Isolation of (-)(2S)-5,6,7,3′,5′-pentahydroxyflavanone-7-O -β D-glucopyranoside, from Lippia graveolens H.B.K. var. berlandieri Schauer, a new anti-inflammatory and cytotoxic flavanone

Article abstract of DOI:10.1080/14786419.2010.488234

A new flavanone glycoside, (-)(2S)-5,6,7,3',5'-pentahydroxyflavanone-7-O - D-glucopyranoside (1), was isolated from the stems of Lippia graveolens H.B.K. (Verbenaceae). The structure of 1 was elucidated based on spectral analysis and chemical transformations. The treatment of 1 with acetic anhydride and pyridine afforded the corresponding per-acetylated derivative 2, while an acid hydrolysis reaction of 1 afforded a 5,6,7,3',5'-pentahydroxy flavanone (3). Additionally, the anti-inflammatory and cytotoxic activities of 1, 2 and 3 were determined.

Full text of DOI:10.1080/14786419.2010.488234

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Natural Product Research: Formerly
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Isolation of (-)(2S)-5,6,7,3′,5′-
pentahydroxyflavanone-7-O-β-
D-glucopyranoside, from Lippia
graveolens H.B.K. var. berlandieri
Schauer, a new anti-inflammatory and
cytotoxic flavanone
M.C. González-Guëreca a , M. Soto-Hernández b & M. Martínez-
Vázquez c
a Instituto Politécnico Nacional, Fraccionamiento 20 de
Noviembre, 34220 Durango, Dgo, México
b Colegio de Postgraduados, Km 36.5 Carretera México-Texcoco
56230, Montecillo, Texcoco, Edo de México, México
c Instituto de Química, Universidad Nacional Autónoma de México,
Circuito Exterior, Ciudad Universitaria, Coyoacán 04510, México
D.F., México
Version of record first published: 09 Sep 2010.
To cite this article: M.C. González-Guëreca , M. Soto-Hernández & M. Martínez-Vázquez (2010):
Isolation of (-)(2S)-5,6,7,3′,5′-pentahydroxyflavanone-7-O-β-D-glucopyranoside, from Lippia
graveolens H.B.K. var. berlandieri Schauer, a new anti-inflammatory and cytotoxic flavanone,
Natural Product Research: Formerly Natural Product Letters, 24:16, 1528-1536
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Natural Product Research
Vol. 24, No. 16, 10 October 2010, 1528–1536
Isolation of (Z)(2S)-5,6,7,30,50-pentahydroxyflavanone-7-O-b-D-
glucopyranoside, from Lippia graveolens H.B.K. var. berlandieri
Schauer, a new anti-inflammatory and cytotoxic flavanone
c
lez-Guerecaa, M. Soto-Herna
´
ndezb and M. Martı
´
nez-Va
*
´
zquez
M.C. Gonza
´
¨
a
´
Instituto Politecnico Nacional, Fraccionamiento 20 de Noviembre, 34220 Durango, Dgo,
Mexico; Colegio de Postgraduados, Km 36.5 Carretera Mexico-Texcoco 56230, Montecillo,
b
´
´
c
´
Texcoco, Edo de Mexico, Mexico; Instituto de Quımica, Universidad Nacional Autonoma de
Mexico, Circuito Exterior, Ciudad Universitaria, Coyoacan 04510, Mexico D.F., Mexico
´
´
´
´
´
´
´
(Received 1 February 2010; final version received 17 April 2010)
A new flavanone glycoside, (ꢀ)(2S)-5,6,7,30,50-pentahydroxyflavanone-
7-O-ꢀ-D-glucopyranoside (1), was isolated from the stems of Lippia
graveolens H.B.K. (Verbenaceae). The structure of 1 was elucidated
based on spectral analysis and chemical transformations. The treatment
of 1 with acetic anhydride and pyridine afforded the corresponding per-
acetylated derivative 2, while an acid hydrolysis reaction of 1 afforded a
5,6,7,30,50-pentahydroxy flavanone (3). Additionally, the anti-inflammatory
and cytotoxic activities of 1, 2 and 3 were determined.
Keywords: Lippia graveolens; flavanone; anti-inflammatory; cytotoxic
1. Introduction
Lippia graveolens H.B.K. (syn. Lippia berlandieri Schauer., Verbenaceae) is an
aromatic species and medicinal plant known as ‘Mexican oregano’. In Mexican
traditional medicine, the aerial parts of this species are used as an antiseptic,
antipyretic, analgaesic, abortive, antispasmodic and anti-inflammatory agent and for
the treatment of menstrual disorders and diabetes (Pascual, Slowing, Carretero,
Sanchez-Mata, & Villar, 2001). The chemical composition of the essential oil of
´
L. graveolens has been determined by gas chromatography mass spectroscopy
(GC–MS). A high content of oxygenated monoterpenes was found, its major
constituents being carvacrol, thymol and p-cymene. In addition, the essential oil
demonstrated significant antimicrobial and antioxidant activity (Rocha-Guzma
et al., 2007). As far as other components are concerned, naringenin, pinocembrin,
lapachenole and 10 iridoids have been isolated (Domınguez, Sanchez, Suarez,
Baldas, & Gonzalez, 1989; Rastrelli, Caceres, Morales, De Simone & Aquino, 1998).
´
n
´
´
´
´
Most of the studies of L. graveolens have focused on the chemical composition
and biological activities of the essential oil, but few scientific reports regarding
chemical characterisation of the polar extracts as well as their biological activities are
˜
found (Arcila-Lozano, Loarca-Pina, Lecona-Uribe, & Gonzalez de Mejıa, 2004).
´ ´
*Corresponding author. Email: marvaz@servidor.unam.mx
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786419.2010.488234
http://www.informaworld.com

Natural Product Research
1529
Despite its ethno-medical importance, the main use of this species is of its
essential oils, which are popular condiments for food. Mexico is the largest exporter
of L. graveolens in the world, with 35–40% of the international market. This high
´
demand is due to the quality of essential oil present in the leaf (Gonzalez-Guereca,
¨
Soto-Hernandez, Kite, Martınez-Vazquez, 2007). The collection of leaves for
´
´
´
extraction of essential oils of L. graveolens is an economic activity which is
complementary to the rain-fed agriculture in the arid and semi-arid zones of North
Mexico. Approximately 4000 tons of leaves of L. graveolens are exported every year.
As a result of this activity, large quantities of stems are produced as ‘useless’
residue. In spite of this abundance, chemical and biological evaluations of the stems
of this species have not been carried out. Recently, we published a study on the
presence of the flavonoids pilosin, cirsimartin, narigenin, kaempferol and
isokaemferide, among other compounds, in the EtOAc extract of L. graveolens
stems (Gonza
Now, as part of our systematic study of the isolation of anti-inflammatory and
cytotoxic compounds from natural sources (Flores-Rosete & Martınez-Vazquez,
´
lez-Guereca et al., 2007).
¨
´
´
2008), we wish to report the isolation and structural elucidation of (ꢀ)(2S)-
5,6,7,30,50-pentahydroxyflavanone-7-O-ꢀ-glucopyranoside (1), a new compound
isolated from the EtOAc extract of L. graveolens stems. The treatment of 1 with
acetic anhydride and pyridine afforded the per-acetylated derivative 2, while an acid
hydrolysis of 1 yielded the flavanone 3 (Figure 1). The anti-inflammatory and
cytotoxic properties of 1 and its derivatives 2 and 3 are also reported.
2. Results and discussion
25
Compound 1 was obtained as yellow amorphous powder (½ꢁꢁD –36.5ꢂ), m.p. 187–
189ꢂC. HRMS of 1 gave the quasimolecular ion [M þ H]þ at m/z ¼ 467.1188 (Calcd
467.1190), corresponding to the molecular formula C21H22O12. Its IR spectrum
showed bands at 3394, 3001–2939, 1651 and 1605 cmꢀ1, which together with its UV
absorptions at 215, 288 and 366 nm suggested that 1 could be a flavonoid compound.
1
This proposal is supported by its H-NMR spectrum, where the typical ABX three
proton system signals of a flavanone nucleus were observed at 5.34 ppm (dd, J ¼ 3,
13), 2.67 ppm (dd, J ¼ 3, 17) and 3.21 ppm (dd, J ¼ 13, 17), assigned to the H-2, H-3
eq. and H-3 ax., respectively. These signals correlated, in the HETCOR spectrum,
with the signals at ꢂ 78.65 (C-2) and at 42.64 (C-3), which were observed in the DEPT
spectrum as methine and methylene signals, respectively. A survey of the literature
revealed that the spectral data of 1 were very similar to those of (2S)-5,7,30,50-
tetrahydroxyflavanone-7-O-ꢀ-D-glucopyranoside isolated from Jasminium lanceolar-
ium (Sun, Yang, & Zhang, 2007). However, the molecular formula of the flavanone 1
showed 16 units of mass, in contrast to those found in J. lanceolarium, indicating the
presence of an additional hydroxyl group in 1. The position of the extra hydroxyl
group at C-6 in 1 was deduced as follows. The impact electron mass spectroscopy
(IEMS) spectrum of 1 showed the ion molecular at M 466. Loss of a glucose residue
from 1 was indicated by the peak at m/z ¼ 303. It also showed peaks at m/z ¼ 136 and
168 assigned to fragments of a retro Diels–Alder breakup from the 303 m/z fragment.
Taking into account the published data of similar flavonoids, the signal of an
aromatic methine atom carbon at 94.20 ppm, in the 13C-NMR spectrum of 1, was

´
M.C. Gonzalez-Guereca et al.
¨
1530
OR
2
R =H
1 R =R O
2
1
1
H
H
OH
HO
H
H
R O
O
O
1
OR
2
O
HO
R O
2
H
OH
OR
2
3 R =R =H
R =Ac
2 R =R O
1
2
2
1
1
H
H
OAc
AcO
H
H
O
AcO
H
OAc
Figure 1. Structures of 1–3.
assigned to C-8 (Kawabata et al., 2003; Saracoglu Varel, Sebnem-Harput, &
Nagatsu, 2004). The signal of a quaternary carbon atom at 153.40 showed that
a glucose residue could be in position C-7, as in all the flavonones presented in
L. graveolens (Lin, Mukhopadhyay, Robbins, & Harnly, 2007). All of these
assignments were confirmed by the correlations shown in the HMBC experiment
(Figure 2). The circular dichroism (CD) spectrum showed a positive effect at 316 nm
and a negative one at 289 nm, consistent with the S-configuration at C-2, as in all the
flavanones isolated from L. graveolens (Flores-Rosete & Martınez-Vazquez, 2008).
´ ´
Then the compound 1 was identified as (ꢀ)(2S)-5,6,7,30,50-pentahydroxyflavanone-
7-O-ꢀ-D-glucopyranoside, which, according to our knowledge, constituted a new
compound isolated from a natural source.
The per-acetylated derivative 2 was obtained by treating 1 with pyridine and
acetic anhydride. Although the molecular ion of 2 was not shown in its EI–MS
spectrum, the peak at m/z ¼ 331 (44%) revealed the presence of a tetra-acetylated–
glucopyranoside residue in 2, while the peak at m/z ¼ 169 (100%) was due to the
1
di-acetylated B ring fragment. The H-NMR spectrum of 2 showed eight singlets
(3H each) at ꢂ 1.95, 2.02, 2.04, 2.06, 2.26, 2.31, 2.32 and 2.37 ppm assigned to the
methyl groups of the acetate residues. All these signals correlated with a complex
signal at 20.4–20.7 ppm in the HETCOR spectrum. The rest of the signals in
1
the H- and 13C-NMR spectra as well as the bands in the IR spectrum support the
structure of 2 (see Section 3).
When 1 was treated with H2SO4 diluted solution, the yellow-greenish solid
aglycon (3) was obtained. The HRMS of this product showed a pseudo-molecular
ion at 305.0661, corresponding to the molecular formula C15H13O7 (Calcd 305.0661),
which is in full agreement with the expected structure. Furthermore, the IEMS
spectrum showed the molecular ion at m/z ¼ 304 Mþ (85%), as well as the expected

Natural Product Research
1531
OH
H
H
HO
H
H
H
H
O
O
O
O
OH
HO
OH
H
H
HO
H
H
HO
H
H
OH
1
Figure 2. Important HMBC correlations of 1.
Table 1. Effect of ME and EAE in the TPA-induced oedema model.
Sample
Doses (mg per ear)
Oedema (mg)
IE (%)
TPAa
15.12 ꢃ 0.34
10.7 ꢃ 0.84**
13.60 ꢃ 0.48
7.92 ꢃ 1.20**
TPA þ MEa
1
1
29.26
41.73
TPAb
TPA þ EAEb
Notes: aSolvent used MeOH–H2O (1 : 1). bSolvent used MeOH–EtOAc
(1 : 1). **All data were analysed by ANOVA followed by Dunnett’s test,
and values of p ¼ 0.01 are considered as statistically significant with respect
to the control.
peaks at m/z ¼ 168 (100%) and 136 (20%) of a retro Diels–Alder fragmentation of 3.
The 1H-NMR spectrum of 3 showed the ABX system at 5.32 ppm (dd, J ¼ 2.5, 12.8),
3.19 ppm (dd, J ¼ 17, 12.8) and 2.61 ppm (dd, J ¼ 17, 3), assigned to the protons H-2,
H-3 ax. and H-3 eq., respectively. These signals correlated, in the HECTOR
spectrum, with the methine carbon at 78.40 (C-2) and the methylene carbon at
42.35 ppm (C-3), respectively, while those singlets at 6.74 (2H) and 6.90 assigned
to the protons H-20, H-60 and H-40, respectively, correlated, in the HECTOR
experiment, with the signals at 114.24, 117.80 and 115.25 ppm, respectively, and the
singlet at 5.93 ppm was assigned to H-8; this signal correlated with the methine
at 94.64 (C-8). The remaining signals at 101.66 (C-10), 126.21 (C-6), 155.68 (C-5),
155.14 (C-9) and 156.39 (C-7) were assigned by comparison with those spectral data
of similar structures (Miyake et al., 2003).
It is well known that several flavonoids possess both cytotoxic and anti-
inflammatory activities, probably by inhibition of the activation of the NF-ꢃB factor
(Liang et al., 1999). Then it was decided to evaluate the cytotoxic and anti-
inflammatory properties of 1–3. Also, both an aqueous extract and an EAE of the
stems were tested. The results showed that both extracts and 1–3 showed anti-
inflammatory activity in the TPA-induced oedema in mice model (Tables 1 and 2).
The EtOAc extract was more active than the ME, while the compounds 1 and 2
showed an oedema inhibition effect (IE) of 34.32% and 30.40% at the doses of

´
M.C. Gonzalez-Guereca et al.
¨
1532
Table 2. Effect of the compounds 1, 2 and 3 in the TPA-induced oedema model.
Doses
(mg per ear)
Doses
(mmol per ear)
Sample
Oedema (mg)
IE (%)
TPA
14.23 ꢃ 0.81
11.63 ꢃ 2.21*
9.90 ꢃ 1.59*
9.18 ꢃ 1.09*
16.24 ꢃ 0.86
1.57 ꢃ 0.33**
TPA þ 1a
TPA þ 2a
TPA þ 3a
TPAb
0.31
0.31
0.31
6.65 ꢄ 10ꢀ4
3.86 ꢄ 10ꢀ4
1.01 ꢄ 10ꢀ3
1.28 ꢄ 10ꢀ3
34.32
30.40
35.50
TPA þ indomethacineb
0.46
89.19
Notes: aSolvent used MeOH–CH2Cl2 (1 : 1). bSolvent used EtOH–acetone (1 : 1). The weight of
ears was presented as the mean ꢃ standard error of mean (SEM) of 3–4 male mice. IE (%) was
calculated from the test sample group with reference to the TPA, and data were analysed using
Student’s t-test; *p ꢅ 0.05 and **p ꢅ 0.01 were considered significant with respect to the
control.
Table 3. Effect of 1–3 ME and EAE on human tumour cell lines.
Cell lines GI (%)
Sample (50 mg mLꢀ1
)
HCT-15
MCF-7
U251
PC-3
K562
ME
EAE
1
2
3
NA
21.57
NA
85.51
55.20
5.11
6.31
NA
98.86
65.10
1.59
2.84
4.94
70.33
69.95
3.26
22.14
5.45
73.16
66.09
60.09
100.0
91.97
97.87
83.39
Notes: NA, not active; ME, methanol extract; EAE, EtOAc extract.
6.65 ꢄ 10ꢀ4 and 3.86 ꢄ 10ꢀ4 mmol by ear, respectively. The flavanone 3 and the
reference drug indomethacine showed IE of 35.50% and 89.19% at the doses of
1.01 ꢄ 10ꢀ3 and 1.28 ꢄ 10ꢀ3 mmol by ear, respectively. These results indicated that
2 was the most active anti-inflammatory, probably because it possesses more
lipophilic character than 1 and 3, and it penetrates more easily through the skin of
the test animals. On the other hand, the results of growth inhibition of human cancer
lines showed that the EtOAc extract exhibits 100% of growth inhibition of the line
K562 at the 50 mg mLꢀ1 dose. Again 2 was more active than 1 and 3 at the
50 mg mLꢀ1 dose level (Table 3). Taking into account these results, it was decided to
establish the IC50 values of 2, against several human cancer lines. The results were
47.36 ꢃ 4.6 (HCT-15), 69.62 ꢃ 2.6 (MCF-7), 53.26 ꢃ 7.0 (U251) and 37.82 ꢃ 4.3
(K562). Our results showed that 2 showed better anti-inflammatory and cytotoxic
properties than the glycoside 1 and the flavanone 3, although, as mentioned before,
its lipophilic properties could account for this anti-inflammatory activity. However,
it is not clear whether this property can account for the growth inhibition of cancer
cells.
Although the anti-inflammatory and cytotoxic activities of 1 were relatively low,
its potential abundance could account for the development of anti-inflammatory and
cytotoxic drugs from this natural resource.

Natural Product Research
1533
3. Experimental
3.1. General
Vacuum column chromatography (VCC) and thin-layer chromatography (TLC)
were carried on silica gel 60 F254 (Merck 1.05554). The spots were observed under
ultraviolet (UV) light at 366 and 254 nm, and the TLC plate was then sprayed with
cerium sulphate tetrahydrate (1%) in sulphuric acid solution. Melting points were
determined with a Fisher Johns apparatus and are uncorrected. The UV spectra were
recorded on a Shimadzu U 160 model spectrophotometer. The infrared (IR) spectra
were recorded on a Nicolet FT-IR5-SX spectrophotometer. Nuclear magnetic
resonance (NMR) spectra were recorded on a Varian-Unity 300 (1H at 300 and
13C at 75.4 MHz) and a Varian-Inova 500 (1H at 500 and 13C at 12 MHz). Chemical
shifts are expressed in ꢂ (ppm) relative to tetramethylsilane (TMS) as the internal
standard, and coupling constants, J, are in Hz. The solvent is indicated for each
compound. Correlation spectroscopy (COSY), nuclear overhauser effect spectros-
copy (NOESY) and heteronuclear correlation (HETCOR) were carried out using the
Varian-Unity 300 spectrometer, while the heteronuclear multiple bond correlation
(HMBC) experiment was performed using the Varian-Inova 500 spectrometer. The
electron ionisation mass spectroscopy (EI–MS) and high resolution mass spectro-
metry (HRMS) were recorded on a Jeol AH505HR mass spectrometer.
3.2. Plant material
The collection of stems was done in Mezquital, Durango, Mexico (at 2230 m altitude
and 23ꢂ430, 24.200N; 104ꢂ240, 0.7900W). A voucher of a complete (leaves and stem)
specimen has been deposited at the Herbarium, CIIDIR-IPN, under register
no. 16543.
3.3. Extraction and isolation
Dried and ground stems (1.7 kg) were successively extracted with hexane, EtOAc
and MeOH at room temperature (9 times ꢄ 48 h each). Evaporation of the solvents
under low pressure afforded 5.3, 37.5 and 184.5 g of the respective extracts. The
aqueous extract was prepared by the addition of 10 g of stems to 90 mL of boiling
water. The resulting solution was lyophilised, affording 1 g of the aqueous
lyophilised extract (WE).
A fraction of the EtOAc extract (12 g) was dissolved in methanol and absorbed
into 10 g of silica gel G (Alltech). Afterwards the solvent was evaporated at room
temperature. The resulting dried mixture was put on the top of a chromatography
column, packed with 130 g of silica gel and eluted with hexane and mixtures of
hexane–EtOAc and EtOAc–MeOH of increasing polarity. A total of 198 fractions
of 200 mL each were obtained. Fractions eluted with EtOAc 100% (162 and 163)
were combined to afford 547 mg of 5,6,30,40-tetrahydroxy flavanone 7-O-ꢀ-D-glucose
(1) (0.033% yield from dried material).
3.3.1. 5,6,7,30,40 pentahydroxy flavanone 7-O-ꢀ-d-glucopyranoside (1)
5,6,7,30,40 pentahydroxy flavanone 7-O-ꢀ-D-glucopyranoside (1) was isolated as
yellow-greenish solid with m.p. 187–189ꢂC. Its HRMS showed a pseudo-molecular

´
M.C. Gonzalez-Guereca et al.
¨
1534
ion at m/z ¼ 467.1188 ([M]þ þ 1), Calcd for C21H23O12. IEMS (70 eV) (rel. int.) m/
z ¼ 466.40, 304, 168 and 136. UV ꢄmax (nm) (EtOH) 215 (log " ¼ 1.52), 288
(log " ¼ 2.1) and 366 (log " ¼ 2.26). IR (KBr) ꢅmax (cmꢀ1) 3394, 1651, 1497 and 1073.
1H-NMR (dimethyl sulphoxide (DMSO)), 500 MHz): ꢂ 2.67 (1H, dd, 2.5, 17.3 Hz, H-
3 eq.), 3.12 (1H, m, H-400), 3.21 (1H, dd, 12.8, 17.3 Hz, H-3 ax.), 3.25 (1H, m, H-200),
3.26 (1H, m, H-500), 3.50 (1H, m, H-300), 3.42 (1H, m, H-6a00), 3.66 (1H, m, H-b00),
4.85 (1H, d, 7.5 Hz, H-100), 5.34 (1H, dd, 2.5, 12.8 Hz, H-2), 6.28 (1H, s, H-8), 6.73
(2H, s, H-20and H-60) and 6.86 (1H, s, H-40); 13C-NMR (DMSO, 125 MHz): ꢂ 78.72
(C-2), 42.66 (C-3), 197.89 (C-4), 149.19 (C-5), 127.74 (C-6), 153.40 (C-7), 94.20 (C-8),
154.51 (C-9), 103.36 (C-10), 129.50 (C-10), 117.97 (C-20), 145.60 (C-30), 114.33 (C-40),
145.10 (C-50), 115.28 (C-60), 100.58 (C-100), 73.09 (C-200), 77.14 (C-300), 69.58 (C-400),
1
75.64 (C-500) and 60.56 (C-600). HETCOR main correlations H/13C: 6.86/114.33,
6.73/117.91, 6.73/115.28, 6.28/94.20, 5.34/78.7, 4.85/110.58, 2.69 and 3.21/42.66.
3.3.2. Per-acetylated compound (2)
To a solution of 1 (100 mg) in of pyridine (1 mL), anhydride acetic (1 mL) was added.
The resulting solution was heated at 60ꢂC for 6 h. After the usual work up, 100 mg
of the per-acetylated derivate 2 as a brown–red powder was obtained. It showed
an m.p. of 114–115ꢂC. IEMS (70 eV) m/z (rel. int.): 331 (45), 169 (100), 109 (40), 43
(44). IR (KBr) ꢅmax (cmꢀ1) 2969, 1622, 1216 and 1075. 1H-NMR (CDCl3, 300 MHz):
ꢂ 2.02 (3H, s, CH3CO), 2.04 (3H, s, CH3CO), 2.05 (3H, s, CH3CO), 2.06 (3H, s,
CH3CO), 2.26 (3H, s, CH3CO), 2.31(3H, s, CH3CO), 2.32 (3H, s, CH3CO), 2.37 (3H,
s, CH3CO), 2.74 (1H, dd, 3, 17 Hz, H-3 eq.), 2.98 (1H, dd, 12.8, 17.3 Hz, H-3 ax.),
3.89 (1H, m, H-500), 4.19 (2H, m, H-600), 5.08 (1H, d, 8 Hz, H-100), 5.11 (1H, m, H-400),
5.30 (2H, m, H-200 and H-300), 5.43 (1H, dd, 2.5, 13), 6.06 (1H, s, H-8), 7.26 (2H, brs,
H-20 and H-60) and 7.29 (1H, brs, H-400); 13C-NMR (CDCl3,75.4 MHz): ꢂ 79.08 (C-2),
44.87 (C-3), 188.03 (C-4), 143.29 (C-5), 136.59 (C-6), 160.40 (C-7), 101.29 (C-8),
154.57 (C-9), 109.35 (C-10), 128.13 (C-10), 124.36 (C-20), 142.56 (C-30), 123.98 (C-40),
142.45 (C-50), 121.54 (C-60), 98.24 (C-100), 70.81 (C-200), 72.29 (C-300), 67.99 (C-400),
72.45 (C-500), 61.63 (C-600), 170.48 (CH3CO), 169.99 (CH3CO), 169.35 (CH3CO),
168.33 (CH3CO), 168.01(CH3CO) and 20.67–19.90 (CH3CO, complex signal).
3.3.3. 5,7,8,30,50Pentahydroxy flavanone (3)
To 100 mg of 1 dissolved in MeOH (20 mL), a solution of H2SO4 4N (5 mL) was
added. The mixture of reaction was refluxed for 24 h. After the usual work up, 22 mg
of 5,7,8,30,40pentahydroxy flavanone (3), as a yellow-greenish powder, was obtained.
It showed an m.p. of 189ꢂC. HRMS showed a pseudo-molecular ion at 305.0661 for
C15H13O7, Calcd 305.0661. IEMS (70 eV) m/z (rel. int.)¼ 304 Mþ (85), 168 (100), 136
(20), 69 (18), 57 (19). 1H-NMR (DMSO-d6, 300 MHz): ꢂ 2.61 (1H, dd, 3, 17 Hz, H-3
eq.), 3.19 (1H, dd, 12.8, 17 Hz, H-3 ax.), 5.32 (1H, dd, 2.5, 12.8 Hz, H-2), 5.93 (1H, s,
H-8), 6.74 (2H, s, H-20and H-60) and 6.90 (1H, s, H-40); 13C-NMR (DMSO, 75 MHz):
ꢂ 78.45 (C-2), 42.35 (C-3), 196.91 (C-4), 155.68 (C-5), 126.21 (C-6), 156.39 (C-7),
94.64 (C-8), 155.14 (C-9), 101.66 (C-10), 129.72 (C-10), 114.24 (C-20), 145.12 (C-30),
115.25 (C-40), 145.57 (C-50) and 117.80 (C-60).

Natural Product Research
1535
3.4. Anti-inflammatory activity
The anti-inflammatory assay of 12-O-tetradecanoylphorbol-13-acetate (TPA)-
induced ear oedema in mice was performed as described previously (De Young,
Kheifets, Ballaron, & Young, 1989). Solutions of samples of the methanol extract
(ME), ethyl acetate extract (EAE), 1–3 and indomethacin, dissolved in mixtures
(water/MeOH 1 : 1, MeOH/EtOAc 1 : 1, MeOH–CH2–Cl2 1 : 1 and EtOH–CH2–Cl2
1 : 1), respectively, were evaluated.
3.5. Cytotoxic activity
The cytotoxic activity of the test compound was assayed in prostate cancer (PC-3),
leukaemia (K562), central nervous system (U251), breast cancer (MCF-7) and colon
cancer (HCT-15) human tumour cell lines using the protein-binding dye
sulphorhodamine B (SRB), as reported previously (Monks et al., 1991).
Acknowledgements
We are grateful to Teresa Ramı
´
Zavala, Hector Rios, Isabel Cha
´
rez-Apam, Antonio Nieto, Javier Perez, Luis Velasco, Nieves
vez, Rocio Patino and Beatriz Quiroz for providing technical
´
˜
assistance.
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