stronger antioxidant activity compared to that by HRE. The Particular EEP through HRE, which

presented lower total polyphenol along with flavonoid contents, exhibited

weaker antioxidant activity.

The antioxidant exercise proven inside Figure 1 seemed to become able to correlate with

the total polyphenol along with flavonoid associated with EEP (Table 1). Positive

correlations were found among total polyphenol along with flavonoid contents

in the actual EEP and their antioxidant activities. Phenolic compounds, such as

flavonoids, are generally the kind of antioxidant that possesses any strong

inhibition effect against lipid oxidation via radical scavenging.

Flavonoids happen to be able to be reported to be one of the most abundant and many effective

antioxidant inside propolis (Isla, 2001). Right now there are numerous papers concerning

the antioxidant activity in the ethanol extract associated with propolis, which has

been attributed towards the higher content involving flavonoids inside propolis (Pascual

et al., 1994; Chen and also Ho, 1995).

Effect of Different EEP Examples about DPPH Totally Free Radical

The DPPH free associated with charge radical scavenging exercise associated with different EEPexamples is

shown inside Figure 2. Your model system regarding scavenging DPPH free radical is a

simple method regarding evaluating your antioxidant activity involving compounds. It

is accepted that the DPPH totally free radical scavenging by simply antioxidants can be due

to their own hydrogen-donating ability (Tang et al., 2002). We evaluated

various EEP examples with the last concentration regarding 10 [micro]g/mL.

[FIGURE two OMITTED]

As shown within Figure 2, EEP biological materials by simply 3 extraction techniques had

strong DPPH totally free radical scavenging activities that could reach over 60%. EEP samples

by HHPE as well as LRT had stronger DPPH free associated with charge radical scavengingactivities

than in which by simply HRE. These kind of EEP samples had substantial total polyphenol and

flavonoid contents (Table 1). EEP sample by simply HRE, which had weaker

antioxidant actions inside the assay system using your discolouration of

[beta]-carotene (Figure 1), exhibited weaker DPPH free radical

scavenging activity. We additionally discovered that the DPPH free regarding charge radicalscavenging

activity demonstrated within Figure two seemed for you to correlate with just about all theantioxidant

activity proven throughout Figure 1. the EEP test along with strong antioxidant

activity even offers strong DPPH totally free radical scavenging activity.

CONCLUSIONS

In this study, your antioxidant exercise of numerous EEP samples

obtained simply by 3 extraction methods (HHPE, LRT along with HRE) was

Gene/bees_b.jpg" width="380" />

investigated. The Particular results showed that the EEP obtained simply by HHPE and LRT

had relatively strong antioxidant activities, which might become correlated

with the high total polyphenol as well as flavonoid contents. Antioxidant

activities regarding EEP obtained by simply HHPE had been exactly the same as these associatedwith EEP

obtained by LRT. LRT typically needs several days or will take much more than

7 d, whilst HHPE requirements http://www.about-bees.com/honey-bees.html only 1 min. These typesof findings further illustrate that

HHPE features a bright prospect regarding extracting flavonoids through propolis.

HHPE works for that extraction regarding flavonoids through propolis,

not only as it is more rapid, safer and also eco-friendly than

conventional extraction methods, but throughout addition since its extract features strong

antioxidant activity. Meals and medicinal industries will benefit from

this emerging technology.

ACKNOWLEDGEMENTS

The author is grateful for the financial assistance in the Jilin

Provincial Institute regarding Drug Manage (2003).

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* Author in order to whom correspondence might become addressed. E-mail address:

xijun@mail.tsinghua.edu.cn

Manuscript received November 18, 2005; revised manuscript received

February 11, 2006; accepted for publication April 3, 2006.

Xi Jun *

School of Aerospace, Tsinghua University, Beijing 100084, Chinaa

Table 1. Total polyphenol and also flavonoid contents associated with EEP obtained

by different extraction methods

Total polyphenol

Extraction Flavonoids content material content associated with EEP

methods associated with EEP (mg/g) (mg/g)

HHPE 230.4 [+ or perhaps -] 6.5 (a) 290.4 [+ or perhaps -] 8.7 (a)

LRT 232.1 [+ or -] 3.2 (a) 296.0 [+ or perhaps -] 6.4 (a)

HRE 167.9 [+ as well as -] 5.5 (b) 247.7 [+ or perhaps -] 7.0 (b)

Values tend to be means [+ or perhaps -] regular deviations involving triplicate

measurement.

For different extraction methods, indicates with assorted letters (a, b)

were significantly distinct (P0.05, Student's t-test).