Antinociceptive and anti-inflammatory activities of Aquilaria sinensis (Lour.) Gilg. Leaves extract

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Journal of Ethnopharmacology 117 (2008) 345–350

Antinociceptive and anti-inflammatory activities of Aquilaria sinensis (Lour.) Gilg. Leaves extract Minhua Zhou a,b , Honggang Wang a , Suolangjiba a , Junping Kou a,∗ , Boyang Yu a,b,∗∗ a

Department of Complex Prescription of TCM, China Pharmaceutical University, 1 Shennong Road, Nanjing 210038, PR China b Key Lab for Modern Traditional Chinese Medicine, Ministry of Education, China Pharmaceutical University, 1 Shennong Road, Nanjing 210038, PR China Received 23 July 2007; received in revised form 14 October 2007; accepted 2 February 2008 Available online 12 February 2008

Abstract Aim of the study: The analgesic and anti-inflammatory activities of the ethanol extract of Aquilaria sinensis (Lour.) Gilg. Leaves were observed in various experimental models related to nociception and inflammation, so as to provide some evidence for its traditional use. Materials and methods: Acetic acid-induced writhing and a hot plate test in mice were used to evaluate its analgesic activity. On the other hand, its anti-inflammatory activity was observed in xylene or carrageenan-induced edema, carboxymethylcellulose sodium (CMC-Na)-induced leukocyte migration in mice and lipopolysaccharide (LPS)-induced nitric oxide (NO) release from mouse peritoneal macrophages in vitro. Results: The ethanol extract significantly inhibited acetic acid-induced writhing after single oral administration at doses of 424 and 848 mg extract/kg, and the response to the thermal stimulus in mice at the dose of 848 mg/kg. Meanwhile, the ethanol extract also remarkably lessened xylene-induced ear swelling, carrageenan-induced paw edema, and CMC-Na-induced leukocyte migration. Furthermore, the extract considerably reduced NO release from LPS-stimulated macrophages with IC50 of 80.4 mg/ml. Conclusion: These findings suggest that Aquilaria sinensis (Lour.) Gilg. Leaves extract present notable analgesic and anti-inflammatory activities, which support its folkloric use for some diseases related with painful and inflammatory conditions such as trauma etc. © 2008 Published by Elsevier Ireland Ltd. Keywords: Aquilaria sinensis (Lour.) Gilg. Leaves (Thymelaeaceae); Analgesic; Anti-inflammatory; Nitric oxide; Macrophage

1. Introduction Lignum Aquilariae Resinatum has been widely used in the treatment of various kinds of pain, cough and anaphylaxis for hundreds of years in Asia, especially in China, Vietnam, and Indonesia, etc. (Okugawa et al., 1993; Ou, 2003; Yagura et al., 2003). Aquilaria sinensis (Lour.) Gilg. (Thymelaeaceae) and Aquilaria agallocha Roxb. (Thymelaeaceae) belonging to the source of Lignum Aquilariae Resinatum are generally consid-

∗

Corresponding author. Tel.: +86 25 85391042; fax: +86 25 85391042. Corresponding author at: Department of Complex Prescription of TCM, Key Lab for Modern Traditional Chinese Medicine, Ministry of Education, China Pharmaceutical University, 1 Shennong Road, Nanjing 210038, PR China. Tel.: +86 25 85391042; fax: +86 25 85391042. E-mail addresses: [email protected] (J. Kou), [email protected] (B. Yu). ∗∗

0378-8741/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2008.02.005

ered as the similar in use (Yang and Chen, 1983). It was found that the essential oil of Lignum Aquilariae Resinatum from the two herbs both possessed the same activities, such as anesthesia and analgesia, etc. (Yang and Chen, 1983), and many chemical ingredients with similar structure have been isolated from both herbs (Yang and Chen, 1983, 1986; Yang et al., 1989a,b, 1990). In addition, the aqueous extract of Aquilaria agallocha stems had been found to suppress the immediate hypersensitivity reaction by the inhibition of histamine release from mast cells (Kim et al., 1997), and linalool, existing in Aquilaria agallocha Roxb. had been proved to be a potentially anti-inflammatory and analgesic agent (Peana et al., 2002, 2003). On the other hand, the leaves of Aquilaria sinensis (Lour.) Gilg., which widely cultivated in Guangdong, Hainan and Taiwan provinces in China are orally reported to be used locally in trauma-related diseases such as fracture, bruise, etc. However, there are few reports about its pharmacological activity

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and chemical constituents, therefore, this study was undertaken to investigate medicinal properties of the plant leaves relating to anti-inflammatory and analgesic effects, so as to provide some pharmacological evidences for its folkloric uses and to promote further exploitation of the rich resources of Aquilaria sinensis (Lour.) Gilg. Leaves. 2. Materials and methods

2.5. Acetic-acid-induced writhing response in mice The test was carried out using the described technique previously (Elisabetsky et al., 1995). Mice were injected intraperitoneally with 0.1 ml/10 g body weight of 0.7% acetic acid solution in saline 1 h after the oral administration of tested drugs. The frequency of writhing occurring was recorded 15 min after the injection of acetic acid. Indomethacin was administrated to mice as a positive control in this experiment.

2.1. Plant material 2.6. Hot plate latency assay in mice The leaves of Aquilaria sinensis (Lour.) Gilg. were harvested from Dianbai County, Guangdong province, China in October 2005 and was authenticated by Dr. Zenglai Xu (Nanjing Zhongshan Arboretum, Jiangsu province, China). The voucher specimen (BYY051020) was deposited at the Herbarium of China Pharmaceutical University. 2.2. Preparation of plant extract The leaves (500 g) were air-dried and extracted under reflux for 2 h with 50% ethanol twice yielding a total extract (84.80 g, 16.96%, w/w). The extract was suspended in normal saline as indicated concentrations for in vivo experiments, and was dissolved in dimethylsulfoxide (DMSO) with the final concentration of 0.1% for in vitro tests. In all experiments, the dosage was recorded as the mass of extract/kg (body weight of mice), or the weight of extract/ml. 2.3. Drugs and reagents Indomethacin tablet (Indo, Linfenqi Pharmaceutical Co. Ltd., China); Dexamethasone Acetate tablet (Dex, Zhejiang Xianju Pharmaceutical Co. Ltd., Zhejiang, China); Hydrocortisone (Hyd, Yangzhou Pharmaceutical Co. Ltd., Yangzhou, China); Carboxymethylcellulose-sodium (CMC-Na) and Xylene (Shanghai Chemical Reagent Company, Shanghai, China); Carrageenan (type I, Sigma Chemical Co., St. Louis, MO, U.S.A.); Thioglycolate broth (Shanghai Insect Technology Development Co., Shanghai, China); RPMI-1640 medium (Gibco BRL, Life Technologies, Inc., New York, U.S.A.); Newborn bovine serum (NBS, Hangzhou Sijiqing Biomaterial Co. Ltd., Hangzhou, China); 3-(4,5-dimethyl-2-thiazol)-2,5diphenyl-2H-tetrazolium bromide (MTT, Ameresco, U.S.A.); Lipopolysaccharide (LPS, FLUCA, U.S.A.). 2.4. Animals Male or female ICR mice weighing 18–22 g were obtained from the Experimental Animal Center of China Pharmaceutical University (Nanjing, China). They were kept in plastic cages at 22 ± 2 ◦ C with free access to pellet food, water and on a 12 h light/dark cycle. This study complied with current ethical regulations on animal research (National Research Council of USA, 1996) and related rules of our school, and all animals used in the experiment received humane care.

Experiments were carried out according to previously described method (Adzu et al., 2003). Female animals were habituated twice (1 min) to the hot plate at 24 h and 20 min prior to the test. For the testing, female mice were placed on hot plate maintained at 55 ± 5 ◦ C. The time that elapsed until the occurrence of either a hind paw licking or a jump off the plate surface was recorded as the hot-plate latency. Mice with baseline latencies of <5 or >30 s were removed from the study. After the baseline determination of response latencies, hot-plate latencies were measured at 1 and 2 h after oral administration of tested samples, and animals delayed not more than 1 min every time on the hot-plate. 2.7. Xylene-induced ear swelling in mice The samples tested including indomethacin (20 mg/kg) as a positive-control were given orally to the mice fasted for 10 h. One hour later, each animal received 30 ␮l of xylene on the anterior and posterior surfaces of the right ear lobe. The left ear was considered as control. Another hour later, the animals were sacrificed by cervical dislocation and both ears were sampled. Circular sections were taken, using a cork borer with a diameter of 8 mm, and weighed. The degree of ear swelling was calculated based on the weight of left ear without applying xylene (Kou et al., 2005a). 2.8. Carrageenan-induced paw edema in mice The initial hind paw volume of the ICR mice was determined volumetrically. A 2% solution of carrageenan in normal saline (0.02 ml/mouse) was injected subcutaneously into the right hind paw 1 h after the test samples had been administered orally. The control group received the vehicle. Paw volumes were measured at 0, 1, 3 and 5 h after injection, and the volume of the edema was measured with a plethysmometer (Kou et al., 2005a). 2.9. CMC-Na-induced leukocyte emigration in mice The test was carried out using the technique described by Kou et al. (2005b). One hour after being orally given tested drugs, fasted mice were injected intraperitoneally with 1.5% CMC-Na solution (375 mg/kg) in normal saline. Four hours later, mice were killed by cervical dislocation, 5 ml of normal saline was injected into the peritoneal cavity, and then the washing solution

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was collected, 50 ␮l of peritoneal fluid was mixed with 0.45 ml of Turk’s solution (0.01% crystal violet in 3% acetic acid) and leukocyte cells were counted under a microscope. Dexamethasone (20 mg/kg) was administrated to mice as a positive-control in this experiment. 2.10. LPS-stimulated NO release from macrophages in vitro 2.10.1. Isolation and culture of macrophages All assays were performed on thioglycollate-elicited mouse peritoneal macrophages prepared according to Li et al. (1997). Then macrophages were washed twice with phosphate buffer saline (PBS) and finally suspended at a density of 2 × 106 /ml in ice-cold phenol red free RPMI-1640 medium supplemented with 10% newborn bovine serum. Cell viability (≥95%) was confirmed by trypan blue exclusion assay. Cells were incubated at 37 ◦ C and 5% CO2 for 2 h to allow macrophages to adhere to the surface of culture plates. The non-adherent cells were removed by vigorously washing with RPMI-1640 and adherent mono-layer of macrophages was maintained in abovementioned medium at 37 ◦ C and 5% CO2 . 2.10.2. Measurement of nitrite production and cell viability The cells were incubated with 5 ␮g/ml LPS in the presence of the ethanol extract (50, 100, and 200 ␮g/ml) or 0.1 ␮M hydrocortisone (positive control) for 48 h. Then the amount of nitrite in cell-free culture supernatant was determined by Griess reagent (Green et al., 1982). Briefly, 100 ␮l of supernatant was mixed with an equal volume of Griess reagent [1:1 (v/v) of 0.1% N-1-naphthyl-ethylenediamine in distilled water and 1% sulfanilamide in 5% phosphoric acid] on a 96-well flat-bottom plate. The absorbance at 550 nm was measured after 10 min using the ELISA reader. The amount of nitrite was calculated from a standard curve created using known concentrations of sodium nitrite (NaNO2 ). Following the aspiration of media for nitrite determination, viability of macrophages was determined by MTT assay at OD570 (Kou et al., 2005a).

Fig. 2. Effect of the ethanol extract of Aquilaria sinensis (Lour.) Gilg. Leaves (EtOH ext.) on a hot plate test in mice. EtOH ext. at doses of 424 and 848 mg/kg and indomethacin 20 mg/kg were administered orally 1 h before recording the hot-plate latencies. Each value represents the mean ± S.E.M. of 10 mice. *P < 0.05, **P < 0.01 vs. the control group.

2.11. Statistical analysis Results were expressed as mean ± S.E.M. Data were analyzed by a one-way ANOVA, followed by Student’s two-tailed t-test for the comparison between test and control, and Dunnett’s test when the data involved three or more groups. P < 0.05 was considered to be significant. 3. Result 3.1. Effect of the ethanol extract on the writhing response induced by acetic acid As shown in Fig. 1, the ethanol extract (424 and 848 mg/kg) of Aquilaria sinensis Leaves (EtOH ext.) restrained the writhing response induced by intraperitoneal injection of acetic acid in mice with inhibition rate of 62.2% and 66.9%. Indomethacin (20 mg/kg), the positive drug, also markedly reduced writhing times. 3.2. Effect of the ethanol extract on hot-plate latency assay The results presented in Fig. 2 showed that the ethanol extract (848 mg/kg) of Aquilaria sinensis Leaves (EtOH ext.) increased pain threshold of mice by 57.1% at 2 h after oral administration. Table 1 Effect of the ethanol extract from Aquilaria sinensis (Lour.) Gilg. Leaves (EtOH ext.) on xylene-induced ear swelling in mice

Fig. 1. Effect of the ethanol extract from Aquilaria sinensis (Lour.) Gilg. Leaves (EtOH ext.) on acetic-acid-induced writhing response in mice. EtOH ext. at doses of 424 and 848 mg/kg and indomethacin 20 mg/kg were administered orally 1 h before the injection of acetic acid. Each value represents the mean ± S.E.M. of 10 mice. *P < 0.05, **P < 0.01 vs. the control group.

Group

Dose (mg/kg)

Ear swelling (%)

Control EtOH ext.

– 424 848 20

74.38 51.04 37.56 26.85

Indo

± ± ± ±

7.48 7.38* 6.19** 5.10**

Inhibition (%) – 31.38 49.50 63.90

Each value represents the mean ± S.E.M. of 10 mice. *P < 0.05, **P < 0.01 vs. the control group.

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Table 2 Effect of the ethanol extract from Aquilaria sinensis (Lour.) Gilg. Leaves (EtOH ext.) on carrageenan-induced paw swelling in mice Group

Dose (mg/kg)

Footpad swelling after injection (%) 1h

Control EtOH ext.

– 424 848 20

Indo

19.99 17.46 13.13 12.58

3h ± ± ± ±

1.97 4.14 2.28* 2.78*

36.01 25.02 17.36 17.18

5h ± ± ± ±

4.49 6.01 2.91* 2.32*

33.21 24.34 16.40 14.76

± ± ± ±

1.90 3.21 2.44** 1.57**

Each value represents the mean ± S.E.M. of 10 mice. *P < 0.05, **P < 0.01 vs. the control group. Table 3 Effect of the ethanol extract from Aquilaria sinensis (Lour.) Gilg. Leaves (EtOH ext.) on CMC-Na-induced peritoneal leukocytes migration of mice Group

Dose (mg/kg)

Leucocyte count (×104 )

Control Model EtOH ext.

– – 424 848 20

97.00 182.14 123.57 105.00 99.69

Dex

± ± ± ± ±

11.72 11.74## 10.4** 8.22** 9.47**

Inhibition (%) – – 68.79 90.60 96.84

3.4. Effect of the ethanol extract on carrageenan-induced paw edema As shown in Table 2, the ethanol extract of Aquilaria sinensis Leaves (EtOH ext.) notably inhibited paw edema in mice when given orally at 1, 3 and 5 h after carrageenan injection at the dose of 848 mg/kg, and its potency is similar to that of indomethacin (20 mg/kg).

Each value represents the mean ± S.E.M. of 10 mice. ## P < 0.01 vs. the control group; **P < 0.01 vs. the model group.

3.5. Effect of the ethanol extract on leukocyte emigration induced by CMC-Na

Meanwhile, indomethacin (20 mg/kg) also markedly increased mice latency.

Carboxymethylcellulose-sodium (1.5% CMC-Na, 0.25 ml/ 10 g, i.e. 375 mg/kg i.p.) significantly enhanced leukocyte emigration in the mice peritoneal cavity. The ethanol extract of Aquilaria sinensis Leaves (EtOH ext.) dose-dependently inhibited leukocyte emigration with inhibition percentage of 90.6% at the dose of 848 mg/kg, which was comparable to that of dexamethasone (20 mg/kg) (Table 3).

3.3. Effect of the ethanol extract on xylene-induced ear swelling The results presented in Table 1 showed that the ethanol extract of Aquilaria sinensis Leaves (EtOH ext.) dosedependently suppressed xylene-induced ear swelling in mice with 51.0% of the inhibition rate at the dose of 848 mg/kg. Indomethacin (20 mg/kg) also exerted significant inhibitory effect.

3.6. Effect of the ethanol extract on NO release in LPS-stimulated macrophages As shown in Fig. 3, the normal, unactivated macrophages produced little nitrite (measured as index of NO) after 48 h of incubation at 37 ◦ C, then exposure to LPS induced a massive amount of NO in macrophages. Against this, the ethanol extract of Aquilaria sinensis Leaves (EtOH ext.) produced a concentration-dependent decrease at concentrations of 50, 100, and 200 ␮g/ml with IC50 of 80.4 ␮g/ml. On the other hand, the tested samples had shown no obvious effect on the viability of macrophages during 48 h period of incubation (data not shown). 4. Discussion

Fig. 3. Effect of the ethanol extract of Aquilaria sinensis (Lour.) Gilg. Leaves (EtOH ext.) on the levels of nitric oxide production by LPS-stimulated peritoneal macrophages in mice. Macrophages were stimulated with LPS (5 ␮g/ml) and incubated for 48 h with EtOH ext. (50, 100, and 200 ␮g/ml) or 0.1 ␮M hydrocortisone. The amount of nitrite in cell-free culture supernatants was determined by Griess reagent. Each value represents means ± S.E.M. of 9 wells from three independent experiments. ## P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the model group.

In the present study, the antinociceptive and antiinflammatory effects of the ethanol extract of Aquilaria sinensis Leaves were investigated in various related models in vivo and in vitro. The analgesic activities were evaluated by two mouse models, which could provide response to two different grades of noxious stimuli including thermal stimulus and chemically induced tissue damage (Victor et al., 2004). The hot-plate test was selected to investigate its central antinociceptive activity, because it had several advantages, particularly the sensitivity to strong analgesics and limited tissue damage. Acetic acid causes an increase in peritoneal fluids of prostaglandins such as PGE2

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and PGF2␣ , serotonin, and histamine involved in part, which was a model commonly used for screening peripheral analgesics (Deraedt et al., 1980). The results (Figs. 1 and 2) showed that the ethanol extract considerably inhibited acetic-acid-induced writhing in mice after oral administration for 1 h at the dose of 848 mg/kg, and also significantly inhibited the response to the thermal stimulus at 2 h after administration at the same dose. Such results suggested that the ethanol extract of Aquilaria sinensis Leaves had a remarked antinociceptive activity. Then, three animal models were chosen for the screening of its anti-inflammatory effects. Xylene-induced neurogenous swelling partially associated with substance P was first selected because it was a common inflammatory model for evaluating vascular permeability (Luber-Narod et al., 1997). The results in Table 1 showed that the ethanol extract of Aquilaria sinensis Leaves effectively inhibited xylene-induced ear swelling in a dose-dependent manner in mice, which suggested it might reduce the release of substance P or antagonize its action. Meanwhile, carrageenan-induced paw edema in mice has been accepted as a useful phlogistic tool for investigating anti-inflammatory agents, which has been demonstrated that bradykinin, substance P and prostaglandin are implicated and a diffuse cellular infiltrate with predominance of neutrophils are revealed in carrageenan mouse paw edema (Garcia-Pastor et al., 1999). It was suggested that there were biphasic effects in carrageenan-induced edema and the early hyperemia result from the release of histamine and serotonin, the delayed phase of carrageenan-induced edema result mainly from the potentiating effects of bradykinin on mediator release, and also of prostaglandins, producing edema after the mobilization of leukocytes (Castro et al., 1968; Kulkarni et al., 1986). According to Table 2, the ethanol extract of Aquilaria sinensis leaves showed obviously inhibitory activity on carrageenan-induced paw inflammation over a period of 5 h, which indicated its action against neutrophils migration and release of histamine, serotonin and kinins in early phase, and prostaglandin in later phase. Furthermore, considering the crucial role of the migration of leukocytes to inflammatory area in the progress of inflammation, CMC-Na-induced leukocytes emigration was also employed in this study. The data in Table 3 showed that the ethanol extract of Aquilaria sinensis Leaves significantly suppressed on leukocytes emigration, which was comparable to that of dexamethasone, and the inhibition was also obviously enhanced with the dose raised. Therefore, such results revealed that the ethanol extract of Aquilaria sinensis Leaves displayed anti-inflammatory activity significantly, via inhibiting vascular permeability and leukocyte transmigration, which might be connected with the reduction of release of inflammatory mediator, etc. On the other hand, various studies suggest macrophages constantly express induced-nitric oxide synthase (iNOS) producing a large amount of NO, which contributes to inflammation by increasing vascular permeability, causing edema formation, inducing synthesis of prostaglandins and due to its direct cytotoxicity (Moncada et al., 1991; Grisham et al., 1999). It has been reported that NO produced by iNOS is involved in the maintenance of the inflammatory response at later time

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points in carrageenan-induced paw edema, and also plays an important role in the peripheral and central pain (Kawabata et al., 1994; Salvemini et al., 1996). Finally LPS-stimulated NO release from macrophages model was used to evaluate the activity of the ethanol extract of Aquilaria sinensis Leaves. As shown in Fig. 3, the ethanol extract potently and concentrationdependently inhibited the elevation of NO level in macrophages with the inhibition percentage of 62.5% at the concentration of 200 ␮g/ml, which further proved the anti-inflammatory and analgesic activities of Aquilaria sinensis Leaves. Its molecular mechanism remained to be elucidated in the future studies. In addition, preliminary acute tests showed that medial lethal dose (LD50 ) of this extract could not be determined after intragastric administration, and its maximally tolerated dose was 20.4 g/kg (the mass of extract/body weight of mice), which showed its comparative safety. Meanwhile, as one kind of tree with green leaves all the seasons, Aquilaria sinensis (Lour.) Gilg. Leaves is very rich in resource. Therefore, the results of these studies not only provided partial experimental evidence for its therapeutic efficacy in the treatment of various conditions like trauma, but also would be beneficial to the future studies and exploitation of Aquilaria sinensis Leaves. 5. Conclusion To summarize, Aquilaria sinensis Leaves have been confirmed to exert potent analgesic and anti-inflammatory actions, which may provide some evidence for its folk use and further exploitation. On the other hand, it is also suggested that to identify the potent fractions or components is essential and remained to be clarified in the further studies. Acknowledgements We would like to thank Jianhong Zhang and Shanshan Han for their assistance in experiments and Dr. Zenglai Xu for his kind help on identification of the herb. References Adzu, B., Amos, S., Kapu, S.D., Gamaniel, K.S., 2003. Anti-inflammatory and anti-nociceptive effects of Sphaeranthus senegalensis. Journal of Ethnopharmacology 84, 169–173. Castro, J., Sasame, H., Sussman, H., Bullette, P., 1968. Diverse effects of SK 17 52 and antioxidants on CCl4 induced changes in liver microsomal P-450 content and ethylmorphine metabolism. Life Sciences 7, 129–136. Deraedt, R., Jougney, S., Delevalcee, F., Falhout, M., 1980. Release of prostaglandins E and F in an algogenic reaction and its inhibition. European Journal of Pharmacology 61, 17–24. Elisabetsky, E., Amador, T.A., Albuquerque, R.R., Nunes, D.S., 1995. Analgesic activity of Psychotria colorata (wild. ex R. & S.) Muell. Arg. alkaloids. Journal of Ethnopharmacology 48, 77–83. Garcia-Pastor, P., Randazzo, A., Gomez-Paloma, L., Alcaraz, M.J., Paya, M., 1999. Effects of petrosaspongiolide M, a novel phospholipase A2 inhibitor, on acute and chronic inflammation. Journal of Pharmacology and Experimental Therapeutics 289, 166–172. Green, L.C., Wanger, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate nitrite and [15 N] nitrate in biological fluids. Analytical Biochemistry 126, 131–138.

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