Cyclooxygenase inhibitory properties of nor -neolignans from Styrax pohlii

Article abstract of DOI:10.1080/14786419.2012.671320

Chemical investigation of the n-hexane and EtOAc fractions of the ethanolic extract from Styrax pohlii (Styracaceae) aerial parts resulted in the isolation of the benzofuran nor-neolignan derivatives egonol (1), homoegonol (2), homoegonol gentiobioside (3), homoegonol glucoside (4) and egonol gentiobioside (5). This is the first report of compounds 1-5 in S. pohlii. Compounds 1-5, the acetyl derivatives 1a and 2a, the ethanolic extract (EE), the n-hexane fraction (HF) and EtOAc fraction (EF) were tested for their inhibitory activities against COX-1 and COX-2. The results showed that EE, HF, EF and compounds 1-5 and 1a-2a shown weak to moderate inhibition of COX-1 and COX-2. Among the assayed nor-neolignans, 4 gave a COX-1 inhibition of 35.7% at 30μM. Compound 5 displayed a COX-2 inhibition of 19.7% at 30μM.

Full text of DOI:10.1080/14786419.2012.671320

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Cyclooxygenase inhibitory properties of
nor-neolignans from Styrax pohlii
Camila S. Bertanha a , Caio G. Braguine a , Ana C.G. Moraes a
Valéria M.M. Gimenez b , Milton Groppo c , Márcio L.A. Silva a
Wilson R. Cunha a , Ana H. Januário a & Patrícia M. Pauletti a
,
,
a Núcleo de Pesquisa em Ciências Exatas e Tecnológicas,
Universidade de Franca , Franca , Brazil
b Centro Universitário Claretiano, Batatais , São Paulo , Brazil
c Department of Biology , Faculdade de Filosofia, Ciências e
Letras, Universidade de São Paulo , Ribeirão Preto , Brazil
Published online: 29 Mar 2012.
To cite this article: Camila S. Bertanha , Caio G. Braguine , Ana C.G. Moraes , Valéria M.M.
Gimenez , Milton Groppo , Márcio L.A. Silva , Wilson R. Cunha , Ana H. Januário & Patrícia
M. Pauletti (2012) Cyclooxygenase inhibitory properties of nor-neolignans from Styrax
pohlii , Natural Product Research: Formerly Natural Product Letters, 26:24, 2323-2329, DOI:
10.1080/14786419.2012.671320
To link to this article: http://dx.doi.org/10.1080/14786419.2012.671320
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Natural Product Research
Vol. 26, No. 24, December 2012, 2323–2329
Cyclooxygenase inhibitory properties of nor-neolignans from Styrax pohlii
Camila S. Bertanhaa, Caio G. Braguinea, Ana C.G. Moraesa, Vale
´
ria M.M. Gimenezb,
rioa and
Milton Groppoc, Marcio L.A. Silvaa, Wilson R. Cunhaa, Ana H. Janua
´
´
a
*
Patrı
´
cia M. Pauletti
a
b
´
Nu´cleo de Pesquisa em Cieˆncias Exatas e Tecnologicas, Universidade de Franca, Franca, Brazil;
Centro Universitario Claretiano, Batatais, Sa˜o Paulo, Brazil; Department of Biology,
c
´
Faculdade de Filosofia, Cieˆncias e Letras, Universidade de Sa˜o Paulo, Ribeira˜o Preto, Brazil
(Received 19 November 2011; final version received 9 January 2012)
Chemical investigation of the n-hexane and EtOAc fractions of the ethanolic
extract from Styrax pohlii (Styracaceae) aerial parts resulted in the isolation of the
benzofuran nor-neolignan derivatives egonol (1), homoegonol (2), homoegonol
gentiobioside (3), homoegonol glucoside (4) and egonol gentiobioside (5). This is
the first report of compounds 1–5 in S. pohlii. Compounds 1–5, the acetyl
derivatives 1 a and 2 a, the ethanolic extract (EE), the n-hexane fraction (HF) and
EtOAc fraction (EF) were tested for their inhibitory activities against COX-1 and
COX-2. The results showed that EE, HF, EF and compounds 1–5 and 1 a–2 a
shown weak to moderate inhibition of COX-1 and COX-2. Among the assayed
nor-neolignans, 4 gave a COX-1 inhibition of 35.7% at 30 mM. Compound 5
displayed a COX-2 inhibition of 19.7% at 30 mM.
Keywords: Styrax pohlii; Styracaceae; cyclooxygenase – 1/2 inhibitory activity
1. Introduction
Prostaglandins (PGs) are mediators of pain, fever and other symptoms associated with
inflammation. Their biosynthesis is rate-limited by the initial conversion of arachidonic
acid to PG G2, and by conversion of the latter to PG H2, which involves a single enzyme,
namely cyclooxygenase (COX). Nonsteroidal anti-inflammatory drugs exert their thera-
peutic effects by inhibiting PG synthesis in COX (Smith, DeWitt, & Garavito, 2000). It is
well established that COXs exist as three isoforms that catalyse the same reaction but
differ in terms of expression regulation. More specifically, COX-1 is constitutively
expressed in most tissues and is thought to be responsible for regulating normal
physiological functions, while COX-2 is induced in macrophages, fibroblasts and several
other cell types by pro-inflammatory stimuli. COX-3 is a splice variant of COX-1
(Chandrasekharan et al., 2002; Smith et al., 2000; Warner & Mitchell, 2002).
Styrax pohlii A. DC. (Styracaceae), known in Brazil as ‘benjoeiro’, ‘estoraqueiro’,
ba’, ‘pindaubuna’ or ‘pindauvuna’, is a tree that grows in the
s, and Mato Grosso do Sul and is commonly
‘a
´
rvore-de-ba
´
o Paulo, Minas Gerais, Goia
lsamo’, ‘pindaı
´
States of Sa
˜
´
employed in folk medicine to relieve fever (Lorenzi, 1998; Rodrigues & de Carvalho, 2008).
The resinous material that is usually secreted, when the barks and trunks of this plant are
*Corresponding author. Email: pmpauletti@unifran.br
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2012.671320
http://www.tandfonline.com

2324
C.S. Bertanha et al.
cut by sharp objects is often utilised in traditional medicine to substitute the benzoin resin,
which is known for its anti-inflammatory properties (Lorenzi, 1998; Pauletti, Teles, Silva,
Arau´ jo, & Bolzani, 2006). Previous studies on this genus have resulted in the isolation of
lignan derivatives of 3,7-dioxabicyclo[3.3.0]octane, butanolide and tetrahydrofuran;
neolignan derivatives of dihydrobenzofuran, nor-neolignan derivatives of benzofuran,
phenylpropanoids, phenolic acids, pentacyclic saponins and triterpenes (Pauletti et al.,
2006). In addition, biological investigations have shown that the nor-neolignans from
Styrax, particularly egonol (1) and homoegonol (2) and their derivatives, display moderate
¨
antimicrobial and cytotoxic actions (Hirano, Gotoh, & Oka, 1994; Ozturk, Akgul, & Anil,
¨
¨
2008; Pauletti, Arau´ jo, Young, Giesbrecht, & Bolzani, 2000; Teles et al., 2005). Moreover,
egonol has been shown to inhibit the hemolytic activity of the complement system, which
could be beneficial in the therapy of inflammatory diseases (Min et al., 2004).
As a part of our ongoing biological and chemical studies on Styrax (Pauletti et al.,
2000; Pauletti, Arau´ jo, Young, & Bolzani, 2002; Teles et al., 2005), as well as our
investigations on the anti-inflammatory action of natural and semi-synthetic compounds
(Coimbra et al., 2004; Neto et al., 2005; Vasconcelos et al., 2006), we now report on the
isolation and structural identification of chemical constituents from the n-hexane and
EtOAc fractions of S. pohlii aerial parts, as well as their COX inhibitory activities.
2. Results and discussion
The spectral data of all the isolated compounds (Figure 1) are in agreement with
previously published data and allowed for identification of egonol (1), homoegonol (2),
homoegonol glucoside (4) and egonol gentiobioside (5) (Hopkins, Ewing, & Chisholm,
1967; Pauletti et al., 2000; Schreiber & Stevenson, 1976; Takanashi & Takizawa, 2002).
Homoegonol gentiobioside (3) had been previously isolated from S. officinalis seeds (Anil,
1980), although no NMR data were provided. To the best of our knowledge, this is the
first report of compounds 1–5 in S. pohlii.
Earlier studies with lignans, neolignans and nor-neolignans have revealed that some
display significant anti-inflammatory activity (Ban et al., 2002; Blunder et al., 2010; Coy,
Cuca, & Sefkow, 2009; Su et al., 2004; Tzeng & Liu, 2004). This has encouraged us to
continue our biological and chemical investigations of lignans, neolignans and nor-
neolignans from Styrax species that display different structural features. To establish the
potential of crude extracts, fractions and isolated compounds as anti-inflammatory agents,
an in vitro screening for their ability to inhibit COX isozymes was accomplished.
Figure 1. Chemical structures of the isolated compounds.

Natural Product Research
2325
Regarding the COX assay (Table 1), incubation of COX-1 and COX-2 with the
ethanolic extract (EE, 50 mg mLꢀ1) inhibited PG production by 4.8 ꢁ 7.7% and
84.1 ꢁ 3.9%, respectively. Considering these values, the crude extract exhibited a
promising selectivity toward COX-2.
Diminished inhibitory activity was detected during assessment of the anti-inflamma-
tory action of the n-hexane fraction (HF, 50 mg mLꢀ1) and EtOAc fraction (EF,
50 mg mLꢀ1) with respect to COX-1 and COX-2. Indeed, HF and EF were completely
unable to inhibit PG production by COX-1. Compared to the crude extract (EE) at the
same concentration, a small inhibitory activity was observed for COX-2 in the case of HF
and EF (19.3 ꢁ 4.8% and 4.1 ꢁ 3.1%, respectively).
Compounds 1–5 and 1 a–2 a were also evaluated for their inhibitory activity toward
COX-1 and COX-2 (Table 1). Each compound was tested at final concentrations of 30, 20,
10 and 5 mM. Egonol (1) and homoegonol (2) at 30 mM gave COX-1 inhibition of
19.1 ꢁ 4.3% and 10.6 ꢁ 3.5%, respectively, and COX-2 inhibition of 4.5 ꢁ 3.8% and
7.1 ꢁ 4.9%, respectively. On the other hand, acetylation of 1 and 2 did not improve COX-1
inhibition by 1 a or 2 a. In fact, 1 a and 2 a were unable to inhibit COX-1 at any extension,
but these derivatives enhanced COX-2 inhibition as compared to 1 and 2 at 30 mM by
11.1 ꢁ 1.6% and 16.0 ꢁ 0.2%, respectively.
The glycosylated compounds homoegonol gentiobioside (3), homoegonol glucoside (4)
and egonol gentiobioside (5) at 30 mM yielded COX-1 inhibition of 1.3 ꢁ 0.6%,
35.7 ꢁ 6.7%, and 23.3 ꢁ 9.1%, respectively. Furthermore, COX-2 inhibition was
6.5 ꢁ 0.1%, 11.9 ꢁ 0.2% and 19.7 ꢁ 3.2%, respectively. Thus, a very small inhibitory
activity was observed for COX-1 and COX-2 in the case of compound 3. Additionally,
among the assayed nor-neolignans, 4, furnished the best value of COX-1 inhibition at
30 mM. As for 5, it provided the greatest percentage of COX-2 inhibition at this same
concentration.
The compounds SC-560 and DuP697 were used to standardise the assay for COX-1
(tested concentration ¼ 20 nM, 48.1 ꢁ 9.0%) and COX-2 (tested concentration ¼ 40 nM,
49.3 ꢁ 3.9%), respectively. The IC50 for Ibuprofen, used as positive control, were
determined to be 2.6 and 1.53 mM for COX-1 and COX-2, respectively.
Table 1. COX-1 and COX-2 inhibitory effects of the extracts, fractions and compounds from
S. pohlii.
COX inhibition (percentage)c
Extract/compound
Concentration
COX-1
COX-2
EE
HF
EF
1
2
3
4
5
1 a
2 a
50a
50a
50a
30b
30b
30b
30b
30b
30b
30b
4.8 ꢁ 7.7
n.a.
n.a.
19.1 ꢁ 4.3
10.6 ꢁ 3.5
1.3 ꢁ 0.6
35.7 ꢁ 6.7
23.3 ꢁ 9.1
n.a.d
84.1 ꢁ 3.9
19.3 ꢁ 4.8
4.1 ꢁ 3.1
4.5 ꢁ 3.8
7.1 ꢁ 4.9
6.5 ꢁ 0.1
11.9 ꢁ 0.2
19.7 ꢁ 3.2
11.1 ꢁ 1.6
16.0 ꢁ 0.2
n.a.
Notes: Conc., mg mLꢀ1
.
a
bConc., mM.
cThe data represent the mean ꢁ S.E.M.
dNot active.

2326
C.S. Bertanha et al.
Compounds 1 and 2 are structurally quite similar, both have a benzofuran moiety and
differ mainly in the presence of a methylenedioxy substituent in 1 versus two methoxyl
groups in 2. An attempt to improve the weak activity showed by 1 and 2 was made by the
production of their acetyl derivatives. Although, 1 a and 2 a lost COX-1 inhibitory activity.
Regarding the glycosylated nor-neolignans, that enhance the hydrosolubility of the
egonol and homoegonol derivatives, the number of glucosyl groups in the homoegonol
moiety suggests that the presence of one glucosyl group, as in the case of compound 4, may
improve the activity of this derivative, since 4 was more active than 3 and 2. Furthermore,
it can be observed that compound 5 is quite similar to egonol (1), differing only in the
presence of two glucose unites, exhibited a slight enhanced COX-2 inhibitory activity.
Considering their previous reported biological activities, egonol (1) and homoegonol
(2), which occur widely in Styrax (Styracaceae), have attracted researchers’ attention
mainly because of their antibacterial, antifungal and moderate cytotoxic properties
(Pauletti et al., 2000; Teles et al., 2005). Additionally, egonol has been shown to be able to
inhibit the hemolytic activity of the complement system with IC50 values of 33 mM, while
homoegonol was inactive. The modulation of the complementary activity might be
beneficial in the therapy of inflammatory diseases (Min et al., 2004). Other nor-neolignans
and neolignans having a benzofuran moiety, such as 90-nor-7,8-dehydro-isolicarin B,
ocophyllals A and ocophyllals B, have been described to exhibit COX-2 selectivity,
affording the best results among other nor-neolignans and neolignans cores, with IC50
lying in the range of 3.32–16.8 mM (Coy et al., 2009).
3. Experimental
3.1. General
1H and 13C NMR spectra were recorded in DMSO-d6 or MeOD-d4 on a Bruker AVANCE
DRX 500 spectrometer, using TMS as internal standard. Both analytical and preparative
HPLC separation analyses were carried out on a Shimadzu LC-6AD system equipped with
a degasser DGU-20A5, a UV–Vis detector SPD-20 A series, a communication bus module
CBM-20 A, and a Rheodyne manual injector. Separations of the compounds were carried
out on a SHIMADZU Shim-pack ODS (particle diameter 5 mm, 250 mm ꢂ 4.60 mm and
250 mm ꢂ 20 mm) columns equipped with a pre-column of the same material. The MeOH
used in the experiments was HPLC grade, J.T. Baker. Ultrapure water was obtained by
passing redistilled water through a Direct-Q UV3 system, Millipore. Silica gel 60 (230–400
mesh, Sigma-Aldrich) and Sephadex LH-20 (Sigma-Aldrich) were employed for column
chromatography, as well as silica on TLC Alu foils with fluorescent indicator 254 nm
(Sigma-Aldrich). Prep-TLC was conducted on silica gel type G (Sigma-Aldrich). Silica gel
90 reverse-phase ODS (Fluka, 230–400 mesh) was utilised for sample preparation prior to
injection into the HPLC system.
3.2. Plant material
Aerial parts of Styrax pohlii A. DC. were collected in Luis Antonio city, (21ꢃ330–21ꢃ370 S
and 47ꢃ450–47ꢃ570 W, in October 2008), and identified by Prof. V.M.M. Gimenez and
Prof. M. Groppo. A voucher specimen (SPFR12168) was deposited in the Herbarium of
the Department of Biology, Laboratory of Plant Systematics, Faculdade de Filosofia
˜ ˜
Cieˆ ncias e Letras de Ribeirao Preto, University of Sao Paulo, Brazil (Herbarium SPFR).
3.3. Extraction and isolation
The air-dried, powdered stems and leaves (2.4 kg) of S. pohlii were extracted with EtOH.
After filtration, the solvents were removed under reduced pressure, to yield 87 g of

Natural Product Research
2327
the extract. The EE (40 g) was then dissolved in MeOH/H2O (2 : 8 v/v) and successively
partitioned with n-hexane, EtOAc and n-BuOH. After solvent removal using a rotary
evaporator, each partition phase yielded 2.8, 8.9, 7.6 and 8.2 g, respectively. The HF (1.4 g)
was chromatographed over silica using a gradient of n-hexane/EtOAc as eluent, which
afforded 111 fractions. Compounds 1 (27 mg) and 2 (22 mg) were isolated from fractions
92–100 and 69–80, respectively, after a prep-TLC silica using CH2Cl2/MeOH (98 : 2 v/v).
The EtOAc residue (3.2 g) was submitted to a Sephadex LH-20 chromatographic column
employing MeOH as eluent, which furnished seven fractions. Fractions 8–10 (336 mg) were
dissolved in MeOH/H2O (68 : 32 v/v), chromatographed over a reverse phase ODS
cartridge, and subsequently submitted to preparative RP-HPLC purification using
MeOH/H2O/AcOH (68 : 31.9 : 0.1 v/v/v), UV detection at 254 nm and 9 mL minꢀ1 flow
rate, which yielded six fractions. Fractions 2, 3 and 5 gave compounds 3 (18 mg, Rt 15.9),
4 (10 mg, Rt 18.8) and 5 (7 mg, Rt 26.2), respectively.
1
Homoegonol gentiobioside (3). H NMR (500 MHz, ꢀ, methanol-d4): 1.98 (m, 2 H,
H-200), 2,82 (t, J ¼ 7.4, 2 H, H-100), 3.19 (d, J ¼ 10.5, 1 H, H-60000), 3.21 (m, 1 H, H-2000 and
H-20000), 3.28 (m, 1 H, H-50000), 3.36 (m, 3 H, H-3000, H-4000 and H-30000), 3.44 (m, 1 H, H-5000),
3.48 (m, 1 H, H-40000), 3.59 (m, 1 H, H-3000), 3.75 (dd, J ¼ 5.4 and 11.4, 1 H, H-6000), 3.86
(dd, J ¼ 5.4 and 11.4, 1 H, H-60000), 3.90 (s, 3 H, 30-OCH3), 3.94 (m, 1 H, H-300), 3.95
(s, 40-OCH3), 4.04 (s, 3 H, 7-OCH3), 4.10 (dd, J ¼ 1.3 and 11.4, 1 H, H-6000), 4.28 (d, J ¼ 7.8,
1 H, H-1000), 4.34 (d, J ¼ 7.5, 1 H, H-10000), 6.79 (s, 1 H, H-6), 7.05 (s, 1 H, H-3), 7.07 (s, 1 H,
H-4), 7.08 (d, J ¼ 8.4, 1 H, H-50), 7.46 (d, J ¼ 1.7, 1 H, H-20), 7.49 (dd, J ¼ 1.7 and 8.4, 1 H,
H-60). 13C NMR (125 MHz, ꢀ, methanol-d4): 32.2 (C-200), 32.5(C-100), 55.6 (30-OCH3), 55.7
(40-OCH3), 55.8 (7-OCH3), 66.0 (C-60000), 68.8 (C-6000), 69.0 (C-300), 70.2 (C-40000), 70.5 (C-4000),
74.0 (C-20000), 74.2 (C-2000), 76.1 (C-5000), 76.9 (C-50000), 76.9 (C-3000), 77.1 (C-30000), 100.7 (C-3),
103.7 (C-1000), 104.7 (C-10000), 107.9 (C-6), 108.7 (C-20), 112.7 (C-4), 112.7 (C-50), 118.1
(C-60), 124.0 (C-10), 131.6 (C-9), 138.4 (C-5), 142.7 (C-8), 145.2 (C-7), 149.9 (C-40), 150.2
(C-30), 156.4 (C-2).
3.4. Acetylation of egonol (1) and homoegonol (2)
Four milligrams of 1 and 2 were treated with acetic anhydride (3 mL) and pyridine (3 mL)
overnight. The reaction was quenched with cold H2O (30 mL) and extracted with CH2Cl2
(2 mL ꢂ 15 mL), HCl 10% (2 mL ꢂ 15 mL) and H2O (3 mL ꢂ 15 mL). Anhydrous sodium
carbonate was added to the organic phase, which was filtered and concentrated under
reduced pressure, to give acetate derivatives 1 a and 2 a.
3.5. In vitro COX-1 and COX-2 inhibitory activity
The extracts, fractions, isolated and acetylated compounds were tested in vitro for their
COX-1 and COX-2 inhibitory activity using a COX inhibitor screening assay kit
(catalogue no. 560131, Cayman Chemicals, MI, USA), according to the manufacturer’s
instructions. COX-1 and COX-2 catalyse the conversion of arachidonic acid to PGH2;
PGF2ꢁ is produced from PGH2 by reduction of the latter with stannous chloride and this
product is quantified via an enzyme immunoassay [Acetylcholinesterase, AChE]. In our
experiments, the samples were dissolved in DMSO and diluted to the desired concentra-
tion using the reaction buffer. The reaction mixtures (1.15 mL) containing 950 mL 0.1 M
Tris-HCl (pH 8.0) as well as 5 mM EDTA, 2 mM phenol, 10 mL heme and 10 mL COX-1
(ovine) or COX-2 (human recombinant) were pre-equilibrated to 37ꢃC in a water bath.
Next, 20 mL of the sample with the desired concentration of the test solution (final con-
centrations of 30, 20, 10 and 5 mM for the pure compounds and 50 mg mLꢀ1 for the
crude extract and fractions) were added to the tube containing the inhibitor

2328
C.S. Bertanha et al.
(Gautam, Srivastava, Jachak, & Saklani, 2010). 20 mL of the buffer were added to the
100% initial activity tubes. The incubation period was 10 min. The reaction was initiated
by addition of 10 mL arachidonic acid at a final concentration of 100 mM to all the test
tubes. After 2 min, the reactions were finalised by addition of HCl 1 M (50 mL), followed
by 100 mL stannous chloride. Then, PGF2ꢁ was quantified by means of the ELISA method.
For this purpose, the contents of the reaction tubes were diluted and transferred to a
96-well plate coated with a mouse anti-rabbit IgG, which was followed by addition of the
PG screening AChE tracer and the PG screening antiserum. Plates were incubated in an
orbital shaker for 18 h, at room temperature. Next, the reaction mixtures were removed,
and the wells were washed five times with buffer containing 0.05% Tween 20. Ellman’s
reagent (200 mL) was then added to each well, and the plate was incubated in an orbital
shaker for 60 min, at room temperature, until the control wells yielded an OD lying
between 0.3–0.8 at 405 nm. A standard curve with PG was generated from the same plate,
which was used to quantify the PG levels produced in the presence of the samples. Results
are expressed as the percentage of inhibition relative to a control (100% initial activity,
solvent-treated samples). All determinations were carried out in duplicate. The compounds
SC-560 (Cayman Chemicals) and DuP697 (Cayman Chemicals) were used to standardise
the assay for COX-1 (tested concentration ¼ 20 nM, 48.1 ꢁ 9.0%) and COX-2 (tested
concentration ¼ 40 nM, 49.3 ꢁ 3.9%), respectively. Ibuprofen (Sigma-Aldrich) was
employed as positive control. The concentration of drug giving 50% of inhibition (IC50)
was calculated by non-linear regression analysis of percentage of inhibition versus
concentration.
4. Conclusions
In summary, chemical investigations of n-hexane and EtOAc fractions of the EE from
S. pohlii resulted in the isolation and identification of compounds 1–5. Additionally,
biological results indicated that EE, HF, EF and compounds 1–5 and 1 a–2 a are able to
weakly or moderately inhibit COX-1 and COX-2.
Acknowledgements
The authors are grateful to Fundac¸
Conselho Nacional de Desenvolvimento Cientı
a
˜
o de Amparo a Pesquisa do Estado de Sa
˜
o Paulo (FAPESP) and
´
is also acknowledged for financial support (Grant # 2008/10.283-1).
fico e Tecnologico (CNPq) for fellowships. FAPESP
´
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