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Investigation of anti-inflammatory, antinociceptive and antipyretic activities of Stahlianthus involucratus rhizome ethanol extract Phornchai Pingsusaen, Puongtip Kunanusorn, Parirat Khonsung, Natthakarn Chiranthanut, Ampai Panthong, Chaiyong Rujjanawate

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S0378-8741(14)00770-3 http://dx.doi.org/10.1016/j.jep.2014.10.060 JEP9115

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Journal of Ethnopharmacology

Received date: 23 July 2014 Revised date: 24 September 2014 Accepted date: 27 October 2014 Cite this article as: Phornchai Pingsusaen, Puongtip Kunanusorn, Parirat Khonsung, Natthakarn Chiranthanut, Ampai Panthong, Chaiyong Rujjanawate, Investigation of anti-inflammatory, antinociceptive and antipyretic activities of Stahlianthus involucratus rhizome ethanol extract, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2014.10.060 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 Investigation of anti-inflammatory, antinociceptive and antipyretic activities of Stahlianthus involucratus rhizome ethanol extract Phornchai Pingsusaena, Puongtip Kunanusorna,*, Parirat Khonsunga, Natthakarn Chiranthanuta, Ampai Panthonga and Chaiyong Rujjanawateb a

Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b School of Medicine, Mae Fah Luang University, Chiang Rai 57100, Thailand *Corresponding author: Asst Prof Puongtip Kunanusorn, Ph.D. Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. Phone: +66 53 945353 Fax: +66 53 945355 Email: [email protected]

Abstract Ethnopharmacological relevance Stahlianthus involucratus (Zingiberaceae) has long been used in traditional medicine to treat inflammation, pain, and fever. However, no pharmacological study of this plant has been reported to confirm these activities. The aim of this study was to investigate the antiinflammatory, antinociceptive and antipyretic activities of S. involucratus rhizome ethanol extract (SiE) in animal models. Materials and methods Anti-inflammatory activity of SiE was investigated in rats using ethyl phenylpropiolate (EPP)-induced ear edema, carrageenan- and arachidonic acid (AA)-induced hind paw edema, and cotton pellet-induced granuloma formation models. Acetic acid-induced writhing response in mice and tail-flick test in rats as well as yeast-induced hyperthermia in rats were used to investigate the antinociceptive and antipyretic activities, respectively. Results SiE significantly inhibited EPP-induced ear edema, carrageenan- and AA-induced hind paw edema. Its inhibitory effect in carrageenan-induced hind paw edema seemed to be in a dosedependent manner. In cotton pellet-induced granuloma formation, SiE showed suppressive effects on granuloma formation but not on body weight gain and dry thymus weight. It could normalize serum alkaline phosphatase activity to nearly normal level. SiE also possessed a significant inhibitory effect, which seemed to be dose-dependent, on acetic acid-induced writhing response, whereas only at the highest dose of SiE could significantly increase test reaction time at all time-points in tail-flick test. However, no antipyretic activity was observed.

2 Conclusions These results suggest that SiE possesses anti-inflammatory and antinociceptive, but not antipyretic, activities. This study therefore rationalizes the traditional use of SiE for the treatment of inflammation and pain. Keywords Stahlianthus involucratus; rhizome; anti-inflammatory; antinociceptive; antipyretic Chemical compounds studied in this article Acetone (PubChem CID: 180); Dimethylsulfoxide (PubChem CID: 679); Diclofenac sodium (PubChem CID: 5018304); Arachidonic acid (PubChem CID: 444899); Ethyl phenylpropiolate (PubChem CID: 91516); Codeine phosphate (PubChem CID: 12303736); Acetic acid (PubChem CID: 176); Prednisolone (PubChem CID: 5755); Lambda carrageenan Abbreviations SiE, Stahlianthus involucratus rhizome ethanol extract; EPP, ethyl phenylpropiolate; AA, arachidonic acid; PG, prostaglandin; LT, leukotriene; gas chromatography/mass spectrometry, GC/MS; DMSO, dimethylsulfoxide; NSS, normal saline solution; AP, alkaline phosphatase; MPE, maximum possible effect; COX, cyclooxygenase; NSAIDs, non-steroidal anti-inflammatory drugs 1. Introduction Stahlianthus involucratus (King) Craib ex Loes, a perennial herbaceous plant of the Zingiberaceae family, is widely distributed in the forest and on the mountains of many countries in Asia, including Thailand (Chaveerach et al., 2007). Its local names are “Wan Phet Yai” in Thai, “Tu Tian Qi” in Chinese and “Easkine” in Bangladesh. In Bangladesh folk medicine, the plant has been claimed to treat inflammatory disorders and fever (Yusuf et al., 2007). Traditional healers in Thailand also topically apply the crushed rhizome to relieve muscle pain and edema. Several pharmacological activities of plants in Zingiberaceae family, including antiinflammatory, analgesic, and antipyretic activities have been shown in many scientific reports (Azam et al., 2014; Thenmozhi et al., 2013; Somchit et al., 2005). The most recognized antiinflammatory effect of rhizomes of several Zingiberaceae species has consistently reported and confirmed. The hexane extract as well as the active constituents of Curcuma xanthorrhiza Roxb. have been shown to inhibit carrageenan-induced paw edema in rats (Claeson et al., 1993). The rhizomes of Zingiber officinale and Alpinia officinarum contain potent inhibitors against prostaglandin (PG) synthetase, an enzyme of PG biosynthesis and arachidonate 5-lipoxygenase, an enzyme of leukotriene (LT) biosynthesis (Kiuchi et al., 1992). The hydroalcoholic extract of Z. officinale rhizomes also reduces carrageenan-induced paw swelling in rats (Penna et al., 2003). Mukophadhyay et al. (1982) demonstrated the activity of curcumin, an active constituent of C. longa L. against carrageenan-induced rat paw edema and cotton pellet granuloma models of inflammation in rats. The biologically active compounds of Zingiberaceae plants that exert anti-inflammatory activity include, for example, gingerol and shogaol of Z. officinale Roscoe extract (Dugasani et al., 2010); diarylheptanoid of A. officinarum extract (Yasukawa et al., 2008; Yadav et al., 2003), and curcuminoids especially curcumin of C. longa L. extract (Jobin et al., 1999; Huang et al., 1991).

3 Despite those traditional claims of S. involucratus, the scientific evidence for its pharmacological as well as phytochemical properties have not been reported yet. The present study aimed to investigate the anti-inflammatory, antinociceptive and antipyretic activities of the ethanol extract from S. involucratus rhizomes in animal models. 2. Materials and methods 2.1. Plant material and extraction The rhizomes of S. involucratus were collected in April 2009 from Chiang Rai Province, Thailand. The plant was authenticated by one of the authors (Dr. Rujjanawate) and the voucher specimen (no. 144) has been deposited at the School of Medicine, Mae Fah Luang University, Chiang Rai, Thailand. The air dried (at room temperature) rhizome powder were macerated with 95% ethanol for two days and filtered. The marc was remacerated two times in the same manner as the initial maceration and filtered. The combined filtrate was concentrated in vacuo at 55 °C and lyophilized to obtain a dry ethanol extract (SiE, 6% w/w yield). 2.2. Gas chromatography/mass spectrometry (GC/MS) analysis GC/MS analysis of SiE was carried out on a gas chromatograph (GC 7890 Agilent Technologies) fitted with a DB-5MS column (30 m x 0.25 mm i.d., 0.25 ȝm film thickness). The GC oven temperature was programmed at 50 oC, held for 5 min, raised to 200 oC at 10 o C/min, then to 250 oC at 5 oC/min and held for 10 min. The injection temperature was 250 o C; the flow rate of carrier gas, helium, was at 1.5 mL/min; 1:25 split ratio. The gas chromatograph was coupled to a mass selective detector (Agilent HP 5973). The MS operating parameters were as follows: ionization voltage, 70 eV; ion source temperature, 230 o C. Identification of the extract components was performed by comparison of their relative retention times and mass spectra with those in the NIST05a.L Database (Agilent Technologies Inc.). 2.3. Experimental animals Male Sprague-Dawley rats and male Swiss albino mice were purchased from the National Laboratory Animal Center, Mahidol University, Nakorn Pathom, Thailand. All animals were acclimatized at least 1 week in a room maintained under environmentally controlled conditions of 23 ± 2 oC and a 12 h light-dark cycle before starting the experiments. They had free access to water and standard diet (Pokphan Animal Feed Co. Ltd., Bangkok, Thailand), but only the diet was withdrawn 12 h before dosing. All experimental procedures were approved by the Animal Ethics Committee of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (Protocol Number 8/2553). 2.4. Drugs and chemicals Diclofenac sodium, lambda carrageenan and arachidonic acid (AA) were purchased from Sigma Chemical Company (St. Louis, U.S.A.). Ethyl phenylpropiolate (EPP) and Brewer’s yeast were purchased from Fluka Chemicals Co. Ltd. (Japan). Codeine phosphate and acetic acid were purchased from the Government Pharmaceutical Organization (Thailand). Prednisolone was purchased from Schering, Bangkok Ltd. (Thailand). All other chemicals were of analytical grade.

4

2.5. Test substance administration SiE and reference drugs were orally administered in an equivalent volume of 0.2 mL/100 g body weight of rats and 0.1 mL/10 g body weight of mice except those rats in the EPPinduced ear edema model of which the test substances were applied topically (20 µL/ear). Tween 80 (5%) was used as vehicle for SiE in all experimental models except in ear edema model in which acetone and 5% dimethylsulfoxide (DMSO) in acetone were used as vehicles for the extract and diclofenac, respectively. In the control group, animals received only the vehicle in the same volume and the same route. 2.6. Anti-inflammatory activity 2.6.1. EPP-induced ear edema The method described by Brattsand et al. (1982) was used with slight modification, to assess topical anti-inflammatory activity of SiE. Male rats of 40–60 g body weight were randomly divided into four groups of three rats each. EPP at the dose of 1 mg/ear was dissolved in acetone and topically applied to the inner and outer surfaces of both ears. The vehicle, SiE (5 mg/ear) or diclofenac (5 mg/ear) was applied to the ear just before EPP application. The thickness of each ear was measured with the digital vernier calipers before and at 15, 30, 60 and 120 min after edema induction. The increase in ear thickness of each test group was compared with its corresponding control group and percent inhibition was calculated. 2.6.2. Carrageenan-induced hind paw edema This method was performed as described by Winter et al. (1962). Male rats of 100–120 g body weight were randomly divided into five groups of six rats each. Carrageenan [(0.05 mL, 1%, w/v in normal saline solution (NSS)] was injected intradermally into the plantar side of the right hind paw 1 h after the administration of the vehicle, diclofenac (10 mg/kg) or SiE (75, 150 and 300 mg/kg). Paw volumes were measured using a plethysmometer (model 7150, Ugo Basile, Italy) before and at 1, 3, and 5 h after carrageenan injection. 2.6.3. AA-induced hind paw edema Male rats of 100–120 g body weight were randomly divided into six groups of six rats each. AA [0.1 mL, 0.5%, w/v in 0.2 M carbonate buffer (pH 8.4)] was injected intradermally into the plantar side of the right hind paw 2 h after the administration of the vehicle, diclofenac (10 mg/kg), prednisolone (5 mg/kg), or SiE (75, 150 and 300 mg/kg). Paw volumes were measured using a plethysmometer (model 7150, Ugo Basile, Italy) before and at 1 h after AA injection (Di Martino et al., 1987). 2.6.4. Cotton pellet-induced granuloma formation The method was slightly modified from that of Swingle and Shideman (1972). Male rats weighing 180-200 g were randomly divided into five groups of six rats each. Two sterilized cotton pellets (20 ± 1 mg) were implanted subcutaneously, one on each side of the abdomen in all groups except in the normal group, under light ether anesthesia. Rats in group I (normal group) and II (control group) received 5% Tween 80. Rats in group III and IV received diclofenac and prednisolone, respectively, at the same dose of 5 mg/kg/day. Rats in group V

5 received SiE at the dose of 300 mg/kg/day. Each test substance was administered in 3 divided doses for 7 days. On the eighth day, each rat was anesthetized and the heart blood was collected for determination of alkaline phosphatase (AP) activity and total protein. The rat was then sacrificed and the implanted pellets as well as the thymus were dissected out and determined for their wet and dry weights (dried at 60 °C for 18 h). The granuloma and transudative weights and the percent inhibition of granuloma formulation of the test compounds were calculated. The body weight gain was also recorded. 2.7. Antinociceptive activity 2.7.1. Acetic acid-induced writhing response The method described by Collier et al. (1968) and modified by Nakamura et al. (1986) was used. Male mice weighing 30-40 g were randomly divided into five groups of six mice each. Acetic acid (0.1 mL/10 g body weight, 0.75%, v/v in NSS) was injected intraperitoneally 1 h after the administration of either 5% Tween 80, diclofenac (10 mg/kg) or SiE (18.75, 37.5, and 75 mg/kg). The number of writhes was counted during continuous observation for 15 min, beginning at 5 min after acetic acid injection. 2.7.2. Tail-flick test The method of D’Amour and Smith (1941) modified by Gray et al. (1970) was used. Male rats weighing 160-180 g were randomly divided into six groups of six rats each. The tail-flick response was measured by placing the rat tail on a photocell window of the tail-flick apparatus (model 7360, Ugo Basile, Italy) which generated heat through a light beam. The time taken for each rat to flick its tail from heat was recorded as the reaction time. The cut-off time of 10 s was a maximum time allowed for an unflicked tail to expose to heat to avoid tissue damage. The reaction time was determined before and at 1, 2, and 3 h after the administration of all test substances in the same manner as those groups in acetic acidinduced writhing response with the additional group of codeine (200 mg/kg). The percentage of maximum possible effect (MPE) was calculated by using the following formula: (Test reaction time) – (Baseline reaction time) % MPE =

× 100 (Cut-off time) – (Baseline reaction time)

2.8. Antipyretic activity Yeast-induced hyperthermia model was performed in male rats (weighing 180-200 g, five groups of six rats each) following the method of Teotino et al. (1963). The baseline rectal temperatures were recorded using a twelve-channel electric thermometer (LETICA, model TMP 812 RS, Panlab S.L., Spain). Hyperthermia was induced by subcutaneous injection of yeast (1 mL/100 g body weight, 25%, w/v in NSS). The rectal temperatures were recorded again 18 h later. Those rats that had more than 1°C rise in the temperature were orally administered with 5% Tween 80, diclofenac (10 mg/kg) or SiE (75, 150, and 300 mg/kg). The rectal temperatures were then recorded at 30, 60, 90, 120, and 180 min following the treatment.

6 2.9. Statistical analysis Data are expressed as mean ± standard error of the mean (S.E.M.). Statistical comparison between groups were analyzed using one-way analysis of variance (ANOVA) followed by post hoc least-significant difference (LSD) test, whereas the comparison between each timepoint in the same group was analyzed using paired samples t test. p < 0.05 was considered significant. 3. Results 3.1. GC/MS analysis of the plant extract The GC/MS chromatogram of SiE is shown in Fig. 1. The analysis of the chromatogram indicated the presence of 19 compounds in which 17 of them could be identified (Table 1). The three most abundant constituents were benzyl benzoate (22.71%); ethane, isothiocyanate (17.28%); and 2-phenanthrenol,7-ethenyl-1,2,3,4,4a,4b,5,6,7,9,10,l0a-dodecahydro-1,1,4a,7tetramethyl-,[2S (2.alpha.,4a.alpha.,4b.beta.,7.beta.,l0a.beta.)] (11.18%). The other 16 compounds collectively constituted 48.84% of the extract. 3.2 EPP-induced ear edema As shown in Table 2, application of EPP topically on rat ears produced ear swelling within 15 min. The maximum effect occurred at 1 h and gradually decreased thereafter. SiE and diclofenac at the dose of 5 mg/ear significantly (p < 0.001) inhibited ear edema formation with comparable percentages of edema inhibition at all evaluation time points (64, 63, 64 and 65% vs 74, 67, 63 and 62%, respectively). 3.3 Carrageenan-induced hind paw edema The inhibitory activity of oral administration of SiE on carrageenan-induced hind paw edema in rats is demonstrated in Table 3. The injection of carrageenan resulted in hind paw edema within 1 h and the maximum effect was reached at 3 h. SiE (75, 150 and 300 mg/kg) as well as diclofenac (10 mg/kg) significantly inhibited hind paw edema formation at all evaluation time points, with maximum inhibition at 3 h after carrageenan injection. This inhibitory effect of SiE seemed to occur in a dose-related manner. 3.4 AA-induced hind paw edema The inhibitory activity of oral administration of SiE on AA-induced hind paw edema in rats is demonstrated in Table 4. SiE at doses of 150 and 300 mg/kg as well as diclofenac (10 mg/kg) and prednisolone (5 mg/kg) significantly inhibited hind paw edema formation by 39%, 58%, 35% and 58% respectively. 3.5 Cotton pellet-induced granuloma formation Diclofenac and prednisolone at a dose of 5 mg/kg/d, and SiE at the dose of 300 mg/kg/d significantly reduced transudative and granuloma weights as shown by their granuloma inhibition of 50%, 72% and 24% respectively (Table 5). However, the effects of SiE were less pronounced when compared to those of diclofenac and prednisolone. It was also found that the body weight gain and dry thymus weight were not significantly different among

7 groups (normal, control, diclofenac, and SiE groups), except in the prednisolone group which revealed a significant decrease from those of both normal and control groups.

(2)

(3)

(4) (7)

(8)

(11) (10)

(6) [(1)-(19)]. Fig. 1. GC-MS chromatogram of SiE showing the presence of 19 compounds

(1)

(5)

(13) (14)

(9) (12)

(15)

(16) (17)

(18) (19)

8

Table 1 Chemical components of SiE as analyzed by GC-MS. Peak Retention time % of Total Compound (min) 1 13.702 6.95 Benzenecarboxylic acid 2 15.562 1.64 Resorcinol 3 19.980 17.28 Ethane, isothiocyanate 4 20.038 9.11 Heptanoic acid 5 22.082 22.71 Benzyl benzoate 6 22.787 0.71 l-Methoxy-3-(2-hydroxyethyl) nonane 7 24.404 2.42 n-Hexadecanoic acid 8 24.935 6.36 Hexadecanoic acid, ethyl ester 9 26.656 4.39 Phytol 10 26.922 1.71 9,12-Octadecadienoic acid (Z,Z) 11 27.014 2.02 cis,cis,cis-7,10,13-Hexadecatriena 12 27.418 3.73 Linoleic acid ethyl ester 13 27.505 2.96 7,10,13-Hexadecatrienoic acid, methyl ester 14 27.898 0.68 D-Gluconic acid, 2,3,4,5-tetra-O-methyl-6-O-(2,3,4,6-tetra-O-methyl-.alpha.-D-glucopyranosyl)-, methyl ester 15 28.729 11.18 2-Phenanthrenol, 7-ethenyl-1,2,3,4,4a,4b,5,6,7,9,10,l0a-dodecahydro-1,1,4a,7-tetramethyl-, [2S(2.alpha.,4a.alpha.,4b.beta.,7.beta.,l0a.beta.)] 16 29.884 2.52 2-Isopropylidenehydrazono-3-methyl-6-chloro-2,3-dihydrobenzothiazole 17 31.554 0.87 No matches found 18 33.621 2.03 1,2-Benzenedicarboxylic acid, di-isooctyl ester 19 34.620 0.74 No matches found

9

5 5

Diclofenac

Control of SiE

SiE

27 ± 3*

75 ± 11

18 ± 3*

70 ± 10

15 min

45 ± 2*

123 ± 7

38 ± 5*

115 ± 7

30 min

52 ± 3*

143 ± 6

50 ± 3*

137 ± 4

1h

10

75

Diclofenac

SiE 0.10 ± 0.02

*

0.15 ± 0.01

*

0.19 ± 0.02

*

0.07 ± 0.01

*

0.27 ± 0.05

63

44

30

74

-

0.17 ± 0.02

*

0.38 ± 0.02

*

0.42 ± 0.02

*

0.15 ± 0.01

*

Edema volume (mL) 0.75 ± 0.05

Edema volume (mL) %Inhibition

Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the control group, p < 0.05.

300

150

-

Control

3h

1h

Table 3 Effects of SiE and diclofenac on carrageenan-induced hind paw edema in rats. Group Dose (mg/kg) Time after carrageenan injection

Values are expressed as mean ± S.E.M. (n = 6). Control of diclofenac: treated with 5% DMSO in acetone. Control of the extract: treated with acetone. * Significantly different from its control group, p < 0.05.

-

Control of diclofenac

(mg/ear)

Table 2 Effects of SiE and diclofenac on EPP-induced ear edema in rats. Group Dose Edema thickness (µm)

77

49

44

81

-

%Inhibition

43 ± 2*

122 ± 7

42 ± 5*

110 ± 11

2h

64

-

74

-

63

-

67

-

30 min

0.30 ± 0.05

*

0.57 ± 0.02

*

0.58 ± 0.02

*

0.24 ± 0.03

*

Edema volume (mL) 0.74 ± 0.08

5h

15 min

Edema inhibition (%)

65

-

62

-

2h

60

23

22

68

-

%Inhibition

64

-

63

-

1h

10

Dose (mg/kg) 0.26 ± 0.01 0.17 ± 0.03* 0.11 ± 0.04* 0.25 ± 0.04 0.16 ± 0.02* 0.11 ± 0.02*

Edema volume at 1 h (mL) 35 58 4 39 58

% Inhibition

Dose (mg/kg/d) TrW (mg)

GrW (mg/mg cotton)

% GI

BW gain (g)

Dry TW (mg/100 g BW)

AP activity (U/mg of TP × 10-4)

Normal 57.33 ± 2.71 57.46 ± 0.71 33.77 ± 1.75 Control 476.42 ± 48.66 3.47 ± 0.33 59.67 ± 3.28 61.20 ± 0.71 50.02 ± 1.33# * * Diclofenac 5 255.88 ± 13.26 1.73 ± 0.09 50 55.67 ± 3.20 61.36 ± 2.62 35.62 ± 2.71* Prednisolone 5 186.54 ± 10.97* 0.96 ± 0.07* 72 6.00 ± 5.75#, * 23.60 ± 0.92#, * 31.89 ± 2.82* * * SiE 300 343.41 ± 11.85 2.63 ± 0.14 24 62.67 ± 2.29 59.08 ± 2.99 39.96 ± 2.14* Values are expressed as mean ± S.E.M. (n = 6). TrW: Transudative weight, GrW: Granuloma weight, GI: Granuloma inhibition, BW: Body weight, TW: Thymus weight, AP: Alkaline phosphatase, U: Unit of enzyme, TP: Total protein # Significantly different from the normal group, p < 0.05. * Significantly different from the control group, p < 0.05.

Group

Table 5 Effects of SiE, diclofenac, and prednisolone on transudative weight, granuloma weight, and percentage of granuloma inhibition on cotton pelletinduced granuloma formation in rats.

10 5 75 150 300 Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the control group, p < 0.05.

Control Diclofenac Prednisolone SiE

Group

Table 4 Effects of SiE, diclofenac, and prednisolone on AA-induced hind paw edema in rats.

11

13 The serum AP activity was significantly elevated in the control group (50.02 ± 3.26 U of enzyme/mg of total protein × 10-4) when compared with the normal group (33.77 ± 4.28 U of enzyme/mg of total protein × 10-4). However, all test drugs could normalize the serum AP activity to nearly normal level. 3.6 Acetic acid-induced writhing response Diclofenac at the dose of 10 mg/kg and SiE at all doses (18.75, 37.5, and 75 mg/kg) possessed inhibitory effect on writhing response as shown in Table 6. The writhing response inhibition of the extract was dose-dependent and at the dose of 75 mg/kg was comparable to that of diclofenac (76% and 81%, respectively). 3.7 Tail-flick test Although both SiE (75 mg/kg) and codeine (200 mg/kg) could significantly increase reaction time in tail-flick test at all time-points but the percentage MPE of SiE was far less than those of codeine (Table 7). 3.8 Antipyretic activity At 18 h after yeast injection, the hyperthermia was generated and maintained at all timepoints in the control group. Diclofenac at the dose of 10 mg/kg significantly reduced hyperthermia at all time-points, whereas SiE at all doses (75, 150, 300 mg/kg) failed to reduce hyperthermia (Table 8).

Table 6 Effects of SiE and diclofenac on acetic acid-induced writhing response in mice. Group Dose (mg/kg) Number of writhes % Inhibition Control Diclofenac SiE

21 ± 1 10 4 ± 1* 18.75 17 ± 0* 37.5 11 ± 0* 75 5 ± 1* Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the control group, p < 0.05.

81 19 48 76

18 h after yeast injection

2.50 ± 0.11 3.63 ± 0.13 8.77 ± 0.60* 2.73 ± 0.18 2.05 ± 0.02 3.25 ± 0.08*

3h 2 76 2 13 25

1h

120 39.0 ± 0.1 36.9 ± 0.1* 39.1 ± 0.1 39.1 ± 0.1 39.2 ± 0.1

1 96 1 2 10

2h

180 39.2 ± 0.2 37.4 ± 0.1* 39.2 ± 0.1 38.9 ± 0.1 39.2 ± 0.1

0 82 0 0 6

3h

%Maximum possible effect

Time after drug administration (min) 30 60 90 39.2 ± 0. 39.2 ± 0.1 39.1 ± 0.1 38.2 ± 0.1* 37.6 ± 0.1* 37.2 ± 0.0* 39.3 ± 0.1 39.1 ± 0.2 39.1 ± 0.1 39.4 ± 0.1 39.2 ± 0.1 39.1 ± 0.1 39.3 ± 0.2 39.2 ± 0.1 39.2 ± 0.1

2.40 ± 0.07 3.68 ± 0.15 9.70 ± 0.30* 2.78 ± 0.15 2.18 ± 0.03* 3.53 ± 0.08*

2h

37.5 ± 0.1 39.0 ± 0.1 10 37.4 ± 0.1 38.9 ± 0.1 75 37.6 ± 0.1 39.2 ± 0.1 150 37.6 ± 0.0 39.1 ± 0.1 300 37.6 ± 0.1 39.2 ± 0.2 Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from rectal temperature at 18 h after yeast injection, p < 0.05.

Control Diclofenac SiE

Baseline

Table 8 Effects of SiE and diclofenac on yeast-induced hyperthermia in rats. Group Dose (mg/kg) Rectal temperature (°C)

2.25 ± 0.11 2.50 ± 0.09 10 3.63 ± 0.13 3.77 ± 0.17 200 3.12 ± 0.08 8.33 ± 0.56* 18.75 2.70 ± 0.11 2.82 ± 0.17 37.5 2.02 ± 0.02 3.05 ± 0.04* 75 2.82 ± 0.15 4.62 ± 0.09* Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the baseline reaction time, p < 0.05.

Control Diclofenac Codeine SiE

1h

Table 7 Effects of SiE, diclofenac and codeine on tail-flick test in rats. Group Dose (mg/kg) Baseline reaction time (s) Test reaction time (s)

14

15 4. Discussion By means of various animal models, this study could demonstrate, for the first time, the antiinflammatory and analgesic but not the antipyretic activities of S. involucratus. The EPP-induced ear edema model has been widely used for screening and evaluating the anti-inflammatory activity of test compounds (Brattsand et al., 1982). EPP causes acute inflammatory response by inducing pro-inflammatory mediator releases, e.g., histamine, serotonin, kinins, and PGs (Carlson et al., 1985) which in turn cause vascular changes, including vasodilatation, and increasing in vascular permeability leading to ear edema formation (Murphy and Ward, 2005). The result of the present study revealed the antiinflammatory effect of SiE in this model. Thus, the possible mechanism of action of SiE may be elicited via the inhibition of the release of these pro-inflammatory mediators. In carrageenan-induced rat hind paw edema model, the injection of carrageenan into the plantar surface of hind paw causes acute inflammatory response leading to biphasic phase of paw edema. The first phase (0-2.5 h after carrageenan injection) results from the concomitant releases of histamine, serotonin, and kinins, whereas the second phase (2.5-6 h) is correlated with the elevation of inducible cyclooxygenase (COX)-2 activity, production of PGs, and oxygen-derived free radicals, as well as the local neutrophil infiltration and activation (Di Rosa, 1972). The anti-inflammatory action of SiE demonstrated in this model may reasonably be derived from inhibition of one or more of pro-inflammatory mediator cascades and PGs biosynthesis. It has been shown that lipoxygenase-inhibitors and steroids, but not COX-inhibitors, are effective in AA-induced hind paw edema (Di Martino et al., 1987). Therefore this model is used to detect the mechanism other than the COX inhibition. In this study, SiE’s inhibition of the paw edema induced by AA was modestly significant. Diclofenac, a non-steroidal antiinflammatory drug (NSAID) with established COX-2 but not lipoxygenase (LOX) inhibition also produced a comparable inhibition of AA-induced paw edema to moderate doses of SiE. Thus, the anti-edema effect of SiE and diclofenac in this model was, at best, minimally mediated through LOX inhibition. In the cotton pellet-induced granuloma formation, the responses can be divided into three phases. The first, transudative phase, 0-3 h after cotton pellet implantation, is defined as the leakage of fluid from blood vessels caused by increasing in vascular permeability. The second, exudative phase, 3-72 h after cotton pellet implantation, is defined as the leakage of protein from bloodstream around granuloma caused by the intensive maintenance in vascular permeability change. The final, proliferative phase, 3-6 d, is defined as the production of granulomatous tissues caused by continuous pro-inflammatory mediator release (Swingle and Shideman, 1972; Sarraf and Sneller, 2005). In these chronic inflammatory responses, there is persistence of inflammatory cells leading to the release of pro-inflammatory mediators and oxygen-derived free radicals, as well as lysosomal enzymes such as AP, which cause subsequent tissue injury. The AP level is increased and can be detected in serum of animals (Murphy and Ward, 2005; Bessey et al., 1946; Sarraf and Sneller, 2005). Swingle and Shideman (1972) reported that steroids can markedly inhibit both transudative and proliferative phases, whereas NSAIDs can modestly exhibit these effects. Steroids and some NSAIDs, such as indomethacin, aspirin, including diclofenac, are known to possess lysosomal membrane stabilization property (Furst and Robert, 2007). Moreover, the long term treatment of steroids can reduce thymus weight and body weight gain. These effects may be due to peripheral catabolism of lymphoid and connective tissues, and fat (Schimmer

16 and Parker, 2006). The present results revealed that although SiE could significantly reduce transudative weight, inhibit granuloma formation, and normalize serum AP activity to nearly normal level but its effects on transudative weight and granuloma formation were less pronounced when compared with those of diclofenac and prednisolone. In addition, SiE did not influence thymus weight and body weight gain. Thus, it seems unlikely that SiE possesses steroidal-like effect. All these results indicated that SiE could inhibit chronic inflammation, and its possible mechanisms of action may be partially similar to those of diclofenac, albeit less effective as mentioned above. The acetic acid-induced writhing response is used for screening of the analgesic activity regardless of the central or peripheral causes. Acetic acid is an irritant which causes the synthesis and release of pro-inflammatory mediators, e.g., bradykinin, serotonin, histamine, PGs, and substance P that provoke pain nerve endings (Nakamura et al., 1986; Raj, 1996). In this model, SiE exhibited analgesic effect in a dose dependent manner, and at the highest dose tested, produced comparable analgesic efficacy as 10 mg/kg diclofenac. The analgesic activity of SiE and diclofenac may be mediated through their anti-inflammatory effects seen in EPP-induced ear edema and caragenan-induced paw edema above. In the tail-flick test, which uses a thermal stimulus, an increase in reaction time is generally considered an important parameter of central analgesic activity (Chang and Lewis, 1989). The flick of tail is explained by reflex arc in spinal cord which is modulated through descending pathway mechanism (Nakamura et al., 1986). The opioid drugs such as morphine can almost completely inhibit this reflex (D’Amour and Smith, 1941; Gray et al., 1970). It was found that SiE and diclofenac exerted minimal and non-significant analgesic effect in this test when compared with codeine. Collectively, the analgesic effect of both SiE and diclofenac is therefore mainly effective against pain that derived from peripheral inflammation. Hyperthermia induced by yeast is believed to be caused by activation of endogenous pyrogen and subsequent secretion of pro-inflammatory mediators (Au et al., 1994; Kevin and Gordon, 2007) which eventually act on the hypothalamus and stimulate PGE2 synthesis in the preoptic area of anterior hypothalamus thermoregulatory centers (Murphy and Ward, 2005; Janeway and Travers, 1999). The antipyretic effect of some NSAIDs, including diclofenac results from the inhibition of PGs synthesis within hypothalamus (Ganong, 2001). In this study, diclofenac could significantly reduce body temperature, whereas SiE could not exert this effect. This finding together with the results from the tail-flick test led to an assumption that SiE might not be able to cross the blood brain barrier to exert central antipyretic and analgesic effects, respectively. Since gastrointestinal side effects of the available anti-inflammatory drugs in the market are their disadvantages, new drugs without these problems will be welcome as attractive alternatives for the patients and physicians alike. In order to find out whether SiE could produce gastric ulcer or not, at the end of cotton pellet-induced granuloma formation study, the stomach of each rat that received SiE at 300 mg/kg/d for 7 days, the highest dose tested, was examined. The gross pathological examination showed no sign of gastric ulcer in all of these animals. This preliminary result demonstrates, in part, the potential safety of SiE on gastrointestinal system which is in line with its safe, continuing uses in traditional medicine.

17 5. Conclusions The present study demonstrates the anti-inflammatory and antinociceptive, but not antipyretic activities of SiE in rats. The inhibition of peripheral pro-inflammatory mediator production and/or activity may be the main possible mechanism of action of SiE. These findings lend support to the traditional use of SiE for the treatment of inflammation and pain. Further and more extensive toxicity studies of SiE should be performed to confirm its safety issues. Acknowledgement This work was supported by the Faculty of Medicine Research Fund, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (Grant Number 59/2553).

References Au, B., Williams, T., Collins, P., 1994. Zymosan-induced IL-8 release from human neutrophils involves activation via the CD11b/CD18 receptor and endogenous platelet- activating factor as an autocrine modulator. Journal of Immunology 152, 5411-5419. Azam, M.G., Noman, M.S., Al-Amin, M.M., 2014. Phytochemical screening and antipyretic effect of Curcuma zedoaria Rosc. (Zingiberaceae) rhizome. British Journal of Pharmaceutical Research 4, 569-575. Bessey, O., Lowry, H., Brock, M., 1946. Method for the determination of alkaline phosphatase with five cubic millimeters of serum. Journal of Biological Chemistry 164, 321-329. Brattsand, R., Thalen, A., Roemple, K., Kallstrom, L., Gruvstad, E., 1982. Influence of 16 Į, 17 Į-acetal substitution and steroid nucleus fluorination on the topical to systemic activity ratio of glucocorticoids. Journal of Steroid Biochemistry 16, 779-786. Carlson, R., O'Neill-Davis, L., Chang, J., Lewis, A., 1985. Modulation of mouse ear edema by cyclooxygenase and lipoxygenase inhibitors and other pharmacologic agents. Agents and Actions 17(2), 197-204. Chaveerach, A., Mokkamul, P., Sudmoon, R., Tanee, T., Garcia, V.F., 2007. A new species of Stahlianthus (Zingiberaceae) from Northeastern Thailand. Taiwania 52, 315-319. Chang, J.Y., Lewis, A.J., 1989. Pharmacological methods in the control of inflammation (Modern methods in Pharmacology), vol. 5, Wiley-Liss, New York, pp. 195–212. Claeson., P., Panthong, A., Tuchinda, P., Reutrakul, V., Kanjanapothi, D., Taylor, W.C., Santisuk, T., 1993. Three non-phenolic diarylheptanoids with anti-inflammatory activity from Curcuma xanthorrhiza. Planta Medica 59, 451-454. Collier, H., Dinneen, L., Johnson, C., Schneider, C., 1968. The abdominal constriction response and its suppression by analgesic drugs in the mouse. British Journal of Pharmacology and Chemotherapy 32, 295-310. D’Amour, F., Smith, D., 1941. A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics 72, 74-79. Di Martino, M., Campbell, G., Wolff, C., Hanna, N., 1987. The pharmacology of arachidonic acid-induced rat paw edema. Agents and Actions 21(3-4), 303-305. Di Rosa, M., 1972. Biological properties of carrageenan. Journal of Pharmacy and Pharmacology 24(2), 89-102. Dugasani, S., Pichika, M., Nadarajah, V., Balijepalli, M., Tandra, S., Korlakunta, J., 2010. Comparative antioxidant and anti-inflammatory effects of [6]-gingerol, [8]-gingerol, [10]-gingerol and [6]-shogaol. Journal of Ethnopharmacology 127, 515-520.

18 Furst, D., Robert, W., 2007. Nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, nonopioid analgesic, and drug used in gout, in: Katzung, B. (Ed.), Basic and clinical pharmacology. 10th ed. McGraw-Hill Companies, New York, pp. 573-598. Ganong, W., 2001. Functions of the nervous system: central regulation of visceral function, in: Ganong, W. (Ed.), Review of medical physiology. 20th ed. McGraw-Hill, New York, pp. 245-247. Gray, W., Osterberg, A., Scute, T., 1970. Measurement of the analgesic efficacy and potency of pentazocine by the D’Amour and Smith method. Journal of Pharmacology and Experimental Therapeutics 172, 154-162. Huang, M.T., Lysz, T., Ferraro, T., Abidi, T.F., Laskin, J.D., Conney, A.H., 1991. Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis. Cancer Research 51, 813-819. Janeway, C., Travers, P., 1999. Host defense against infection, in: Walport, M., Capra, J. (Eds.), Immunobiology: the immune system I health and disease. 4th ed. Garland Publishing, New York, pp. 363-415. Jobin, C., Bradham, C.A., Russo, M.P., Juma, B., Narula, A.S., Brenner, D.A., Sartor, R.B., 1999. Curcumin blocks cytokine-mediated NF-kappa B activation and proinflammatory gene expression by inhibiting inhibitory factor I-kappa B kinase activity. The Journal of Immunology 163, 3474-3483. Kevin, M., Gordon, D., 2007. The role of the beta-glucan recrptor Dectin-1 in control of fungal infection. Journal of Leukocyte Biology 82, 253-258. Kiuchi, F., Iwakami, S., Shibuya, M., Hanaoka, F., Sankawa, U., 1992. Inhibition of prostaglandin and leukotriene biosynthesis by gingerols and diarylheptanoids. Chemical & Pharmaceutical Bulletin (Tokyo) 40, 387-391. Mukophadhyay, A., Basu, N., Ghatak, N., Gujral, P.K., 1982. Anti-inflammatory and irritant activities of curcumin analogues in rats. Agents and Actions 12, 508-515. Murphy, H., Ward, P., 2005. Inflammation, in: Rubin, E. (Ed.), Rubin’s pathology: clinicopathologic foundations of medicine. 4th ed. Lippincott Williams & Wilkins, Philadelphia, pp. 40-83. Nakamura, H., Shimoda, A., Ishii, K., Kadokawa, T., 1986. Central and peripheral analgesic action of non-acidic non-steroidal anti-inflammatory drugs in mice and rats. Archives internationales de pharmacodynamie et de thérapie 282,16-25. Penna, S.C., Medeiros, M.V., Aimbire, F.S., Faria-Neto, H.C., Sertie, J.A., Lopes-Martins, R.A., 2003. Anti-inflammatory effect of the hydroalcoholic extract of Zingiber officinale rhizomes on rat paw and skin edema. Phytomedicine 10, 381-385. Raj, P., 1996. Pain mechanisms, in: Raj, P., (Ed.), Pain Medicine: A Comprehensive Review. Mosby-Year Book, Missouri, pp.12-23. Sarraf, P., Sneller, M., 2005. Pathogenesis of Wegener's granulomatosis: current concepts. Expert Reviews in Molecular Medicine 7(8), 1-19. Schimmer, B., Parker, K., 2006. Adrenocorticotropic hormone; Adrenocortical steroids and their synthetic analogs; Inhibitors of synthesis and actions of adrenocortical hormones, in: Brunton, L., Lazo, J., Parker, K. (Eds.), Goodman & Gilman's the pharmacological basis of therapeutics. 11th ed. Mc Graw-Hill, New York, pp. 15871612. Somchit, M.N., Shukriyah, M.H.N., Bustamam, A.A., Zuraini, A., 2005. Anti-pyretic and analgesic activity of Zingiber zerumbet. International Journal of Pharmacology 1, 277-280.

19 Swingle, K., Shideman, F., 1972. Phases of the inflammatory response to subcutaneous implantation of a cotton pellet and their modification by certain anti-inflammatory agents. Journal of Pharmacology and Experimental Therapeutics 183, 226-234. Teotino, U., Friz, L., Gandini, A., Bella, D., 1963. Thio derivatives of 2, 3-dihydro-4H-1, 3benzoazin-4-one synthesis and pharmacological properties. Journal of Medicinal Chemistry 6, 248-250. Thenmozhi, S., Chaturvedi, M., Dwivedi, S., Subasini, U., 2013. Investigation of antiinflammatory, analgesic and antipyretic activity of rhizome volatile oil of Alpinia speciosa. Asian Journal of Medical and Pharmaceutical Researches 3, 82-84. Winter, C., Risley, E., Nuss, G., 1962. Carrageenan-induced edema in hind paw of the rat as an assay for anti-inflammatory drug. Proceedings of the Society for Experimental Biology and Medicine 11, 544-547. Yadav, P.N., Liu, Z., Rafi, M.M., 2003. A diarylheptanoid from lesser galangal (Alpinia officinarum) inhibits proinflammatory mediators via inhibition of mitogen-activated protein kinase, p44/42, and transcription factor nuclear factor-kappa B. Journal of Pharmacology and Experimental Therapeutics 305, 925-931. Yasukawa, K., Sun, Y., Kitanaka, S., Tomizawa, N., Miura, M., Motohashi, S., 2008. Inhibitory effect of the rhizomes of Alpinia officinarum on TPA-induced inflammation and tumor promotion in two-stage carcinogenesis in mouse skin. Journal of Natural Medicines 62, 374-378. Yusuf, M., Wahab, M.A., Yousuf, M.D., Chowdhury, J.U., Begum, J., 2007. Some tribal medicinal plants of Chittagong hill tracts, Bangladesh. Bangladesh Journal of Plant Taxonomy 14, 117-128.

20 Table 1 Chemical components of SiE as analyzed by GC-MS. Peak Retention % of Compound time Total (min) 1 13.702 6.95 Benzenecarboxylic acid 2 15.562 1.64 Resorcinol 3 19.980 17.28 Ethane, isothiocyanate 4 20.038 9.11 Heptanoic acid 5 22.082 22.71 Benzyl benzoate 6 22.787 0.71 l-Methoxy-3-(2-hydroxyethyl) nonane 7 24.404 2.42 n-Hexadecanoic acid 8 24.935 6.36 Hexadecanoic acid, ethyl ester 9 26.656 4.39 Phytol 10 26.922 1.71 9,12-Octadecadienoic acid (Z,Z) 11 27.014 2.02 cis,cis,cis-7,10,13-Hexadecatriena 12 27.418 3.73 Linoleic acid ethyl ester 13 27.505 2.96 7,10,13-Hexadecatrienoic acid, methyl ester 14 27.898 0.68 D-Gluconic acid, 2,3,4,5-tetra-O-methyl-6-O-(2,3,4,6-tetra-Omethyl-.alpha.-D-glucopyranosyl)-, methyl ester 15 28.729 11.18 2-Phenanthrenol, 7-ethenyl-1,2,3,4,4a,4b,5,6,7,9,10,l0adodecahydro-1,1,4a,7-tetramethyl-, [2S(2.alpha.,4a.alpha.,4b.beta.,7.beta.,l0a.beta.)] 16 29.884 2.52 2-Isopropylidenehydrazono-3-methyl-6-chloro-2,3dihydrobenzothiazole 17 31.554 0.87 No matches found 18 33.621 2.03 1,2-Benzenedicarboxylic acid, di-isooctyl ester 19 34.620 0.74 No matches found

50 ± 3* 52 ± 3*

75 ± 11

38 ± 5*

137 ± 4

1h

SiE 27 ± 3* 45 ± 2* Values are expressed as mean ± S.E.M. (n = 6). Control of diclofenac: treated with 5% DMSO in acetone. Control of the extract: treated with acetone. * Significantly different from its control group, p < 0.05.

5

Control of SiE

18 ± 3*

115 ± 7

30 min

143 ± 6

5

Diclofenac

70 ± 10

15 min

123 ± 7

-

Control of diclofenac

(mg/ear)

Table 2 Effects of SiE and diclofenac on EPP-induced ear edema in rats. Group Dose Edema thickness (µm)

43 ± 2*

122 ± 7

42 ± 5*

110 ± 11

2h

64

-

74

-

15 min

63

-

67

-

30 min

Edema inhibition (%)

64

-

63

-

1h

65

-

62

-

2h

21

10

75

Diclofenac

SiE 0.10 ± 0.02

*

0.15 ± 0.01

*

0.19 ± 0.02

*

0.07 ± 0.01

*

0.27 ± 0.05

63

44

30

74

-

0.17 ± 0.02

*

0.38 ± 0.02

*

0.42 ± 0.02

*

0.15 ± 0.01

*

Edema volume (mL) 0.75 ± 0.05

Edema volume (mL) %Inhibition

Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the control group, p < 0.05.

300

150

-

Control

3h

1h

Table 3 Effects of SiE and diclofenac on carrageenan-induced hind paw edema in rats. Group Dose (mg/kg) Time after carrageenan injection

77

49

44

81

-

%Inhibition

0.30 ± 0.05

*

0.57 ± 0.02

*

0.58 ± 0.02

*

0.24 ± 0.03

*

Edema volume (mL) 0.74 ± 0.08

5h

60

23

22

68

-

%Inhibition

22

10 5 75 150 300 Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the control group, p < 0.05.

Control Diclofenac Prednisolone SiE

0.26 ± 0.01 0.17 ± 0.03* 0.11 ± 0.04* 0.25 ± 0.04 0.16 ± 0.02* 0.11 ± 0.02*

Table 4 Effects of SiE, diclofenac, and prednisolone on AA-induced hind paw edema in rats. Group Dose (mg/kg) Edema volume at 1 h (mL) 35 58 4 39 58

% Inhibition

23

10 18.75 37.5 75 Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the control group, p < 0.05.

Control Diclofenac SiE

21 ± 1 4 ± 1* 17 ± 0* 11 ± 0* 5 ± 1*

Table 6 Effects of SiE and diclofenac on acetic acid-induced writhing response in mice. Group Dose (mg/kg) Number of writhes

81 19 48 76

% Inhibition

Normal 57.33 ± 2.71 57.46 ± 0.71 33.77 ± 1.75 Control 476.42 ± 48.66 3.47 ± 0.33 59.67 ± 3.28 61.20 ± 0.71 50.02 ± 1.33# Diclofenac 5 255.88 ± 13.26* 1.73 ± 0.09* 50 55.67 ± 3.20 61.36 ± 2.62 35.62 ± 2.71* Prednisolone 5 186.54 ± 10.97* 0.96 ± 0.07* 72 6.00 ± 5.75#, * 23.60 ± 0.92#, * 31.89 ± 2.82* * * SiE 300 343.41 ± 11.85 2.63 ± 0.14 24 62.67 ± 2.29 59.08 ± 2.99 39.96 ± 2.14* Values are expressed as mean ± S.E.M. (n = 6). TrW: Transudative weight, GrW: Granuloma weight, GI: Granuloma inhibition, BW: Body weight, TW: Thymus weight, AP: Alkaline phosphatase, U: Unit of enzyme, TP: Total protein # Significantly different from the normal group, p < 0.05. * Significantly different from the control group, p < 0.05.

Table 5 Effects of SiE, diclofenac, and prednisolone on transudative weight, granuloma weight, and percentage of granuloma inhibition on cotton pelletinduced granuloma formation in rats. Group Dose TrW (mg) GrW (mg/mg cotton) % GI BW gain (g) Dry TW AP activity (mg/kg/d) (mg/100 g BW) (U/mg of TP × 10-4)

24

26 Table 7 Effects of SiE, diclofenac and codeine on tail-flick test in rats. Group Dose Baseline Test reaction time (s) (mg/kg) reaction time (s) 1h 2h 3h

%Maximum possible effect 1h 2h 3h

Control

-

-

-

2

1

0

76

96

82

2

1

0

13

2

0

25

10

6

-

2.25 ± 0.11

2.50 ± 2.40 ± 2.50 ± 0.09 0.07 0.11 Diclofenac 10 3.63 ± 0.13 3.77 ± 3.68 ± 3.63 ± 0.17 0.15 0.13 Codeine 200 3.12 ± 0.08 8.33 ± 9.70 ± 8.77 ± 0.56* 0.30* 0.60* SiE 18.75 2.70 ± 0.11 2.82 ± 2.78 ± 2.73 ± 0.17 0.15 0.18 37.5 2.02 ± 0.02 3.05 ± 2.18 ± 2.05 ± 0.04* 0.03* 0.02 75 2.82 ± 0.15 4.62 ± 3.53 ± 3.25 ± 0.09* 0.08* 0.08* Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from the baseline reaction time, p < 0.05.

Table 8 Effects of SiE and diclofenac on yeast-induced hyperthermia in rats. Group Dose Rectal temperature (°C) (mg/kg) Baseline 18 h after Time after drug administration (min) yeast injection 30 60 90 120 180 Control 37.5 ± 39.0 ± 0.1 39.2 ± 39.2 ± 39.1 ± 39.0 ± 39.2 ± 0.1 0. 0.1 0.1 0.1 0.2 Diclofenac 10 37.4 ± 38.9 ± 0.1 38.2 ± 37.6 ± 37.2 ± 36.9 ± 37.4 ± 0.1 0.1* 0.1* 0.0* 0.1* 0.1* SiE 75 37.6 ± 39.2 ± 0.1 39.3 ± 39.1 ± 39.1 ± 39.1 ± 39.2 ± 0.1 0.1 0.2 0.1 0.1 0.1 150 37.6 ± 39.1 ± 0.1 39.4 ± 39.2 ± 39.1 ± 39.1 ± 38.9 ± 0.0 0.1 0.1 0.1 0.1 0.1 300 37.6 ± 39.2 ± 0.2 39.3 ± 39.2 ± 39.2 ± 39.2 ± 39.2 ± 0.1 0.2 0.1 0.1 0.1 0.1 Values are expressed as mean ± S.E.M. (n = 6). * Significantly different from rectal temperature at 18 h after yeast injection, p < 0.05.

27

(5)

(15) (8) (4)

(1)

(3)

Fig. 1. GC-MS chromatogram of SiE showing the presence of 19 compounds [(1)-(19)].

(9) (12)

(7)

(2)

(6)

(11) (10)

(13) (14)

(16)

Graphical Abstract (for review)

Investigation of anti-inflammatory, antinociceptive and antipyretic activities of Stahlianthus involucratus rhizome ethanol extract.

Stahlianthus involucratus (Zingiberaceae) has long been used in traditional medicine to treat inflammation, pain, and fever. However, no pharmacologic...
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