Kaempferol glycosides from Lobularia maritima and their potential role in plant interactions

Article abstract of DOI:10.1002/cbdv.200800097

Six kaempferol glycosides, four of them characterized for the first time, were isolated from the leaf extract of Lobularia maritima. The structural elucidation was performed by a combined approach using Electrospray-Ionization Triple-Quadrupole Mass-Spect

Full text of DOI:10.1002/cbdv.200800097

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Kaempferol Glycosides from Lobularia maritima and Their Potential Role in
Plant Interactions
by Antonio Fiorentino*, Andreina Ricci, Brigida DꢀAbrosca, Annunziata Golino, Angelina Izzo,
Maria Teresa Pascarella, Simona Piccolella, and Assunta Esposito
`
Dipartimento di Scienze della Vita, Laboratorio di Fitochimica, Seconda Universita degli Studi di
Napoli, via Vivaldi 43, I-81100 Caserta
(phone: þ39-0823-274576; fax: þ39-0823-274571; e-mail: antonio.fiorentino@unina2.it)
Six kaempferol glycosides, four of them characterized for the first time, were isolated from the leaf
extract of Lobularia maritima. The structural elucidation was performed by a combined approach using
Electrospray-Ionization Triple-Quadrupole Mass-Spectrometric (ESI/TQ/MS) techniques, and 1D- and
2D-NMR experiments (1H, 13C, DEPT, DQ-COSY, TOCSY, ROESY, NOESY, HSQC, HMBC, and
HSQC-TOCSY). The isolated kaempferol derivatives have different disaccharide substituents at C(3)
and four of them have a rhamnose unit at C(7). To evaluate their potential allelopathic role within the
herbaceous plant community, the compounds, as well as the aglycone obtained from enzymatic
hydrolysis, have been tested in vitro on three coexisting plant species, Dactylis hispanica, Petrorhagia
velutina, and Phleum subulatum. The results obtained allow us to hypothesize that the type of the sugar
modulates the biological response. The bioassay data, analyzed by a multivariate approach, and grouping
the compounds on the basis of the number of sugar units and the nature of carbohydrates present in the
disaccharide moiety, indicate a structure–activity relationship.
1. Introduction. – Plant–plant interactions play a fundamental role in the
mechanisms regulating plant community composition and structure. In fact, these
interactions should be considered among the most important factors affecting seed
germination, seedling growth, and mortality, thus influencing plant population
dynamics. It is well-known that these interactions could be responsible for inhibiting
or stimulating effects, in relation to target species and environment, which are essential
in niche regeneration and biodiversity preservation [1][2]. Although it is generally
accepted that the coexistence of plant species depends mainly on the competition
mechanisms between plants and on abiotic factors, there is increasing evidence of the
involvement of allelopathy in this phenomenon [1][3]. In fact, compounds secreted by
plants (allelochemicals) can significantly affect the biological processes of neighboring
plant individuals [1][4][5]. However, very few studies have investigated the species-
specific effects on coexisting plants, and on the structural and functional processes at
the population and community level [2][6][7].
In recent years, our studies on allelopathy have dealt with the structural
characterization of allelochemicals [8] and with the evaluation of their potential
ecological role in regulating the high level of biodiversity in herbaceous plant
communities of Mediterranean vegetation [9].
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Recently, we reported the allelopathic interference of secondary metabolites
isolated from Melilotus neapolitana, a stenomediterranean species frequently occurring
in this plant community, on three coexisting species, Dactylis hispanica, Petrorhagia
velutina, and Phleum subulatum. Results evidenced the relationship between the
distribution pattern observed in the field and the phytotoxic effect of coumarin, the
most abundant metabolite isolated [10].
This work aims to investigate the potential allelopathic interference of Lobularia
maritima (Brassicaceae), a stenomediterranean and chamaephyte subfructicose
species, characteristic of the pioneer stage of the same Mediterranean plant community.
Previous phytochemical investigations on L. maritima and other Brassicaceae species
led to the discovery of the presence of acylated cyanidin 3-sambubioside-5-glucosides
[11], glucosinolates [12], and fatty acids [13].
In particular, in this article, we report: 1) the isolation and structural character-
ization of kaempferol glycosides from L. maritima, four of them isolated for the first
time; 2) the in vitro evaluation of their potential interference role on the three coexisting
herbaceous species Dactylis hispanica, Petrorhagia velutina, and Phleum subulatum.
All of the structures were elucidated by using an effective approach based on the
combination of spectroscopic 1D- and 2D- (1H, 13C, DEPT, DQ-COSY, TOCSY,
ROESY, NOESY, HSQC, HMBC, and HSQC-TOCSY) NMR, and mass spectrometric
(ESI/TQ/MS) techniques.
2. Results and Discussion. – 2.1. Structure Elucidation. The phytochemical
investigation of Lobularia maritima leaf extract led to the isolation of six kaempferol
glycosides. The joint application of NMR (DQ-COSY, TOCSY, HSQC, HMBC,
NOESY, and ROESY) and mass-spectrometric techniques (ESI/TQ/MS) allowed us to
identify four new flavonol glycosides, 1–4, together with two known flavonol
glycosides, i.e., 5, previously isolated from the Crassulaceae Sedum telephium, ssp.
maximum [13], and 6 reported as a component of Astragalus shikokianus [14].
On the basis of the molecular mass and the structural information obtained by ESI-
MS and 13C-NMR techniques, a molecular formula C26H28O15 was attributed to
compound 1.
In the aromatic region of the 1H-NMR spectra, besides two meta-coupled doublets
at d 6.47 (HꢀC(8)) and 6.21 (HꢀC(6)), an AA’BB’ spin system was evident as two
doublets at d(H) 8.06 (HꢀC(2i), HꢀC(6i)) and 6.91 (HꢀC(3i), HꢀC(5i)).
In the upfield region of the spectrum, there were signals due to a saccharidic unit as
two anomeric H-atoms (d 5.58 HꢀC(1ii) and 4.35 HꢀC(1iii)) and further eleven H-
atoms (d(H) 3.4 to 4.2).
The 13C-NMR spectrum (Table 1) showed 24 signals for 26 C-atoms (two signals
were generated by two homotopic C-atoms), identified, on the basis of DEPT
experiments, as two CH2, 13 CH groups, and nine tetrasubstituted C-atoms. The HSQC
experiment allowed a complete assignment of all H- and C-atom resonances. The
1
values of the aromatic signals of both the H- and 13C-NMR spectra suggested the
presence of kaempferol (¼ 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-
one) as aglycone, which was confirmed by the HMBC experiment, and by chromato-
graphic and spectroscopic comparison of the product obtained by acidic hydrolysis with
an authentic sample.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Useful structural information was supplied by mass spectrometry. MS/MS Analysis
for the structural characterization of glycosyl flavonoids has been recently reviewed
[15][16].
Collisionally activated dissociation (CAD) mass-spectrometric experiments were
performed on protonated, deprotonated, and sodiated ions. In Table 2, the CAD mass
fragments observed and their relative abundances are listed. The [MþH]þ CAD
spectrum of compound 1 (m/z 581) showed fragments at m/z 419 and 287,
corresponding to the loss of an hexose (162 Da) and a pentosylhexose residues
(132þ162 Da), respectively. The CAD mass spectrum of the ions at m/z 287, generated
in the ESI source by using higher cone voltage, displayed the characteristic fragments of
protonated kaempferol [17][18].
The fragmentation pathway observed for the [MþNa]þ and the [MꢀH]ꢀ ions
reported supported the structural hypothesis regarding the presence of the disaccharide
moiety and the sugar sequence obtained by the [MþH]þ CAD spectrum. In the [Mꢀ
H]ꢀ CAD mass spectrum of compound 1, both ions were observed at m/z 285 and 284.
Their intensities ratio suggests 3-O-glycosylation of the kaempferol [19].
MS Evidence was confirmed by 2D-NMR experiments. In fact, the HSQC-TOCSY
experiment showed correlations between the two spin systems of the disaccharide H-
atoms and the C-atoms C(1iii), C(2 iii), C(3iii), C(4iii), C(5iii), and C(6iii) (d(C) 104.0,
73.6, 76.8, 69.6, 76.7, and 60.8, resp.), of an hexose moiety, and the C-atoms C(1ii),
C(2ii), C(3ii), C(4ii), and C(5ii) of a pentapyranose unit. The chemical-shift values of the
H- and C-atoms of the monosaccharide units were in good agreement with those of a
glucopyranose and a xylopyranose. To confirm the identity of the sugar moiety, the
glycoside 1 was hydrolyzed with 2n CF3COOH (TFA). The HPLC analysis of the
reaction products, compared with pure standards, indicated the presence of a glucose

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
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Table 1. 13C-NMR Data of the Flavonol Glycosides 1–6 from L. maritima Recorded in (D6)DMSO
Position
1
2
3
4
5a)
6b)
2
3
4
5
6
7
8
9
156.1
133.9
177.5
160.1
98.9
164.3
93.6
156.2
104.0
120.4
131.0
115.4
161.1
115.4
131.0
98.7
78.8
76.8
68.7
64.1
–
104.0
73.6
76.8
69.6
76.7
60.8
–
–
–
–
–
156.2
133.9
177.4
160.0
99.2
164.2
93.7
156.2
104.0
120.6
130.8
115.2
161.2
115.2
130.8
98.7
74.6
72.6
66.3
64.0
–
100.7
70.6
70.6
71.8
68.6
17.5
–
–
–
–
–
155.9
133.2
177.7
160.9
99.4
155.9
133.2
177.6
160.2
99.4
155.9
133.0
177.5
160.9
99.4
155.9
133.0
177.5
160.9
99.4
161.6
94.4
161.5
94.4
161.6
94.5
161.5
94.5
156.1
105.5
120.7
131.1
115.3
160.2
115.3
131.1
98.4
80.4
77.0
69.8
75.9
60.7
104.2
74.4
77.0
69.7
76.6
60.0
98.4
70.1
156.1
105.6
120.7
131.1
115.3
160.2
115.3
131.1
98.4
80.3
74.3
69.8
75.9
60.7
104.2
74.4
77.0
70.0
76.5
60.0
98.4
70.1
156.6
105.7
120.7
130.9
115.1
160.1
115.1
130.9
98.3
77.6
77.5
69.8
77.2
60.8
100.6
70.6
70.6
71.6
68.3
17.2
98.4
70.0
156.6
105.6
120.7
130.9
115.1
160.1
115.1
130.9
98.7
75.1
73.9
69.8
75.7
60.2
100.6
70.6
70.6
71.6
68.2
17.2
98.4
70.1
10
1i
2i
3i
4i
5i
6i
1ii
2ii
3ii
4ii
5ii
6ii
1iii
2iii
3iii
4iii
5iii
6iii
1iv
2iv
3iv
4iv
5iv
6iv
70.2
71.6
67.6
17.9
70.2
71.6
67.6
17.9
70.2
71.8
68.3
17.9
70.2
71.8
68.5
17.9
–
–
a) Data assigned in accordance with Mulinacci et al. [20]. b) Data assigned in accordance with Yahara
et al. [14].
and xylose. The coupling constant of the anomeric H-atom of the glucose (7.8 Hz) was
in accordance with that of the b anomer, while the coupling constant of the anomeric H-
atom of the pentose (2.1 Hz) was in accordance with an a-xylose. This latter H-atom (d
5.58; HꢀC(1ii)) correlated, in the HMBC experiment, with C(3) (d(C) 133.9) while, in
the DQ-COSY, it showed cross-peaks with the H-atom (d 4.28, HꢀC(2ii)) bonded to
C(2ii) (d(C) 78.8). Both HꢀC(2ii) (d(H) 4.28) and C(2ii) (d(C) 78.8) showed, in the
HMBC experiment, cross-peaks with C(1iii) (d(C) 104.0) and HꢀC(1ii) (d(H) 4.35
HꢀC(1iii) of the b-glucose, respectively. Furthermore, the NOE observed in NOESY
and ROESY experiments between the HꢀC(2ii) (d(H) 4.28) and HꢀC(1iii) (d(H)
4.35) confirmed the presence of a 1!2 glycosidic bond. All these data unequivocally