Phytochemistry, Vol. 31, No. 12, pp. 4125 4128, 1992 0031-9422j92 $S.OO+O.

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Printed in Great Britain. @ 1992 Pergamon Press Ltd

ISOLATION, SPECTROSCOPY AND SELECTIVE PHYTOTOXIC EFFECTS OF

POLYPHENOLS FROM VEGETABLE WASTE WATERS

R. CAPASSO, G. CRISTINZIO,* A. EVIDENTE and F. SCOGNAMIGLIO

Dipartimentodi Scienze Chimico-Agrarie, Universitd degli Studi di Napoli “Federico II”, 80055-Portici, Italy; *Istituto di Patologia
Vegetale, Universit$ degli Studi di Napoli ‘“Federico II”, 80055-Portici, Italy

(Received in wevised form 15 May 1992)

Key Word Index--Olea europea; Oleaceae;olive oil waste waters; polyphenols; acetates; chromatographic isolation;
phytotoxicity.

Abstrac&--Catechol, 4-methylcatechol, tyrosol and hydroxytyrosol were isolated and characterized as the main
pol~h~nols from olive oil mill waste waters. In addition, the corresponding acetates were prepared. In ph~otoxicity
assays carried out on tomato (Lycopersicon esc~~enru~) and vegetable marrow (Cucurbita pepo) plants, the compounds
were selectively toxic, except for 4-methylcatechol and its acetate. The vegetation waters remained phytotoxic even
after total extraction of the polyphenols, suggesting that other chemical products contribute to the overall
phytotoxicity.

Su~quently, compounds 1-4 were ~a~fo~ed into

the corresponding acetates 58. The derivatization was
The waste waters from olive oil mill (VW) are character-
performed in order to compare the biological behaviour
ized by a strong polluting activity [l-S]. Many articles
of the phenolic compounds l-4 with that of their acetates
describe marked phytotoxic effects of these waste waters
5-8. Compounds 1-8 were identified and characterized
[S-9], causing a very serious problem owing to their
by ‘H and ‘%NMR, EI- and FAB-MS, UV and IR
spreading on land suitable for agriculture. The study of
spectroscopic analysis (see Experimental). For the first
ecological problems raised by the disposal of these waste
time 4-methyIcat~hol(2) has been found to occur in olive
waters and researches to find methods for their
oil mill waste waters.
detoxification and recycling, have prompted the authors
All the phenols identified were assayed for phytotox-
to study both the organic extracts and the resulting
icity on tomato and vegetable marrow. As indicated in
exhausted solution of VW, in order to identify the
Table 2, 2 and the corresponding diacetate (6) showed
compounds responsible of the antimicrobial [ 1.01,insect
maximum phytotoxicity and aspecificity. Hydroxytyr-
ovipositional deterrent [l l] and phytotoxic activity
osol (4) and its triacetate (8) were harmful only to the
[5-91. This paper reports the ~hromatographic isolation,
vegetable marrow plants, while catechol (1), tyrosol (3)
spectroscopic characterization and phytotoxic effects,
and the diacetates 5 and 7 were toxic only to tomato
assayed on tomato and vegetable marrow testing plants,
plants. In general, the most harmful compounds were
of the main polyphenols which occur in the VW organic
catechol, 4-methylcatechol and their diacetates.
extracts and of the corresponding acetates.
The aspecific biological behaviour of 2 and 6 can be
explained by their higher lipophilicity in comparison with
RESUL’B AND DISCUSSION

From the three chromatographic methods used to purify

the polyphenols occurring in the organic extracts of olive
oil mill waste waters [12], the second method proved to
be the best, a procedure consisting of chromatography
under low pressure of an ethyl acetate extract of VW on a
C-8 reverse phase column. Furthermore, the procedure
permitted the separation, in a single step, of the four main
phenolic components: catechol (l), 4-methylcate- R1 R3
chol (2), tyrosol (3) and hydroxytyrosol (4). By a further
separation on the same column, phenols 1, 2 and 4 were 1 OH H
obtained as chromatographically pure and spectroscopi- 2 OH
l?fs2
cally homogeneous compounds, while 3 was crystallized 3 OH CH2(7-kSJH
from chloroform. The other two chromatographic meth- 4 OH &t&H2OH
ods (Table 1) required more time and yielded polyphenols
5 UAc H
in smaller amounts. When using the first method, only
tyrosol and hydroxytyrosol could be purified, a further 6 OAC
P
purification step on a C-8 reverse phase column was 7 OAC C&&2-
necessary to separate catechol and 4-methylcatechol. 8 OAc &i$H,c

4125
PXY31: 12-6
4126 R. CAPASSO et al.

Table 1. Isolation of polyphenols 1-4 (comparison percolation, and on a prepacked silica gel column (240 x 10 mm,
of the etTrciency, mg/comp. of three chromatographic Merck, Lobar Ltchroprep Si 60 mesh, 40-60 pm) under Iow
methods for purifying the extracts from 300 ml veg- pressure (3 bar). The columns were &ted with C6H,-
etation waters) EtOAc-MeOH f6:3 : 1) (eluent A). The purifications were also
performed on a przpacked C-8 reverse phase column j240
Chromatographic x 10 mm, Merck, Lobar Lichroprep RP-8. 40- 60 urn) &ted
method* 1 2 3 4 with H,Q-MeCN (4: 1) (eluent B) under low pressure (3 bar).
Analytical TLC was performed on silica gel plates (20 x 20 cm,
First 34 12 20 60 Merck, Kieselgel 60 F,,,. 0.25 mm) with soivent system A and
Second 126 58 63 144 on reverse phase plates (20 x 20 cm, Whatman Stratocrom C-18,
Third 37 IS 28 42 0.2 mm) with mixture B. The spots were visualized by exposure
to UV light selected at 254 nm and/or by spraying the plates first
*See Experimental with 10% H,SO, in MeOH and then with 3% phosphomolibdic
acid in MeOH, followed by heating for 5 min at 105‘. In
Table 2. Effects of the phenolic compounds and corresponding addition, the chromatograms were also sprayed with a solution
acetates on tomato and vegetable marrow after 48 hr at 25” of 1% FeCI, in H,O, and then heated for 5 mm at 105”.
LR spectra were recorded in CHCI, or neat. UY spectra were
obtained in MeOH. rH NMR and “CPND (Briton-Norse
Wilt index* Decoupled) and DEPT (Distorsionless Enhancement by Polar-
ization Transfer) spectra were recorded in CDCl, or in CD,OD
Tomato Vegetable marrow at 270 and 67.92 MHz, respectively. EI mass spectra were
Compound 10 - 2 M 1O-3 M IO-* M 1O-3 M recorded at 70 eV, FAB mass spectra were recorded using Xe as
the ionizing beam. Mp: uncorr.
3.85- a: 3.8 a 0.1 c 0.0 c Extraction and c~ro~uzo~~~~~i~idatim. Fresh olive oil mill
3.8 a 3.0 b 0.1 c 0.0 c waste waters (VW) were supplied by mills from the Avellius
3.8 a 3.8 a 3.6 a 3.5 a province (Italy). Fresh VW (400 ml, pH 5.5) were acidified to pH
3.8 a 3.8 a 3.6 a 3.5 a 4.5 with cone HCI and extracted with EtOAc (4 x 400 ml). The
3.6 a 1.0 c 0.1 c 0.0 c organic extracts were combined, dried and evapd under red. pres.
3.6 a 1.0 c 0.0 c 0.0 c The residue (3.175g) was divided into 3 ahquots, which were
0.1 b 0.0 d 2.1 b 1.9 b purified using 3 chromatographic methods. First rne~~o~. An
0.1 b 0.0 d 3.6 a 2.0 b aliquot of the above extract (2 g corresponding to 300 ml VW)
was ch~omatographed on a silica gel column (120x 3.5 cm)
*The Wilt index is determined on a scale O-4, where O=no eluted with solvent system A. Eight groups of homogeneous frs
symptoms, 1% 5-15% collapse; 2 = 2650% collapse; 3 = 51-75% were collected; the second fr. (128 mg) was further purified on a
collapse; 4 = 76-100% collapse. reverse phase C-8 prepacked column eluted with mixture B
tEach value is the average of four experiments (four replicates (under low pressure; flow rate af 1 ml/IO set). Two frs were
per experiment). collected and lyophili~d. The co~~ponding residues, which
fMeans in the same column followed by the same letter are, were shown to be homogeneous by reverse phase TLC analysis,
according to Duncan’s test, not statistically different (P=O.l). were, respectively, catechol, (1,33.8 mg) and 4-methylcatechoi (2.
11.7 mg). The fourth (150 mg) fr. from the initial silica gel column
was further sepd using another silica gel column (50 x 2 cm),
the other compounds. Moreover, the acetates 5-g show- eluted with solvent system A to produce 3 groups of homogen-
ed the same phytotoxic behaviour as the parent com- eous fractions: the third of these frs (42.9 mg) was crystallized
pounds 1-4, suggesting a possible enzymatic hydrolysis from CHCI,, producing a crystalline product (20 mg) which was
in the plant of 5-8 into the corresponding 1-4. Such identified as tyrosol(4-hydroxyethylphenot, 3). by analysis. The
biological behaviour is frequently called ‘lethal metabol- sixth (142.1 mg) of the 8 frs mentioned above was purified by a
ism’ [I3]. further step on a silica gel column (50 x 2 cm) giving rise to a
In order to support this hypothesis, the acetates 5-8 product (60 mg) which was homogeneous by silica gel TLC. This
were dissolved at 10Vz and toe3 M in sterilized water compound was identifi~ as hydroxytyrosol (4-hydroxyethyl-
and the solutions were kept for 48 hr under the same 1,2-benzenediol, 4).
ex~rimental conditions as those used for the phytotoxic Second period. A second aliquot of the extract (145 mg), was
assays. TLC analysis of these samples, performed as chromatographed on a C-S reverse phase column (240
indicated above, did not reveal any chemical modihca- x 100 mm) eluted with mixture I3 under low pressure, at a flow
tion. rate of 1ml/10 sec. Four groups of homogeneous frs were
Moreover, the exhausted waste waters, deprived of collected. The residues obtained from the first, third and fourth
their polyphe~o~s by repeated extractions with ethyl fr. (14.1, 18.8 and 7.2 mg, respectively) were further purified on
acetate and n-butanol (TLC control), still showed phyto- the same column, eluted with mixture B yielding 3 pure com-
toxicity. This result suggests that the ph~otoxi~ity of the pounds, which were, respectively, identified as hydroxytyrosol
waste waters should be attributed not only to the poly- (3.5 mg), eatechol(7 mg) and 4-methylcatechol(3.2 mg), by TLC
phenofs, which were shown to be selectively toxic, but comparison with authentic samples. Finaily, the residue of the
also to other organic and/or inorganic compounds. second fr. group (7.1 mg) was crystallized from CHCI,, giving
rise to a crystalline compound (3.5 mg), identified as tyrosol.
Third me?~o~. A third aliquot of the extract (690 mg) was
purified on a prepacked silica gel column eluted with mixture A
General, The chromato~aphjc purifications were carried out under medium pressure, at a flow rate of 1 ml/20 sec. Six
on silica gel columns (Merck, Kieselgel 60,0.063-0.2 mm) by homogeneous fractions were collected. The residue left from the
 Phytotoxic effects from vegetable waste waters
second group (144.3 mg) was chromat~8raphed on a C-8 reverse
phase column eluted with solvent system B under medium
pressure, at a flow rate of l mlj2Osec. Three frs were collected.
The residues obtained from the first (12-4 mg) and the second
(6.0 mg) fraction were chromatographically pure and by TLC
comparison with authentic samples identified as catechol and 4-
methylcatechol. An ahquot (97 mg) of the third fr. group, origin-
ally obtained by silica gel CC, was fur&her purified on another
silica gel column (33 x 3 cm) and 3 groups of homogeneous
fractions collected_ The residue of the first fr. (~rystaI1ized from
CHC1,) and that of the third fr. were ~hromatographi~~l1y pure
and were, respectively, identified as tyrosol (9.4 mg) and
hydrox~yrosol (14mg), by TLC comparison with authentic
samples.
Care&of (1). ‘HNMR, IR, EIMS and UV data were in
accordance with the literature [14-17”“.
4-~~~~~~~a~~chol(2). ‘H NMR, IR, BIMS and UV data were
in accordance with the titerature f14-171.
Tyrosol(3). ‘H NMR, IR, EIMS and WV data were in accard-
ante with the Iiterature 1181. In addition, the authors report the
following new data: *3CNMR (CD,GD), 6: 156.7 (s, C-l), 130.9
(s, C-4); 130.8 (cJ,C-3 and C-5), 116.0 (d. C-2 and C-6), 64.5 (t, C-
2), 39.3 0, C-1’).
~y~rox~~o~o~ (4). UV and EIMS data were in accordance
with the literature 1191. In addition the authors report the
following new data: “H NMR (CDCI,): 6.67 (lH, d, J,,, x8.0 Hz,
H-6), 664(lH, d&,=2.1 Hz, H-3), 6.52 (lH, dd, J,,,=2.1 Hz,
J,.,=S.O Hz, H-S), 3.66(2H, t, J,.*,.=7.3 Hz, H&Y), 2.66(2H, t,
J ,.,,?=7.3 Hz, Hz-l’); IR vgi cm-‘: 3387 (phenohc and aleo-
hohc GHX 16O7,1548 and 1447 (benzene ring).
~~~~y~~~~c~o~ (5). Mp 63-64” (CHCl& ‘H NMR (CDCIsk
6: 7.25 (2H, m, H-4 and H-S), 7.19 (2H, rpt,H-3 and H-6), 2.29 (6H,
s, 2Ac); IR v$;;i, cm-‘: 1762 (C=O, ester), 1372 (Me of acetyl
groups), 1209 (C-G, ester); EIMS, m/z (tel. int.): 194 [M] * (l4),
152 [M-CH,CO]+ (33), 110 [M-ZCH,CO]+ (lOO), 92 [M
-2CH,CO-H@]’ (3), 81 [M-2CH,CO-CHO]+ (14), 64
[C,Hd+ (lo), 55 [CsH,OH]+ (14); UV >wXnm (a): 209 (44503,
260 (280).
Diacetyl-Pmethylcatefhot (6). Mp 52-53” (CHCI,); ‘H NMR
(CD&), 6 7.03 (2H, br d, J5,6 = 8.0, H-5 and H-6), 6.97 (lH, br s,
H-3), 2.33 (3H, s, AC),2.27 (6H, s, Me and AC);IR vE$iz cm-i:
1771 (C=O, ester), 1507 and 1271 (Me of acetyl groups), X214
(C-O, ester); EIMS, m/z (re1. int.): 208 [MI’ (5), 166 CM
-CHaCG]* (15)s 124[M-2CH,CO]” (10O),77 [C,H,]* (IO),
65 [C,H,J* (5), 43 [AC]’ (Sot; UV 2tiXI nm (s): 271 (4O8), 266
(440) and 203 (6900).
Diacetyityrosol (7). ‘HNMR (CDCl&, 6: 7.22 (2H, hd, J,,,
=J,,, =8.0 Hz, H-3 and H-S), 7.02 (ZH, brd, Jzqs =J5,6 ~8.0 Hz,
H-2 and H-6), 4.27 (2H, t, J , 2.= 7.3 Hz, H,-2’), 2.93 (2H, t, Jrs,x.
= 7.3 Hz, Hz-l’), 2.29 (3H, s, kc), 2.04 f3H, s, AC);IR vale cm - ‘:
1765 (C =O, phenolic ester), 1737 (C=O, alcoholic ester), 1509
and 1368 (Me of acetgl groups), 1595 and 1400 (benzene ring),
1200 (C-O, ester); EIMS, m/z frel. int.): 162 [M -AcOW]’ (52),
120 [M-AcOH-CH,CO]+ (loo), 107 [M-CH,CG-
CH,OAc]+ (90),91 [C&H,]+ (23),77 [C,HS]+ (25),65 [C&J+
(10); UV v,,. nm (8): 212 (9140) and 263 (507).
TriacetylRydrrtxytyrosoI (4-aeetoxyethyf-1,2-diacetoxy6errz-
ime_ 8). ‘HNMR, (CD,OD), 6: 7.13 (lH, d, Js_,=8.0 Hz, H-6),
7.lOflH, d, Js,x==Z.f Hz, H-3), 7.08 (lH, dd, J,,,=2.1 Hz, J,,,
=8.0 Hz, H-5), 4.27 (2H, r, J1,2.=7.3 Hz, H,-2’), 2.92 (ZH, t,
J 1,,2,=7.3 Hz,&l’),2.29(3H,s,Ac),2.28(3H,s,Ac),2.04(3H,s
Ack i3CNMR (CDCI,), 6: 170.9 (s, C&G), 168.2 (s, C=G), 168.1
(s, C-O), 142.0(s, C-2), 140.8 (s, C-l), 136.7 (s, C-4), 126.7 (d, C-5),
123.8 (d, C-3), 123.3 id, C-Q, 64.3 (t, C-2’), 34.40, Cl’), 20.8,20.6
and 20.6 fs, 3Ac); IR vz3 em-‘: 1770 (C=O, phenolie ester),
t735 (C=O, alcohdic ester), 1508 and 137l (methyt of acetyf
4127
groups), 1259 (-0, ester); EIMS, m/z (rel. int.): 279 [M-H) +
(12). 238 CM-CH,CO]+ (14) 220 CM-AcGH]+ (lo), 1% [M
-ZCH,CO]’ (8X 178 [M-CR&O-A&H]+ (13), 136 EM
-ZCH,CG--AcOH]’ (10O), 107 [M-AcOH-2CH,CO
-HCG] + Es), 77 CC,H,]+ (St, 65 [C,H,]+ (5), 43 [AC]’ (90);
FAB-MS (positive ions): 281 [MH]+, 239 [MB-CH,CO]+,
221 [MH-AcGH]+, 179 [MH-CH,CO-AcOH]*; UV
A,,,,,,nm (8): 267 (380), 264 (420), 205 (7030)
Bioassay. The phytotoxicity of the compounds on cv “Mar-
mande” tomato and cv ‘S. Pasquafe” vegetabb marrow phurts
was tested using the technique described in ref. CZO].First leaf
stage plant cuttings were dipped with their cut ends in 2 ml of the
test soln and stored at 25”. Wilt symptoms were evaluated after
48 hr and rated on a wilt index scale of O-4, where 0= no
symptoms, l-5-25% collapse, 2=26-500/o collapse, 3
= 51-75% collapse and 4= 76-100% &lapse. B~dist~1~ Hz0
or 5% MeOH solns were used as controf. Data were analysed
using ANOVA and means were compared using Duncan’s
multiple range test. Seedlings without roots were immersed in
vials containing 2 ml of the test solution at 25” and wilting was
evaluated after 48 hr.
Tesring by TLC analysis of the efiemkaf stability of aceryfderi-
vatitvs oj the stated ~uiy~~e~~~s. &Ins 10-a and 10e3 M of
~orn~~ds 5-B in 2 mf sterilized H,O were analysed using TLC
(silica gel p1ates, eluent A and the described chromogenic sprays),
after 24 and 48 hr.
Ex~~ti~e extraction of phenok from vegetation waters. A
sample of vegetation waters (14 ml) was extracted with EtOAc (4
x 20 ml) and then with n-BuOH (4x 19 ml). The combined
extracts were dried on Na,SO, and evapd under red. pres.,
Ieaving a brown residue. This was redissolved in MeGH ft. m1)
and analysed by TLC (silica gel plates, eiuent A and the
described chromogenic sprays). The remaining aq. soln was dried
under N, and the residue obtained was redissolved in sterilized
H,O, and both analysed by TLC for phytotoxicity.
~~~W~d~e~~f~Th~s jnvest~at~on was supported in part by
grants from the Italian Ministry of University and Scientific and
T~hnolo~ca1 Research and in part by the Italian National
Research Council, Comitato Science Agrarie. MS were provided
by the Servizio di Spettromctria di Massa de1 CNR e
de1~Un~vers~t~di Napoli “Federico II”, the assistance of staff is
gratefully acknowledged.
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