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Piper longum Linn. Extract inhibits TNF-[alpha]-induced expression of cell adhesion molecules by inhibiting NF-[kappa]B activation and microsomal lipid peroxidation.


Leukocytes are major players in the inflammatory response because of their antimicrobial, secretory and phagocytic activities. They are recruited to the inflamed tissue by sequential adhesive interactions between leukocytes and the endothelium which are mediated by cell adhesion molecules (CAMs) on the surface of the interacting cells (Bochner et al., 1991). Various inflammatory mediators induce the expression of CAMs (Mantovani et al., 1992) on the endothelial cells. The increased expression of CAMs alters the adhesive property of the vasculature leading to indiscriminate infiltration of the leukocytes across the blood vessels(Krieglstein and Granger, 2001). The transcriptional regulation of expression of CAMs is controlled by transcription factor kappa B (NF-[kappa]B), which plays pivotal role in the immune responses (Baldwin, 2001). The blockade of adhesion pathways are being actively explored as a potential strategy to therapeutically manage various inflammatory diseases including asthma and allergic diseases (Gorski, 1994; Weiser et al., 1997).

TNF-[alpha]-induced free radical generation like [H.sub.2][O.sub.2] activates inflammatory signalling pathway, including NF-[kappa]B in vascular cells (Garg and Agrawal, 2002; Takacs et al., 2001), and regulating the expression of CAMs on endothelial cells and hence play an important role in various inflammatory diseases (Rahman and MacNee, 1998). Many compounds that inhibit lipid peroxidation and influence the generation of ROS, in turn lead to decrease in the expression of the CAMs and subsequently decrease inflammation, hence are found to be useful therapeutic agents in various inflammatory diseases (Cuzzocrea et al., 2001). Treatment of endothelial cells with antioxidants is also shown to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells (Marui et al., 1993; Walther et al., 1999).

Several medicinal herbs have been shown to augment specific cellular and humoral immune responses (Duke, 1985). Piper longum is a component of Indian traditional medicine reported to be used as a remedy for treating gonorrhea, menstrual pain, tuberculosis, sleeping problems, respiratory tract infection, chronic gut-related pain and arthritic conditions (Ghoshal et al., 1996; Choi and Hwang, 2003; Mata et al., 2004). Other reported beneficial effects of P. longum include analgesic and diuretic effects, relaxation of muscles tension and alleviation of anxiety (Vedhanayaki et al., 2003). Piper extracts and piperine possess inhibitory activities on prostaglandin and leukotrienes COX-1 inhibitory effect and thus exhibit anti-inflammatory activity (Stohr et al., 2001). Recently, biochemical activities of some important medicinal plants including Piper species and their metabolites have been described (Prasad et al., 2005; Kumar et al., 2005a). However, very little is done to elucidate the possible targets of its action.

In the present study, we have evaluated the inhibitory activity of P.longum chloroform extract (PICE) on the adhesion of neutrophils to TNF-[alpha]-induced HUVECs. We have also demonstrated that this inhibition is due to the ability of PICE in blocking the TNF-[alpha]-induced expression of ICAM-1, VCAM-1 and E-selectin on HUVECs. Furthermore, as the induction of these CAMs is taking place at the level of transcripts by transcription factor, NF-kB, we checked the status of NF-[kappa]B in PICE-treated cells. We found that PICE inhibited the NF-[kappa]B activation. This extract, therefore, could be useful for the identification of small molecule(s) towards the development of anti-inflammatory molecule(s).

Materials and methods


Anti-ICAM-1, anti-VCAM-1, anti-E-selectin antibodies were purchased from Pharmingen (USA). M-199, L-glutamine, penicillin, streptomycin, amphotericin, endothelial cell growth factor, trypsin, 0-phenylenedia-mine dihyrochloride, anti-mouse IgG-HRP were purchased from Sigma Chemical Co. (USA). Fetal calf serum was purchased from Biological Industries (Israel). Sodium phosphate, sodium chloride, citric acid and other chemicals were purchased from Merck.

Procurement of plant material

Plant material (dry fruits) was collected from Khari Baoli, Delhi. The plant material was identified as fruits of P.longum Linn., which is commonly known as Pipli or Indian long pepper by Dr. H.B. Singh, Head, Raw Materials Herbarium and Museum, National Institute of Science Communication and Information Resources (CSIR), Delhi. Specimen sample has been deposited in the herbarium, voucher no. NISCAIR/ RHM/F-3/2005/ Conslt/619/99.

Preparation of P.longum extracts

P.longum fruit powder (100g) was dissolved in 150ml of 50% ethanol and incubated at room temperature (28-30[degrees]C) for 16h. The supernatant (140ml) collected by centrifugation at 14,000 rpm was dried in vacuum (3.5g), designated as ethnolic extract (F001). This was further fractionated using hexane (35ml, b.p. 68-70[degrees]C), soluble fraction dried under vacuum and designated as hexane extract (F002). The insoluble fraction was further dissolved in chloroform (40ml, b.p. 65[degrees]C), the supernatant was separated by using a separatory funnel. The lower fraction was dried under vacuum, and designated as PICE (F003). Finally, all the extracts were dissolved in DMSO individually, and used for testing ICAM-1 inhibitory activities. Among various extracts prepared, PICE was found to be more inhibitory in TNF-[alpha]-induced ICAM-1 expression (data not shown), and was chosen for further study. The phytochemical profile of PICE was determined by HPLC.

HPLC system and chromatographic condition

The HPLC system consisted of an Agilent 1100 series isocratic pump and an Agilent 1100 series diode array (DAD) detector. A Hypersil-C18 column (4.6mm x 150mm) (Thermo, USA) was used. The mobile phase was methanol--[H.sub.3][PO.sub.4] (75:25, v/v), filtered through a 0.2[mu]m filter and degassed prior to use. The flow rate was l ml/min. Detection was performed at a wavelength of 254nm at room temperature. A 10 [micro]l volume of the sample was injected for each separation.

Isolation and culture of endothelial cells

Primary endothelial cells were isolated from human umbilical cord as described before (Gupta and Ghosh, 1999). Endothelial cells were cultured in M-199 medium supplemented with 15% fetal calf serum, 2mM L-glutamine, 100 units/ml antibiotic and antimycotic solution.

Neutrophils isolation

Neutrophils were isolated from peripheral blood as described before (Madan et al., 2000). Blood from healthy individuals was collected, red blood cells were removed by sedimentation. The white blood cells rich in plasma were collected and layered on ficoll-hypaque solution and centrifuged the sample at 2000 rpm for 20min at 20[degrees]C. Following centrifugation, the top layer and the ficoll-hypaque layer were removed and neutrophil layer was collected.

Cell adherence assay

The adherence assay was performed as described before (Madan et al., 2000). To assay the adhesion of neutrophils, endothelial monolayer was prepared. The cells were incubated with or without PICE for 2h and induced with TNF-[alpha] for 6h. The endothelial cells were washed with PBS and incubated with neutrophils (6 x 10[sub.4]/well) for 1 h at 37[degrees]C. Adherent cells were assayed colorimetrically by adding substrate solution and the absorbance was read at 490nm.

Modified ELISA for measurement of ICAM-1, VCAM-1 and E-selectin

Cell-ELISA was used for measuring the expression of ICAM-1, VCAM-l and E-selectin on the surface of endothelial cells (Madan et al., 2000). Briefly endothelial cells were incubated with or without PICE at desired concentrations for 2h followed by induction with TNF-[alpha] (10ng/ml) for 16h for ICAM-1 and VCAM-1 expression and for 4h for E-selectin expression. After fixation and blocking, cells were incubated with anti-ICAM-1, anti-VCAM-1 mAb or anti-E-selectin mAbs. Cells were further incubated with peroxidase-conjugated goat anti-mouse secondary Ab. The cells were exposed to the peroxidase substrate and absorbance at 490nm.

Preparation of nuclear extracts

Nuclear extracts were from PICE-treated endothelial cells (Madan et al., 2000). Endothelial cells (2 x [10.sub.6]) were incubated with or without 17.5 [micro]g/ml of PICE for 2h followed by induction with TNF-[alpha] (10ng/ml) for 30min. The cells were incubated in cell lysis buffer allowed to swell on ice for 5min, centrifuged at 3300g for 15 min, the supernatant was collected as cytoplasmic extract and stored at-70[degrees]C. The nuclear pellet was resuspended in nuclear extraction buffer and incubated for 30min at 4[degrees]. The extracted nuclei were pelleted at 25,000g (15 min at 4[degrees]C) and the supernatant was collected as nuclear extract.

NF-[kappa]B activation assay

NF-[kappa]B activation assay, electrophoretic mobility shift assay (EMSA) was performed as previously published (Madan et al., 2000). Briefly, 10[micro]g of nuclear extract was incubated with 40-80fmole of [.sup.32]P-end labelled double-stranded NF-[kappa]B oligonucleotide in binding buffer for 30 min at RT. The DNA-protein complexes were analyzed by electrophoresis on a 4% native polyacrylamide gel using Tris-glycine buffer pH 8.5, and visualized by autoradiography.

Preparation of rat liver microsomes and the assay of initiation of lipid peroxidation

Rat liver microsomes used for the lipid peroxidation studies were prepared adopting the method of Kumar et al. (2005b). Male rats of wistar strain weighing around 200g were used for the preparation of liver microsomes. The assay of the initiation of lipid peroxidation has been described previously (Kumar et al., 2005b).

Statistical analysis

Results are given as mean [+ or -]SD. Independent two-tailed Student's t-test was performed. Differences were considered statistically significant for p<0.05. All statistical analyses were performed by using Microcal Origin software (version 3.0; Microcal Software Inc., Northampton, MA, USA).


PICE is non-toxic and reversible to the endothelial cells

The cytotoxic effect of PICE on endothelial cells was examined, the cell morphology was observed undermicroscope and viability of cells was determined by trypan blue exclusion test. It was observed that the time of incubation (upto 24h) and the concentration used for subsequent experiments did not affect the viability and morphology of the endothelial cells (data not shown). Furthermore, this was also proved by reversible effect of PICE on endothelial cells. It has been examined whether cells were preincubated with 17.5 [micro.g/ml] of PICE has any permanent effect on endothelial cells, the cells were preincubated with 17.5[micro].g/ml of PICE for varying time periods ranging from 1 to 6h. As detected by cell-ELISA the effect of PICE was found to be reversible (data not shown).

HPLC profile of PICE

Attempt was made to identify active principles in the PICE using HPLC. The analysis was performed using C18 normal column. The chromatogram of PICE was recorded at 254 nm and compared with piperine, it showed at least 10 peaks with different intensities (Fig. 1A and B). Interestingly, there are four major peaks present in the extract of PLCE. It remains to be determined whether all these components or a combination of these single components are required for its activity. Nevertheless, the HPLC profile could be utilized to identify active principle(s) from the extract.


PICE inhibits TNF-[alpha]-induced adhesion of neutrophils to endothelium monolayer

The effect of PICE on adhesion of neutrophils to endothelial monolayer was determined, the endothelial cells were incubated with or without PICE at various concentrations ranging from 5 to 17.5 [micro-g/ml], following the adhesion assay as described in Materials and methods. Our results demonstrate that the adhesion of neutrophils to the unstimulated endothelial cells was low as compared to the adhesion to TNF-[alpha]-stimulated endothelial cells. Treatment with TNF-[alpha] increased the adhesion of neutrophils by three-fourfolds. Pre-treatment of endothelial cells with PICE inhibited the adhesion of neutrophils to TNF-[infinity-stimu-lated ] lated endothelial cells in a concentration-dependent manner with almost 60.0% inhibition at a concentration of 17.5 [micro.g/ml](Fig.2). However. PICE alone did not affect the adhesion of neutrophils to unstimulated endothelial monolayers. These results suggest that PICE is effective in blocking adhesion of neutrophils to the endothelial cells. Pipeine, a reference compound was also tested for the inhibited the adhesion of neutrophils to endothelial monolayer. The maximum inhibition was comparable with PICE but at higher concentrations (unpublished data). Although piperine is an active constituent of P. longum and the HPLC chromatogram of PICE showed the presence of piperine, however, it may not the major constituent.



PICE inhibits TNF-[apha]-induced ICAM-1, VCAM-1 and E-selectin expression

As the adhesion of neutrophils to endothelial cells requires an increased expression of CAMs, namely ICAM-1, VCAM-I and E-selectin (Essani et al., 1996), we investigated the effect of PICE on TNF-[alpha]-induced expression of these CAMs. Our results using cell-ELISA demonstrate that ICAM-1, VCAM-1 and E-selectin were expressed at low levels on unstimulated endothelial cells and there was over three-four-fold increase in their expression after stimulation with TNF-[alpha]. Pretreatment of endothelial cells with PICE alone had no effect on constitutively expressed levels of ICAM-1, VCAM-1 and E-selectin. However, PICE at a concentration of 17.5 [micro] g/ml significantly inhibits (55%) TNF-[alpha]-induced expression of ICAM-1. Similary, PICE significantly inhibits TNF-[alpha]-induced expression of VCAM-1 and E-selectin (Fig. 3). The inhibition by ethnolic and hexane extracts of P. longum on the TNF-[alpha]-induced ICAM-1 expression was found to be 30% and 40%, respectively. Furthermore, the time kinetics study of ICAM-1 inhibition on endothelial cells showed that PICE inhibited the induced expression of ICAM-1 when PICE was added prior to induction with TNF-[alpha]. However, when it was added simultaneously or after induction, the inhibition of ICAM-1 expression was not significant (data not shown). These results, therefore, indicate that PICE interferes with early signalling events in response to stimulus. The inhibition by PICE remains unchanged if HUVECs were stimulated with LPS instead of TNF-[apha] (data not shown).


PICE inhibits TNF-[alpha]-induced NF-[kappa]B activation

Cytokine-induced expression of CAMs takes place at the level of transcription factor through the action of nuclear transcription factor-KB (NF-kB). To know whether PICE inhibits NF-[kappa]B activation or not, nuclear extracts were prepared from PICE-treated endothelial cells and assayed for NF-[kappa]B activation by EMSA. As shown in Fig. 4, free probe (oligonucleotide) migrates freely (lane 1). There was a low level of NF-[kappa]B in the nucleus of unstimulated cells (lane 2), no retardation. Upon stimulation with TNF-[alpha], there was an increase in levels of NF-kB, thus causing substantial retardation in the mobility of the labelled oligonucleotide (lane 3), high intensity of shifted band. PICE alone had no effect on the retardation of labelled oligos (lane 5). In contrast, the treatment of cells with PICE (17.5 [micro.g/ml] prior to the induction with TNF-[alpha] caused a significant decrease in the intensity of shifted band (compare lane 3 vs. Lane 4). The specificity of the NF-kB--DNA complex was confirmed in control experiments, where nuclear extracts were incubated with excess unlabelled oligos. Unlabelled NF-[kappa]B oligos inhibited the formation of the complex (lane 7), whereas competition with an excess of an irrelevant oligonucleotide, Oct-1 or SP-1 did not inhibit the complex (compare lane 6 and 8 with lane 7).


PICE inhibits NADPH-catalyzed rat liver microsomal lipid peroxidation

TNF-[alpha]-induced free radical generation like H2O2 activates inflammatory signalling pathway, including NF-[kappa]B in vascular cells (Garg and Agrawal, 2002). Normally the antioxidant property of a compound is attributed to its (a) oxygen radical scavenging ability, (b) the ability to inhibit cellular microsomal P-450 linked mixed function oxidases (MFOS) ability to suppress the formation of reactive oxygen species (ROS) (Parmar et al., 1999). As PICE inhibits TNF-[alpha]-induced NF-[kappa]B activation, therefore, we examined the effect of PICE on inhibition of Microsomal lipid peroxidation as mentioned in Materials and methods, to assure its antioxidant activity. We have observed that PICE inhibits lipid peroxidation. The maximum inhibition in micro-somal lipid peroxidation was estimated 62.2% at 17.5 [micro].g/ml. Thus the inhibition of CAMs and NF-[kappa]B may be mediated through inhibition free radical generation in the form of lipid peroxidation.


Small molecules from natural and synthetic sources have been shown to be useful for downnregulating the induced expression of CAMs both in vitro and in vivo (Madan and Ghosh, 2003; Madan et al., 2003; Kumar et al., 2005a,b). The earlier phytochemical studies indicate the presence of various long-chain esters and amides, alkaloids, lignans, neolignans, amides, terpenes, steroids, chalcones, flavones and flavanones in P. longum extract (Parmar et al., 1997; Stohr et al., 2001; Kumar et al., 2005a).

Here, we demonstrated that extracts of P. longum inhibited the TNF-[alpha]-induced expression of ICAM-1. Out of these extracts, PICE was found to be more active (data not shown). We found that PICE inhibited the adherence of neutrophils to endothelial monolayer by inhibiting the TNF-[alpha]-induced expression of ICAM-1, VCAM-1 and E-selectin in a dose-and time-dependent manner. Furthermore, this inhibition was found to be reversible (data not shown). The transcriptional regulation of CAMs is controlled by NF-[kappa]B, thus it prompted us to check the status of NF-[kappa]B in the presence of PICE. Interestingly, we found that PICE significantly inhibited the TNF-[alpha]-induced activation of NF-[kappa]B (Fig. 4). The inhibition of CAMs and NF-[kappa]B by PICE may be due to its effect as antioxidant activity, as shown by the inhibition as antioxidant activity, as shown by the inhibition of micorosomal lipid peroxidation.

Extracts from various plants have been reported to be effective in inhibiting the induced expression of CAMs on endothelial cells (Gupta and Ghosh, 1999; Madan et al., 2003; Chen et al., 2001, 2003; Ren et al., 2002). For example, Ginkgo biloba extract inhibits TNF-[alpha]-induced expression of ICAM-1 and VCAM-1 by 69% and 63%, respectively, at a concentration of 50 [micro]g/ml, whereas PICE inhibits these molecules at a lower concentration to almost the same level (Chen et al., 2003). Furthermore, we compared our results with well-known compound, diferuloylmethane, which inhibited the TNF-[alpha]-induced expression of CAMs on endothelial cells (Gupta and Ghosh, 1999). We found that our results are comparable to diferuloylmethane as well as the plant extract Curcuma longa (Gupta and Ghosh, 1999). Not only PICE used here is non-toxic to endothelial cells, its activity is also found to be reversible. In other words, it is not causing any permanent changes to endothelial cells.


In conclusion, we demonstrated that PICE inhibited the adherence of neutrophils to endothelial monolayer by inhibiting the TNF-[alpha]-induced expression of ICAM-1, VCAM-1 and E-selectin in a dose-dependent manner. Further, we found that it also inhibited the activation of NF-kB. The inhibition of CAMs as well as NF-[kappa]B is most probably mediated through its antioxidant activity. Thus, PICE could be useful for controlling various pathological conditions associated with increased endothelial leukocyte adhesion molecules.


Authors acknowledge the help of St. Stephan Hospital, New Delhi for providing the umbilical cord. Council of Scientific and Industrial Research, India supported this work (Task Force Project SMM0006).


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Naresh Singh (a), Sarvesh Kumar (a), Prabhjot Singh (c), Hanumantharao G, Raj(c), Ashok K. Prasad(b), Virinder S. Parmar(b), Balaram Ghosh(a), *

(a) Molecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, University of Delhi Campus (North), Mall Road, Delhi 110 007, India

(b) Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India Department of Biochemistry, V.P. Chest Institute, University of Delhi, Delhi 110 007, India

Received 23 December 2005; accepted 17 April 2007

* Corresponding author. Tel.: +911127662580; fax: +911127667471.

E-mail address: (B.Ghosh).
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Author:Singh, Naresh; Kumar, Sarvesh; Singh, Prabhjot; Raj, Hanumantharao G.; Prasad, Ashok K.; Parmar, Vir
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Apr 1, 2008
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