We tested baicalin for its antiinflammatory and analgesic effects (and the mechanisms) in a rat model of carrageenan-evoked thermal hyperalgesia. Pre- or posttreatment with baicalin (10, 30, or 100 mg/kg intraperitoneally) caused a significant analgesic effect with a similar effect of dose-matched ibuprofen. Furthermore, baicalin dose-dependently attenuated tumor necrosis factor-α (from 3510 ± 150 pg/mL to 2860 ± 148 pg/mL to 1480 ± 210 pg/mL), interleukin (IL)-1β (from 3210 ± 210 pg/mL to 2200 ± 140 pg/mL to 750 ± 95 pg/mL), and IL-6 (from 58.5 ± 9.8 pg/mL to 38.5 ± 9.0 to 21.0 ± 8.1 ng/mL) formation but enhanced IL-10 (from 18.1 ± 2.5 pg/mL to 36.1 ± 5.5 pg/mL to 71.2 ± 9.5 pg/mL) production in paw exudates at 4 h after carrageenan injection. Prostaglandin E2 (PGE2) and nitrate formation in the carrageenan-injected paws were dose-dependently inhibited by baicalin (10–100 mg/kg intraperitoneally) (PGE2: from 15.9 ± 2.1 ng/mL to 12.1 ± 1.6 ng/mL to 6.2 ± 1.8 ng/mL; nitrate: from 39.8 ± 4.8 μM to 27.5 ± 3.0 μM to 17.2 ± 1.6 μM) at 4 h but not at 1.5 h after carrageenan injection. Increased myeloperoxidase activity in carrageenan-injected paws was also dose-dependently reduced by baicalin. These findings suggest that the antiinflammatory and analgesic mechanisms of baicalin may be associated with the inhibition of critical inflammatory mediators, including nitric oxide, PGE2, and proinflammatory cytokines, accompanied by an increase in IL-10 production, as well as neutrophil infiltration at sites of inflammation.
IMPLICATIONS: Our results showed that baicalin possesses an analgesic effect in carrageenan-evoked thermal hyperalgesia. The possible mechanisms of action of baicalin may be associated with the inhibition of proinflammatory mediator overproduction, including cytokines, nitric oxide, and prostaglandin E2, as well as neutrophil infiltration. This implies that baicalin may be a potential therapeutic analgesic for inflammatory pain.
*Department of Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan, Republic of China; and
†Department of Radiation Oncology,
‡Department of Anesthesiology, and
§Division of Cardiology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China
Supported in part by a research grant from the National Science Council of Taiwan (NSC 88-2314-B016-072) and a grant from Tri-Service General Hospital (TSGH-C91-15), Republic of China.
Accepted for publication June 27, 2003.
Address correspondence and reprint requests to Tz-Chong Chou, PhD, Department of Physiology and Biophysics, National Defense Medical Center, No. 161, Min-Chuan E. Rd., Sec. 6, Taipei, Taiwan, ROC. Address e-mail to firstname.lastname@example.org.
Scutellaria baicalensis Georgi (Huang Qui), a Chinese traditional medicinal herb, is widely used as an antiinflammatory, antibacterial, and hepatoprotective drug (1,2). Baicalin (7-glucuronic acid,5,6-dihydroxy-flavone), a flavonoid compound isolated from Huang Qui, possesses antioxidant properties (3) and has an inhibitory effect on carrageenan-induced rat paw edema (1), suggesting that it may be a potential antiinflammatory drug.
The carrageenan-evoked thermal hyperalgesia resulting from the combined effect of the release of proinflammatory cytokines, cyclooxygenase (COX) products, and sympathomimetic amines (4) is a common model used to study inflammatory pain. Tumor necrosis factor-α (TNF-α) has an early and crucial role in the cascade of proinflammatory cytokine production and subsequent inflammatory processes (5). COX catalyzes the conversion of arachidonic acid to many biologically active mediators, such as prostaglandin E2 (PGE2). It has been reported that overproduction of inflammatory prostaglandins by an inducible form of COX (COX-2) plays an important pathophysiological role in the development of inflammatory pain (6,7).
Nitric oxide (NO), synthesized by the enzyme NO synthase (NOS), is an important mediator in the regulation of cell functions (8). However, overproduction of NO derived from inducible NOS (iNOS) activated by proinflammatory cytokines, free radicals, and lipopolysaccharide (LPS) may cause the pathogenesis of inflammatory diseases, including carrageenan-evoked inflammatory pain (6,9). Inhibition of COX-2 activity in a castor-oil-induced diarrhea model, as well as NO formation and iNOS expression in LPS-treated RAW 264.7 macrophages by baicalin (10,11), suggests that baicalin may suppress the COX-2 and iNOS pathways, a critical mediator accounting for the carrageenan-induced inflammatory pain.
In this study, we first demonstrate that baicalin exhibits an analgesic effect in a rat model of carrageenan-evoked thermal hyperalgesia. To further investigate the analgesic mechanisms involved, the effect of baicalin on the formation of important inflammatory mediators, including cytokine, NO, and PGE2 formation, as well as neutrophil infiltration at inflammatory sites, was also studied.
Male Sprague-Dawley rats (8–9 wk) weighing 200–250 g, purchased from the National Animal Center (Taipei, Taiwan), were used in this study. This study was approved by the local institutional animal care and use committee. Animals were housed in a standard environment and maintained on tap water and rodent food ad libitum throughout the investigation.
To induce local inflammation, 2 mg of λ-carrageenan (Sigma, St. Louis, MO; 100 μL of 2% [wt/vol] in saline) was injected subcutaneously into the plantar surface intraplantar injection (i.pl.) of rat right hind paws at Time 0 (T = 0). Rats were allowed 30 min to acclimate to the device before testing. Baicalin, purchased from Aldrich Chemical Co. (Milwaukee, WI), was dissolved in saline and delivered in a volume of 0.2 mL. Baicalin (10, 30, or 100 mg/kg intraperitoneally [IP]), vehicle (saline, IP), or ibuprofen (30 mg/kg IP) (Sigma) was injected at 30 min before (T = −30) or 165 min after (T = 165) carrageenan injection. Hyperalgesia was assessed by measuring the paw withdrawal latency with a nonnoxious heat stimulus by using a device (7370 Plantar Test; Ugo Basile, Comerio, Italy). The paw withdrawal latencies were evaluated every 30 min for 240 min starting 60 min after carrageenan injection, and the baseline latencies were assessed at 40 min before (T = −40) carrageenan injection. Each test was calculated as a mean of three repeated measurements. Each rat was used once. The vehicle (IP)-treated and the saline (i.pl.)-injected rats acted as a control group. Each group contained six rats.
After the analgesic experiments were finished, rats were killed by exsanguination. Then the hind paws were cut at the level of the calcaneus bone and centrifuged at 400 g for 15 min at 4°C to collect the exudates (edema fluid) for measuring the levels of TNF-α, interleukin (IL)-10, IL-1β, and IL-6 in paw exudates by using rat enzyme immunoassay kits (Genzyme Corp., Cambridge, MA). Concentrations of nitrate and PGE2 were determined in paw exudates collected as described previously, 1.5 or 4 h after carrageenan injection. The levels of nitrate (total nitrite and nitrate) in paw exudates were measured by using a Sievers nitric oxide analyzer (280 NOA; Sievers, Boulder, CO). The levels of PGE2 were measured by a rat enzyme immunoassay kit.
For myeloperoxidase (MPO) activity assay, 4 h after the injection of carrageenan, rats were killed. The tissues of carrageenan-injected paws were removed, washed with sterile normal saline, and homogenized in ice-cold 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer (pH 6.0; 5 mL of HTAB per gram of tissue) on ice by using a homogenizer (Pro Scientific Inc., Pro Model 200; Monroe, CT). They were then sonicated and centrifuged at 15,000 g for 15 min at 4°C. Then the supernatant was mixed 1:30 (supernatant/assay buffer) and read at 460 nm. The assay buffer consisted of 100 mM potassium phosphate (pH 6.0), 0.083 mL of H2O2 (Sigma; 30% stock diluted 1:1000), and 0.834 mL of o-dianisidine hydrochloride (Sigma; 10 mg/mL). MPO activity was calculated and expressed as ΔA460 per minute per milligram of protein.
Data were expressed as mean ± sem. Statistical analysis was performed by one-way analysis of variance. If significant results were revealed by this analysis, then differences between group means were evaluated by the Student’s t-test for unpaired observations. A P value <0.05 was considered statistically significant.
Basal withdrawal latencies for all experimental groups were similar, at approximately 22.0 ± 0.3 s. Contralateral left (carrageenan-uninjected) hind paw withdrawal latencies remained constant at basal levels for the entire experiment. Injection of carrageenan into the right hind paw of rats evoked thermal hyperalgesia with a significant decrease of withdrawal latencies that began at 60 min and reached the smallest value of 3.9 ± 0.5 s at 3 h after carrageenan injection. The administration of baicalin (10–100 mg/kg IP) either 30 min before or 165 min after carrageenan injection significantly inhibited carrageenan-evoked thermal hyperalgesia (P < 0.01 versus vehicle-treated and carrageenan-injected rats) in a dose-dependent manner (Fig. 1). Pre- or posttreatment with the same dose (30 mg/kg IP) of baicalin or ibuprofen, a nonsteroidal antiinflammatory drug (NSAID), had a similar analgesic effect.
The administration of baicalin 30 min before carrageenan injection dose-dependently inhibited the increased formation of TNF-α, IL-6, and IL-1β but enhanced the IL-10 formation in paw exudates at 4 h after carrageenan injection compared with that of vehicle-treated and carrageenan-injected rats (Fig. 2). Injection of carrageenan (i.pl.) led to a time-dependently increased production of nitrate and PGE2 in the paw exudates. The administration of baicalin 30 min before carrageenan injection dose-dependently inhibited the nitrate and PGE2 production at 4 h rather than at the early phase (1.5 h) after carrageenan injection (Fig. 3). Four hours after the injection of carrageenan, the increased MPO activity in carrageenan-injected paws was dose-dependently depressed by baicalin (Fig. 4).
In carrageenan-evoked inflammatory pain, the proinflammatory cytokines—including TNF-α, IL-1β, and IL-6—play an early and crucial role in the subsequent inflammatory responses (5). In contrast, an “antagonist cytokine,” IL-10, exerts its antiinflammatory effect by inhibiting the production of proinflammatory cytokines (12) and by inducing the formation of the IL-1 receptor antagonist (13). Injection of a monoclonal antibody to mouse IL-10 enhances carrageenan- or TNF-α-evoked inflammatory pain (14), suggesting that IL-10 may be a potential therapeutic drug for inhibiting inflammatory responses. In this study, we first demonstrated that baicalin has a preventive and therapeutic analgesic effect in inflammatory pain. In addition, baicalin inhibits the formation of TNF-α, IL-1β, and IL-6 but enhances the production of IL-10, thus resulting in an overall attenuation of the proinflammatory/antiinflammatory cytokine ratio in carrageenan-injected paws, which may contribute to its antiinflammatory and analgesic effects.
It was proposed that peripheral COX-1 plays a primary role in early inflammatory responses; after inflammation is fully established (when COX-2 is activated), peripheral prostaglandin production synthesized by COX-2, and perhaps also COX-1, may account for the maintenance of carrageenan-evoked thermal hyperalgesia (15). Systemic administration of COX-2 inhibitors or PGE2 monoclonal antibodies resulted in an attenuation of carrageenan-induced inflammatory hyperalgesia (7,16), further indicating that overproduction of PGE2 derived from COX-2 plays a critical role in the generation and maintenance of inflammatory pain. Similarly, NO produced by constitutive NOS is involved in the early phase and NO produced by iNOS is involved in the late phase during the development of the carrageenan-evoked inflammatory pain (9). Thus, overproduction of NO and PGE2 mainly derived from iNOS and COX-2, respectively, may play an important pathophysiological role in the development of carrageenan-induced inflammatory pain. Our results demonstrated that baicalin inhibited PGE2 and nitrate overproduction in carrageenan-injected paws only at the late phase (4 hours) (when COX-2 and iNOS activity were activated) rather than at the early phase (1.5 hours), suggesting that baicalin may be a relatively selective inhibitor for COX-2 and iNOS, which is possibly due to baicalin’s initial attenuation of the formation of proinflammatory cytokines. The increased MPO activity (an indicator of neutrophil infiltration) seen at sites of inflammation exerting an important source of oxygen-derived free radicals and many proinflammatory mediators (17) was significantly inhibited by baicalin, which may be another mechanism of its antiinflammatory effect.
Interestingly, the analgesic effect of baicalin was similar to that of ibuprofen, a traditional NSAID. Although NSAIDs are the most commonly used remedy for treating inflammation, they cause several serious adverse effects because of their nonselective inhibition of COX (18). Many studies have shown that the effect of ibuprofen on the production of proinflammatory cytokines is controversial. The administration of ibuprofen led to a pronounced increase of TNF-α and IL-6 levels in serum and to increased mortality in murine endotoxic shock (19,20). Ibuprofen also increased secretion of IL-6 and TNF-α but reduced production of IL-10 in LPS-treated mononuclear cells (21). Systemic administration of iloprost, a prostacyclin analog, inhibited serum TNF-α levels after IP LPS (21), indicating that TNF-α production is modulated by endogenous prostaglandins in vivo. The enhanced production of TNF-α by COX inhibitors may lead to exacerbation of some inflammatory processes. However, two studies (22,23) reported that treatment with ibuprofen attenuated cardiopulmonary dysfunction and LPS-induced lung injury through inhibiting TNF-α and macrophage inflammatory protein-2 production and proinflammatory cytokine gene expression but increased lung iNOS messenger RNA expression without affecting the MPO activity in the lung. The reasons causing the inconsistent effects of ibuprofen are still unknown. On the basis of these differences between baicalin and ibuprofen, we proposed that the analgesic effect of the two drugs may be through different mechanisms.
Baicalin has been used as an antiinflammatory and antiallergic drug in a variety of inflammatory diseases (2). Baicalin also possesses therapeutic activity against prostate and bladder cancers (24,25), as well as the infection of human peripheral blood mononuclear cells by acquired human immunodeficiency virus Type 1 (26). No serious side effects of baicalin have been reported. Recently, a study of metabolic pharmacokinetics of baicalin in rats showed that when baicalin was administered orally, glucuronides and sulfates of baicalein were exclusively circulating in the plasma, implying that baicalin is absorbed as conjugated metabolites of baicalein by the gastrointestinal tract (27).
This study is the first to demonstrate that baicalin exhibits a potent analgesic effect on carrageenan-evoked thermal hyperalgesia. Furthermore, we propose that the mechanisms of baicalin may be associated with the inhibition of inflammatory mediator overproduction, including proinflammatory cytokines, NO, and PGE2. The other possibilities are an increase of IL-10 and an inhibition of neutrophil infiltration in carrageenan-injected paws. These findings suggest that baicalin may be therapeutically useful for mitigating inflammatory pain.
We thank Ting-Hui Chu for technical assistance.
1. Lin CC, Shieh DE. The anti-inflammatory activity of Scutellaria rivularis
extracts and its active compounds, baicalin, baicalein and wogonin. Am J Chin Med 1996; 24: 31–6.
2. Kubo M, Matsuda H, Tanaka M, et al. Studies on Scutellariae radix
. VII. Anti-arthritic and anti-inflammatory actions of methanolic extract and flavonoid components from Scutellariae radix.
Chem Pharm Bull 1984; 32: 2724–9.
3. Gao Z, Huang K, Yang X, et al. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis
Georgi. Biochem Biophys Acta 1999; 1472: 643–50.
4. Nakamura M, Ferreira SH. A peripheral sympathetic component in inflammatory hyperalgesia. Eur J Pharmacol 1987; 135: 145–53.
5. Cunha FQ, Poole S, Lorenzetti BB, et al. The pivotal role of tumour necrosis factor alpha in the development of inflammatory hyperalgesia. Br J Pharmacol 1992; 107: 660–4.
6. Vane JR, Mitchell JA, Appleton I, et al. Inducible isoforms of cyclooxygenase and nitric oxide synthase in inflammation. Proc Natl Acad Sci U S A 1994; 91: 2046–50.
7. Portanova JP, Zhang Y, Anderson GD, et al. Selective neutralization of prostaglandin E2
blocks inflammation, hyperalgesia, and interleukin 6 production in vivo. J Exp Med 1996; 184: 883–91.
8. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43: 109–42.
9. Salvemini D, Wang ZQ, Wyatt PS, et al. Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation. Br J Pharmacol 1996; 118: 829–38.
10. Kase Y, Saitoh K, Makino B, et al. Relationship between the antidiarrhoeal effects of Hange-Shashin-To and its active components. Phytother Res 1999; 13: 468–73.
11. Chen YC, Shen SC, Chen LG, et al. Wogonin, baicalin and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide. Biochem Pharmacol 2001; 61: 1417–27.
12. Oswald IP, Wynn TA, Sher A, et al. Interleukin-10 inhibits macrophage microbicidal activity by blocking the endogenous production of tumor necrosis factor α required as costimulatory factor for interferon-γ-induced activation. Proc Natl Acad Sci U S A 1992; 89: 8676–80.
13. Jenkins JK, Malyak M, Arend WP. The effects of interleukin-10 on interleukin-1 receptor and antagonist and interleukin-1 production in human monocytes and neutrophils. Lymphokine Cytokine Res 1994; 13: 47–54.
14. Poole S, Cunha FQ, Selkirk S, et al. Cytokine-mediated inflammatory hyperalgesia limited by interleukin-10. Br J Pharmacol 1995; 115: 684–8.
15. Dirig DM, Isakson PC, Yaksh TL. Effect of COX-1 and COX-2 inhibition on induction and maintenance of carrageenan-evoked thermal hyperalgesia in rats. J Pharmacol Exp Ther 1998; 285: 1031–8.
16. Zhang Y, Shafferm A, Portanova J, et al. Inhibition of cyclooxygenase-2 rapidly reverses inflammatory hyperalgesia and prostaglandin E2
production. J Pharmacol Exp Ther 1997; 283: 1069–75.
17. Fantone JC, Ward PA. Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 1982; 107: 397–418.
18. Wallace JL, Carter L, McKnight W, et al. ML 3000 reduces gastric prostaglandin synthesis without causing mucosal injury. Eur J Pharmacol 1994; 271: 525–31.
19. Coran AG, Drongowski RA, Paik JJ, et al. Ibuprofen intervention in canine septic shock: reduction of pathophysiology without decreased cytokines. J Surg Res 1992; 53: 272–9.
20. Pettipher ER, Wimberly DJ. Cyclooxygenase inhibitors enhance tumor necrosis factor production and mortality in murine endotoxic shock. Cytokine 1994; 6: 500–3.
21. Sirota L, Shacham D, Punsky I, et al. Ibuprofen affects pro- and anti-inflammatory cytokine production by mononuclear cells of preterm newborns. Biol Neonate 2001; 79: 103–8.
22. Daphtary KM, Heidemann SM, Gilbetic M. Ibuprofen attenuates early lung injury in endotoxemic, neutropenic rats. Prostaglandins Leukot Essent Fatty Acids 2001; 65: 59–65.
23. Qiu HB, Pan JQ, Zhao YQ, et al. Effects of dexamethasone and ibuprofen on LPS-induced gene expression of TNF alpha, IL-1 beta, and MIP-1 alpha in rat lung. Zhongguo Yao Li Xue Bao 1997; 18: 165–8.
24. Ikezoe T, Chen SS, Heber D, et al. Baicalin is a major component of PC-SPES which inhibits the proliferation of human cancer cells via apoptosis and cell cycle arrest. Prostate 2001; 49: 285–92.
25. Ikemoto S, Sugimura K, Yoshida N, et al. Antitumor effects of Scutellariae radix
and its components baicalein, baicalin and wogonin on bladder cancer cell lines. Urology 2000; 55: 951–5.
26. Kitamura K, Honda M, Yoshizaki H, et al. Baicalin, an inhibitor of HIV-1 production in vitro. Antiviral Res 1998; 3: 131–40.
27. Lai MY, Hsiu SL, Tsai SY, et al. Comparison of metabolic pharmacokinetics of baicalin and baicalein in rats. J Pharm Pharmacol 2003; 55: 205–9.