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Biochimie 92 (2010) 1213e1221

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Biochimie

journal homepage: www.elsevier .com/locate/biochi

Research paper

b-sitosterol among other secondary metabolites of Piper galeatumshows inhibition of TNFa-induced cell adhesion moleculeexpression on human endothelial cells

Pankaj Gupta a,1, Sakshi Balwani b,1, Sarvesh Kumar b, Neha Aggarwal a, Miriam Rossi c,Sarah Paumier c, Francesco Caruso d, Paolo Bovicelli d, Luciano Saso e, Anthony L. DePass f,Ashok K. Prasad a,*, Virinder S. Parmar a, Balaram Ghosh b,**

aBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, IndiabMolecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology (IGIB), Mall Road, Delhi University Campus, Delhi-110 007, IndiacVassar College, Department of Chemistry, Poughkeepsie, NY 12604-0484, USAd Instituto di Chimica Biomolecolare, CNR, Piazzale Aldo Moro 5, Rome 00185, ItalyeDepartment of Physiology and Pharmacology ‘Vittorio Erspamer’, Sapienza University, Piazzale Aldo Moro 5, Rome 00185, ItalyfDepartment of Biology, Long Island University-Brooklyn, 1 University Plaza, Brooklyn, NY 11201, USA

a r t i c l e i n f o

Article history:Received 26 February 2010Accepted 4 June 2010Available online 15 June 2010

Keywords:Piper galeatum1-(30-hydroxy-50-methoxycinnamoyl)-piperidineAnti-inflammatory activityICAM-1VCAM-1E-Selectin

* Corresponding author for Chemistry Section. Tel.:** Corresponding author for Biology Section. Tel.: þ(011)27667471.

E-mail addresses: ashokenzyme@yahoo.com (A.K(B. Ghosh).

1 These authors contributed equally to this work.

0300-9084/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.biochi.2010.06.005

a b s t r a c t

A phytochemical investigation of the stems of Piper galeatum yielded one novel amide, 1-(30-hydroxy-50-methoxycinnamoyl)-piperidine (5) along with four known compounds, i.e. b-sitosterol (1), cyclo-stachine-A (2), piperine (3) and piperolein-B (4). The structures of all the five compounds, isolated for thefirst time from this plant were unambiguously established on the basis of their detailed spectral analysis.The structure of cyclostachine-A (2) was confirmed by X-ray crystallographic studies and structures ofknown compounds were confirmed by comparison of their physical and/or chemical data with thosereported in the literature, which were in complete agreement. Additionally, the crude extracts as well asthe isolated pure compounds were screened for their activity to inhibit TNFa (tumour necrosis factor-a)-induced expression of cell adhesion molecule ICAM-1 (intercellular adhesion molecule-1) on the surfaceof human umbilical vein endothelial cells (HUVECs). Among all, b-sitosterol (1) was found to be the mostactive compound, which was taken for further studies. b-sitosterol also significantly inhibited theTNFa-induced expression of VCAM-1 and E-selectin, which also play key role in various inflammatorydiseases. The functional correlation of cell adhesion molecules inhibition was assessed by cell adhesionassay using human neutrophils. We found that b-sitosterol significantly blocks the adhesion of neutro-phils to endothelial monolayer. To elucidate the molecular mechanism of inhibition of cell adhesionmolecules, we investigated the status of nuclear transcription factor-kB (NF-kB) and were able toestablish that b-sitosterol significantly blocked the TNFa-induced activation of NF-kB.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

The genus Piper (Piperaceae), largely distributed in the tropicaland subtropical regions of the world, are used extensively intraditional medicine [7,15,30,33,38]. Chemical investigations have

þ91(011) 27662486.91(011) 27662580; fax: þ91

. Prasad), bghosh@igib.res.in

son SAS. All rights reserved.

shown that the genus Piper has different classes of compoundsincluding amides, lignans, flavonoids, terpenes, steroids, aristo-lactams, propenylphenols, esters, alkaloids, oxygenated cyclohex-anes, hydroquinone and benzoic acid derivatives [8,22,24,25,35,36].Piper galeatum is indigenous to India and grows wild in the forestsof Wayanad, Kerala region. Previous phytochemical study reportedthe isolation and characterization of ten sesquiterpenes andmonoterpenes namely, b-elemene, d-elemene, a-humulene, b-car-yophyllene, a-copaene, a-ionone, 10-(acetylmethyl)-(�)-3-carene,dihydrocarvyl acetate, 1-p-menthen-8-yl acetate and linalyl acetatefrom the fruits extract of P. galeatum [12]. In continuation of ourprevious investigations on various Indian Piper species, we

P. Gupta et al. / Biochimie 92 (2010) 1213e12211214

undertook the phytochemical examination of the stems ofP. galeatum which led to the isolation and characterization of fourknown compounds, i.e. b-sitosterol (1) [1,3], cyclostachine-A (2)[13,14], piperine (3) [6], piperolein-B (4) ([15b]) and a novel ami-de, namely 1-(30-hydroxy-50-methoxycinnamoyl)-piperidine (5)which has been reported for the first time from P. galeatum (Fig. 1).

Inflammation involves various immune cells including leuko-cytes and many inflammatory mediators. Localized leukocyteaccumulation is the cellular hallmark of inflammation. Endothelialcells are key players in the recruitment of leukocytes to the site ofinflammation. The migration of the leukocytes to the site ofinflammation is regulated in part by the expression of cell adhesionmolecules such as intercellular adhesion molecule-1 (ICAM-1),VCAM-1 and E-Selectin [4]. These cell adhesion molecules areinduced on endothelial cells by various pro-inflammatory cytokineslike IL-1b and TNFa and also by bacterial LPS [10,32]. The increasedexpression of cell adhesionmolecules on the endothelial cells altersthe adhesive property of the vasculature leading to indiscriminateinfiltration of the leukocytes across the blood vessels and hencecauses inflammation [21]. A promising therapeutic approach forthe management of various inflammatory conditions would be toinhibit the cytokine induced expression of cell adhesion molecules[16]. Although various synthetic drugs, antibodies and peptideshave been demonstrated to inhibit the expression of these mole-cules, they have their own limitations for usage because ofunwanted side effects [9].

Recently, considerable efforts are being focused to identify anti-inflammatory compounds from natural sources [2,18,20,23,31].Earlier, we have reported the biochemical activities of somemedicinally important plants including Piper species [26]. Recently,the ICAM-1 inhibitory activity of an aromatic ester and its analogsincluding piperine has also been reported by our group [17]. In thepresent study, we have evaluated the inhibitory activity of crudeextracts (petroleum ether, chloroform and methanol) of the stemsof P. galeatum together with their pure natural isolates on the TNFa-induced expression of ICAM-1 on endothelial cells with an aim ofidentifying the active constituents.

β -Sitosterol

1

Piperine

3

1-(3'-Hydroxy-5'-methoxyci

5

2

3

1 '2 '

3 '4 '

5 ' 6 '

H

H

HOH

N

O

O

O

O

O

OH

M eO

Fig. 1. Structures of isolated compound

2. Results and discussion

2.1. Isolation and purification of the compounds 1e5from the stems of P. galeatum

Compounds 1e4 were isolated from petroleum ether extractand compound 5was isolated frommethanol extract of P. galeatumusing column chromatography over silica gel, followed by crystal-lization of the fractions containing compounds (Fig. 1); thecompounds 1e5were evaluated for their ICAM-1 inhibitory activityon human endothelial cells.

The novel amide 5was isolated from the methanol extract of thestems of P. galeatum as a white solid, m.p. 73e75 �C, it gave the [M]þ

ion peak atm/z 261.1356 in its HRMS spectrum, which correspondedwith the molecular formula C15H19O3N. In its UV spectrum, itexhibited a strong absorption at 289 nm indicating the presence ofa conjugateddouble bond system,whichwas further supported by itsIR spectrum that showed a strong absorption at 2940 cm�1 for anextended a,b-unsaturated amide, and at 1601 cm�1 (for transconjugated olefinic double bond) and 3434 cm�1 (for hydroxylgroup). The 1H NMR spectrum of 5 showed two multiplets at d 1.87(integrating for six protons) and at d 3.53 (integrating for fourprotons) due to themethylene protons C-300H, C-40 0H and C-500H& C-200Hand C-60 0H, respectively of the piperidinemoiety. The compoundshowed the presence of a methoxy group as indicated by a singlet atd 3.73 integrating for three protons in its 1H NMR spectrum. Twobroad singlets in the aromatic region at d6.39 and 6.59 integrating forone proton and two protons were assigned to the C-40H and C-20H &C-60H, respectively. The signals at d 6.64 and 7.53 appeared as a broadsinglet and as a doublet (J¼ 15.4 Hz) integrating for one proton eachdue to the C-2H and C-3H, respectively. The 13C NMR spectrum ofcompound 5 exhibited all relevant signals. The characteristic reso-nances for carbonyl carbon and a,b-unsaturated carbons wereexhibited at d 168.80, 142.00 and 119.81, respectively in the 13C NMRspectrum of compound 5. Further, all the CH3, CH2 and CH carbonspresent in compound 5were confirmed by the DEPT experiments at90� and 135�, these results were endorsed by its 1He1H COSY and

Cyclostachine-A

2

Piperolein-B

4

nnamoyl)-piperidine

1 '' 2 ''3 ''

4 ''5 ''6 ''

1

N

O

N

O

H

HON

O

O

s from the stems of Piper galeatum.

Table 1Effect of Piper galeatum compounds on the TNFa-induced expression of ICAM-1 onendothelial cells.

S.No. Compound Maximumtolerableconc. (mM)

Cell viabilityat maximumtolerableconc. (%)

ICAM-1inhibitionat maximumtolerableconc. (%)

ICAM-1inhibitionIC50 (mM)

1. b-sitosterol (1) 50 99 86 262. Cyclostachine-A (2) 57 97 66 363. Piperine (3) 175 98 70 1584. Piperolein-B (4) 58 98 43 >585. 1-(30-Hydroxy-50-

methoxycinnamoyl)-piperidine (5)

77 97 14 >77

P. Gupta et al. / Biochimie 92 (2010) 1213e1221 1215

1He13C COSY spectral data as well. The identity of compound 5 wasthus unambiguously established as 1-(30-hydroxy-50-methox-ycinnamoyl)-piperidine, this is the first report of its isolation fromany natural source and has not been synthesized earlier.

The structures of known compounds, i.e. b-sitosterol (1) [1,3],cyclostachine-A (2) [13]; piperine (3) [6], piperolein-B (4) [15b]were established on the basis of their detailed spectral data (IR,1H, 13C NMR, 1He1H COSY NMR, 1He13C COSY NMR spectra andHRMS) analysis and further confirmed by the comparison of thedata with those reported in literature. The structure of cyclo-stachine-A was also confirmed by X-ray crystallographic studies(Fig. 2). In the literature, using spectroscopic studies, the absoluteconfiguration of cyclostachine-A was first described [13]. In addi-tion, the same group published the crystal structure of cyclo-stachine-A as a sulfate salt [14]; however the carbonyl oxygen ismissing from the figure and there are no published or depositedatomic coordinates of the X-ray study. Moreover, the publishedpicture [14] shows the juncture in the decalin system having both Hatoms trans displayed, instead of the expected cis arrangement ofcyclostachine-A, as established by us (Fig. 2). Because of the lack ofhydrogen bond donors, there are no hydrogen bonds among theintermolecular forces in the crystal.

ICAM-1 VCAM-1 E-Selectin

2.2. ICAM-1 inhibitory activity

The purified compounds from the stems of P. galeatum, i.e. b-sitosterol (1), cyclostachine-A (2), piperine (3), piperolein-B (4), and1-(30-hydroxy-50-methoxycinnamoyl)-piperidine (5) were evalu-ated for their inhibitory activity against TNFa-induced ICAM-1expression. The concentrations of the compounds were selected onthe basis of maximum tolerable dose determined by the cytotox-icity assays (Table 1).

To determine the effect of P. galeatum extracts on ICAM-1expression on endothelial cells, the endothelial cells were incu-bated with or without extracts at different concentrations for 2 hprior to treatment with TNFa for 16 h.

The ICAM-1 inhibitory activities of the five compounds frompetroleum ether and methanol extracts are tabulated in Table 1.b-sitosterol (1) showed an IC50 value of 26 mM with 86% inhibitionof ICAM-1 at the maximum tolerant dose of 50 mM (Fig. 3). On theother hand, cyclostachine-A (2) showed an IC50 value of 36 mMwith66% inhibition of ICAM-1 at a concentration of 57 mM. Piperine (3)showed an IC50 value of 158 mM with 70% inhibition of ICAM-1 at

Fig. 2. X-ray of one molecule of cyclostachine-A (compound 2).

a concentration of 175 mM which is in accordance with our earlierreport [17]. Although the maximum tolerable dose of other naturalisolate, piperolein-B (4), is almost similar to that of b-sitosterol (1),its inhibitory activity is lower than that of the compound 1. Theactivity screening data (Table 1, Fig. 3) revealed that b-sitosterol (1)is most potent in inhibiting the ICAM-1 expression on TNFa-induced human endothelial cells.

2.3. b-sitosterol inhibits the expression of VCAM-1 and E-Selectinon endothelial cells

The expression of cell adhesion molecules VCAM-1 andE-Selectin along with ICAM-1, upon TNFa induction to the humanendothelial cells, is also required for the firm adhesion and trans-migration of lymphocytes to the site of tissue injury. Thus we havetested the ability of b-sitosterol to inhibit the TNFa-inducedexpression of VCAM-1 and E-Selectin. It was found that b-sitosterolinhibits the TNFa-induced expression of VCAM-1 and E-Selectinsignificantly with IC50 values of 30 and 28 mM, respectively (Fig. 3).

2.4. b-sitosterol inhibits the adhesion of neutrophils to theendothelium

Neutrophil adhesion to endothelial monolayer requires a seriesof interactions that are mediated by cell adhesion molecules

0.00

25.00

50.00

75.00

100.00

3 6 12.5 25 50

Conc. of βββ-sitosterol (µM)

% I

CA

M-1

Inh

ibit

ion

Fig. 3. b-sitosterol significantly inhibits the expression of cell adhesion molecules onhuman endothelial cells: HUVECs were seeded in 96-well plates at a cell density of2 � 104 cells/well. Cells were pre-treated with various concentrations of b-sitosterol for2 h, followed by induction with TNFa (10 ng/ml) for 16 h for ICAM-1 and VCAM-1, and4 h for E-Selectin. The cell surface expression of cell adhesion molecules was measuredby ELISA. The data presented are representative of three independent experiments.Values are means � the standard error of mean (sem) of quadruplicate wells.

P. Gupta et al. / Biochimie 92 (2010) 1213e12211216

including ICAM-1, VCAM-1 and E-Selectin. To check the functionalconsequence of inhibition of cell adhesion molecules by b-sitos-terol, neutrophil adhesion assay was performed. As detected bycolorimetric assay, there was low adherence of neutrophils onunstimulated endothelium. This adherence was increased to morethan 3-fold upon stimulation of endothelial cells by TNFa (data notshown). Interestingly, b-sitosterol significantly inhibited theadhesion of neutrophils to the endothelium in a concentrationdependent manner with an IC50 of 22 mM (Fig. 4).

2.5. The transcript levels of ICAM-1, VCAM-1 and E-Selectinare decreased by b-sitosterol

As discussed earlier, b-sitosterol significantly inhibited theinduced protein levels of ICAM-1, VCAM-1 and E-Selectin and it isknown that the promoter region of their genes contain binding site(s) for the transcription factor, NF-kB (nuclear transcription factor),responsible for their activation. The results revealed low levels ofICAM-1 and almost undetectable levels of VCAM-1 and E-SelectinmRNAs in uninduced cells treated with or without b-sitosterol(Fig. 5). The faint band which is visible in case of b-sitosterol givenalone corresponds to the basal levels of ICAM-1 present in theendothelial cells in absence of induction. It is well documented thatICAM-1 protein and mRNA basal levels are present in unstimulatedHUVECs whereas the basal levels of VCAM-1 and E-Selectin areundetectable in unstimulated conditions ([39,40]). Upon stimula-tion of cells with TNFa, there was an upregulation in ICAM-1,VCAM-1 and E-Selectin mRNA levels (3-, 9- and 9-fold, respec-tively). However, treatment of cells with b-sitosterol beforeinductionwith TNFa significantly reduced the inducedmRNA levelsof ICAM-1, VCAM-1 and E-Selectin (Fig. 5A). Interestingly, the co-treatment of cells with b-sitosterol appreciably decreased the levelsof mRNA transcripts of cell adhesion molecules (Fig. 5B). However,post-treatment of cells with b-sitosterol did not have any effect onthe levels of mRNA transcripts of cell adhesion molecules (Fig. 5C).These results indicated that b-sitosterol may be acting at thetranscriptional level to modulate the expression of ICAM-1,VCAM-1 and E-Selectin.

0.00

25.00

50.00

75.00

100.00

3 6 13 25 50

Conc. of βββ-sitosterol (µM)

% I

nhib

itio

n of

neu

trop

hils

adhe

sion

to

End

othe

lial c

ells

Fig. 4. b-sitosterol inhibits the adhesion of neutrophils to the endothelium: HUVECswere seeded in 96-well plates at a cell density of 2 � 104 cells/well. Cells were pre-treated with various concentrations of b-sitosterol for 2 h, followed by induction withTNFa (10 ng/ml) for 6 h. Neutrophils, isolated from human blood, were added onto theendothelial cells monolayer and allowed to adhere for 1 h at 37 �C. The number of cellsremained adhered to the monolayer were estimated by measuring the peroxidaseactivity. Data is expressed as mean � sem. Results are representative of three inde-pendent experiments.

Fig. 5. b-sitosterol decreases the transcript levels of ICAM-1, VCAM-1 and E-Selectin:The endothelial cells were (A) pre-treated with or without 50 mM concentration of b-sitosterol before induction with TNFa (10 ng/ml), (B) co-treated with or without 50 mMconcentration of b-sitosterol and TNFa (10 ng/ml) and (C) post-treated with or without50 mM concentration of b-sitosterol after induction with TNFa (10 ng/ml), for 4 h. Thetotal RNA of the cells was isolated and analyzed by RT-PCR. The intensity of transcriptswere densitometrically scanned using AlphaImager gel documentation system (Biorad,USA) and normalized with that of b-Actin levels expressed under similar conditions.

P. Gupta et al. / Biochimie 92 (2010) 1213e1221 1217

2.6. b-sitosterol inhibits TNFa-induced p65 translocationto the nucleus

In order to elucidate its mechanism of action, we furtherinvestigated b-sitosterol for its inhibition of nuclear transcriptionfactor-kB (NF-kB). Inflammatory cytokine TNFa has been shown tocause rapid phosphorylation and degradation of IkBa, a cytoplasmicinhibitor of NF-kB, resulting in translocation of the activated p50/p65 heterodimer from the cytoplasm to the nucleus. For theinduction of cell adhesion molecules the activation of NF-kB isrequired. To examine whether b-sitosterol inhibits the activation ofNF-kB, we measured the levels of p65 in the cytoplasmic and thenuclear extracts prepared from b-sitosterol treated cells. It wasobserved that low levels of p65 were present in the nucleus of theunstimulated cells or cells treated with b-sitosterol alone, whilecomparatively higher levels were observed in the cytoplasm(Fig. 6). Upon stimulation with TNFa, the levels of p65 in thecytoplasm were decreased (3-fold) while its levels were increasedin the nucleus (3.5-fold). However, upon pre-treatment of cells withb-sitosterol, the levels of p65 did not decrease significantly in thecytoplasm and there was no concomitant increase in the p65 levelsin the nucleus (Fig. 6A). Interestingly, the co-treatment of cells withb-sitosterol moderately upregulated the levels of p65 in the cyto-plasm and resulted in slight decrease of its levels in the nucleus(Fig. 6B). However, post-treatment of cells with b-sitosterol resul-ted in no significant effects on the levels of p65 either in thecytoplasm or in nucleus (Fig. 6C). These results therefore indicatethat b-sitosterol blocks the translocation of NF-kB p65 subunit fromcytoplasm to the nucleus when given prior to or along withinduction with TNFa, and hence may be responsible for preventingthe induction of cell adhesion molecules expression by TNFa.

b-sitosterol, a naturally occurring plant sterol was chemicallydefined in 1922 and has only recently enjoyed biological impor-tance as the natural approach to control plasma cholesterol levels[5]. However, isolated reports through the years have ascribedother properties to these molecules isolated from different plantsources, including their ability to reduce inflammation in animalmodels [37], their insulin-releasing activity (hence their potentialuse in diabetes) [11], and their inhibitory activity in cancer cell lines[27]. Recently, its cardioprotective effects on HAECs and its inhibi-tory effect on oxidized LDL-stimulated ICAM-1 expression onHUVECs have also been demonstrated [41,42]. These propertiesof b-sitosterol could be attributed, at least in part, to inhibition ofNF-kB and cell adhesion molecules such as ICAM-1, VCAM-1 andE-Selectin. In our study, b-sitosterol at a concentration of 50 mM isfound to be a potent inhibitor of NF-kB activation and cell adhesionmolecule expression. Some of the known compounds inhibit NF-kBand cell adhesionmolecule expression at higher concentrations, forexample, diclofenac inhibits at 750 mM [28], N-acetyl cysteine(NAC) and pyrrolidone dithiocarbamate (PDTC) are most effectiveat concentrations of 100 mM and 1 mM, respectively [34]. b-sitos-terol, therefore, works well as an inhibitor of NF-kB activation andcell adhesion molecule expression at a comparatively lowerconcentration. These results with b-sitosterol open avenues fortherapeutic management of various pathological conditions asso-ciated with upregulation of endothelial leukocyte adhesion mole-cules and NF-kB.

3. Experimental section

3.1. General

The 1H and 13C NMR spectra were recorded on a Bruker AC-300Avance spectrometer at 300 and at 75.5 MHz, respectively. Thechemical shift values are in d ppm relative to TMS used as internal

standard and the coupling constants (J) are measured in Hz.Analytical TLCs were performed on precoated Merck silica gel60F254 plates; the spots were detected by viewing under UV light.The IR spectra were recorded as KBr pellets on a PerkineElmermodel 2000 FT-IR spectrophotometer and UV was recorded usingBeckman DU-64 spectrophotometer. The FAB-HRMS spectra wererecorded on a JEOL JMS-AX505W high-resolution mass spectrom-eter in positive ion mode using the matrix HEDS (bis-hydroxy-ethylsulphide) doped with sodium acetate while the ESIMS spectrawere recorded on a Micromass KC 455 TOF mass spectrometer inpositive ion mode. Column chromatography was carried out usingsilica gel (100e200 mesh). Melting points were determined usingH2SO4 bath and are uncorrected.

Anti-ICAM-1, anti-VCAM-1, anti-E-Selectin and recombinanthuman TNFa were purchased from BD Pharmingen, USA. M 199media, L-glutamine, endothelial cell growth factor (ECGF), trypsin,dextran, ficoll-hypaque, o-phenylenediamine (OPD), DAB (dihy-drobenzidine tetrahydrochloride), MTT (methylthiazolydiphenyltetrazolium bromide), anti-mouse IgG-HRP, anti-rabbit IgG-HRP,anti-Lamin B1 and anti-b-tubulin were purchased from SigmaChemical Co., USA. Fetal calf serum (FCS) was purchased fromBiological Industries, Israel. Anti-p65 NF-kB was purchased fromSanta cruz Biotechnology, USA.

3.2. Plant material

The stems of P. galeatum were collected from the wild plantsgrowing in the forests of Wayanad, Kerala. Voucher Herbariumspecimens of this plant have been deposited in the Herbarium ofDepartment of Botany, M.S. University, Baroda (BARO).

3.3. Extraction and isolation

Dried and finally powdered plant material (1.5 kg) was extractedfor several hours separately with hot petroleum ether (2 L), hotchloroform (2 L) and hotmethanol (2 L) in succession using a soxhletapparatus. Organic solvent was removed from all the extracts sepa-rately on a rotavapour under reduced pressure and the residue(petroleum ether extract 38 g, chloroform extract 16 g and methanolextract 28 g, respectively) left was subjected to column chromatog-raphy over silica gel. Crude petroleum ether extract was fractionatedon silica gel column using petroleum ether and petroleum ether/ethyl acetate mixture as eluents in increasing polarity. Elution withpetroleum ether/ethyl acetate (96: 4), (88: 12), (86: 14) and (80: 20)resulted in the fractions that contained compounds 1, 2, 3 and 4,respectively. Repeated column chromatography and re-crystalliza-tions of these fractions led to the isolation of four compounds in thepure form, viz. b-sitosterol (1) (100 mg) from fraction 1, cyclo-stachine-A (2) (800 mg) from fraction 2, piperine (3) (80 mg) fromfraction 3 and piperolein-B (4) (80 mg) from fraction 4. Crudemethanolic extract was fractionated over silica gel column usingchloroform and chloroformemethanol mixture as eluent inincreasing polarities. Elution with chloroformemethanol (96:4) ledto the fraction 5. Repeated recrystallization led to the isolation ofnovel compound 1-(30-hydroxy-50-methoxycinnamoyl)-piperidine(5) (45 mg) from fraction 5 of methanolic extract.

3.4. Cell culture

Primary endothelial cells were isolated from human umbilicalcord using mild trypsinization. The cells were grown in M199medium supplemented with 15% heat inactivated fetal calf serum,2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin,0.25 mg/ml amphotericin B, endothelial cell growth factor (50 mg/ml)and heparin (5 U/ml). At confluence, the cells were subcultured

Fig. 6. b-sitosterol blocks NF-kB p65 translocation: The endothelial cells were (A) pre-treated with or without 50 mM concentration of b-sitosterol before induction with TNFa(10 ng/ml), (B) co-treated with or without 50 mM concentration of b-sitosterol and TNFa (10 ng/ml) and (C) post-treated with or without 50 mM concentration of b-sitosterol afterinduction with TNFa (10 ng/ml), for 30 min. The cytoplasmic and nuclear extracts were subjected to SDS-PAGE followed by probing with NF-kB p65 antibody. The intensity of bandswere densitometrically scanned using AlphaImager gel documentation system (Biorad, USA) and normalized with that of b-Tubulin and Lamin B1 for cytoplasmic and nuclearextracts, respectively. The data presented are representative of three independent experiments.

P. Gupta et al. / Biochimie 92 (2010) 1213e12211218

P. Gupta et al. / Biochimie 92 (2010) 1213e1221 1219

using 0.05% trypsin-0.01 M EDTA solution and were used betweenpassages three to four.

3.4.1. Preparation of compound stocksStock solutions of the isolated pure compounds were prepared

in dimethylsulphoxide (DMSO). All the compounds were diluted toworking concentrations, from the above stocks, in aqueous cellculture medium. The maximum DMSO concentration was 0.5%,and, in control experiments, this was without effect on themorphology and viability of the cells (data not shown).

3.4.2. Cell viability assayThe cytotoxicity of these compounds was analyzed by colori-

metric MTT assay. Briefly, endothelial cells were treated withdifferent concentrations of b-sitosterol for 24 h. Four hours beforethe end of incubation, medium was removed and 100 ml MTT(2.5 mg/ml in phosphate buffered saline, pH 7.4) was added to eachwell. The MTT solutionwas removed after 4 h and 100 ml DMSOwasadded to each well to dissolve water insoluble formazan crystals.Absorbance was recorded at 570 nm in an ELISA reader (Bio-Rad,Model 680, USA). All experiments were performed at least 3 timesin triplicate wells. From this assay, the % viability of the cells atvarious concentrations of each compound was determined bynormalization to cells incubated in vehicle (0.5% DMSO in cellculture medium) and which were considered 100% viable. Thehighest concentration at which the viability of the cells was >95%was denoted as the maximum tolerable concentration for thatcompound.

3.4.3. Cell-ELISA for measurement of cell adhesion moleculeexpression

Cell-ELISA was used for measuring the expression of ICAM-1,VCAM-1 and E-Selectin on surface of endothelial cells [19]. Endo-thelial cells were incubated with or without the b-sitosterol atdifferent concentrations for 2 h, followed by treatment with TNFa(10 ng/ml) for 16 h for ICAM-1 and VCAM-1 and 4 h for E-Selectinexpression. The cells were fixed with 1.0% glutaraldehyde at 4 �C.Non-specific binding of antibody was blocked by using skimmedmilk (3.0% in PBS). Cells were incubated overnight at 4 �C with anti-ICAM-1 or anti-VCAM-1 or anti-E-Selectin primary Abs, diluted inblocking buffer, the cells were further washed with PBS and incu-bated with peroxidase-conjugated goat anti-mouse secondary Abs.After washings, cells were exposed to the peroxidase substrate(o-phenylenediamine dihydrochloride 40 mg/100 ml in citratephosphate buffer, pH 4.5). Reaction was stopped by the addition of2 N sulfuric acid and absorbance at 490 nm was measured usingmicroplate reader (Spectramax 190, Molecular Devices, USA).

3.5. Neutrophil isolation

Neutrophils were isolated from peripheral blood of healthy indi-viduals [19]. Blood was collected in heparin solution (20 U/ml) anderythrocytes were removed by sedimentation against 6% dextransolution. Plasma, rich in white blood cells, was layered overFicolleHypaque solution, followed by centrifugation (300 g for20 min, RT). The top saline layer and the FicolleHypaque layer wereaspirated leaving neutrophils/RBC pellet. The residual red blood cellswere removedbyRBC lysis buffer (0.83%ammoniumchloride in0.1MTris-Cl, pH8.0) at room temperature. Isolated cellswerewashedwithPBS and resuspended in PBS containing 5 mM glucose, 1 mM CaCl2,and 1 mMMgCl2 at a final concentration of 6 � 105 cells/ml.

3.5.1. Neutrophil adhesion assayNeutrophil adhesion assay was performed under static condi-

tions as described previously [19]. Briefly, endothelial cells plated in

96-well culture plates were incubated with or without b-sitosterolat different concentrations for 2 h, followed by inductionwith TNFa(10 ng/ml) for 6 h. Endothelial monolayers were washed with PBSand neutrophils (6 � 104/well) were added over it and wereallowed to adhere for 1 h at 37 �C. The non-adherent neutrophilswere washed with PBS and neutrophils bound to endothelial cellswere assayed by adding a substrate solution consisting of o-phe-nylenediamine dihydrochloride (40mg/100ml in citrate phosphatebuffer, pH 4.5), 0.1% cetrimethyl ammonium bromide, and 3-amino-1,2,4 triazole (1 mM). The absorbance was read at 490 nmusing an automated microplate reader (Model 680, Bio-Rad, USA).

3.6. Treatment of endothelial cells with b-sitosterol

In order to examine whether b-sitosterol blocked the inductionof transcription of cell adhesion molecules genes and the trans-location of NF-kB p65 subunit from the cytosol into the nucleus, theendothelial cells were incubated with 50 mM b-sitosterol for 2 hbefore inducing the cells with TNFa (pre-treatment), simulta-neously at the time of inductionwith TNFa (co-treatment) and after30 min of induction with TNFa (post-treatment), followed byRT-PCR and western blot.

3.6.1. Preparation of cytoplasmic and nuclear extractsAfter the treatment with b-sitosterol, the cells were washed

with PBS, dislodged using a cell scraper, and centrifuged at 300 g for10 min. The cytoplasmic and nuclear extracts were prepared asdescribed previously [19].

3.6.2. Western blots of NF-kB p65The cytoplasmic and nuclear extracts were subjected to 10%

SDSePAGE and transferred to nitrocellulose membrane in Transferbuffer containing 25mMTris,192mM glycine, and 20%methanol at35 V for 16 h. After blocking in 3% skimmed milk, membrane wasincubated with anti-NF-kB p65 (Santa Cruz biotechnology) or anti-b-tubulin antibody (Sigma) or anti-Lamin B1 antibody (Sigma) for2 h at RT. Immunoreactive proteins were detected by HRP-conju-gated anti-rabbit secondary antibody (Santa Cruz biotechnology)incubated for 2 h at RT. After intensive washing, membrane wasdeveloped with DAB (dihydrobenzidine tetrahydrochloride) asa substrate.

3.7. Total RNA isolation and Reverse Transcription-PolymeraseChain Reaction (RT-PCR)

RNA was isolated from b-sitosterol treated cells (Section 3.6)according to a modified guanidinium thiocyanate procedure. Theexpression of the transcripts for ICAM-1, VCAM-1 and E-Selectinwas evaluated by RT-PCR. The primers were synthesized accordingto the published cDNA sequences to yield products of length 555,533 and 479 base pairs, respectively. As a control, b-actin mRNAwas also amplified by RT-PCR and a product of 661 base pairs wasobtained. The RT-PCR was performed following the manufacturer’sprotocol (Access RT-PCR system; Promega). Briefly, 100 ng of thetotal RNA was reverse transcribed using AMV reverse transcriptaseat 48 �C for 45 min followed by amplification using Tfl polymerasefor 25 cycles. The conditions for PCR were as follows: denaturationat 92 �C for 1 min, primer annealing at 52 �C for 90 s, extension at72 �C for 2 min, and a final extension at 72 �C for 10 min. The PCRproducts were analyzed in 1% agarose gel electrophoresis andvisualized by ethidium bromide staining. The intensity of ICAM-1,VCAM-1 and E-Selectin transcripts were densitometrically scannedusing AlphaImager gel documentation system (Biorad, USA) andnormalized with that of b-Actin levels expressed under similarconditions.

Table 2Experimental X-ray structural data of cyclostachine-A.

Chemical formula C22H27NO3

Fw 353Crystal system monoclinicSpace group P21a, Ǻ 10.4118(1)b, Ǻ 11.5853(2)c, Ǻ 15.6888(2)V, Ǻ3 1835.60(4)Z 4Crystal size, mm3 0.25 � 0.20 � 0.20d,g/cm3 1.279m,cm�1 0.084Reflns[I > 2s(I)] 9826R(Rw) 0.0496 (0.1068)

P. Gupta et al. / Biochimie 92 (2010) 1213e12211220

3.8. 1-(30-Hydroxy-50-methoxycinnamoyl)-piperidine (5)

It was obtained as a white solid (45 mg), mp 73e75 �C. Rf : 0.32(65% ethyl acetate/petroleum ether, v/v). IR (KBr) nmax: 3434 (OH),2940, 1653 (CO), 1601, 1421, 1353, 1296, 1204, 1153, 1058, 979, 920and 826 cm�1. 1H NMR (300 MHz, CDCl3): d 1.87 (6H, m, C-300H,C-400H and C-500H), 3.53 (4H, m, C-20 0H and C-60H), 3.73 (3H, s,OCH3), 6.39 (1H, br s, C-40H), 6.59 (2H, br s, C-2’H and C-600H), 6.64(1H, br s, C-2H) and 7.53 (1H, d, J ¼ 15.4 Hz, C-3H). 13C NMR(75.5 MHz, CDCl3): d 24.61 and 26.50 (C-30 0, C-400 and C-50 0), 46.42and 46.90 (C-20 0 and C-600), 55.72 (OCH3), 101.91 (C-40), 106.20 (C-20

and C-60), 119.81 (C-2), 137.73 (C-10), 142.00 (C-3), 161.32 (C-30),164.91 (C-50) and 168.80 (CO). HRMS:m/z calculated for C15H19O3N261.1365, observed 261.1356.

3.9. X-ray diffraction study

A Bruker Apex-2 diffractometer was used with Mo-Ka graphite-monochromatized radiation to collect data on a suitable crystal at125K, see Table 2. The structure was solved with ShelX [29]. Themolecule appears in space group P21 with two molecules perasymmetric unit; the two molecules differ only in slight disorder ofthe twist in the pyrrolidine ring. Only one conformer is shown inFig. 2. As a result of this disorder, the hydrogen atoms are incalculated positions with distances of 0.99 Å for sp3 and 0.95 Å forsp2 CeH hydrogens. Coordinates of this crystal structure have beendeposited in the Cambridge Crystallographic database (CSD) withcode CCDC763648.

4. Conclusion

In the present study, we have isolated one novel and fourknown compounds from the stems of P. galeatum. This is the firstreport of the isolation of these compounds from P. galeatum, andthese compounds showed good to moderate inhibition of theTNFa-induced expression of ICAM-1. Among them, b-sitosterol(1) was the most active and significantly inhibited the expressionof ICAM-1, VCAM-1 and E-Selectin on the surface of TNFa-stim-ulated endothelial cells. Interestingly, b-sitosterol significantlyinhibited the adhesion of neutrophils to the endothelium ina concentration dependent manner. b-sitosterol was also foundto significantly reduce the transcript levels of ICAM-1, VCAM-1and E-Selectin. In addition, b-sitosterol blocked the translocationof NF-kB p65 from cytoplasm to the nucleus to inhibit the TNFa-induced expression of cell adhesion molecules. These findingssuggest that sterol constituents, particularly b-sitosterol ofP. galeatum are promising lead compounds for the developmentof anti-inflammatory agents.

Acknowledgements

We thank the University of Delhi and Department of Science andTechnology (DST), New Delhi, India & the Italian Ministry forEducation, University and Research, General Management forIndustrial Research, Rome, Italy for financial support to this workunder their special program “Italy-India Executive Program ofScientific and Technological Co-operation for the Years 2008e2010”.SB and SK thank CSIR (Council of Scientific and Industrial Research,New Delhi, India) for the award of fellowships. We thank NishaSinha for her assistance.

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