Inhibitory Effect of the root of Polygala tenuifolia on Bradykinin and COX 2-Mediated Pain and Inflammatory Activity

Purpose: To gain insight into the mechanisms of analgesic and anti-inflammatory activities of the root extract of Polygala tenuifolia. Methods: Polygala tenuifolia was extracted with 70 % methanol and tested for analgesic and antiinflammatory activities (0.1, 1, 10 and 100 mg/kg) using the following models: acetic acid-induced writhing, rat paw edema, bradykinin inhibtion with rat ileum, and prostaglandin assay. Results: Administration of the Polygala tenuifolia extract at 100 mg/kg dose produced significant analgesic effect on acetic acid-induced writhing (97 % inhibition) but its effect in the tail-flick test was not significant (p < 0.05). In addition, the extract exerted significant anti-inflammatory effect in the rat paw edema model (8 to 33 % inhibition) at doses ranging from 0.1 - 100.0 mg/kg). A significant inhibitory action (53%) on the bradykinin-mediated contractions of rat ileum was also observed. Furthermore, the extract significantly (p < 0.05) inhibited the production of lipopolysaccharides-induced 6-keto-PGF 1  by 28% in macrophage cultures. Conclusion: These results provide evidence that the Polygala tenuifolia root extract exerts analgesic and anti-inflammatory effects via its significant inhibitory effect on acetic acid writhing test, bradykinin-mediated actions as well as on 6-keto-PGF 1  induction.


INTRODUCTION
Medicinal plants are believed to be an important source of products with potential therapeutic effects on various diseases.Searching for new active extracts or components derived from various natural plants can be useful in the management of inflammation and pain [1].Polygalae radix, the root of Polygala tenuifolia Willdenow (Polygalaceae), is a well-known traditional medicine used as an expectorant, tonic, tranquillizer, antipsychotic agent and functional diet for improving memory in China and Korea [2,3].It was widely used in the Chinese medicine prescription for 2000 years to treat insomnia, dream-disturbed sleep, forgetfulness and palpitation; promoting expectoration; used in cough; causing subsidence of swelling, used on boils and sores, swelling and pain [4].

EXPERIMENTAL Materials
Polygalae radix was purchased from Kyung-Dong Oriental Medicine Market, Seoul, Korea.They were authenticated by Professor Emeritus Chang-SooYok, Department of Oriental Pharmacy, Kyung Hee University, Seoul, Republic of Korea.A voucher specimen (no.98-02) was deposited at the herbarium of the Department of Pharmacology, School of Dentistry, Kyung Hee University.Organic solvent such as methanol, ether and n-butanol were molecular biology grade and purchased from Duksan Chemical Co, Seoul, Korea.Other materials used were purchased from Sigma Chemical Company, USA.

Animals
Male Sprague-Dawley rats (200-250 g) and male ICR mice (15-25 g) were purchased from Hanlim Experimental Animals Co. Korea.Animals were housed in a room with controlled temperature (22  2 C) under a 12 h light / dark cycle with free access to standard certified rodent diet and tap water.All experiments were performed according to the guidelines for the care and use of laboratory animals [ 14].

Preparation of Plant Extracts
Polygalae Radix (250 g) was cut into small pieces and extracted with 70 % methanol (750 ml) three times sequentially for 3 h on each occasion.The combined methanol extracts was concentrated in a rotary evaporator (Eyela N-N series) and dried in a freeze-dryer (FD5510 Freeze Dryer, Ilshin Lab, Republic of Korea).

Paw-edema test
Anti-inflammatory activity was determined by carrageenin-induced paw edema test in rats [17].Briefly, the rats was treated with extract or vehicle orally (0.1, 1, 10, 100 mg/kg).After 1 h, 0.1 ml of 1 % carrageenan was injected subplantarly into one of the hind paw and the contralateral paw was injected with 0.1 ml saline as control.Edema measurements were made with Ugo-Basile Plethysmograph prior and 60 min after carrageenan injection.

Evaluation of contractile responses of rat ileum
Contractile responses of rat ileum were measured as essentially described by Schapoval et al. with slight modification [18].The rats were scarificed by exposure to CO 2 gas [19], and their ilea excised and mounted in an organ bath, oxygenated with 95 % O 2 and 5 % CO 2 .The organ bath was suspended at 37 C in Tyrode solution.Contractile responses were recorded isotonically with Grass  Low Level DC Amplifier (model 7P122P).After the equilibration period of 30 min, concentration-response curves were generated by adding contractile agonist (bradykinin, 50 ng/ml) to the organ bath.After washing the ileum and the baseline tension was restored, the extract (40 g/ml,) was incubated in the organ bath for 15 min.,bradykininwas added and contractile responses recorded by MacLab 8E data acquisition system (AD Instruments)

Isolation of mouse peritoneal macrophage and cultures
Phosphate buffered saline (pH 7.4) (5 ml) was injected into ICR (Imprinting Control Region) mouse and peritoneal fluids were collected by using a needle.The peritoneal fluids were subjected to centrifuge to pellet macrophages.The resulting cell pellets were washed three times with PBS and resuspended with RPMI media at the concentration of 10 6 cells/ml.500 M of aspirin was added to the cells to irreversibly inhibit endogenous cyclooxygenase (COX) activity.The cell suspensions were applied to a 96-well dish (100 l/well) and cultured at 37 C with 5 % CO 2 atmosphere for 2 h.Thereafter, the cells were washed with PBS three times and used for experiments [1]

COX-2 inhibition assay
The cells were treated with 200 l of 3 % FBS-RPMI1640 media containing10 g/ml of LPS.The test materials were added to the wells at a final concentration of 1 or 10 g/ml and incubated for 16.Thereafter, the cell culture medium was collected and used to measure 6keto-PGF 1 by ELISA [1].

Statistical analysis
The values are expressed as mean  standard deviation (SD).The results were computed statistically (Graphpad Prism 5, Graphpad Software, Inc, La Jolla, CA, USA) using Student t-test or one-way analysis of variance.Post hoc testing was performed for intergroup comparisons using least significance test.

Anti-noticeptive activity
Anti-noticeptive activity was evaluated by acetic acid-induced writhing and tail-flick methods and the results are shown in Figs 1 and 2. Polygalae radix extract treatment significantly (p < 0.05) reduced the number of writhes by 29, 39, 68 and 97 % at doses of 0.1, 1, 10 and 100 mg/kg, respectively as compared to control.In the tailflick test, however, extract treatment produced little effect on latency time upon infra-red light exposure.

Effect of Polygalae radix extract on rat ileum contraction
To gain insight into the mechanisms of analgesic and anti-inflammatory actions, the effect of Polygalae radix extract on the rat ileum contraction using bradykinin test was investigated.The extract (40 g/ml) induced significant inhibition of bradykinin-induced contractions of rat ileum by 53 % compared to control.

Effect of Polygalae radix extract on COX-2 induction in macrophages
The effect of 10 g/ml of the extract on macrophages significantly inhibited LPS-mediated production of 6-keto-PGF 1 by 28 % compared to control.

DISCUSSION
Polygala radix, the toot of Polygala tenuifolia, is a folk medicine widely applied to achieve amelioration of several neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and psychosis in Korea [20] In the present study, we evaluated the methanol extract of Polygalae radix for analgesic and antiinflammatory effects and also sought to gain an insight into the mechanism of its actions.There is evidence that the extract has remarkable analgesic and anti-inflammatory actions with inhibitory effects on bradykinin-mediated contraction of rat ileum as well as on the LPSinduced production of 6-keto-PGF 1 in mouse peritoneal macrophages.
Acetic acid induced writhing assay is used to detect both central and peripheral analgesia whereas tail-flick assay is more sensitive to centrally acting analgesics [21].Our results indicate that the extract exerted significant dosedependent inhibitory effect with maximal effect observed at 100 mg/kg in acetic acid writhing assay but had no inhibitory effect in tail-flick test.In our previous report, we found that aspirin at 200 mg/kg has the ability to inhbit by 71% as compared to control in acetic acid writhing assay [22].These results suggest that the Polygala tenuifolia extract is more potent than aspirin in terms of analgesic action.In terms of antiinflammation, it has been identified that ibuprofen (50 mg/kg) causes 63% inhibition of rat paw edema [22] whereas the Polygala tenuifolia extract inhibited rat paw edema by 33 % at 10 mg/kg.Thus, it is reasonable to suggest that the extract has a strong inhibitory effect on peripheral noticeptive reaction rather than central action on pain transmission [23,24] In the carrageenan-induced rat paw edema test, the extract also exerted significant anti-inflammatory effect.These results indicate that the extract contains active components responsible for analgesic and anti-inflammatory actions.
Pain and inflammation are mediated by a number of chemical entities such as prostaglandins, histamine and bradykinin [25,26].In the present study, the extract inhibited prostaglandin synthesis and bradykinin-induced rat ileum contraction, thus strongly suggesting that it exerts anti-inflammatory action via inhibition of prostaglandin and bradykininmediated inflammation.It is reasonable to speculate that the Polygala tenuifolia extract exerts analgesic and anti-inflammatory activity by partly inhibiting bradykinin receptor-mediated reactions.
Cyclooxygenase (COX) is a key enzyme responsible for the production of prostaglandins and exists as isozymes including COX-1 and COX-2 [27].COX-1 is a constitutively present enzyme in cells under physiological condition, whereas COX-2 is induced by some cytokines and mitogens which are endotoxins produced during inflammation [28].
COX-1 has cytoprotective action while COX-2 play important role in inflammation.LPS-induced synthesis of prostaglandins and thromboxanes in macrophages is known to be due to selective expression of COX-2.One of the major drawbacks of currently available NSAIDs (nonsteroidal anti-inflammatory drugs) such as aspirin is stomach irritation due to the inhibition of COX-1.The selective inhibitory effect on the COX-2 is essential for the development of a new generation of anti-inflammatory drugs.The present result suggests that the Polygala tenuifolia extract has the ability to inhibit the production of 6-keto-PGF 1 , which may play an important role in its analgesic and antiinflammatory actions.Thus, there is the interesting possibility that the extract could be used effectively to treat pain and inflammation without the side effect of stomach irritation.Since inflammatory reactions induce many reactive oxygen radicals, our results suggest that the extract could be beneficial in the protection of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.With regard to the active components responsible for the extract's analgesic and anti-inflammatory activities, a thorough analytical investigation by employing activity-guided fractionation would be required.Interestingly, however, it has been proposed that sinapic acid is a probable active component of Polygalae radix responsible for antiinflammation, and hence its action could produce neuroprotection and memory improvement [20].

CONCLUSION
The present study strongly suggests that the methanol extract of Polygala tenuifolia exerts significant analgesic and anti-inflammatory effects.The extract can be used as functional food.It protects cells against oxidative stress and some inflammatory reactions, and therefore could be useful in the protection and treatment of some diseases caused by inflammation.

Fig 2 :
Fig 2: Effect of Polygalae radix extract on on tail flicks.Data are expressed as mean  SD (n = 5).The effect of the extact was not significant at all doses tested (p < 0.01), compared with controlAnti-inflammatory activityThe results of the rat paw edema test are shown in Fig 3.The extract inhibited the carrageenininduced paw edema by 11, 16, 33 and 8 % at doses of 0.1, 1, 10 and 100 mg/kg, respectively, after 60 min, compared to control.The effect was significant at only the first three doses..