Effect of leaf extract of Seabuckthorn on lipopolysaccharide induced inflammatory response in murine macrophages

International Immunopharmacology 6 (2006) 46 – 52 www.elsevier.com/locate/intimp

Effect of leaf extract of Seabuckthorn on lipopolysaccharide induced inflammatory response in murine macrophages Yogendra Padwad, Lilly Ganju *, Monika Jain, Sudipta Chanda, Dev Karan, Pratul Kumar Banerjee, Ramesh Chand Sawhney Immunomodulation Laboratory, Defence Institute of physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi, 110054, India Received 25 May 2005; received in revised form 28 June 2005; accepted 25 July 2005

Abstract Nitric oxide (NO) is synthesized in large quantities by activated inflammatory cells and has been demonstrated to be involved in the pathogenesis of acute and chronic inflammatory conditions. Seabuckthorn (SBT) has been used in traditional medicine systems for the treatment of various diseases like cardiovascular, pain relief, oral inflammation and promotion of tissue regeneration. The present study focuses on the effects of SBT leaf extract on NO production induced by lipopolysaccharide (LPS) in the murine macrophage cell line RAW 264.7. In addition, cell viability, free radical-scavenging activity and inducible nitric oxide synthase (iNOS) expression were also evaluated. Seabuckthorn leaf extract significantly inhibited the enhanced production of NO induced by LPS in a dose dependent manner. Treatment with SBT did not reduce cell viability at any dose used. The extract showed significant scavenging of NO radicals released by the NO donor. Treatment of macrophages with SBT leaf extract also caused a significant inhibition of iNOS activation. These observations suggest that the inhibition of net NO production by SBT leaf extract may be due to its scavenging activity and/or its inhibitory effects on iNOS activation. The study suggests that SBT leaf extract has significant anti-inflammatory activity and has potential for the treatment of inflammatory diseases. D 2005 Elsevier B.V. All rights reserved. Keywords: Inflammation; Nitric oxide; Macrophage; Seabuckthorn

1. Introduction During the last decade, the discovery of the endogenous formation of nitric oxide (NO) has lead to an explosion in research on NO induced cellular injury * Corresponding author. Tel.: +91 11 23985033; fax: +91 1 11 23914790. E-mail address: [email protected] (L. Ganju). 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.07.015

[1,2]. Nitric oxide is synthesized from l-arginine in numerous mammalian cells and tissues and is a well established marker of inflammation. It has diverse physiological roles and may also contribute towards pathological processes. When NO is synthesized in large quantities by activated inflammatory cells, it has cytotoxic properties and may be involved in the pathogenesis of acute and chronic inflammatory conditions [3]. This NO production is mediated by indu-

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cible nitric oxide synthase (iNOS), which is induced by several stimuli including bacterial lipopolysaccharide (LPS) and interferon-gamma. iNOS is present in macrophages and hepatocytes. During inflammation associated with different pathogens, NO production increases significantly [4] and may become cytotoxic. Moreover, the free radical nature of NO and its high reactivity with oxygen to produce peroxynitrite (ONOO ) makes NO a potent pro-oxidant molecule able to induce oxidative damage, and to be potentially harmful towards cellular targets [5]. Thus inhibition of NO production in response to inflammatory stimuli might be a useful therapeutic strategy in inflammatory diseases [6,7]. Seabuckthorn (SBT) (Hippophae rhamnoides, Family—Elaeagnaceae) is a popular medicinal plant used in various parts of the world for preparation of vitamin and nutrient products [8,9]. All parts of the plant are rich in various biologically active compounds. Its fruits are preferably used for the commercial scale production of medically important fatty oil (Oleum Hippophae) [10,11]. Seabuckthorn leaves have been reported to have marked anti-bacterial, anti-viral and anti-tumor activities [12,13] and have been demonstrated to enhance wound healing effect after chemical burns and plain wounds [14]. Recent studies from our laboratory have shown that SBT leaves also possess the anti-oxidant and immunomodulatory properties [15] besides having a significant anti-inflammatory activity [16]. However the possible mechanism responsible for its anti-inflammatory effects remains unknown. As NO production has been accepted as a marker of inflammation [3], therefore the efficacy of SBT leaf extract in curtailing NO production, cell viability, iNOS activation and NO scavenging property were studied in-vitro in murine macrophages RAW 264.7 during LPS induced inflammation. 2. Methods

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extract were carried out in sterile double distilled water. Identical procedure was followed for the preparation of extracts from Amla (Emblica officinalis), Bahera (Terminalia belerica), Sacred basil (Ocimum sanctum), and Shankhpushpi (Evolvulus alsinoides) to compare with SBT. 2.2. Cell culture and stimulation of macrophages with LPS RAW 264.7 cell line was obtained from ATCC, Atlanta (USA) and was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA), 100 Ag/ml Penicillin and 100 mg/ml Streptomycin at 37 8C under 5% CO2 humidified air. Cells were harvested by gentle scraping and passaged every 3–6 days by 1 : 10 dilutions of a suspension of the cells in fresh medium. For stimulation with LPS, cells were seeded into 24 well plates at 1  105 cells/well and allowed to adhere for 12 h at 37 8C under 5% CO2. The medium was replaced with fresh medium without FBS for 5 h, then replaced again with fresh medium containing 10% FBS and 1 Ag/ml LPS. The SBT leaf extract was compared with four other herbal extracts of known medicinal value like Amla, Bahera, Sacred basil and Shankhpushpi [17–19]. To evaluate the effect of all the five extracts, cells were preincubated for five h with 100 Ag/ ml of each extract, followed by addition of LPS for 24 h as above. Another experiment was further carried out in a similar way to determine the dose response stimulation of SBT extract (12.5, 25, 50, 100 and 200 Ag/ml). Controls were run in parallel and anti-inflammatory steroid dexamethasone (Wockhardt Ltd, India) was used as a positive control. 2.3. Measurement of nitrite production As an indicator of NO production, nitrite concentration in the culture medium was determined by Griess reaction [20]. In brief, cell supernatant was mixed with equal volume (100 Al) of Griess reagent (1% sulphanilamide, 0.1% N-(1naphthyl ethylenediamine) that produces a chromophoric azo-derivative molecule that absorbs light at 540 nm, which was measured in terms of optical density (OD) in a microplate reader (Molecular Dynamics, USA). A range of dilutions of sodium nitrite was used to generate a standard curve with each assay.

2.1. Plant material 2.4. MTT assay for cell viability Leaves of SBT were collected from hilly regions of Western Himalayas, India, and dried under shade. The extraction was carried out using powdered leaf material with 70% ethanol overnight. The extract was dried at 50 8C and then dissolved in 70% alcohol. The dilutions of

Cell viability was assessed by the mitochondrial respiration-dependent 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction method. Cells (1  104 cells/well) in 96 well plates were incubated with

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two doses of test compound (50 and 100 Ag/ml) of SBT leaf extract and 10 AM of dexamethasone at 37 8C in 5% CO2 for 24 h. After treatment, 10 Al of MTT solution was added to each well followed by incubation for 4 h at 37 8C. The formazan crystals in viable cells were solubilized with 100 Al of lysis buffer (10% SDS in 0.01 M HCl) for 1 h. The OD of each well was then read at 540 nm.

applicable. The post hoc test was done with Newman Keul’s test where appropriate. Significance level was set at p b 0.01.

3. Results 3.1. Effect of SBT leaf extract on NO production

2.5. Assay for scavenging for NO radical All the five extracts (100 Ag/ml) were dissolved in phosphate buffered saline (PBS) to a total volume of 250 Al and incubated with 250 Al of sodium nitropruside (SNP) in PBS at 37 8C for 3 h. After incubation, the concentration of nitrite was measured by the Griess reagent method as described above. Once the scavenging activity of all the five extracts was determined, SBT leaf extract was further evaluated at various concentrations (12.5, 25, 50, 100 and 200 Ag/ml) to obtain a dose response scavenging activity.

Incubation of murine macrophages with LPS resulted in a significant increase in NO production in the culture medium after 24 h of incubation (Fig. 1). Pretreatment of macrophages with addition of different extracts like Bahera, Sacred basil, Shankhpushpi, Amla and SBT leaf extract caused an inhibition in NO production, but SBT leaf extract showed maximum significant inhibition (Fig. 1). Dose response of SBT leaf extract showed a maximum inhibition at 100 Ag/ml (Fig. 2). Therefore 100 Ag/ml dose of SBT leaf extract was used for further investigations.

2.6. Immunoblotting for iNOS expression

3.2. Scavenging of NO

RAW cell line was grown in a 90 mm petri dish as described above. The cells were treated with SBT leaf extract (100 Ag/ml) and 5 h later with LPS (1 Ag/ml) for 24 h. Cells were harvested and resolved on sodium-dodecylsulphate-polyacryl-amide gel electrophoresis (SDS-PAGE) followed by Western blotting using specific monoclonal antibody (Moab) against iNOS, essentially following the method described by Laemmli and Towbin et al. [21,22]. Briefly, harvested cells were subjected to protein extraction by repeated freeze thaw and protein was estimated by the method of Lowry et al. [23]. Samples were prepared in SDSPAGE sample buffer before being separated on 10% gel and transferred on to nitro cellulose membrane (Schiecher and Schuell, SDassel, FRG). For immunoblotting, membrane was blocked by incubation with Tris–buffered saline with 0.1% Tween20 (TBST20) and supplemented with 1% bovine serum albumin for 1 h at room temperature, washed thrice in TBST20 buffer and incubated with Moab against iNOS (Santa Cruze, USA) at 37 8C for 1 h. Membrane was again washed thrice with TBST20 and incubated with peroxidase conjugated secondary antibody for 1 h at room temperature. After washing the membrane thrice, protein bands were detected by incubating with diamino-benzedine + H2O2 for 5 min.

The NO radical-scavenging activity of the SBT leaf extract was compared with Sacred basil, Shankhpushpi, Bahera and Amla extracts. All the five extracts tested showed a significant ( p b 0.01) scavenging activity (Fig. 3). The NO scavenging activity of SBT leaf extract was found to be significantly higher ( p b 0.001) as compared to other four extracts. The scavenging activity of SBT leaf extract was further evaluated at various concentrations (12.5, 25, 50 and 100 Ag/ml). The observed effect was dose dependent and maximum scavenging was observed at 100 Ag/ml (60%) as compared to control (100%) (Fig. 4).

2.7. Statistical analysis The results are expressed as mean F SEM and all the statistical comparisons were carried out using Analysis of Varience (ANOVA) for repeated measurements where

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NO (µmol)

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Plant extracts Fig. 1. Effect of pretreatment with 100 Ag/ml concentration of five different herbal preparations on nitric oxide production by LPS stimulated murine macrophages. Values are expressed from six independent experiments performed in triplicate. **Vs LPS, p b 0.01, aa Vs Amla, p b 0.01. (LPS=Lipopolysaccharide, B=Bahera, SB= Sacred basil; SP=Shankhpushpi, A=Amla, SBT=Seabuckthorn,).

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NO radical (%)

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Fig. 2. Effect of pretreatment with four different concentrations of SBT leaf extract on nitric oxide production by LPS stimulated murine macrophages. Values are expressed from six independent experiments performed in triplicate. **Vs LPS, p b 0.01 (LPS=Lipopolysaccharide; SBT=Seabuckthorn).

3.3. Effect of SBT leaf extract on cell viability Addition of dexamethasone used as control caused a significant reduction ( p b 0.001) in cell viability when studied using MTT assay (Fig. 5). SBT leaf extract did not affect the cell viability at low concentration (50 Ag/ml) but when added at a higher concentration (100 Ag/ml), it significantly ( p b 0.001) increased cell viability.

Fig. 4. Scavenging of nitric oxide by different concentrations of SBT leaf extract. Values are expressed from six independent experiments performed in triplicate. Control is taken as 100% **Vs control, p b 0.01. (SBT=Seabuckthorn).

lane 1). In contrast, 24 h incubation with LPS (lane 2) induced a significant increase in iNOS expression in terms of binding to Moab vs. control (lane 1). Treatment of cells with SBT leaf extract significantly reduced iNOS expression (lane 4) with respect to the LPS treated cells (lane 2). Whereas dexamethasone treated (lane 3) showed almost a minimal expression. Relative density of bands was measured by Labworks software (UVP Bioimaging System, USA) and expressed in terms of OD. These are the results of experiments repeated three times.

3.4. Effect of SBT on iNOS expression at translation level

120 100

4. Discussion The results from the present study suggest that pretreatment with SBT leaf extract significantly inhibited LPS induced NO production in murine macrophage cell line model. While evaluating cell viability using MTT assay it was observed that the SBT leaf extract did not affect cell viability at lower concentration, but increased the viability when the macrophages

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To investigate whether SBT leaf extract affects iNOS expression, at translation level, Western blotting was carried out using specific Moab against iNOS. Non-stimulated RAW cells had barely detectable iNOS expression (Fig. 6,

0.3 0.2 0.1

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Fig. 3. Scavenging of nitric oxide by different herbal extracts (100 Ag/ml). Values are expressed from six independent experiments performed in triplicate. Control is taken as 100%. **Vs control, p b 0.01, aa Vs Amla, p b 0.01 (SB=Sacred basil; SP=Shankhpushpi; B=Bahera, A=Amla, SBT=Seabuckthorn).

Fig. 5. Effect of two different concentrations of SBT leaf extract and Dexa on viability of macrophages. Results shown are from six independent experiments performed in triplicate. **Vs control, p b 0.01 (SBT=Seabuckthorn, Dexa=Dexamethasone).

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A

β-Actin iNOS 1

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Fig. 6. Inhibition of LPS induced iNOS expression at translation level in activated RAW 264.7 macrophages following pre-treatment with SBT extract and Dexamethasone. Data represents one of the three experiments performed separately with similar results. Panel A—Western blot analysis on probing with monoclonal antibodies against iNOS whereas h-Actin was taken as control and Panel B—density plot of the probed bands. Lane 1, control; 2, LPS stimulated cells (*Vs control p b 0.001); 3, LPS stimulated cells pretreated with Dexamethasone (aVs LPS stimulated cells p b 0.001); 4, LPS stimulated cells pretreated with SBT leaf extract (aaVs LPS stimulated cells p b 0.01). (C = cells; L, LPS=Lipopolysaccharide; Dexa=Dexamethasone, SBT=Seabuckthorn).

were treated with higher concentration of the extract. All the five extracts tested showed marked scavenging of NO radicals released by the NO generator. However, the SBT leaf extract showed maximum scavenging activity. Besides scavenging NO activity, the SBT leaf extract also inhibited iNOS expression in the macrophages. The LPS treated RAW 264.7 macrophage model is widely used in studies of mechanisms of iNOS induction and NO production [24,25]. The iNOS induced by microbial products like LPS accounts for the sustained generation of NO and it is well known that NO plays an important role in immunological responses such as inflammation and autoimmunity. This is also known that some chemical constituents of medicinal plants show biological activity affecting different aspects of the inflammation process. Many flavanoids and phenylethanoids are antioxidants [26,27] protecting biological system against oxidative stress [27,28]. This is mainly due to their redox properties, which can play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxidase [29]. In this study all the five plant extracts showed NO scavenging activity, whereas the most significant effect was seen in SBT

leaf extract. This could be due to the fact that the scavenging activity of phenolic compounds require a high concentration of phenol groups [30,31] and SBT is known to possess anti-oxidant activity [15] due to high concentration of phenol. The increase in cell viability at a higher concentration of SBT leaf extract indicates that the observed inhibition of NO production by the extract cannot be attributed at least at high concentration like 100 Ag/ml to cytotoxicity. Activation of RAW 264.7 macrophages with LPS resulted in accumulation of nitrite in culture medium, reflecting increased NO production due to activation of iNOS. NO production by macrophages depends on iNOS, which can be activated by various agents, including LPS, IFN-g, TNF-a etc. [32]. The onset of the NO production cascade induced by LPS in macrophages requires a number of steps, including the activation of nuclear factor (NF)-kh and subsequent iNOS mRNA expression [32]. Although the cellular mechanism of suppression of iNOS induction by flavanoids is not clearly understood, it has been demonstrated that some flavanoids decrease iNOS induction and thus inhibit NO production [33,27]. Lin and Lin [34] and Vigo et al. [35] have also demonstrated that some polyphenols decrease the

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iNOS levels and activity by reducing the expression of iNOS mRNA, possibly through prevention of binding of NF-kh to the iNOS promoter. It is possible that inhibition of NO production by pretreatment with SBT leaf extract could be due to inhibition of transcription of the iNOS gene which is quite evident at translation level on probing with Moab against iNOS. Further investigations at mRNA level are required to confirm these observations. Similar findings have been reported for dexamethasone, which decreased the activity of the iNOS promoter [36]. In conclusion, these results demonstrate that SBT leaf extract show NO scavenging activity and that pretreatment with this extract inhibit NO production without affecting the cell viability in LPS stimulated RAW 264.7 murine macrophage. In accordance with these findings, we can speculate that the extract may modulate net NO production, through scavenging activity. Although this hypothesis cannot be resolved on the basis of the current experimental results, all data obtained indicate that SBT leaf extract has antiinflammatory properties without affecting cell viability, and therefore may possibly be useful in the treatment of inflammatory pathologies, in line with its traditional use.

References [1] Abramson SB, Attur M, Amin AR, Clancy R. Nitric oxide and inflammatory mediators in the perpetuation of osteoarthritis. Curr Rheumatol Resp 2001;3:535 – 64. [2] Brown GC. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett 1995;369:136 – 9. [3] Moilanen E, Whittle B, Moncada S. Nitric oxide as a factor in inflammation. In: Gallin JI, Snyderman R, editors. Inflammation: basic principles and clinical correlates. Philadelphia7 Williams and Wilkins; 1999. p. 787 – 800. [4] Kharitonov S, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994;343:133 – 5. [5] Epe B, Ballmaier D, Roussyn I, Brivida K, Sies H. DNA damage by peroxynitrite characterized with DNA repair enzyme. Nucleic Acids Res 1996;24:4105 – 10. [6] Hobbs A, Higgs A, Moncada S. Inhibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol 1999;39:191 – 220. [7] Sautebin L. Prostaglandins and nitric oxide as molecular targets for anti-inflammatory therapy. Fitoterapia 2000;71:S48 – 57. [8] Beveridge S, Li T, Oomah BD, Smith A. Seabuckthorn pro-

[9] [10] [11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

51

ducts: manufacture and composition. J Agric Food Chem 1999;47:3480 – 8. Xu MY, Sun XX, Tong WX. Medical research and development of Seabuckthorn. Hippophae 1994;7:32 – 40 [Chinese]. Bazaron EG, Tsybikova DTs. Seabuckthorn is the remedy of Indo-Tibet medicine. Rastit Resur 1978;14(1):67 – 9. Shapiro DK, Garanovich IM, Anikhimovskaya LV, Narizhnaya TI, Vereskovskii VV. Biochemical and morphological characteristics of prospective forms of Seabuckthorn population of North Azerbaijan. Rastit Resur 1978;14(1):60 – 4. Vermenichev SM. Experimental study of toxicity and antitumor activity of natural and synthetic compounds of pyrone series. Phenolic compounds and their physiological properties, Proceedings of the 2nd All-Union Symposium on phenolic compounds held 17–21 May 1971 in Alma-Ata, ’Nauka’ Kazakh. SSR; 1973. p. 210 – 4. Tsybikova DTs, Rasputina DB, Zalykeeva DN, Darzhapova GZh, Khundanova LL. A study of leaves and the oil cake of Seabuchthorn. Biology, chemistry and pharmacology of Seabuckthorn. Novosibirsk7 Nauka Sibirdiv; 1983. p. 107 – 9. Tsybikova DTs, Malakshinova MM, Zylykeeva DN, Rasputina DB, Nazaro V, Rygdylov VE. Wound healing drugs from SBT leaves. Ecological pathology and its pharma correction. 3rd International conference, Abstracts of report, 26–27 June, 1991, Part II, Chita; 1991. p. 82. Geetha S, SaiRam M, Singh V, Illavazhagan G, Sawhney RC. Anti-oxidant and immunomodulatory properties of Seabuckthorn (Hippophae rhamnoides)—an in-vitro study. J Ethnopharmacol 2002;79:373 – 8. Ganju L., Padwad Y., Singh R., Karan D., Chanda S., Chopra M.K., Bhatnagar P., Kashyap R., Sawhney R.C.. Antiinflammatory activity of Seabuckthorn (Hippophae rhamnoides) leaves. Int Immunopharmacol 2005;5(12):1675 – 84. Singh S, Majumdar DK, Rehan HM. Evaluation of antiinflammatory potential of fixed oil of Ocimum sanctum (Holy Basil) and its possible mechanism of action. J Ethnopharmacol 1996;46(3):195 – 9. SaiRam M, Neetu D, Yogesh B, Anju B, Dipti P, Pauline T, et al. Cyto-protective and immunomodulatory properties of Amla (Emblica officinalis) on lymphocytes: an in-vitro study. J Ethnopharmacol 2002;81:5 – 10. Pandey VK, Sharma AK. Evaluation of Vatahari Guggulu and Nadivaspa-Sweda in the management of rheumatic disease. Rheumatism 1986;22(1):1 – 7. Dirsch VD, Stuppner H, Vollmar AM. The Griess assay: suitable for a bio-guided fractionation of anti-inflammatory plant extracts. Planta Med 1998;64:423 – 6. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680 – 5. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci 1979; 76:4350 – 4. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagent. J Biol Chem 1951;193:265 – 75.

52

Y. Padwad et al. / International Immunopharmacology 6 (2006) 46–52

[24] Szabo C, Mitchell JA, Gross SS, Thiemermann C, Vane JR. Nifedipine inhibits the induction of nitric oxide synthase by bacterial lipopolysaccharide. J Pharmacol Exp Ther 1993; 265:674 – 80. [25] Swierkosz TA, Mitchell JA, Warner TD, Botting RM, Vane JR. Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids. Br J Pharmacol 1995;114:1335 – 42. [26] Rice-Evans CA, Miller NJ, Pagana G. Structure–antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med 1996;20:933 – 56. [27] Xiong Q, Tezuka Y, Kaneko T, Li H, Tran L, Hase K, et al. Inhibition of nitric oxide by phenylethanoids in activated macrophages. Eur J Pharmacol 2000;400:137 – 44. [28] Kandaswami C, Middleton E. Free radical scavenging and antioxidant activity of plant flavonoids. Adv Exp Med Biol 1994;366:351 – 76. [29] Osawa T. Novel nature oxidants for utilization in food and biological system. In: Uritani I, Gareia V, Mendoza EM, editors. Post harvest biochemistry of plant food materials in the tropics. Tokyo7 Japan Scientific Societies Press; 1994. p. 241 – 51. [30] Wang PF, Kang JH, Zheng RL, Yang ZH, Lu JF, Gao JJ, et al. Scavenging effects of phenylpropanoid glycosides from Pedicularis on superoxide anion and hydroxyl radical by the spin trapping method (95) 02255-4. Biochem Pharmacol 1996; 51:687 – 91.

[31] Xiong Q, Kadota S, Tani T, Namba T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull 1996;19:1580 – 5. [32] Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev 1991;43: 109 – 42. [33] Cheon BS, Kim YH, Son KS, Chang HW, Kang SS, Kim HP. Effects of prenylated flavonoids and biflavonoids on lipopolysaccharide-induced nitric oxide production from the mouse macrophage cell line RAW 264.7. Planta Med 2000; 66:596 – 600. [34] Lin YL, Lin JK. Epigallocatechin-3-gallate blocks the induction of nitric oxide synthesis by down regulating lipopolysaccharide-induced activity of transcription factor nuclear factorkh. Mol Pharmacol 1997;52:464 – 72. [35] Vigo E, Cepeda A, Gualillo O, Fernandez RP. In-vitro antiinflammatory effect of Eucalyptus globules and Thymus vulgaris: nitric-oxide inhibition in J774 A.1 murine macrophages. J Pharm Pharmacol 2004;56:257 – 63. [36] Kleinert H, Euchenhofer C, Ihrig-Biedert I, Forstermann U. Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-kB. Mol Pharmacol 1996;49:15 – 21.