The Particular antioxidant actions in the ethanolic extracts regarding propolis obtained simply by d...

1. Comparison of antioxidant activity of ethanolic extracts of
propolis obtained by different extraction methods.
The Particular antioxidant actions in the ethanolic extracts regarding propolis obtained simply by
different 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

2. 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,

3. 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 to
polyphenol
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 employed
in
extracting flavonoids from propolis (Jun, 2005). Experiment outcomes have
shown HHPE has many advantages, like shorter occasion (only one min),

4. 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.

5. (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 involving
75%
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

6. 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 the
particular same
conditions and the http://en.wikipedia.org/wiki/Honey_bee supernatants were pooled. Supernatants
obtained 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.

7. 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

8. 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.

9. 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 had
been 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 spray
at
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.

10. 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 EEP
obtained
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]

11. 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 EEP
examples 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

12. 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 scavenging
activities
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 radical
scavenging
activity demonstrated within Figure two seemed for you to correlate with just about all the
antioxidant
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
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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

13. activities regarding EEP obtained by simply HHPE had been exactly the same as these associated
with 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 types
of 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

18. 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).