Author's Accepted Manuscript

Compounds from Geijera parviflora with pros-taglandin E2 inhibitory activity May explainits traditional use for pain relief

Linda K. Banbury, Qingyao Shou, Dane E.Renshaw, Eleanore H. Lambley, Hans J. Gries-ser, Htwe Mon, Hans Wohlmuth

PII: S0378-8741(15)00048-3DOI: http://dx.doi.org/10.1016/j.jep.2015.01.033Reference: JEP9280

To appear in: Journal of Ethnopharmacology

Received date: 14 July 2014Revised date: 8 January 2015Accepted date: 25 January 2015

Cite this article as: Linda K. Banbury, Qingyao Shou, Dane E. Renshaw,Eleanore H. Lambley, Hans J. Griesser, Htwe Mon, Hans Wohlmuth,Compounds from Geijera parviflora with prostaglandin E2 inhibitory activityMay explain its traditional use for pain relief, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.01.033

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Compounds from Geijera parviflora with prostaglandin E2 inhibitory activity

may explain its traditional use for pain relief

Linda K. Banburya,1, Qingyao Shoua,2, Dane E. Renshawa,3, Eleanore H. Lambleya,

Hans J. Griesserb, Htwe Monb and Hans Wohlmutha,c, *

a Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore

NSW 2480, Australia

b Mawson Institute, University of South Australia, Mawson Lakes SA 5095, Australia

c Integria Healthcare, Gallans Rd, Ballina NSW 2478, Australia

Email addresses:

LKB: lkbanbury63@gmail.com; QS: sanqi_0617@126.com; DER:

dane@rkeenanconsult.com.au; EHL: eleanore.lambley@scu.edu.au; HJG:

hans.griesser@unisa.edu.au; HM: htwe.mon@unisa.edu.au; HW:

hans.wohlmuth@scu.edu.au

* Corresponding author. Current address: Integria Healthcare, Gallans Rd., Ballina

NSW 2478, Australia. E-mail address: hans.wohlmuth@integria.com, Tel: +61-2-

66205180 (H. Wohlmuth).

1 Current address: Division of Pathogenic Biochemistry, Institute of Natural Medicine,

University of Toyama, Sugitani 2630, Toyama 930-0194, Japan

2 Current address: Department of Chemistry, University of Florida, Gainesville, FL,

USA

3 Current address: R Keenan Consulting, Hindmarsh St, East Ballina NSW 2478,

Australia

ABSTRACT

Ethnopharmacological relevance: Australian Aboriginal people used crushed leaves

of Geijera parviflora Lindl. both internally and externally for pain relief, including for

toothache (Cribb and Cribb, 1981). This study tested the hypothesis that this

traditional use might be at least in part explained by the presence of compounds with

anti-inflammatory activity..

Materials and Methods: A crude extract (95% EtOH) was prepared from powdered

dried leaves. From the CH3Cl fraction of this extract compounds were isolated by

bioassay-guided fractionation and tested for: (1) cytotoxicity in RAW 264.7 murine

leukemic monocyte-macrophages, (2) prostaglandin E2 (PGE2) inhibitory activity in

3T3 Swiss albino mouse embryonic fibroblast cells, as well as (3) nitric oxide (NO)

and (4) tumour necrosis factor alpha (TNFα) inhibitory activity in lipopolysaccharide

(LPS)-stimulated RAW 264.7 cells. Isolated compounds were also tested for (5)

antibacterial activity against a panel of Gram-positive (Staphylococcus aureus ATCC

29213 and ATCC 25923, S. epidermidis ATCC 35984, biofilm-forming) and Gram-

negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853)

strains by broth microdilution.

Results: Eleven compounds were isolated, including one new flavone and one new

natural product, with a further four compounds reported from this species for the first

time. Some of the compounds showed good anti-inflammatory activity in vitro. In

particular, flindersine (1) and N-(acetoxymethyl) flindersine (3) inhibited PGE2

release with IC50 values of 5.0 µM and 4.9 µM, respectively, without any significant

cytotoxicity. Several other compounds showed moderate inhibition of NO (5, 6, 7)

and TNFα (6), with IC50 in the low micromolar range; however much of this apparent

activity could be accounted for by the cytotoxicity of these compounds. None of the

compounds showed anti-bacterial activity.

Conclusions The inhibition of PGE2, an important mediator of inflammation and pain,

by flindersine and a derivative thereof, along with the moderate anti-inflammatory

activity shown by several other compounds isolated from G. parviflora leaf extract,

support the traditional use of this plant for pain relief by Australian Aboriginal people.

KEYWORDS: Geijera parviflora, anti-inflammatory, pain relief, prostaglandin E2, flavones, flindersine, Australian plants

1. INTRODUCTION

Geijera parviflora Lindl., known as wilga or native willow, is an endemic Australian

shrub or small tree up to 7 m high, found in inland regions of Eastern Australia

(Porteners, 2014). The species was first described by English botanist John Lindley

in 1848. The genus Geijera Schott is a member of the family Rutaceae and

comprises approximately five species native to Australia, New Guinea and New

Caledonia. The Australian species are G. parviflora, G. salicifolia Schott and G.

linearifolia (DC.) J.M. Black. One species, G. paniculata F. Muell., has recently being

reclassified by Hartley in the genus Coatesia (Hartley, 2001).

Geijera parviflora was used in Australian Aboriginal ceremonies to induce

drowsiness or intoxication. The leaves were baked, powdered and mixed with other

narcotic plant material before being smoked (Lassak and McCarthy, 1983). Leaves

of the plant were also crushed and used externally and internally for pain relief,

including for toothache (Cribb and Cribb, 1981). Australian pastoralists value the tree

as a shade tree and as stock fodder, in particular in times of drought, although its

palatability is variable. The foliage has a protein content of around 15% and is

favoured by sheep but less so by cattle (Cunningham et al., 1992; Wilson and

Harrington, 1980). Plants containing the coumarin geiparvarin (6) are apparently

eaten by sheep, whereas those containing the related compound dehydrogeijerin are

not (Lassak and McCarthy, 1983).

Although the phytochemistry of the plant has been studied since the 1930s and a

number of bioactive compounds have been isolated, the constituents responsible for

the reported traditional uses have not been identified. The composition of the

essential oil from the leaves of G. parviflora was first studied in the 1930s (Penfold,

1932). More recent studies have identified a number of essential oil chemotypes of

G. parviflora (Brophy et al., 2005; Sadgrove et al., 2014; Sadgrove and Jones,

2013). The latter group speculated that minor constituents of the essential oils such

as the monoterpenes 1,8,-cineole, borneol and linalool may be responsible for the

analgesic effects reported, but considered their concentrations to be too low in all but

one geographically restricted chemotype with a high linalool content. It is also not

clear how these relatively simple and widespread compounds could account for the

observed physiological effects.

Other reported major phytochemical constituents of G. parviflora are the coumarin

geiparvarin and the 2-quinoline alkaloid flindersine (Dreyer and Lee, 1972). The

coumarins geiparvarin and dehydrogeijerin were isolated from leaves (Lahey and

Macleod, 1967). There has been considerable research on methods of synthesis of

geiparvarin and its analogues, because these compounds have displayed cytotoxic,

cytostatic and selective antitumour activity (Baraldi et al., 1989; Chimichi et al., 2009;

Viola et al., 2004). The mechanism of action has been proposed to be via disruption

of microtubule formation (Bocca et al., 2001; Miglietta et al., 2001) Inhibition of

monoamine oxidase has also been reported (Carotti et al., 2002). Extracts of leaves

and the smoke of smouldering leaves were found to contain saponins, triterpenoids

and alkaloids but these have not been tested for bioactivity (Sadgrove and Jones,

2013).

7-Geranyloxycoumarin and its derivatives (marmin, 6’-dehydromarmin, geiparvarin,

2’,3’-dihydrogeiparvarin) as well as flindersine have been isolated from the fruit of G.

parviflora (Dreyer and Lee, 1972). Flindersine, originally isolated in 1914 from the

wood of Flindersia australis R.Br. (Matthes and Schreiber, 1914) and since found in

other Rutaceae species, was reported to have antifungal and antimicrobial activity by

one group (Duraipandiyan and Ignacimuthu, 2009) but was considered inactive by

another group (O'Donnell et al., 2010). Moderate antibacterial activity has also been

reported for the leaf essential oil (Sadgrove et al., 2014; Sadgrove and Jones, 2013).

Our group has previously reported five new anthranilic acid derivatives from G.

parviflora. A mixture of three of these (11′-hexadecenoylanthranilic acid, 9′-

hexadecenoylanthranilic acid, and 7′-hexadecenoylanthranilic acid) and the new

natural product hexadecanoylanthranilic acid all showed good inhibition of several

Gram-positive bacterial strains but no anti-inflammatory activity (Shou et al., 2014).

Several novel alkaloids were also isolated, with one of these, parvifloranine A,

inhibiting NO production to a moderate degree (Shou et al., 2013).

During initial screening, the chloroform soluble fraction of the 95% EtOH extract of

the leaves of Geijera parviflora was found to significantly inhibit nitric oxide (NO) and

tumour necrosis factor-α (TNF-α) production in vitro, at a concentration of 50 µg/mL.

This finding led us to hypothesise that the use of the plant for pain relief by

Australian Aboriginal people might be at least in part explained by the presence of

compounds with anti-inflammatory activity. We tested this hypothesis by assaying 11

isolated compounds for in vitro inhibitory activity of PGE2, NO and TNF-α, which are

well established mediators of inflammation. PGE2 is involved in numerous processes

that lead to the classic signs of inflammation: pain, swelling and redness. The

production of PGE2 is controlled by the cyclo-oxygenase (COX) enzymes, which are

the targets of non-steroidal anti-inflammatory drugs (NSAIDs) (Ricciotti and

FitzGerald, 2011). NO plays a key role in inflammation by regulating the expression

of pro-inflammatory cytokines (Kobayashi, 2010), while TNF-α is considered a

central regulator of inflammation by inducing the production of other inflammatory

mediators, including IL-1β, IL-6 and PGE2 (Schaible et al., 2010; Sommer and Kress,

2004). The compounds were also tested for anti-bacterial activity, since infection can

cause pain and inflammation.

2. MATERIALS AND METHODS 2.1 General Experimental Procedures. UV spectra were measured on a Hewlett

Packard 8453 polarimeter at room temperature. The IR spectra were acquired using

a Bruker Vector 33 Spectrometer. High resolution electrospray ionisation (HRESIMS)

accurate mass measurements were carried out on a Bruker micrOTOF-Q instrument

with a Bruker ESI source. NMR spectra were acquired on a Bruker AVANCE 500

MHz spectrometer with TMS as the internal standard. Column chromatography (CC)

separations were carried out using silica gel (Silica-Amorphous, precipitated, 200–

425 mesh, Sigma-Aldrich), Sepra C18-E (50 µm, 65A; Phenomenex Torrance, CA,

USA) and MCI gel CHP20P (Supelco, Bellafonte, PA, USA). Preparative HPLC was

performed on a Gilson 322 system with a UV/Vis-155 detector and a FC204 fraction

collector using a Phenomenex Luna 5 µm (150 × 21.2 mm i.d.) C-18 column.

2.2 Plant Material. The leaves of Geijera parviflora Lindl. were collected near

Lightning Ridge, New South Wales, Australia (29° 25′ S, 147° 59′ E) in December

2011 and authenticated by one of the authors (HW). A voucher specimen

(PHARM110063) has been deposited in the Medicinal Plant Herbarium at Southern

Cross University.

2.3 Extraction and Isolation. The powdered dried leaves of G. parviflora (2 kg)

were extracted with 95% ethanol at room temperature. The ethanol extract was

suspended in H2O and extracted using CHCl3 (3 × 1L). The CHCl3 portion was

evaporated under reduced pressure to afford a crude extract (167.5 g). The crude

CHCl3 extract was subjected to MCI gel (CHP20P) CC, eluted with a gradient of

MeOH/H2O (80:20–100:0) to give five fractions (A–E). After recrystallization in

methanol, Compound 1 (9.6 g) and 6 (1.6 g) were obtained from fraction B and

fraction C respectively. Fraction A was further separated by a C18-E column (4 × 50

cm), eluted with MeOH/H2O (50–70%) to give compound 8 (80 mg) and compound 9

(120 mg). Fraction B (18.4 g) was subjected to silica gel CC, eluted with a gradient of

hexane-EtOAc (4:1, 2:1) to give nine subfractions (BI–BIX), BI was further separated

by preparative HPLC [Phenomenex Luna C18 column (150 × 21.20) 5 µm; mobile

phase acetonitrile and H2O containing 0.05% TFA (0-5 min: 40% acetonitrile, 5–15

min: 40%–95% acetonitrile, 15–20 min: 95% acetonitrile); flow rate 20 mL/min; UV

detection at 210 and 280 nm] to give compound 3 (820 mg). BVIII was applied to a

C18-E column (5 × 40 cm) with a stepwise gradient of MeOH/H2O (40%–70%) to

give compound 2 (16 mg) and compound 11 (16 mg). BIX was subjected to a C18-E

column (5 × 40 cm) eluted with a gradient of MeOH/H2O (40%–60%) to give

compound 4 (8 mg) and compound 7 (12 mg). Fraction D (240 mg) was further

separated by preparative HPLC [Phenomenex Luna C18 column (150 × 21.20) 5 µm;

mobile phase acetonitrile and H2O containing 0.05% TFA (0–5 min: 40% acetonitrile,

5–15 min: 40%–95% acetonitrile, 15–20min: 95% acetonitrile); flow rate 20 mL/min;

UV detection at 210 and 280 nm] to give compound 10 (80 mg). Fraction E was

subjected to a silica gel column, with hexane-EtOAc (8:1, 4:1) as eluent to give six

subfractions (EI–EVI). Compound 5 was obtained from EIV by preparative HPLC

[Phenomenex Luna C18 column (150 × 21.20 mm) 5 µm; mobile phase acetonitrile

and H2O containing 0.05% TFA (0–5 min: 50% acetonitrile, 5–17 min: 50%–95%

acetonitrile, 17–20 min: 95% acetonitrile); flow rate 20 mL/min; UV detection at 210

and 280 nm]. Luminescence was measured on a Wallac 1450 Microbeta

luminescence counter (Wallac, Turku, Finland).

Compound (11). A yellow solid; UV (MeOH) λmax (log ε) 204.0 (4.30), 228.0 (4.11),

282.0 (4.13), 341.0 (4.07), 385.0 (3.90) nm; IR (neat) νmax 3343, 1652, 1607, 1513,

1487, 1440, 1381, 1201, 1178, 1023, 840 cm-1; 1H NMR (Table 1); 13C NMR (Table

1); HREIMS m/z 369.0581 [M + Na]+ (Calc. 369.0586 for C17H28NaO5); APCI-MS m/z

347.1 [M+H]+.

2.4 Bioassays

2.4.1 Cytotoxicity assay. Cytotoxicity in RAW 264.7 murine leukemic monocyte-

macrophages (ATCC, Manassas, VA, USA) was assayed in 96-well plates using the

ATPlite™ Assay kit (PerkinElmer, Glen Waverley, Australia) with chlorambucil

(Sigma-Aldrich, St Louis, MO, USA) as a positive control, as previously described

(Shou et al., 2012). Samples were assayed in triplicate.

2.4.2 PGE2 assay. The effect of the compounds on calcium ionophore A23187-

induced PGE2 production in 3T3 Swiss albino mouse embryonic fibroblast cells

(ATCC, Manassas, VA, USA) was performed as previously described (Shou et al.,

2012). The percentage inhibition of PGE2 production by each sample (assayed in

triplicate) was calculated relative to the DMSO control.

2.4.3 Nitrite (Griess) assay. All compounds were tested for their ability to inhibit

LPS-induced nitric oxide production by RAW 264.7 cells, The experimental

procedure has been reported previously (Shou et al., 2012). Samples and controls

were assayed in triplicate.

2.4.4 TNF-α assay. This assay was performed on a portion of the same cell

supernatants as was used for the nitrite assay in 2.4.3. TNF-α was quantified using a

Quantikine Mouse TNF-α immunoassay kit (R&D Systems, Minneapolis, MN, USA)

according to the manufacturer’s instructions. The percentage inhibition of TNF-α

production by each sample (assayed in triplicate) was calculated relative to the

DMSO control.

2.4.5 Selectivity Index. This index was calculated as the ratio between the IC50

value for cytotoxic activity and the IC50 value for the assay of interest (Jabit et al.,

2009).

2.4.6 Antibacterial assays. All compounds were tested for antibacterial activity

against a panel of Gram-positive (Staphylococcus aureus ATCC 29213 and ATCC

25923, S. epidermidis ATCC 35984, biofilm-forming) and Gram-negative

(Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) strains as

previously described (Shou et al., 2012).

3. RESULTS AND DISCUSSION

A total of four alkaloids (1–4), six coumarins (5–10), and one flavonoid (11) were

isolated from the leaves of G. parviflora (Figure 1). Among them, compound 11 was

a new flavonol, compound 7 was a new natural product, whereas compounds 2–5

were isolated from this species for the first time.

Compound 11 was obtained as a yellow solid. Its HRESIMS exhibited an ion peak at

m/z 369.0581 [M + Na]+, which suggested a molecular formula of C17H28O8. The

characteristic UV absorptions at 282 nm, 341 nm and 385 nm indicated a flavonol

nature of compound 11 (Fang, 1985). The 1H NMR spectrum exhibited two aromatic

doublets at δH 8.24 (2H, d, J = 8.0) and δH 7.03 (2H, d, J = 7.9) in accord with a

flavonol with 3, 5, 6, 7, 8, 4′- oxygenation pattern. A downfield proton at δH 11.65

assignable to the C-5-OH chelated to the carbonyl group of C-4 was also observed.

Two singlets at δH 4.01 and 3.91 (3H, each) suggested the presence of two methoxyl

groups. In the HMBC spectrum of 11, the correlations of methoxyl protons (δH 3.91,

3H, s) and the proton of C-5-OH (δH 11.65) with C-6 (δc 136.8) were observed

(Figure 2), suggesting one of the methoxyl groups being located at C-6. Irradiation of

the protons of one methoxyl group gave rise to a NOE of the other methoxyl group,

which suggested the latter was unequivocally placed at C-7. Thus the structure of

compound 11 was established as 3, 5, 8, 4′-tetrahydroxy-6,7-dimethoxyflavone. All

the 1H and 13C NMR spectroscopic signals of 11 were assigned on the basis of 13C

NMR, 1H-1H COSY, HSQC and HMBC spectra.

Compounds 1-10 (Figure 1) were determined to be flindersine (1) (Jung et al.,

2010), 8-(methoxyl) flindersine (2) (Thangavel et al., 2007), N-(acetoxymethyl)

flindersine (3) (Brader et al., 1993), haplaphine (4) (Ahmed et al., 2010), (R)-6-O-(4-

geranyloxy-2-hydroxy) cinnamoylmarmin (5) (Masuda et al., 1992), geiparvarin (6)

(Lahey and Macleod, 1967), 6-(methoxyl) geiparvarin (7) (Brophy et al., 2005), 6′-

dehydromarmin (8) (Lahey and Macleod, 1967), marmin (9) (Brophy et al., 2005),

and 7-geranyloxycoumarin (10) (Brophy et al., 2005).

All of the compounds (1–11) were investigated for their cytotoxic and anti-

inflammatory activities in vitro. Cytotoxic tests were included to allow for the

calculation of the Selectivity Index (the ratio of the ED50 for cytotoxicity and the IC50

for the activity of interest) (Jabit et al., 2009). The results are summarized in Table 2.

Compound 1 and 3 inhibited the synthesis of PGE2 in calcium ionophore-stimulated

3T3 fibroblast cells with IC50 values of 5.0 µM and 4.9 µM respectively (Figure 3).

Compound 2 showed greatly decreased activity compared to compound 1, with an

IC50 of 122.7 µM, while compound 4, which lacks the dimethyl pyrane ring of

compound 1, showed no activity in this assay, indicating that the presence of this

structural feature is crucial for this activity. It is noteworthy that the Selectivity Index

was relatively high for both compounds 1 and 3 (12.7 and 18.8, respectively),

suggesting the observed inhibitory activity was not a product of cytotoxic effects.

This is the first report of PGE2 inhibitory activity of flindersine and its derivatives.

PGE2 is considered the principal pro-inflammatory prostanoid and contributes to pain

in the peripheral regions as well as the central nervous system (Chen et al., 2013;

Kawabata, 2011). Nonsteroidal anti-inflammatory drugs (NSAIDs), which target

PGE2 synthesis by inhibiting cyclooxygenase enzymes, are widely used but have

undesirable gastrointestinal and cardiovascular side effects (Chen et al., 2013).

Consequently the inhibition of prostaglandin synthases, particularly microsomal

PGES-1, has become the focus of drug development (Chen et al., 2013), as safe

and effective inhibitors of PGE2 production are highly sought after to relieve pain and

inflammation. Further investigation of the mechanism of action of these two

compounds, in particular to determine if they inhibit COX-2 or PGE synthase

enzymes, would be valuable. The results of this study also warrant further

investigation of the structure-activity relationships within this group of compounds.

Compound 6, and to a lesser extent compound 10, inhibited the production of TNF-α

in LPS-stimulated RAW 264.7 macrophages, with IC50 values of 4.1 µM and 19.8

µM, respectively (Table 2). However, the selectivity indices for these compounds

were low (2.0 and 2.2, respectively), suggesting that at least some of the TNF-α

inhibitory effect can be accounted for by their cytotoxic effects. Compounds 5, 6 and

7 were fairly potent inhibitors of NO production in LPS-stimulated RAW 264.7 cells

with IC50 values in the low micromolar range. However, these compounds also

showed considerable cytotoxicity and consequently their selectivity indices were low,

ranging from 2.1 to 2.5, suggesting that cytotoxic effects account for at least some of

the observed NO inhibitory activity. The observed cytotoxicity is consistent with

previous reports of geiparvarin (6) and its analogues with a 3(2H) furanone ring

having antitumour activity (Miglietta et al., 2001; Simoni et al., 1991).

None of the compounds showed anti-bacterial activity against the panel of Gram-

negative and Gram-positive bacteria, i.e. Staphylococcus aureus (MRSA and MSSA

strains), S. epidermidis, Pseudomonas aeruginosa, and Escherichia coli.

The chemical constituents and biological activity of the vast majority of Australian

native plants used traditionally as medicines remain poorly known. The present work

demonstrates that in vitro, two alkaloids found in the leaves of G. parviflora inhibit the

release of PGE2, an important mediator of pain and inflammation. Given this plant

has been used traditionally by Australian Aboriginal people to relieve pain, we

suggest the presence of these anti-inflammatory compounds may at least in part

explain this traditional use. Further work could potentially examine specific

antinociceptic activities in animal models, and structure/activity studies of the

identified compounds may enable the design and synthesis of compounds with

enhanced activity.

Acknowledgments

This work was supported by the Wound Management Innovation CRC (established

and supported under the Australian Government’s Cooperative Research Centres

Program). We thank Mr Graham Macfarlane University of Queensland, School of

Chemistry & Molecular Biosciences, for determining the accurate mass of the new

compound.

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Table 1 The 1H, 13C NMR Data of Compound 11 in Acetone-d6 (δ, ppm; J, Hz)

No. 13C No. 1H 13C

2 147.9 1′ 123.5

3 136.6 2′ 8.24, d (8.0) 130.9

4 177.6 3′ 7.03, d (7.9) 116.4

5 145.1 4′ 160.4

6 136.8 5′ 7.03, d (7.9) 116.4

7 148.0 6′ 8.24, d (8.0) 130.9

8 131.8 6-Me 3.91, s 61.1

9 141.3 7-Me 4.01, s 61.9

10 106.7

Table 2 Cytotoxic and anti-inflammatory activities of compounds 1–11

Compound

no. Cytotoxicity

ECD50 (95% CI)

(µM)

Nitric oxide

inhibition

IC50 (95% CI)

(µM)

TNFα inhibition

IC50 (95% CI)

(µM)

PGE2

inhibition

IC50 (95% CI)

(µM)

Selectivity

Index

1 63.4(48.0–

83.3)

64.0 (54.5–

75.1)

38.4 (21.9–

67.3)

5.0 (3.1–8.3) 12.7 (PGE2)

2 >200 169.6 (135.9–

211.7)

>390 122.7 (77.3–

195.0)

-

3 92.0 (79.6–

106.6)

111.5 (99.8–

124.6)

60.3( 46.3–

78.6)

4.9 (3.1–7.8) 18.8 (PGE2)

4 151.0 (121.4–

187.9)

79.2 (56.6–

110.1)

173.6 (151.7–

198.6)

NA -

5 11.9 (10.1–

13.9)

5.8 (4.1–8.2) 92 (60–139) NA 2.1 (NO)

6 8.3 (6.4–11.0) 3.8 (3.7–4.0) 4.1 (2.2–8.0) NA 2.2 (NO), 2.0

(TNF)

7 12.6 (11.2–

14.1)

5.0 (3.8–6.7) >280 NA 2.5 (NO)

8 43.7 (37.0–

51.5)

57.6 (52.7–

62.9)

>300 NA -

9 79.8 (66.2–

96.1)

105.2 (99.7–

111.8)

>300 NA -

10 43.6 (35.0–

54.3)

62.7 (49.9–

78.8)

19.8 (8.0–48.7) NA 2.2 (TNF)

11 > 300 141.0(119.2–

166.8)

NA NA -

Positive

control

Chlorambucil

31.5 (26.4–

37.6) µM

Dexamethasone

20.8(13.9–31.9)

nM

Dexamethasone

37.0(20.1–67.8)

nM

Indomethacin

6.8 (4.4–10.7)

nM

NA: not active

NH

O

O

NH

O

O

O

N O

O

O

O

NH

O

O

O OO

OH

O

O

HO O

1 2 3 4

5

OOO

O

O

OO O

OOO

O

O

O

6

10

OO O

HO

OH

OO OHO

O

7

89

11

O

O

OH

OH

MeO

MeO

OHOH

Figure 1. Chemical structures of compounds 1–11

Figure 2. Key HMBC and NOE correlations of compound 11

Figure 3. Inhibition of prostaglandin E2 synthesis in 3T3 Swiss albino

cells: dose response curves for compounds 1 and 3.

Figure captions

Figure 1. Chemical structures of compounds 1-11.

Figure 2. Key HMBC and NOE correlations of compound 11.

Figure 3. Inhibition of prostaglandin E2 synthesis in 3T3 Swiss albino cells: dose

response curves for compounds 1 and 3.

Graphical abstract