Antimicrobial and anti-lipase activity of quercetin and its C2-C16 3-O-acyl-esters

Article abstract of DOI:10.1016/S0968-0896(01)00275-9

Neither quercetin (Q), nor 3-O-acylquercetines, up to 100 μg/mL, had any significant activity on selected gram-positive strains (Staphylococcus aureus, Bacillus subtilis, Listeria ivanovi, Listeria monocytogenes, Listeria serligeri), gram-negative strains

Full text of DOI:10.1016/S0968-0896(01)00275-9

Bioorganic & Medicinal Chemistry 10 (2002) 269–272
Antimicrobial and Anti-Lipase Activity of Quercetin and Its
C2-C16 3-O-Acyl-Esters
Maria Teresa Gatto,a Serena Falcocchio,a Eleonora Grippa,a Gabriela Mazzanti,a
Lucia Battinelli,a Giovanni Nicolosi,b Daniela Lambustab and Luciano Sasoa,*
aDepartment of Pharmacology of Natural Substances and General Physiology, University of Rome ‘‘La Sapienza’’, P.le Aldo Moro 5,
00185 Rome, Italy
bIstituto CNR per lo Studio delle Sostanze Naturali di Interesse Alimentare e Chimico-Farmaceutico, Via del Santuario 110,
95028 Valverde (CT), Italy
Received 25 May 2001; accepted 26 July 2001
Abstract—Neither quercetin (Q), nor 3-O-acylquercetines, up to 100 mg/mL, had any significant activity on selected gram-positive
strains (Staphylococcus aureus, Bacillus subtilis, Listeria ivanovi, Listeria monocytogenes, Listeria serligeri), gram-negative strains
(Escherichia coli, Shigella flexneri, Shigella sonnei, Salmonella enteritidis, Salmonella tiphymurium) and yeasts (Candida albicans and
Candida glabrata). In addition, we confirmed the known anti-HIV activity of Q (80% inhibition at 40 mM), which might depend on
the free hydroxyl in the C-3 position, as suggested by the lack of activity of the 3-O-acylquercetines. Finally, we described an
interesting inhibitory activity on Candida rugosa lipase by Q (IC16=10ꢀ4 M) and its esters (3-O-acylquercetines) which, in vivo,
could play an important role against lipase producing microorganisms. In particular, 3-O-acyl-quercetines, being more active
(IC16=10ꢀ4–10ꢀ6 M) and more lipophilic, could be more effective than Q when applied to the skin or mucosae, and deserve to be
studied further. # 2001 Elsevier Science Ltd. All rights reserved.
Quercetin (Q), one of the most studied flavonoids, has
been reported to have antioxidant,1 anti-cancer,2
antithrombotic,3,4 antimicrobial, antiviral and other
activities. In particular, concerning the two latter
properties:
Thus, the aim of the present study was to evaluate the
effect of Q and its C2–C16 3-O-acyl derivatives on
selected bacteria (five gram-positive and five negative
strains), yeasts (four Candida strains) and HIV-1. Fur-
thermore, we decided to examine whether these com-
pounds could affect the activity of Candida rugosa
lipase, using a chromatographic method that has
recently been developed in this laboratory.8
1. Q, at high concentrations, appeared active against
different microorganisms including Bacillus sub-
tilis, Micrococcus luteus, Staphylococcus aureus
and Staphylococcus epidermidis;5 Aspergillus flavus
and Aspergillus parasiticus.6 Additionally, the in
vivo antimicrobial activity of certain natural sub-
stances could be due to their ability to interfere
with virulence factors such as lipase.7,8 Indeed, the
anti-lipase activity of a number of flavonoids,9 but
not of Q, has been described.
Materials and Methods
Reagents
C. rugosa lipase (Crl, 700–1500 U/mg at pH 7.2using
olive oil, cat. No. L-1754), b-naphthyl-laurate (b-NL,
essentially naphthol-free, cat. No. N-9375), b-naphthol
(b-N, cat. No. N-1250), phenacetin (cat. No. A-2375),
taurocholic acid (cat. No. T-4009), acetone (cat. No. A-
4206), Tris (cat. No. T-1503), Quercetin (Q, cat. No. Q-
0125), 30-azido-30-deoxythymidine (AZT, cat. No. A-
2169) and tetracycline hydrochloride (T, cat. No. T-
3383) were purchased from Sigma Chemical Co. (St.
Louis, MO, USA). Trifluoroacetic acid (cat. No.
2. Q and other flavonoids appeared active against
different viruses,10 including HIV,11 probably due
to the inhibition of reverse transcriptase.12
*Corresponding author. Tel.: +39-06-4991-2479; fax: +39-06-4991-
2480; e-mail: luciano.saso@uniroma1.it
0968-0896/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00275-9

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M. T. Gatto et al. / Bioorg. Med. Chem. 10 (2002) 269–272
108262) and sodium chloride were from Merck (Darm-
stadt, Germany). Methanol, ethanol, acetonitrile, and
water for high performance liquid chromatography
(HPLC), were purchased from Lab-Scan (Dublin, Ire-
land). Ethyl acetate (EA) was obtained from Carlo Erba
(Milan, Italy). All reagents were used without further
purification.
1, Q-Bu-1, Q-Is-1 and Q-DE-1 were dissolved at the
concentration of 100 mg/mL in Triton 5% and serially
diluted and filtered through a 0.22 mm filter. The mini-
mal inhibitory concentration (MIC) was determined by
the classical microdilution method using 96-well culture
plates, inoculated with the microorganisms suspended
(105 cells/mL) in Muller–Hinton broth (Becton-Dick-
inson, Oxnard, CA, USA). The plates were incubated
for 24 h (or 48 h for the yeasts) at 37 ꢁC. The cultures
that did not present growth were used to inoculate
plates of solid medium in order to determine the mini-
mal bactericidal concentration (MBC). Proper blanks
were assayed simultaneously. All samples were tested in
triplicate.
Preparation of 3-O-acylquercetines
Q esters (Fig. 1) have been prepared as previously
reported for the acetyl derivative13 and the following
procedure is representative: lipase from Mucor miehei
(immobilised, Lipozyme1 IM, 0.50 g) was added to a
solution of the peracylated quercetine of choice (0.50 g)
in t-BME containing n-butanol (10 equiv). The suspen-
sion was shaken (300 rpm) at 45 ꢁC until analysis of the
reaction mixture by thin layer chromatography showed
the presence of a single product (12–96 h). The reaction
was then quenched, filtering off the catalyst and the fil-
trate evaporated to dryness under reduced pressure. The
residue was purified by column chromatography on
silica gel using a gradient of acetone in petroleum ether
as the eluent to give the relevant 3-O-acylquercetine.
Anti-HIV activity
A primary strain of HIV-1 was isolated from plasma of
an HIV-infected patient and expanded in peripheral
blood mononuclear cells (PBMC) according to standard
protocols.14 Briefly, PBMC from healthy donors were
isolated by Ficoll-Hypaque centrifugation, suspended
(1ꢂ106 cells/mL) in RPMI 1640 complete medium [10%
fetal calf serum, penicillin-G (50 U/mL), streptomycin
(50 mg/mL) and l-glutamine (2mM)] containing phy-
tohemagglutinin (5 mg/mL) and incubated for 72h at
37 ꢁC in a 5% CO2 atmosphere. Then, cells were cen-
trifuged, resuspended in complete medium, infected
with HIV-1 by addition of 500 tissue culture infective
dose (TCID) 50/106 PBMC (multiplicity of infec-
tion=0.05), incubated for 2h, suspended (1 ꢂ106 cells/
mL) in complete medium containing IL-2(5 U/mL),
and seeded in 48-well plates in the presence or not
of AZT (3.7 mM), Q (40 mM), Q-Ac (40 mM) and Q-Pa
(40 mM). After 72h, the viral production was deter-
mined by HIV-1 p24 antigen ELISA (Immunogenetics).
Antimicrobial activity
Microbiological assays were performed using different
clinically isolated microorganisms, including five gram-
positive strains (S. aureus, B. subtilis, Listeria ivanovi,
Listeria monocytogenes, Listeria serligeri), five gram-
negative strains (Escherichia coli, Shigella flexneri, Shi-
gella sonnei, Salmonella enteritidis, Salmonella tiphy-
murium) and four yeasts (two strains of Candida
albicans and two strains of Candida glabrata). Q, Q-Ac-
Anti-lipase activity
Lipase activity in presence or not of the substances
under study was evaluated by HPLC as previously
described.8 Briefly, b-NL was dissolved in acetone at 3
mg/mL and this solution was diluted 1:20 in lipase buf-
fer [50 mM Tris at pH 7.4 (at 22 ꢁC), containing 3.5 mM
NaCl, 1.5 mM CaCl2, and 1 mM sodium taurocholate]
to obtain a final volume of 2mL of a stable substrate
suspension. Then Crl, dissolved at 0.1% w/v in 100 mM
Tris pH 7.4 (at 22 ꢁC), was added to the substrate sus-
pension to a final concentration of 0.001% w/v and
incubated for 30 min at 37 ꢁC under mild mixing. Then,
20 mL of the internal standard phenacetin, dissolved in
methanol at the concentration of 15 mg/mL, were added
and the enzymatic reaction was ended by adding 2mL
of the immiscible solvent (EA), which efficiently extrac-
ted the product of the reaction b-naphthol (b-N) and
the internal standard phenacetin. Afterwards a 500 mL
portion of the organic phase was withdrawn, evapo-
rated under a nitrogen stream at room temperature and
redissolved in 1 mL of methanol. Hence, aliquots of 50
mL were analyzed by HPLC using a C-18 reversed-phase
column (4.6ꢂ250 mm; 5-mm particle size; 90 A pore size;
Vydac cat. No. 201HS54) equilibrated at the flow rate
of 1 mL/min, with a mobile phase consisting of 40:60
Figure 1. Chemical structure of quercetin and abbreviations used for
3-O-acylquercetines.

M. T. Gatto et al. / Bioorg. Med. Chem. 10 (2002) 269–272
271
Table 1. Effect of quercetine and its esters on Candida rugosa lipase
where AUC is the area under the chromatographic peak
of b-N extracted from suspensions containing Crl,
incubated with (AUCc), or without (AUC0) the sub-
stance X at the concentration c, or devoid of the enzyme
(AUCblank). All experiments were done at least in
duplicate.
a
SMAX [mg/mL (M)]
IC16 (M)b
IC50 (M)b
Tetracycline
1.2(2.5 ꢂ10ꢀ3
)
1.6ꢂ10ꢀ4
2.5ꢂ10ꢀ4
ꢀ4
Q
2
ꢂ10ꢀ1 (8.0ꢂ10.7 )
1ꢂ10ꢀ4
(ꢃSMAX
)
Q-Ac
Q-Bu
Q-Is
Q-Ca
Q-De
Q-La
Q-Mi
Q-Pa
1.5ꢂ10ꢀ1 (4.3ꢂ10ꢀ4
1.5ꢂ10ꢀ1 (4.0ꢂ10ꢀ4
1.5ꢂ10ꢀ1 (4.0ꢂ10ꢀ4
)
)
)
2
2
ꢂ10ꢀ.94
1ꢂ10ꢀ4
ꢂ10ꢀ.75
4ꢂ10ꢀ4
2.6ꢂ10ꢀ4
2ꢂ10ꢀ4
The concentrations yielding a lipase inhibition of 16%
(IC16) and 50% (IC50) were calculated from the I (%)
versus concentration curves according to Tallarida and
Murray.15
1.0ꢂ10ꢀ1 (2ꢂ10ꢀ4
)
9.7ꢂ10ꢀ7
1.1ꢂ10ꢀ5
5.7ꢂ10ꢀ6
4.5ꢂ10ꢀ6
1.3ꢂ10ꢀ6
2ꢂ10ꢀ5
1.5ꢂ10ꢀ1 (3.7ꢂ10ꢀ4
1.5ꢂ10ꢀ2 (3.3ꢂ10ꢀ5
1.5ꢂ10ꢀ2 (3.1ꢂ10ꢀ5
)
)
)
1.8ꢂ10ꢀ4
(ꢃSMAX
(ꢃSMAX
)
)
1.1ꢂ10ꢀ2 (1ꢂ10ꢀ5
)
5.2ꢂ10ꢀ6
Statistics
aHighest concentration at which each substance was tested expressed
both as mg/mL and mol/L (M).
bInhibitory concentrations (IC) 16 and 50% calculated from the
inhibition versus concentration curves, according to Tallarida and
Murray.15
Linear regression analysis was performed using the
software Sigma-Plot 6.0 (SPSS, Chicago, IL, USA).
Results
Preparation of 3-O-acylquercetines
3-O-Acetylquercetine (1). Isolated in 94%. Anal. calcd
for C17H12O8: C, 59.31; H 3.51, found: C 59.71; H 3.70.
3-O-Butirrylquercetine (2). Isolated in 90%. Anal. calcd
for C19H16O8: C, 61.29; H 4.33, found: C 61.70; H 4.60.
3-O-iso-Butirrylquercetine (3). Isolated in 92%. Anal.
calcd for C19H16O8: C, 61.29; H 4.33, found: C 61.72; H
4.58. 3-O-Hexanoylquercetine (4). Isolated in 90%.
Anal. calcd for C21H20O8: C, 63.00; H 5.03, found: C
63.31; H 5.20. 3-O-Decanoylquercetine (5). Isolated in
76%. Anal. calcd for C25H28O8: C, 65.78; H 6.18,
found: C 65.99; H 6.32. 3-O-Lauroylquercetine (6). Iso-
lated in 75%. Anal. calcd for C27H32O8: C, 66.93; H
6.66, found: C 67.20; H 6.85. 3-O-Miristoylquercetine
(7). Isolated in 78%. Anal. calcd for C29H36O8: C,
67.95; H 7.08, found: C 68.22; H 7.29. 3-O-Palmi-
toylquercetine (8). Isolated in 82%. Anal. calcd for
C31H40O8: C, 68.87; H 7.46, found: C 69.16; H 7.66.
Figure 2. Anti-lipase activity of quercetin its esters. The 3-O-acyl-
quercetines examined here were more active (IC16=inhibitory con-
centration 16) against Candida rugosa lipase than the parent
compound quercetin (Q). In particular, the inhibitory activity of the 3-
O-acylquercetines increased with the length of the acyl chain, as indi-
cated by the good linear correlation (r2=0.91).
Antimicrobial activity
Q and its esters, up to the concentration of 100 mg/mL, did
not show any activity on the microorganisms examined.
acetonitrile/water, containing 0.1% trifluoroacetic acid.
The eluate was monitored at the wavelength of 230 nm
with a sensitivity of 0.8 A.U.F.S. using a recorder chart
speed of 0.5 cm/min. All chromatographic analyses were
done at room temperature. The chromatographic sys-
tem consisted of a controller (Pharmacia-LKB, model
LC 2152), a precision pump (Pharmacia-LKB, model
2150), a high pressure mixer, a variable wavelength
monitor (Pharmacia-LKB, model 2151), and a peak
area integrator (Waters, model 749).
Anti-HIV activity
Q, but not its acetyl or palmitoyl esters, at the con-
centration of 40 mM, inhibited about 80% of HIV-1
reproduction, similarly to AZT at 3.7 mM.
Anti-lipase activity
Q and selected 3-O-acylquercetines inhibited C. rugosa
lipase with IC16 in the range 10ꢀ4–10ꢀ6 M (Table 1).
When the IC16 were plotted against the length of the
acyl chain, a linear correlation was found (Fig. 2).
Analysis of lipase inhibitors. The substances under eva-
luation were dissolved at their maximum solubility in
methanol, serially diluted and analyzed as described
above. Lipase inhibition (I) was calculated by measur-
ing free b-N resulting from the enzymatic reaction,
according to the following formula:
Discussion
AUCc ꢀ AUCblank
In the present study, a good antiviral (against HIV-1) but
not antimicrobial activity of quercetin (Q) was observed.
½Ið%ފ ¼ 1 ꢀ
ꢁ 100
X;c
AUC0 ꢀ AUCblank

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M. T. Gatto et al. / Bioorg. Med. Chem. 10 (2002) 269–272
. Q was reported by other authors to inhibit differ-
favorable pharmacokinetic properties, when applied to
the skin or mucosae, which deserve to be further studied.
ent microorganisms such as E. coli, Klebsiella
pneumoniae, Bacillus cereus, A. parasiticus, A. fla-
vus,6 S. aureus, S. epidermidis, B. subtilis, M. luteus
and E. coli.5 However, these authors used higher
concentrations (100–200 and 500 mg/mL, respec-
tively) than the maximum concentration tested
here (100 mg/mL), at which no significant inhibi-
tion of five gram-positive strains (S. aureus, B.
subtilis, L. ivanovi, L. monocytogenes, L. serligeri),
five gram-negative strains (E. coli, S. flexneri, S.
sonnei, S. enteritidis, S. tiphymurium) and four
yeasts (two strains of C. albicans and two strains
of C. glabrata) was observed. Besides, our attempt
to augment the activity by introducing an acyl
group on the C-3 position, thus generating highly
lipophilic derivatives, did not succeed.
In conclusion, we have described here for the first time
the anti-lipase activity of Q, and its C2–C16 3-O-acyl-
esters, which could be of pharmacological interest for
the development of new antimicrobic agents.
References and Notes
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. In addition, we confirmed the known anti-HIV
activity of Q,11 which might depend on the pre-
sence of a free hydroxyl in the C-3 position, as
suggested by the lack of activity of the 3-O-acyl-
quercetines examined.
Moreover, for the first time to our knowledge, we
described the inhibition of C. rugosa lipase by Q and its
esters (3-O-acylquercetines), which appeared even more
active (Table 1, Fig. 2). The reason for the latter phe-
nomenon is not clear yet, but in our opinion the trend
observed in the experiments reported in Table 1 could
be explained in terms of lipophilicity degree of the sub-
strates considered. It is plausible that, as observed for
other lipases,16 the inhibitory effect of Q may be related
to the phenolic hydroxyls located on its flavane frame-
work. The introduction of an acyl group on the C-3
position did not affect the inhibitory faculty, improving
the lipophilicity. Consequently, by elongating the alkyl
chain in the acyl group, a better affinity is favored for
the space around the active site of the lipase, whose
nature is hydrophobic.17
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In vivo, the anti-lipase properties of Q could increase its
efficacy against lipase producing microorganisms. In
fact, it is known that the production of extracellular
lipases improves the ability of certain microorganisms
to colonize the external surfaces of the host: (a) free
fatty acids derived from the hydrolysis of endogenous
lipids can increase the adherence of S. epidermidis18 and
Propionibacterium acnes19 to the skin; (b) lipase-produ-
cing fungal dermatophytes can efficiently colonize the
keratinized layers of the skin.20,21 Thus, lipase inhibitors
such as Q could be useful adjuvants in the therapy of
infective diseases due to lipase-producing microorgan-
isms. In particular, the 3-O-acyl-quercetines not only
appeared more active than both Q and the reference
compound tetracycline22ꢀ25 but being more hydro-
phobic than Q and tetracycline they could have more
17. Grochulski, P.; Bouthillier, F.; Kazlauskas, R. J.; Serreqi,
A. N.; Schrag, J. D.; Ziomek, E.; Cygler, M. Biochemistry
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1997, 40, 465.
22. Hassing, G. S. J. Invest. Dermatol. 1971, 56, 189.
23. Weaber, K.; Freedman, R.; Eudy, W. W. Appl. Microbiol.
1971, 21, 639.
24. Mates, A. J. Invest. Dermatol. 1973, 60, 150.
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D. R. Invest.Ophthalmol. Vis. Sci. 1991, 32, 2970.