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DOI: 10.1055/s-2003-45107
Inhibitory Effects of a New Iridoid, Patridoid II and its Isomers, on Nitric Oxide and TNF-α Production in Cultured Murine Macrophages
This research was supported by a grant (PF0320302-00) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Korea GovernmentHyeun Wook Chang
College of Pharmacy
Yeungnam University
712-749 Gyongsan
Korea
Phone: +82-53-810-2811
Fax: 82-53-811-3871
Email: hwchang@yu.ac.kr
Publication History
Received: March 21, 2003
Accepted: July 26, 2003
Publication Date:
02 December 2003 (online)
Abstract
Patridoids I, II and IIA, are new iridoids isolated from the whole plants of Patrinia saniculaefolia. These compounds were examined by assessing their effects on the production of tumor necrosis factor-α (TNF-α) as well as by investigating the expression of two enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in the lipopolysaccharide (LPS)-stimulated murine macrophage-like cell line, Raw 264.7. Among them, patridoid II consistently inhibited nitric oxide (NO) and TNF-α production in a dose-dependent manner, with IC50 values of 14.1 and 17.6 μM, respectively. Western Blotting probed with specific anti-iNOS antibodies showed that the decrease in the quantity of the NO product was accompanied by a decrease in the iNOS protein level. However, all three patridoids did not affect the COX-2 protein expression level in the LPS-stimulated macrophages. In addition, the C-5 isomer of patridoid II, patridoid I, only slightly affected the production of NO. Moreover, the C-3 isomer of patridoid II, patridoid IIA, did not inhibit proinflammatory cytokines and NO production. These results suggest that the orientations of the C-3 and C-5 methoxy groups in the patridoids have a significant role in the expression of their biological activity.
Iridoids exhibit various biological activities such as antimicrobial [1], antitumoral [2], hemodynamic [3], and anti-inflammatory functions [4], [5], [6], [7], [8]. Patrinia saniculaefolia Hemsley (Valerianaceae) is a perennial herb and endemic in Korea. The roots of the genus Patrinia have been used in Korean traditional medicine for treating various diseases including inflammation and edema, appendicitis and abscesses [9]. However, the underlying mechanisms accounting for its anti-inflammatory actions have not yet been elucidated.
Lipopolysaccharide (LPS), which is a component of the cell walls of Gram-negative bacteria, is one of the most potent macrophage activators. LPS induces the production of pro-inflammatory cytokines and nitric oxide (NO) in macrophages. Increasing evidence also suggests that NO, a bioactive free radical, is involved in various pathophysiological processes [10], [11], [12]. Therefore, NO production might reflect the inflammation process and provide a measure for assessing the effects of drugs on the inflammatory process. NO production is regulated by NO synthases (NOSs) and three NOS isoforms have been identified and cloned. A third member of the NOS family (inducible NOS, iNOS) is produced in large quantities in response to inflammatory stimuli, such as LPS and interferon-γ (IFN-γ) [13], [14].
Proinflammmatory cytokines (TNF-α, IL-1β and IL-6) have been suggested to induce tissue damage, and are considered to be an important initiator of the inflammatory responses. Although the evidence accumulated thus far shows that iridoids and their glycosides exert anti-inflammatory effects in vivo, the mechanism underlying these actions is still unclear. In the previous study, we isolated and determined structures of new three iridoids, patridoids I, II and IIA, from the whole plants of P. saniculaefolia (Fig. [1]) [15]. This study examined their effects on NO and TNF-α production, iNOS and COX-2 protein expression in LPS-stimulated Raw 264.7 macrophages.
The experiments were designed to test the overall effects of the patridoids on NO production in Raw 264.7 cells stimulated by LPS. Among the three compounds, only patridoid II exhibited a dose-dependent inhibition of NO production with an IC50 value of 14.1 μM (Fig. [2]). The inhibitory effect of the patridoids on inducible NO production was examined to determine if it is a direct effect on iNOS protein or mediated by some other mechanism. In this experiment, we used ginkgetin as positive control [16]. As shown in Fig. [3], LPS-induced iNOS protein expression was inhibited by patridoid II at a concentration of 15 μM, but neither patridoid I nor patridoid IIA affected the iNOS protein expression level. In addition, a known NOS inhibitor, L-N ω-nitroarginine methyl ester (L-NAME) significantly inhibited NO production (data not shown) but did not inhibit iNOS protein expression under the same conditions. It is well known that LPS also induces COX-2 protein production in Raw 264.7 cells. However, the COX-2 protein expression induced by LPS (200 ng/mL) was not inhibited by the three patridoids under the same conditions. On the other hand, iNOS and COX-2 protein expression was completely inhibited by ginkgetin (Fig. [3]).
Several reports have shown that TNF-α is the most important cytokine mediator linked to the pathogenesis of septic shock [17], [18], [19]. In order to determine if the patridoids can modulate TNF-α production, the cells were pretreated with the patridoids at various concentrations for 30 min prior to LPS stimulation. When the Raw 264.7 cells were stimulated with LPS (200 ng/mL) in the presence of the patridoids and ginkgetin. Patridoid II showed the strongest inhibition on TNF-α production with an IC50 value of 17.6 μM. Patridoid I exhibited a less potent action than patridoid II while patridoid IIA showed no inhibition and 5 μM ginkgetin inhibited by about 70 % TNF-α production (Table [1]). At this assay condition in which the cells were treated with the patridoids, the cell viability was > 95 %. The inhibitory effect of patridoid II was mediated through a blockade of NF-κB activation (data not shown) like iridoid glycoside, aucubin [20].
The overall experimental results suggest that patridoid II exhibited the most potent inhibitory effects on TNF-α and NO production among the three compounds. Patridoid I, which is a C-5 isomer of patridoid II, affected the production of NO and TNF-α production slightly. Patridoid IIA, which is a C-3 isomer of patridoid II, did not inhibit TNF-α and NO production. In the viewpoint of a structure-activity relationship, these results suggest that the 3β- and 5a-orientations of the C-3 and -5 in the patridoids play an important role in their biological activity. These results may serve as an additional rationale for the use of P. saniculaefolia Hemsly in inflammatory disorders.
Fig. 1 Structures of the patridoids.R1 R2 R3 R4Patridoid I: H OCH3 H OCH3 II: OCH3 H H OCH3 IIA: OCH3 H OCH3 H
Fig. 2 Dose-dependent inhibition of NO production and iNOS protein expression by patridoid II in the Raw 264.7 cells. The Raw 264.7 cells (1 × 106 cells/ml) were preincubated with the patridoids for 30 min prior to the addition of LPS (200 ng/mL), after which the cells were incubated for 24 h with the vehicle, LPS (200 ng/mL) plus patridoid II. The samples were processed by SDS-PAGE and transferred to a nitrocellulose filter. The immunoblot was then probed with anti-iNOS or anti-COX-2 antibodies at a dilution 1 : 1,000. Similar results were obtained from 3 independent experiments.
Fig. 3 Inhibition of iNOS and COX-2 protein expression by patridoids in the LPS-stimulated Raw 264.7 cells The cells (1 × 106 cells/mL) were pretreated with either 50 μM of L-NMMA or 15 μM of the patridoids and 5 μM of ginkgetin for 30 min followed by stimulation with LPS (200 ng/mL) for 24 h with the vehicle (Un), LPS + the patridoids. After 24 h, the iNOS and COX-2 expression levels were measured in the cells. The samples were processed by SDS-PAGE and transferred to a nitrocellulose filter. The immunoblot was then probed with anti-iNOS and anti-COX-2 antibodies. The error bars represent the mean ± SD of 3 individual experiments.
| Compounds (μM) | TNF-α production (pg/mL) |
| Patridoid I 5 10 20 |
1 095.6 ± 76.1 890.0 ± 67.8 732.3 ± 32.1 |
| Patridoid II 5 10 20 |
946.3 ± 6.90 861.8 ± 28.1 433.7 ± 11.3 |
| Patridoid IIA 5 10 20 |
1 200.1 ± 70.5 1 190.3 ± 21.4 1 180.3 ± 19.8 |
| Ginkgetin 5 |
382.3 ± 5.1 |
Material and Methods
Patridoids I, II and II-A were isolated from the dried whole plant of P. saniculaefolia as previously reported [15]. All compounds are above 97 % purity as analysed by HPLC. RPMI 1640 and phosphate-buffer saline were obtained from GIBCO-BRL (Grand Island, NY). Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). Rabbit polyclonal COX-2 antibody, rabbit polyclonal iNOS antibody, and anti-rabbit IgG peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LPS (from Escherichia coli 0111:B4, γ-irradiated), and other chemicals were from Sigma Chemical Co. (St. Louis, MO). Patridoids were dissolved in dimethyl sulfoxide (DMSO) before addition to cell cultures: the final concentration of DMSO was 0.05 %. Control with DMSO alone was run in all cases.
Cell culture: Raw 264.7 cells were cultured in RPMI supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 10 % fetal calf serum. Cells were grown at 37 °C, 5 % CO2 in fully humidified air and subcultured 2 times a week. Cells were seeded in 96-well plates at 1 × 106 cells/mL or 6-well plates at 1 × 106 cells/mL. The cells were stimulated for intervals ranging from 1 to 24 h in the presence of LPS with or without patridoids. LPS was diluted with culture medium to a final concentration of 200 ng/mL.
Assay of NO synthesis: Synthesis of NO was determined by assaying culture supernatants for nitrite, the stable reaction product of NO with molecular oxygen. Briefly, 100 μL of culture supernatants were allowed to react with 100 μL of Griess reagent (1 % sulfanilamide, 0.1 % naphthylethylenediamine dihydrochloride, and 2.5 % phosphoric acid) at room temperature for 10 min. The optical density of the assay sample was measured spectrophotometically at 570 nm. Fresh culture medium served as the blank in all experiments. Nitrite concentration was calculated from a standard curve derived from the reaction of NaNO2 under the assay conditions.
TNF-α determination: Raw 264.7 cells were preincubated with patridoids and ginkgetin for 30 min before the addition of LPS (200 ng/mL) unless otherwise stated. TNF-α levels in the cell culture supernatants were measured with a mouse cytokine ELISA kit according to the manufacture’s instructions (Genzyme).
SDS-PAGE/immunoblot analysis: Raw 264.7 cells were plated in 6-well plates (1 × 106 cells/well) and treated with LPS for 24 h. The cells were washed and scraped into cold phosphate-buffered saline (PBS) and centrifuged at 500 × g at 4 °C. The cell pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM ethylenediaminetetraacetic acid [EDTA], 150 mM NaCl, 0.5 % Nonidet-40, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL pepstatin, and 1 μg/mL leupeptin) and centrifuged to yield whole cell lysates. The proteins (20 μg) were separated by 8 % reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred in 20 % methanol, 25 mM Tris, and 192 mM glycine to a nitrocellulose membrane (Schleicher and Schull, Dassel, Germany). The nitrocellulose membrane was then blocked by incubation in TTBS (25 mM Tris-HCl, 150 mM NaCl, and 0.2 % Tween 20) containing 5 % non-fat milk. Subsequently, the membrane was incubated with anti-iNOS antibody or anti-COX-2 antibody for 4 h, washed, and finally incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase. The protein bands were visualized using an enhanced chemiluminesence (ECL) system (Amersham Corp., Newark, NJ, USA).
#References
- 1 Ishiguro K, Yamaki M, Takagi S. Studies on iridoid-related compounds, II. The structure and antimicrobial activity of aglucones of galioside and gardenoside J Nat Prod. 1983; 46 532-6
- 2 Ishiguro K, Yamaki M, Takagi S, Ikeda Y, Kawakami K, Ito K, Nose T. Studies on iridoid-related compounds. V. Antitumor activity of iridoid derivatives: periodate oxidation products. J Pharmacobiodyn. 1988; 11 131-6
- 3 Circosta C, Occhiuto F, Ragusa S, Trovato A, Tumino G, Briguglio F, de Pasquale A. A drug used in traditional medicine: Harpagophytum procumbens DC. II. Cardiovascular activity. J Ethnopharmacol. 1984; 11 259-74
- 4 Recio M C, Giner R M, Manez S, Rios J L. Structural considerations on the iridoids as anti-inflammatory agents. Planta Med. 1994; 60 232-4
- 5 Diaz A M, Abad M J, Fernandez L, Recuero C, Villaescusa L, Silvan A M, Bermejo P. In vitro anti-inflammatory activity of iridoids and triterpenoid compounds isolated from Phillyrea latifolia L. Biol Pharm Bull. 2000; 23 1307-13
- 6 Suksamrarn A, Kumpun S, Kirtikara K, Yingyongnarongkul B, Suksamrarn S. Iridoids with anti-inflammatory activity from Vitex peduncularis . Planta Med. 2002; 68 72-3
- 7 Delaporte R, Sanchez G, Cuellar A, Giuliani A, Palazzo de Mello J. Anti-inflammatory activity and lipid peroxidation inhibition of iridoid lamiide isolated from Bouchea fluminensis (Vell.) Mold. (Verbenaceae). Ethnopharmacology. 2000; 82 127-30
- 8 Lanhers M C, Fleurentin J, Mortier F, Vinche A, Younos C. Anti-inflammatory and analgesic effects of an aqueous extract of Harpagophytum procumbens . Planta Med. 1992; 58 117-23
- 9 Lee T B. Illustrated Flora of Korea. Hyangmoon Publ. Co Seoul; 1980: p 714
- 10 Ialenti A, Ianaro A, Moncada S, Di Rosa M. Modulation of acute inflammation by endogenous nitric oxide. Eur J Pharmacol. 1992; 211 177-82
- 11 Weinstein S L, Gold M R, DeFranco A L. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc Natl Acad Sci USA. 1991; 88 4148-52
- 12 Symeonides S, Balk R A. Nitric oxide in the pathogenesis of sepsis. Infect Dis Clin North Am. 1999; 13 449-63
- 13 Baugh J A, Bucala R. Mechanisms for modulating TNF alpha in immune and inflammatory disease. Curr Opin Drug Discov Devel. 2001; 4 635-50
- 14 Bredt D S. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res. 1999; 31 577-96
- 15 An R B, Na M K, Min B S, Chang H W, Son K H, Kim H P, Kang S S, Bae K H. Iridoid esters from Patrinia saniculaefolia . Chem Pharm Bull. 2003; 51 583-5
- 16 Baek S K, Yun S S, Kwon T K, Kim J R, Chang H W, Kwak J Y, Kim J H, Kwun K B. The effect of two new antagonists of secretory PLA2 on TNF, iNOS, and COX-2 expression in activated macrophages. SCHOCK. 1999; 12 473-8
- 17 Marks J D, Marks C B, Luce J M, Montgomery A B, Turner J, Metz CA Murray J F. Plasma tumor necrosis factor in patients with septic shock. Mortality rate, incidence of adult respiratory distress syndrome, and effects of methylprednisolone administration. Am Rev Respir Dis. 1990; 141 94-7
- 18 Spinas G A, Bloesch D, Kaufmann M T, Keller U, Dayer J M. Induction of plasma inhibitors of interleukin 1 and TNF-alpha activity by endotoxin administration to normal humans. Am J Physiol. 1990; 259 R993-7
- 19 Cannon J G, Tompkins R G, Gelfand J A, Michie H R, Stanford G, van der Meer J W, Endres S, Lonnemann G, Corsetti J, Chernow B. Circulating interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever. J Infect Dis. 1990; 161 79-84
- 20 Jeong H J, Koo H N, Na H J, Kim M S, Hong S H, Eom J W, Kim K S, Shin T Y, Kim H M. Inhibition of TNF-alpha and IL-6 production by aucubin through blockade of NF-kappaB activation RBL-2H3 mast cells. Cytokine. 2002; 18 252-9
Hyeun Wook Chang
College of Pharmacy
Yeungnam University
712-749 Gyongsan
Korea
Phone: +82-53-810-2811
Fax: 82-53-811-3871
Email: hwchang@yu.ac.kr
References
- 1 Ishiguro K, Yamaki M, Takagi S. Studies on iridoid-related compounds, II. The structure and antimicrobial activity of aglucones of galioside and gardenoside J Nat Prod. 1983; 46 532-6
- 2 Ishiguro K, Yamaki M, Takagi S, Ikeda Y, Kawakami K, Ito K, Nose T. Studies on iridoid-related compounds. V. Antitumor activity of iridoid derivatives: periodate oxidation products. J Pharmacobiodyn. 1988; 11 131-6
- 3 Circosta C, Occhiuto F, Ragusa S, Trovato A, Tumino G, Briguglio F, de Pasquale A. A drug used in traditional medicine: Harpagophytum procumbens DC. II. Cardiovascular activity. J Ethnopharmacol. 1984; 11 259-74
- 4 Recio M C, Giner R M, Manez S, Rios J L. Structural considerations on the iridoids as anti-inflammatory agents. Planta Med. 1994; 60 232-4
- 5 Diaz A M, Abad M J, Fernandez L, Recuero C, Villaescusa L, Silvan A M, Bermejo P. In vitro anti-inflammatory activity of iridoids and triterpenoid compounds isolated from Phillyrea latifolia L. Biol Pharm Bull. 2000; 23 1307-13
- 6 Suksamrarn A, Kumpun S, Kirtikara K, Yingyongnarongkul B, Suksamrarn S. Iridoids with anti-inflammatory activity from Vitex peduncularis . Planta Med. 2002; 68 72-3
- 7 Delaporte R, Sanchez G, Cuellar A, Giuliani A, Palazzo de Mello J. Anti-inflammatory activity and lipid peroxidation inhibition of iridoid lamiide isolated from Bouchea fluminensis (Vell.) Mold. (Verbenaceae). Ethnopharmacology. 2000; 82 127-30
- 8 Lanhers M C, Fleurentin J, Mortier F, Vinche A, Younos C. Anti-inflammatory and analgesic effects of an aqueous extract of Harpagophytum procumbens . Planta Med. 1992; 58 117-23
- 9 Lee T B. Illustrated Flora of Korea. Hyangmoon Publ. Co Seoul; 1980: p 714
- 10 Ialenti A, Ianaro A, Moncada S, Di Rosa M. Modulation of acute inflammation by endogenous nitric oxide. Eur J Pharmacol. 1992; 211 177-82
- 11 Weinstein S L, Gold M R, DeFranco A L. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc Natl Acad Sci USA. 1991; 88 4148-52
- 12 Symeonides S, Balk R A. Nitric oxide in the pathogenesis of sepsis. Infect Dis Clin North Am. 1999; 13 449-63
- 13 Baugh J A, Bucala R. Mechanisms for modulating TNF alpha in immune and inflammatory disease. Curr Opin Drug Discov Devel. 2001; 4 635-50
- 14 Bredt D S. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res. 1999; 31 577-96
- 15 An R B, Na M K, Min B S, Chang H W, Son K H, Kim H P, Kang S S, Bae K H. Iridoid esters from Patrinia saniculaefolia . Chem Pharm Bull. 2003; 51 583-5
- 16 Baek S K, Yun S S, Kwon T K, Kim J R, Chang H W, Kwak J Y, Kim J H, Kwun K B. The effect of two new antagonists of secretory PLA2 on TNF, iNOS, and COX-2 expression in activated macrophages. SCHOCK. 1999; 12 473-8
- 17 Marks J D, Marks C B, Luce J M, Montgomery A B, Turner J, Metz CA Murray J F. Plasma tumor necrosis factor in patients with septic shock. Mortality rate, incidence of adult respiratory distress syndrome, and effects of methylprednisolone administration. Am Rev Respir Dis. 1990; 141 94-7
- 18 Spinas G A, Bloesch D, Kaufmann M T, Keller U, Dayer J M. Induction of plasma inhibitors of interleukin 1 and TNF-alpha activity by endotoxin administration to normal humans. Am J Physiol. 1990; 259 R993-7
- 19 Cannon J G, Tompkins R G, Gelfand J A, Michie H R, Stanford G, van der Meer J W, Endres S, Lonnemann G, Corsetti J, Chernow B. Circulating interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever. J Infect Dis. 1990; 161 79-84
- 20 Jeong H J, Koo H N, Na H J, Kim M S, Hong S H, Eom J W, Kim K S, Shin T Y, Kim H M. Inhibition of TNF-alpha and IL-6 production by aucubin through blockade of NF-kappaB activation RBL-2H3 mast cells. Cytokine. 2002; 18 252-9
Hyeun Wook Chang
College of Pharmacy
Yeungnam University
712-749 Gyongsan
Korea
Phone: +82-53-810-2811
Fax: 82-53-811-3871
Email: hwchang@yu.ac.kr
Fig. 1 Structures of the patridoids.R1 R2 R3 R4Patridoid I: H OCH3 H OCH3 II: OCH3 H H OCH3 IIA: OCH3 H OCH3 H
Fig. 2 Dose-dependent inhibition of NO production and iNOS protein expression by patridoid II in the Raw 264.7 cells. The Raw 264.7 cells (1 × 106 cells/ml) were preincubated with the patridoids for 30 min prior to the addition of LPS (200 ng/mL), after which the cells were incubated for 24 h with the vehicle, LPS (200 ng/mL) plus patridoid II. The samples were processed by SDS-PAGE and transferred to a nitrocellulose filter. The immunoblot was then probed with anti-iNOS or anti-COX-2 antibodies at a dilution 1 : 1,000. Similar results were obtained from 3 independent experiments.
Fig. 3 Inhibition of iNOS and COX-2 protein expression by patridoids in the LPS-stimulated Raw 264.7 cells The cells (1 × 106 cells/mL) were pretreated with either 50 μM of L-NMMA or 15 μM of the patridoids and 5 μM of ginkgetin for 30 min followed by stimulation with LPS (200 ng/mL) for 24 h with the vehicle (Un), LPS + the patridoids. After 24 h, the iNOS and COX-2 expression levels were measured in the cells. The samples were processed by SDS-PAGE and transferred to a nitrocellulose filter. The immunoblot was then probed with anti-iNOS and anti-COX-2 antibodies. The error bars represent the mean ± SD of 3 individual experiments.