Journal of Ethnopharmacology 67 (1999) 197 202

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Isolation and antibacterial activity of phenylpropanoid

derivatives from Ballota nigra
Nicole Didry a, Veronique Seidel a, Luc Dubreuil b, Francois Tillequin c,
Francois Bailleul a,*
a
Laboratoire de Pharmacognosie, Faculte des Sciences Pharmaceutiques et Biologiques, B.P. 83, 59006 Lille Cedex, France
b
Laboratoire de Bacteriologie, Faculte des Sciences Pharmaceutiques et Biologiques, B.P. 83, 59006 Lille Cedex, France
c
Laboratoire de Pharmacognosie, Faculte des Sciences Pharmaceutiques et Biologiques, U.R.A. au C.N.R.S. no. 1310, 4,
A6enue de lObser6atoire, 75006 Paris, France
Received 20 November 1998; received in revised form 1 February 1999; accepted 8 February 1999

Abstract

In addition to the previously isolated phenylpropanoid glycosides verbascoside 1, forsythoside B 2, arenarioside 3

and ballotetroside 4, another four compounds were isolated from generative aerial parts of Ballota nigra: three
phenylpropanoid glycosides, alyssonoside 5, lavandulifolioside 6 and angoroside A 7 and a non-glycosidic derivative
( +)-(E)-caffeoyl-L-malic acid 8.The antibacterial activity of the five major compounds (1 4 and 8) was tested against
Gram-positive and Gram-negative bacteria. Three of them (1 3) exhibited a moderate antimicrobial activity against
Proteus mirabilis and Staphylococcus aureus including one methicillin-resistant strain. 1999 Elsevier Science Ireland
Ltd. All rights reserved.

Keywords: Ballota nigra; Phenylpropanoid non-glycosidic ester; Phenylpropanoid glycosidic esters; Antimicrobial activity

1. Introduction and their aqueous and hydroalcoholic extracts are

The genus Ballota L. consists of about 33 spe-
cies of Lamiaceae belonging to the tribe Stachy-
dae, subtribe Ballotae. Most of them grow in the
Mediterranean region and adjoining Europe and
Asia Minor. Only four species originate from
Eastern and Southern Africa (Patzak, 1958, 1959,
1961).
Ballota nigra L. is commonly distributed in
Western Europe where the generative aerial parts
* Corresponding author.
used in medicine for treating influenza, cough,
and more especially to evoke neurosedative
activities.
Previously chemical investigation of aerial parts
resulted in isolation of the flavonoids apigenin-7-
glucoside, vicenin-2 (Darbour et al., 1986) and
tangeretin (Kisiel and Piozzi, 1995) and the lab-
dane diterpenoids ballotinone (Savona et al.,
1976a), ballonigrine (Savona et al., 1976b), 7a-
acetoxymarrubiin (Savona et al., 1977a), bal-
lotenol (Savona et al., 1977b), preleosibirin
(Bruno et al., 1986) and 13-hydroxyballonigrino-

0378-8741/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 0 1 9 - 7
198 N. Didry et al. / Journal of Ethnopharmacology 67 (1999) 197202
lide (Seidel et al., 1996a). Recent studies of a
hydroalcoholic extract of aerial parts led to the
isolation of four phenylpropanoid glycosides: ver-
bascoside 1, forsythoside B 2, arenarioside 3, and
ballotetroside 4 (Seidel et al., 1996b, 1997).
The present work deals with the isolation of
four other known phenylpropanoid derivatives
from a polar extract of aerial parts of Ballota
nigra, including three glycosides: alyssonoside 5,
lavandulifolioside 6, angoroside A 7, and a non-
glycosidic derivative: (+ )-(E)-caffeoyl-L-malic
acid 8 (see Fig. 1).
The well-known antibacterial properties of nu-
merous phenolics and especially of several natural
phenylpropanoids (Jimenez and Riguera, 1994)
prompted us in a second time to investigate the
antimicrobial activity of the five major com-
pounds 14 and 8. They were evaluated against
several strains of both Gram-positive and Gram-
negative bacteria.
Fig. 1. Structures of phenylpropanoid derivatives of Ballota
nigra.
2. Materials and methods
2.1. Plant material
Ballota nigra L. was collected in 1996 in the
vicinity of Angers (France) and identified in com-
parison with an authentic sample cultivated in the
Botanical Garden of the University of Lille.
A voucher specimen is kept in the Department
of Pharmacognosy of that University (No. B.n.
5).
2.2. Instruments and chemicals
Mass spectra were recorded on a Nermag R
10-10C mass spectrometer. NMR spectra were
recorded at 300 MHz (1H) and 75 MHz (13C) on
a Bruker AC 300 spectrometer using TMS as
internal standard. 1H 1H COSY, 13C 1H HET-
COR and 13C 1H COLOC were performed using
Bruker standard microprograms.
2.3. Isolation of phenylpropanoids
The dried powdered generative aerial parts (1
kg) were exhaustively lixiviated with 50% ethanol
(EtOH) at room temperature and the EtOH was
evaporated under reduced pressure. The water-in-
soluble material was removed by filtration.
An aliquot of the aqueous solution (4/5) was
extracted with petroleum ether to remove lipidic
substances. The aqueous phase was then extracted
twice with n-butanol, and the combined organic
phases evaporated in vacuo to dryness to afford a
residue (32 g). This crude extract was submitted
to repeated separations on Sephadex LH 20 using
ethyl acetate (EtOAc)methanol (MeOH) of in-
creasing polarity. Different fractions containing
18 were further purified separatively by repeated
separations on silica gel H using mixtures of
EtOAcMeOHH2O of increasing polarity to
yield the main pure compounds 1 (1.2 g), 2 (1.0
g), 3 (1.1 g), and 4 (1.0 g). Further preparative
TLCs on silica gel afforded pure minor com-
pounds 5 (20 mg), 6 (25 mg), and 7 (17 mg).
Peracetylated derivatives were obtained by treat-
 N. Didry et al. / Journal of Ethnopharmacology 67 (1999) 197202
ment of 5 7 (10 mg each) with acetic anhydride
(1 ml) and pyridine (1 ml).
The remaining aqueous solution (1/5) was ad-
justed to pH 2 (HCl) and extracted twice with
ether. The ether solutions were concentrated in
vacuo and the residue (3.4 g) was submitted to
column chromatography (CC) on Sephadex LH
20 using EtOHH2O 1:1 as eluent. Fractions
containing 8 were further purified by CC on sil-
ica gel H using EtOAc MeOH H2O, 70:40:20
to give pure 8 (0.9 g).
2.4. Total phenylpropanoid content
Total phenylpropanoids of Ballota nigra aerial
parts were determined by a colorimetric method
based on orthodihydroxycinnamic derivatives es-
timation (Arnow, 1937). 1 ml of sample hydroal-
coholic solution was added to 2 ml of aqueous
0.5 N HCl, 2 ml of 10% (w/v) aqueous solution
of sodium nitrite and 10% (w/v) aqueous solu-
tion of sodium molybdate (Arnow reagent) and 2
ml of aqueous 2 N NaOH. The solution was
adjusted to 10 ml with water. The absorption
was measured at 525 nm. Result is expressed in
g/100 g of dry plant material with respect to
verbascoside 1.
2.5. Microorganisms
Bacteria (24 strains) were isolated from human
clinical samples in the Dron Hospital (135, rue
President Coty, F-59200 Tourcoing).
The following tested microorganisms were
Staphylococcus aureus (five strains including
one methicillin-resistant strain [MRSA]) and En-
terococcus faecalis (five strains) for Gram-posi-
tive bacteria, Pseudomonas aeruginosa (three
strains), Escherichia coli (five strains), Proteus
mirabilis (three strains) and one of each strain
of Enterobacter aerogenes, Klebsiella pneu-
moniae, Klebsiella oxytoca for Gram-negative
bacteria.
Staphylococcus aureus were isolated from cuta-
neous pus, other strains from urinary tract
infections.
199
2.6. Culture media
Mueller-Hinton broth (Difco) was used as nu-
trient broth for all the bacteria tested.
Antimicrobial activity determination was per-
formed onto Mueller-Hinton agar (Difco).
2.7. Bacterial suspensions
Bacterial suspensions were obtained from a
broth culture of 24 h. The actively growing cul-
tures were diluted in a sterile 9 sodium chlo-
ride solution to reach and match the turbidity of
0.5 McFarland Standard.
The inocula were approximatively 108 CFU/
ml. The previous inocula diluted 10-fold in
the same diluent were used as bacterial suspen-
sions.
2.8. Material used in microbiological procedure
For antimicrobial activity determination bacte-
rial suspensions were delivered with a Steers
replicator (Mast Systems, London, United King-
dom).
2.9. Antimicrobial susceptibility testing
The antimicrobial activity of compounds was
assessed by determining MIC values obtained by
a reference agar dilution method according to
standard M7 A4 method of National Committee
for Clinical Laboratory Standards (NCCLS,
1997).
Phenylpropanoids were solubilized in dimethyl-
sulfoxide (DMSO) and diluted in sterile distilled
water by a 2-fold serial dilutions. 1 ml of dilu-
tions was incorporated into 19 ml of agar so that
plates contained increasing concentrations of
compounds ranging from 128 to 0.5 mg/ml.
Bacterial suspensions (2 or 3 ml) were delivered
with the Steers replicator and led to a final in-
oculum of 104 CFU/ml per spot of inoculation
on the agar plates.
After incubation for 24 h at 37C, the MIC
values were defined as the lowest concentration
of compound to prevent visible growth.
200 N. Didry et al. / Journal of Ethnopharmacology 67 (1999) 197202
An agar plate without phenylpropanoids was
inoculated and incubated to determine the
viability of the organisms and to serve as a
control for the comparison of growth. Previous
control with DMSO indicated that the growth of
the bacterial strains was not affected by the
DMSO.
3. Results and discussion
Seven phenylpropanoid glycosidic esters 1 7
and one phenylpropanoid non-glycosidic ester 8
were isolated from aerial parts of Ballota nigra.
The structures of 1 4 which were previously
described in this species (Seidel et al., 1996b;
Seidel et al., 1997) were determined by direct
comparison ([a]D, ms, 1H and 13C NMR) with
authentic samples.
The structures of compounds 5 8 which are
described for the first time in the genus Ballota
were deduced from their physical and spectral
data ([a]D, ms, 1H and 13C NMR) and from
those of their peracetylated derivatives in the
case of glycosides 5 7. The complete spectro-
scopic assignment of the different moieties of
these three phenylpropanoid glycosides as well as
the determination of interglycosidic linkages have
been confirmed by extensive experiments using
1
H 1H COSY, 13C 1H HETCOR and COLOC
techniques and in comparison with previously re-
ported data (Calis et al., 1987, 1992; Basaran et
al., 1988; Hahn and Nahrstedt, 1993).
The total phenylpropanoid derivatives in aerial
parts of Ballota nigra determined colorimetrically
accounted for 5.5% of dry weight. Quantitatively,
compounds 14 and 8 which are present in al-
most equal amounts are the major phenyl-
propanoids, compounds 5 7 being present in
very small amounts. Therefore each of com-
pounds 14 and 8 accounts for ca 1% of the
dried aerial parts.
Compounds 14 and 8 were evaluated with
the agar dilution method at concentrations up to
128 mg/ml against a panel of gram-positive and
gram-negative bacteria. Amongst this last type of
microorganisms, we chose three strains of each
Pseudomonas aeruginosa and Proteus mirabilis,
five strains of Escherichia coli and one strain of
Enterobacter aerogenes, Klebsiella pneumoniae
and Klebsiella oxytoca.
Regarding gram-positive bacteria, five strains
of each Enterococcus faecalis and Staphylococcus
aureus (one of them methicillin-resistant) were
used for testing the compounds named above.
It is interesting to take in account that some
infections produced by Staphylococcus aureus are
commonly very difficult to eradicate. Therapy
with several type of antibiotics (Hiramatsu et al.,
1997) is frequently accompanied by the appear-
ance of unexpected side effects and acquisition of
resistance. Methicillin-resistant Staphylococcus
aureus (MRSA) are one of the major nosocomial
pathogens in hospitals (Voss and Doebbeling,
1995) and although some studies devoted to
looking for new anti-MRSA agents have been
reported (Tsuchiya et al., 1996; Chambers, 1997),
other compounds alternative to existing antibi-
otics are still needed.
Results obtained with the five major phenyl-
propanoids tested 14 and 8 showed that 4 and
8 do not possess antimicrobial activity up to 128
mg/ml. Compounds 13 possess a moderate in-
hibitory effect against Staphylococcus aureus and
Proteus mirabilis (see Table 1). Compounds 1
and 2 inhibited one out of three strains of Pro-
teus mirabilis and two out of five strains of
Staphylococcus aureus including the methicillin-
resistant strain, at 128 mg/ml. The more signifi-
cant results were observed with compound 3:
three out of five strains of Staphylococcus aureus
were inhibited at a concentration of 128 mg/ml
and one out of three strains of Proteus mirabilis
at 64 mg/ml.
Although the moderate antimicrobial activity
of verbascoside, forsythoside B and arenarioside
could not allow their use as single agents
we hope that results reported here open the pos-
sibility of further investigations on their antimi-
crobial properties and studies of their com-
binations with other antibiotics. In addition, the
fact that compound 3 shows inhibition of the
methicillin-resistant Staphylococcus aureus strain,
could aid in the development of new agents
against one of the major nosocomial pathogens
in hospitals.
 N. Didry et al. / Journal of Ethnopharmacology 67 (1999) 197202 201

Table 1
Antimicrobial activity of compounds 1, 2, 3 at concentrations of 64 mg/ml and 128 mg/ml against Staphylococcus aureus and Proteus
mirabilis a

Bacterial strains Compounds

1 2 3 1 2 3

64 mg/ml 128 mg/ml

Staphylococcus aureus 1 + + + + + +
2 + + + + + +
3 + + + + +
4 + + +
5 (MRSA) + + +
Proteus mirabilis 1 + + + + + +
2 + + + + + +
3 + +

a
MRSA, methicillin-resistant Staphylococcus aureus; +, growth; , no visible growth.

Acknowledgements feuilles de Ballota foetida Lamk. (Labiees). Pharmazie 41,

The authors would like to thank N. Derensy
and E. Singer, Department of Bacteriology, Uni-
versity of Lille, for their technical assistance in the
course of this work and Professor I. Calis,
Hacettepe University, Faculty of Pharmacy of
Ankara, for supplying authentic samples.
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