204

ACTA FACULTATIS PHARMACEUTICAE UNIVERSITATIS COMENIANAE

Tomus LII 2005





ANTIOXIDATIVE PROPERTIES OF METHANOL EXTRACTS
FROM LEAVES AND FRUITS OF LIGUSTRUM VULGARE L.


1
ere, F.
2
Muaji, P.
2
Granai, D.
2
Nagy, M.

1
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava
2
Department of Pharmacognosy and Botany, Faculty of Pharmacy,
Comenius University, Bratislava



This work deals with antioxidative properties of methanol extracts from leaves and fruits of
Ligustrum vulgare L. It was found that these extracts can scavenge HO

and DPPH radicals. The

effectiveness of the leaves extract to scavenge of DPPH was as 1.4-times higher as that one from
fruits. The effectiveness of the leaves extract to scavenge HO

radicals was lower than the one to
scavenge DPPH. However, the leaves extract was as 1.2-times higher as that one from fruits. The
both extracts exhibited as lower antioxidative effectiveness as vitamin C: 6.7- 9.3-times (DPPH
assay) and 200- 240-times (HO

radicals assay). We assume that phenolic compounds, mainly
flavonoids and secoiridoids and its different content in plant parts (leaves, fruits) are responsible
for the observed effect.

Key words: Ligustrum vulgare L. scavenger activity DPPH hydroxyl radicals


INTRODUCTION

Leaves from Ligustrum vulgare L. are used for the diseases prevention or treatment
in folks medicine in southern Europe [1]. Most of them can be connected with reactive
oxygen species balancing in all human tissues. Phenolic antioxidants are recognized as
main active principles.
It has been shown that infusions and teas from L. vulgare L. leaves have high
antioxidant activity estimated by the 1,1-diphenyl-2-picrylhydrazyl method (DPPH
radical scavenging assay). In addition to the well-known antioxidant properties of
secoiridoids, their role in ecological biochemistry is known also. Oleuropein activated by
-glucosidase had very strong protein-denaturating, protein-crosslinking and lysine-
-alkylating activities suggesting an effective defense mechanism against herbivores [2].
Flavonoids may scavenge reactive oxygen containing species that are produced under
severe stress conditions and protect plant cell metabolism from oxidative damages [3].

205
Flavonoids have been also shown to protect animal cell metabolism and may have an
important role in human health. Secoiridoids accounted for nearly the 76 % of the total
leaf polyphenols content of Ligustrum vulgare L., while content of kaempferol
glycosides were around 23 % suggesting, that secoiridoids and flavonoids may have a
central role in both the ecology and the biology of Ligustrum vulgare L. [4].
Measurement of radical scavenging activity using discoloration of 1,1-diphenyl-2-
-picrylhydrazyl radicals has been widely used due to its stability, simplicity, and
reproducibility. It correlates with real antioxidative activities of different substances [5].
Another type of scavenger activity measurement is EPR method by DMPO spin trap use
[6].
The objective of the present work was therefore to determine the scavenging capacity
of methanolic extracts from Ligustrum vulgare L. fruits and leaves by both methods.


MATERIAL AND METHODS

Reagents and equipments

Methanol, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5,5-dimethyl-3,4-dihydro-2H-
-pyrrole-1-oxid (DMPO), and 30% H
2
O
2
were purchased from Sigma-Aldrich Ltd. The
EPR spectra were registered on ERS 230 (ZWG Akad. Wiss. Berlin, Germany), which
operates in X-band (~9.3 GHz), at modulation amplitude 0.1 mT, and microwave power
5 mW. The absorption spectra were scanned on Specord UV-VIS (C. Zeiss Jena,
Germany).

Preparation of extracts

Methanol extracts from leaves and fruits of Ligustrum vulgare L. were prepared from
the plant material harvested at the end of September in Bratislava. Fresh leaves (100 g)
and fresh fruits (50 g) were extracted with MeOH at room temperature for 30 days.
Extracts were filtered and 200 l of each extract was evaporated in vacuo to dryness at
temperature which not exceeded 40 C. The dry rests were weighted, redissolved in
MeOH and used for DPPH and hydroxyl radical assay.

DPPH assay

Into 10
-4
moldm
-3
of a methanolic DPPH solution different amount of the tested
substance was added. After 20 min, absorbance at 517 nm was measured and SC
50

values (i.e. concentration causing 50 % decrease of absorption) were obtained from the
inhibition curves [7,8].

Hydroxyl radical assay

The scavenging activity of studied samples was determined by EPR spectroscopy.
Spontaneous degraded hydrogen peroxide at 25 C was used as a source of hydroxyl
radicals.

206
H
2
O
2
2HO

Because of HO

radicals possess very short lifetime they can not be detected readily
by the continuous wave EPR spectrometer. Therefore, they were captured by a DMPO
spin trap and the EPR spectra of resulted spin adducts were subsequently recorded. The
aqueous reaction solution contained 0.05 moldm
-3
of DMPO, 0.05 moldm
-3
of H
2
O
2
and
different amount of studied extract.






DMPO SPIN ADDUCT



RESULTS AND DISCUSSION

The effect of studied extracts on the methanolic DPPH solution was accompanied by
a reaction mixture bleaching. In Figure 1 the effect of various concentration of
methanolic extract from leaves of Ligustrum vulgare L. is shown. It is evident this one
causes an evident intensity decrease of absorption band at 517 nm becuse of its
scavenging activity.
The free radical scavenging activity was calculated by using the following equation:

scavenging activity (%) = [A
a
- (A
b
- A
c
)] / A
a
100

where A
a
is the absorbance of the incubation DPPH solution without addition of the
tested samples, A
b
is the absorbance of the incubation mixture containing both the tested
samples and DPPH, and A
c
is the absorbance of the blank solution without DPPH. The
values of SC
50
calculated from a regression curve denote the concentration of sample
required to scavenge 50 % of DPPH radical. The Table 1 presents the SC
50
values for all
studied extracts and SC
50
value for the vitamin C. The values of the average square
deviation (r
2
) were also calculated. It is evident the leaves extract is as better scavenger
of DPPH radicals as the fruit one, whereby vitamin C is 6.7- 9.3-times stronger
scavenger of DPPH than both extracts.
An EPR spectrum, which consists of four lines with the intensity ratio 1:2:2:1, was
observed after H
2
O
2
addition into aqueous DMPO solution. This signal is in the range of
free radicals with g = 2.005 and distances between lines are 1.48 mT (Fig. 2). The signal
belongs to the spin adduct DMPO with hydroxyl radical. Its hyperfine splitting constants
are A
N
= A
H
= 1.48 mT [9]. The signal intensity linearly increased up to 30 min later, till
90 min decreased during 90 min. After adding of the studied extract into above
mentioned H
2
O
2
solution the intensity of EPR spectra of spin adducts DPMO with HO

radicals did not reach a level of control sample.
N
O
C H
3
C H
3
HO
N
O
C H
3
C H
3
OH
H

207

a

b
c

d

e


f


g




Fig. 1. The absorption spectra of 110
-4
mol dm
-3
of DPPH (in methanol) in the presence of:
a) 0, b) 1.05, c) 3.15, d) 5.25, e) 10.5, f) 21 and g) 26.25 g (from top to bottom) of dry
mass (DM) of leaves extract from Ligustrum vulgare L.
























Fig. 2. The EPR spectra of the spin adduct DMPO with HO

without (A)
and after adding 1 mg DM of the fruit extract (B). The spectra were registered 30 min
after adding of H2O2

In order to characterize a scavenging effectiveness of studied samples we were
looking for the lowest extracts concentration, which caused no EPR signal in measured
samples after their addition into above mentioned DMPO with H
2
O
2
water solution. The
Table 2 presents these concentrations. It is evident the leaves extract was as better

332,
5
335,
0
337,
5
magnetic induction [mT]
A
B

208
scavenger of hydroxyl radicals as the fruit extract, however vitamin C was the best
scavenger of HO

radicals (as ca. 200-times as leaves extract and as 240-times as fruit

extract).

Table 1. The SC50 values of the DPPH scavenging by samples from Ligustrum vulgare

Sample leaves fruits vitamin C
SC
50
[g DM/ml]
r
2

14.75
0.99
20.58
0.98
2.21
0.99


Table 2. The values of the lowest concentration (c) of studied compounds

Substance leaves fruits vitamin C
c [mg DM/ml] 2.0 2.4 0.01


CONCLUSION

It was found the methanol extracts from leaves and fruits of Ligustrum vulgare L.
exhibit scavenging properties. We suggest phenolic compounds (flavonoids, hydro-
xycinnamates) and secoiridoids as main extracts constituents which are responsible for
this effect.

Acknowledgement: This work was supported by a VEGA projects of Ministry of Education of
Slovak Republic No. 1/0516/03 and No. 1/1185/04.


REFERENCES

1. PIERONI, A. PACHALY, P.: Isolation and structure elucidation of ligustroflavone, a new
apigenin triglycoside from the leaves of Ligustrum vulgare L. Pharmazie 55, 2000, p. 78-80.
2. KONNO, K. HIRAYAMA, CH. YASUI, H. NAKAMURA, M.: Enzymatic activation of
oleuropein: a protein crosslinker used as a chemical defense in the privet tree. Proc. Natl. Acad.
Sci. USA 96, 1999, p. 9159-9164.
3. TATTINI, M. GALARDI, C. PINELLI, P. MASSAI, R. REMORINI, D. AGATI, G.:
Differential accumulation of flavonoids and hydroxycinnamates in leaves of Ligustrum vulgare
under excess light and drough stress. New Physiologist 163, 2004, p. 547-561.
4. ROMANI, A. PINELLI, P. MULINACCI, N. VINCIERI, F.F. GRAVANO, E.
TATTINI, M.: HPLC analysis of flavonoids and secoiridoids in leaves of Ligustrum vulgare L.
(Oleaceae). J. Agric. Food Chem. 48, 2000, p. 4091-4096.
5. KATSUBE, T. TABATA, H. OHTA, Y. YAMASAKI, Y. ANUURAD, E.
SHIWAKU, K. YAMANE,Y.: Screening for antioxidant activity in edible plant products:
comparison of low-density lipoprotein oxidation assay, DPPH radical scavenging assay, and
Folin-Ciocalteu assay. J. Agric. Food Chem. 52, 2004, p. 2391-2396.
6. YOSHIOKA, H. OHASHI, Y. AKABOSHI, M. SENBA, Y. YOSHIOKA, H.: A novel
method of measuring hydroxyl radical-scavenging activity of antioxidants using gamma-
-irradiation. Free Radic. Res. 35, 2001, p. 265-271.

209
7. DEBY, C. MARGOTTEEAUX, G.: Relationship between essential fatty acids and tissue
antioxidant levels in mice. C. R. Soc. Biol. 164, 1970, p. 2675-2681.
8. JOYEUX, M. MORTIER, F. FLEURENTIN, J.: Antilipoperoxidant and hepatoprotective
effects of nine plant extracts used in Caribbean folk medicine. Phytother. Res. 9, 1995, p. 225-
230.
9. LI, A.S.W. CUMMINGS, K.B. ROETHLING, H.P. BUETTNER, G.R. CHIGNELL,
C.F.: A spintrapping database implemented on the IBM PC/AT". J. Mag. Resonance 79, 1988,
p. 140-142. The database is available through internet at http://epr.niehs.nih.gov/.


Registered: February 11, 2005 Ing. Frantiek ere, CSc.
Accepted: March 18, 2005 Institute of Chemistry
Faculty of Natural Sciences
Comenius University
Mlynsk dolina
842 15 Bratislava
Slovak Republic
sersen@fns.uniba.sk


ANTIOXIDAN VLASTNOSTI METANOLOVCH EXTRAKTOV LISTOV
A PLODOV LIGUSTRUM VULGARE L.

1
ere, F.
2
Muaji, P.
2
Granai, D.
2
Nagy, M.

1
Chemick stav, Prrodovedeck fakulta, Univerzita Komenskho, Bratislava
2
Katedra farmakognzie a botaniky, Farmaceutick fakulta,
Univerzita Komenskho, Bratislava

Prca sa zaober antioxidanmi vlastnosami metanolovch extraktov listov a plodov
Ligustrum vulgare L. Zisten bolo, e tieto extrakty vychytvaj HO

a DPPH radikly. Vychy-

tvanie DPPH radiklov extraktami listov bolo 1,4-krt innejie ako v prpade extraktov plodov.
innos vychytvania HO

radiklov extraktami listov bola niia ako v prpade DPPH radik-

lov, ale 1,2-krt vyia ako v prpade extraktov plodov. V porovnan s vitamnom C oba extrakty
vykazovali niiu aktivitu: 6,7- a 9,3-krt (DPPH metdou) resp. 200- a 240-krt v teste vy-
chytvania hydroxylovch radiklov. Predpokladme, e za antioxidan inky a rozdielnu
aktivitu jednotlivch extraktov vtieho zobu zodpovedaj fenolick ltky (flavonoidy) a sekoiri-
doidy a ich rozdielny obsah v jednotlivch astiach rastliny.

Acta Facult. Pharm. Univ. Comenianae 52, 2005, p. 204-209.