Antinociceptive and anti-arthritic effects of aqueous whole plant extract of Trianthema portulacastrum in rodents: Possible mechanisms of action

Accepted Manuscript Antinociceptive and anti-arthritic effects of aqueous whole plant extract of Trianthema portulacastrum in rodents: Possible mechanisms of action T. Falade, I.O. Ishola, M.O. Akinleye, J.A. Oladimeji-Salami, O.O. Adeyemi PII:

S0378-8741(18)34090-X

DOI:

https://doi.org/10.1016/j.jep.2019.111831

Article Number: 111831 Reference:

JEP 111831

To appear in:

Journal of Ethnopharmacology

Received Date: 4 November 2018 Revised Date:

16 March 2019

Accepted Date: 19 March 2019

Please cite this article as: Falade, T., Ishola, I.O., Akinleye, M.O., Oladimeji-Salami, J.A., Adeyemi, O.O., Antinociceptive and anti-arthritic effects of aqueous whole plant extract of Trianthema portulacastrum in rodents: Possible mechanisms of action, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.111831. 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 proof before it is published in its final 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.

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Antinociceptive and anti-arthritic effects of aqueous whole plant extract of Trianthema portulacastrum in rodents: Possible mechanisms of action

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Falade Ta., Ishola IO*a., Akinleye MOb, Oladimeji-Salami JAc, Adeyemi OOa. Department of Pharmacology, Therapeutics and Toxicology, College of Medicine, University of

Lagos, Idi-Araba, Lagos, Nigeria. b

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Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, Idi-Araba,

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Lagos, Nigeria.

Bioresources Development Centre, Ogbomoso, National Biotechnology Development Agency,

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Oyo State, Nigeria

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* Corresponding Author: Ishola IO (Ph.D)

Department of Pharmacology, Therapeutics and Toxicology, Faculty of Basic Medical Sciences,

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College of Medicine, University of Lagos, Idi-Araba Campus, P.M.B. 12003, Lagos, Nigeria. Email: [email protected] Phone: +2348033018908

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Antinociceptive and anti-arthritic effects of aqueous whole plant extract of Trianthema portulacastrum in rodents: Possible mechanisms of action

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Falade Ta., Ishola IOa., Akinleye MOb, Oladimeji-Salami JAc, Adeyemi OOa. Department of Pharmacology, Therapeutics and Toxicology, College of Medicine, University of Lagos,

Idi-Araba, Lagos, Nigeria.

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, Idi-Araba, Lagos,

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Nigeria. b

Bioresources Development Centre, Ogbomoso, National Biotechnology Development Agency, Oyo

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State, Nigeria

* Corresponding Author: Department of Pharmacology, Therapeutics and Toxicology, Faculty of

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Basic Medical Sciences, College of Medicine, University of Lagos, Idi-Araba Campus, P.M.B. 12003, Lagos, Nigeria. Email: [email protected] Phone: +2348033018908

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Abstract Ethnopharmacological relevance: Trianthema portulacastrum L. (Aizoaceae) is used in traditional

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African Medicine for the treatment of various illnesses including dropsy, inflammation and rheumatism. Aim of the study: This study was designed to investigate the anti-nociceptive and anti-arthritic properties of the aqueous whole plant extract of Trianthema portulacastrum (AETP), possible mechanisms of action and characterize some of the active constituents

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Materials and methods: Antinociceptive activity was evaluated using the acetic acid-induced writhing and hot plate tests in mice. The carrageenan test was used to induce a transient inflammation while arthritis was induced with complete Freund’s adjuvant (CFA) in rats. On completion of CFA-induced

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arthritis macroscopic observations, the rats were euthanized to isolate the spleen, liver and limbs for estimation of oxidative stress and histological analysis

Results: AETP (10, 50, or 250 mg/kg; p.o.) produced significant (p<0.05) and dose-dependent inhibition (41.10, 50.40, and 67.10 %, respectively) of writhing response elicited by acetic acid. Also, increased pain threshold of supraspinally mediated nociceptive behaviour, with peak maximum possible effect

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(MPE) obtained at 250 mg/kg (22.98%; 30 min post-treatment). However, the pre-treatment of mice with Nitro-L-arginine (L-NNA) or naloxone reversed AETP-induced antinociception. In another experiment, AETP produced time course inhibition of carrageenan-induced paw oedema with peak effect (50.60%) at 250 mg/kg as well as significant reduction in CFA-induced arthritis by 58.56%, on

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day 27 and arthritic index (26.84%). Similarly, AETP attenuated CFA-induced MDA generation and deficit in antioxidant enzyme activities. Histological analysis of rat joints revealed a reduction in the

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synovial hyperplasia and mononuclear infiltration induced by CFA in AETP treated groups. Conclusion: This study suggest that AETP possesses anti-nociceptive action through nitrergic and opioidergic signalling as well as anti-arthritic effect through enhancement of antioxidant defense system and inhibition of release or actions of inflammatory mediators. Keywords: arthritis; antioxidant; opioid receptor; nitric oxide, Freund’s adjuvant; hot plate

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1. Introduction Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory disease, affecting more than 1% of

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adult population (Smolen et al., 2016), characterised by persistent synovitis, systemic inflammation, and autoantibodies (particularly to rheumatoid factor and citrullinated peptide) (Scott et al., 2010). People with RA identify pain as the most important symptom, one that often persists despite optimal control of

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inflammatory disease (Walsh and McWilliams, 2014). A number of extra-articular manifestations and comorbidities are present in patients with RA, which result in increased mortality (Gibofsky, 2012).

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Uncontrolled active RA causes joint damage, disability, decreased quality of life, cardiovascular and other comorbidities (Scott et al., 2010; Gibofsky, 2012; Smolen et al., 2016). Disease-modifying antirheumatic drugs (DMARDs), the key therapeutic agents, reduce synovitis, systemic inflammation and improve function but not without adverse effects (Singh et al., 2016). Thus, the need for a novel drug with better efficacy and tolerability for the treatment of both pain and inflammatory components of RA.

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Trianthema portulacastrum L. (Aizoaceae) commonly called giant pigweed, is a yearly herb, utilized as pain relieving, purgative, stomachic, laxative, treatment of blood disease, anaemia, rheumatoid arthritis, and night blindness (Sukalingam et al., 2017). It is called "Akisan" in the indigenous Yoruba language

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(Southwest, Nigeria). Several studies have reported the pharmacological properties of T.

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portulacastrum such as; antigenotoxic (Sarkar et al., 1999), antifungal (Nawaz et al., 2001), antioxidant (Mandal et al., 1997; Yaqoob et al., 2014), and hepatoprotective activities (Mandal et al., 1998). The principal constituent of T. portulacastrum include; ecdysterone, trianthenol, 3-acetylaleuritolic acid, 5,2’-dihydroxy-7-methoxy-6,8-dimethylflavone, leptorumol, 3,4-dimethoxy cinnamic acid, 5-hydroxy2-methoxybenzaldehyde, p-methoxybenzoic acid, and beta cyanin (Vohora et al., 1983). This study sought to investigate the possible anti-nociceptive and anti-inflammatory effects of the whole plant

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extract of T. portulacastrum which could be an indicator for the discovery of new therapeutic agents

2.0

Materials and methods

2.1

Plant material and extraction

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useful in the management of rheumatoid arthritis.

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The whole plant of Trianthema portulacastrum was collected from Ikorodu, Lagos state, Nigeria. Botanical Identification and authentication was done by Mr. T.K Odewo (a forestry expert in the

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herbarium section), Department of Botany, University of Lagos, Akoka, Lagos State, Nigeria. A voucher specimen LUH 9703 was deposited in the herbarium for reference. The whole plant of T. portulacastrum was air dried and pulverized. One kilogram of pulverized T. portulacastrum was infused in 1.55L of distilled water for 4 h at 40°C, then left for 24 h at room temperature for further extraction.

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The extract was filtered and the pooled extract was evaporated to dryness in an oven at 40°C. The percentage yield was 5.13±0.3%w/w. 2.2 Drugs and chemicals

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Complete Freund’s adjuvant (CFA), ethanol, prazosin, formalin, carrageenan, yohimbine, L-arginine,

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reserpine, naloxone, glibenclamide, methylene blue, gallic acid, atropine, rutin, quercetin (Sigma Aldrich, Louis, MO, USA), celecoxib (Pfizer manufacturing Deutschland GmbH, Illertissen, German), ibuprofen (Ranbaxy Pharmaceutical, India), morphine (Martindale Pharma, Essex, United Kingdom). 2.3

Experimental animals

The male albino rats (220-250 g, 12 weeks old) or mice (20-25g, 8 weeks old) used in this study were obtained from the Laboratory Animal Centre of the College of Medicine, University of Lagos, Lagos,

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Nigeria.

The animals were housed in plastic cages with wooden shavings as beddings, at room

temperature under standard environmental conditions (12 hours light/dark cycle), received standard rodent diet (Livestock feed Plc, Lagos, Nigeria) and drinking water ad libitum. The experimental

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procedures adopted were in accordance with the College of Medicine, University of Lagos, Health Research Ethics Committee approval (CMUL/HREC/10/17/447) and United States National Institutes of Health Guidelines for Care and use of laboratory Animals in Biomedical Research (2001). Animals were

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acclimatized for 2 weeks before the commencement of the experiment. Doses were selected based on

mg/kg. 2.4

Phytochemical screening

2.4.1 Quantitative analysis

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our preliminary findings. Moreover, the median effective dose in acetic acid-induced writhing was 50

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The preliminary quantitative estimations of total phenolic, tannins, alkaloid, saponins and cardiac glycosides contents in AETP were assayed using the methods of El-Olemy et al. (1994) and Senguttuvan et al. (2014).

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profiling

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2.4.2 Preparation of Rutin/Quercetin stock standard and concentrations calibration for HPLC

Rutin (10 mg) and Quercetin (10 mg) standards were separately weighed into 10 mL volumetric flasks and dissolved in pure methanol and made up to volume to obtain 1 mg/mL stock solutions. 10 – 100 µg/mL and 100 – 500 µg/mL, respectively, calibration concentrations of rutin and quercetin standards were prepared from the stock solutions. One millilitre of rutin and quercetin stock solution were mixed together for simultaneous elution of the two standards in one chromatogram. Twenty microlitre of the solutions generated were injected into HPLC using the method of Zu et al. (2006).

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2.4.3 Preparation of sample solution from AETP AETP (5 g) was weighed into 10 mL volumetric flask and dissolved in methanol by sonication for 30

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minutes after making up to volume with methanol. The resulting sample solution (5 mg/mL) was filtered using 0.45 µm syringe filter and 20 µL of the filtered sample was injected into the HPLC for analysis.

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2.4.4 HPLC Chromatographic conditions and Analysis.

Chromatographic analysis was done using Agilent Technologies® HPLC 1200 series, Binary pump,

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micro-vacuum degasser, standard and preparative autosampler, Thermostatted Column Compartment, Diode Array and multiple Detector with ChemStation software. The Column used was Agilent® Eclipse XDB-C18, 4.6 × 150 mm, 5µm diameter particle size. The mobile phase was methanolacetonitrile-water (40:15:45,v/v/v) containing 1.0 % v/v glacial acetic acid. The mobile phase was filtered through a 0.45µm membrane filter, then deaerated ultrasonically prior to use. The flow rate, injection volume and wavelengths were 1.0 mL/minute, 20 µL and 257, 334 nm respectively. The

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identification was done using the retention times obtained from the chromatograms of standard compared with that obtained from the plant extract. The quantification was determined from the regression equations obtained from calibration curves of rutin and quercetin standard solutions. All chromatographic operations were done at 25o C temperature.

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MS)

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2.4.5 Identification of Bioactive Constituents using gas chromatography mass spectrometry (GC-

The extract was analyzed by GC-MS detection GC-MS Ultra (Shimadzu, model GC-2010 Plus, Japan) equipped with a capillary column Rtx-5 (30 m × 0.25 mm × 0.25 µm), helium carrier gas, with flow rate of 1.0 ml/min. The temperature of the column was programed as follows: from 60°C to 200°C with a heating rate of 10°C/min and then from 200°C to 280°C with a heating rate of 20°C/min. The injection volume was 1.0 µl. The temperatures of injection and detection were 260°C and 280°C, respectively (Carneiro et al., 2017).

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2.5

Acute toxicity test

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Forty-five male albino mice (20-25 g) fasted for 12 h were randomly divided into 9 groups (n=5) and treated as follows; vehicle (10ml/kg, p.o., normal control), AETP (500, 1000, 2000 or 5000 mg/kg, p.o.) or vehicle (10 ml/kg, i.p., normal control), AETP (10, 500 or 1000 mg/kg, i.p.). Animals were observed

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for 2 h post treatment for behavioural changes, toxicity symptoms and mortality. Mortality was also recorded 24 h later and the surviving mice were observed for a further 14 days for signs of delayed

(Randhawa, 2009).

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toxicity. The median lethal dose (LD50) was estimated by the log dose probit analysis method

2.6

Antinociceptive activity

2.6.1

Acetic acid-induced mouse writhing test

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Acetic acid-induced writhing test was carried out to evaluate possible peripheral antinociceptive effect of AETP in mice. Thirty male albino mice were fasted for 12 h and assigned randomly to five groups (n=6); Group I: vehicle (10ml/kg, p.o.), Group 2-4: AETP (10, 50 or 250 mg/kg, p.o., respectively), and

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Group 5: ibuprofen (100 mg/kg, p.o.). One hour post-treatment acetic acid 0.06%v/v (10 ml/kg, i.p.)

2011). 2.6.2

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was administered and number of cumulative writhing behaviour in 30 min was recorded (Ishola et al.,

Hot plate analgesia test

This test was carried out to investigate central antinociceptive effect of AETP in mice using Columbus hot plate analgesia meter (Columbus Instrument, 1440-E54, Ohio, USA) using our earlier reported protocol (Ishola et al., 2012). Briefly, thirty selected mice were randomly divided into 5 groups (n=6);

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Group I: vehicle (10ml/kg, p.o.), Group 2-4: AETP (10, 50 or 250 mg/kg, p.o., respectively), and Group 5: morphine (5 mg/kg, s.c.). Pre-dosing base-line latencies were 3–4 sec. The reaction latency was recorded at 30, 60, 90, 120, 150 and 180 min post drug treatment (Ishola et al., 2012). A maximum

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hotplate latency of 10 s was used to prevent tissue damage to the paws. Pre-dosing latencies were determined on at least two occasions (5 min apart with the two measurements being within ± 0.5 and ± 1 sec before the administration of drugs or vehicle (South and Smith, 1998).

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[Maximum latency – Pre drug latency]

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%MPE: [Post drug reaction latency-Pre drug reaction latency]×100

2.6.3 Elucidation of possible mechanism of antinociception in mice

To investigate possible mechanism of AETP-induced antinociception, mice (n =6) were pretreated with naloxone (5 mg/kg, s.c, non-selective opioid receptor antagonist) (Ishola et al., 2014); prazosin (1 mg/kg, i.p.; α1-adrenoceptor antagonist) or yohimbine (1 mg/kg, i.p.; α2-adrenoceptor antagonist) (Ishola

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et al., 2015a); ondansetron (0.5 mg/kg, i.p.; non-selective 5HT3 receptor antagonist) (Alchaider, 1991), glibenclamide (10 mg/kg, i.p; AETP-sensitive potassium channels blocker) (Ishola et al., 2014); Larginine (150 mg/kg, i.p., nitric oxide precursor) (Ishola et al., 2015a); L-NNA (10 mg/kg; i.p.);

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methylene blue (MB) (2 mg/kg, i.p.) or vehicle. Fifteen minutes later, AETP (250 mg/kg, p.o.), or

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vehicle (10 ml/kg, p.o.) was administered. One hour post-treatment, acetic acid-induced writhing test was carried out.

2.7 Anti-inflammatory activity 2.7.1. Carrageenan-induced rat paw oedema This test was carried out to evaluate the effect of AETP on acute inflammation induced by carrageenan (Ishola et al., 2011). Thirty male albino rats fasted for 12 h were randomly assigned to 5 groups (n=6). 8

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Group I: vehicle (10 ml/kg, p.o.), Group 2-4: AETP (10, 50 or 250 mg/kg, p.o., respectively), and Group 5: ibuprofen (100 mg/kg, p.o.). One hour post-treatment, 100µL carrageenan 1% v/w in normal saline was subcutaneously injected into the right hind paw. Changes in paw circumference was recorded

2.7.2

Complete Freund’s adjuvant induced arthritis

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for 6 h and at 24 h post-carrageenan injection (Ishola et al., 2012; 2015a).

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Arthritis was induced by subplantar injection of 100µL of complete Freund’s adjuvant (CFA) into the right hind paw of rats (Ishola et al., 2015b). The adjuvant contained Mycobacterium tuberculosis (10

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mg/ml in paraffin oil) (Sigma Aldrich, MO, USA). Forty eight male albino rats fasted for 12 h were randomly divided into six groups (n=8) and initial paw circumference determined using cotton thread method (Ishola et al., 2015b); Group 1: vehicle (10 ml/kg, p.o., normal), Group 2: vehicle (10 ml/kg, p.o., experimental control), Group 3-5: AETP (10, 50 or 250 mg/kg, p.o.) and Group 6: celecoxib (50

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mg/kg, p.o). One hour post-treatment on day 1, animals in groups 2-6 were given CFA (100µL, intraplantar). The paw circumference was measured and treatment was given on a daily basis for 28

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days. Arthritis was evaluated using the scoring system of Whiteley and Dalrymple. (2001.

2.7.3 Assay for loss of function

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Chronic inflammation do compromise the integrity of limbs and joints due to pain and oedema, hence, we investigated the effect of AETP on CFA-induced loss of function in open field test (OFT) and staircase test (Ishola et al., 2015b). The animals were observed in the OFT for 5 min and the number of crossings was recorded as an index of locomotor activity (Ishola et al., 2015b). Locomotor activity was measured on days 8, 16 and 24.

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In the staircase test, rats were fasted for 4 h, then trained to retrieve the food pellets from the ascending steps of the staircase from days 0-3. Each rat was placed on the floor of the box with its back to the staircase, then the number of steps climbed were recorded for 3 min. A step is considered to be climbed

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only if the rat had placed all four paws on the step, the number of steps descended was not taken into

2.8

Collection of organs and blood samples

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account. The number of stairs climbed was measured on days 8, 16 and 24.

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On day 28, the animals were anaesthetized with chloral hydrate (300 mg/kg, i.p.) and blood samples collected via ocular puncture into EDTA (haematological assay) and plain bottle for serum analysis. The stomach and liver were isolated for possible ulcerative or liver injury effect of AETP. The number of lesions or haemorrhage was recorded and average ulcer index was calculated. The liver, spleen and

2.8.1

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limbs were isolated for oxidative stress analysis.

Haematological analysis

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The total white blood cell count (leucocyte count) was determined using Roche-Hitachi 904 automated analyser (Minnesota, USA) while erythrocyte sedimentation rate was assayed using the Wintrobe et al.

2.8.2

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(1974) protocol.

Antioxidants and oxidative stress analysis

The levels of malondialdehyde (MDA) generation, an end-product of lipid peroxidation in the liver, spleen and limbs were measured using the method of Ohkawa et al. (1979), glutathione (GSH) levels based on the protocol developed by Rahman et al. (2006), catalase (CAT) activity was measured by the method of Weydert and Cullen (2010), superoxide dismutase (SOD) activity was assayed following the 10

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protocol of Hamar et al. (2012) while the protein content was quantified with Bradford reagent, using bovine serum albumin as standard. Histological analysis

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2.8.3

The left paw of all groups rats were cut above and below the 0.5 cm of joints. The muscle and skin of joints were trimmed away to leave the intact synovial membrane. All tissues were processed with the

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3% hydrochloric acid (HCl) solution for 5–7 days, with periodic change of HCL on every 24 h for complete decalcification of joints. After that, the joints were fixed in the neutral buffered formalin

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(10%) for 2 days. Decalcified joints of rats were again dehydrated in different series of alcohol and joints were cleared and embedded in liquid paraffin. Decalcified joints were processed for paraffin embedding tissue sections (5 µm) and stained with hematoxylin and eosin (H&E). H&E stained joint slides were examined for bone and cartilage destruction by synovial proliferation, cellular infiltration,

Statistical analysis

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and cartilage erosion.

All data were expressed as the mean ± SEM and statistical analysis was performed using GraphPad

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prism 6 software. Statistical level of significance was assayed using one or two way analysis of variance (ANOVA) followed by Tukey post hoc multiple comparison tests. 3.0

Results

3.1

Phytochemical Analysis

3.1.1 Quantitative analysis

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The preliminary quantitative phytochemical assay revealed the presence of phenolic compounds in AETP expressed as mg gallic acid equivalent (GAE) per gram dry extract weight, with a total phenolic content of 12.10 ± 0.02 mg/100g gallic acid equivalent (GAE). The saponins content of the extract was

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27.60 mg/100g diosgenin equivalent (DE) while precipitation of the extract by aluminium chloride showed the presence of flavonoid compounds expressed as mg rutin equivalent (RE) per gram dry extract weight, indicating a total flavonoid content of 15.80±0.01 mg/100g rutin equivalent (RE). The

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content of alkaloids was measured in terms of atropine equivalent (AE) per gram dry extract weight, the total alkaloids were determined to be 14.60± 0.02 mg/100g atropine equivalent (AE) and total tannin

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content was 8.52±0.01 mg/100g gallic acid equivalent (GAE) of AETP.

3.1.2 HPLC profiling

The regression equations and coefficient of determination (r2) obtained were y = 17.903x – 86.565 and y

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= 24.808x + 253.120; 0.998 and 0.994 for rutin and quercetin, respectively. Where y is the mean peak area and x is the concentration (Fig. 1A). The chromatogram got from combination of rutin and quercetin standards is as shown in figure 1B. The concentrations of rutin and quercetin obtained from

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the dried extract were 0.095 mg/g (9.46 %) and 0.146 mg/g (14.58 %) respectively.

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3.1.3 Gas chromatography-mass spectrometry (GC/MS) analysis About 44 peaks were observed in the chromatogram of the aqueous whole plant extract of T. portulacastrum extract as shown Fig. S1). Peak area offers a suitable approximation of the relative amounts of components, when all components respond equally in the detector and are eluted. The GCMS analysis of aqueous whole plant extract of T. portulacastrum extract demonstrated the presence of epoxyδ-lactone ring and cyclohexane moieties (Fig. S1). 3.2

Acute toxicity test 12

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The median lethal dose for orally and intraperitoneally administered AETP were 2454.71 and 1548.81 mg/kg, respectively. The observed toxicity behaviours includes; abdominal writhes, sedation and

3.3

Anti-nociceptive activity

3.3.1

Effect of AETP on acetic acid-induced mouse writhing.

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reduced motor activity.

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Intra-peritoneal injection of 0.6% acetic acid produced time course abdominal writhing (37.60±2.79) (Fig. 2. However, the pre-treatment of mice with AETP reduced the cumulative number of writhes, with

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peak effect obtained at 250 mg/kg (12.40±3.08; 67.10% inhibition) (Fig. 2). Similarly, the pretreatment of mice with ibuprofen reduced the mean number of writhes by 74.10% in comparison to vehicle treated control. 3.3.2

Effect of AETP on nociceptive behaviour in hot plate test

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Table 1 shows the time course reaction latencies of mice exposed to thermally induced nociception. The pretreatment of mice with AETP produced dose dependent and time course significant increase in pain threshold, with peak effect obtained at AETP 250 mg/kg (22.98% maximum possible effect; 30 min

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post-treatment). Similarly, the standard drug, morphine (10 mg/kg) increased the reaction latency from

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1.94 in control to 6.20 (44.20% maximum possible effect) at 90 min post-treatment. 3.3.3 Elucidation of possible mechanism of the anti-nociceptive effect of AETP in mice. As shown in Fig. 3A-E, intraperitoneal injection of 0.6%v/v acetic acid induced writhing reflex (63.75±5.41) in vehicle treated control group. However, the pretreatment of mice with AETP 250 mg/kg reduced the writhing behaviour by 66.30% indicative of antinociceptive activity. To investigate the involvement of opioidergic system in the antinociceptive effect of AETP, mice were pretreated with

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naloxone. The pretreatment of mice with naloxone before oral administration of AETP, reduced the antinociceptive effect of AETP indicative of opioidergic involvement in its mechanism of action. Fig. 3A shows that the AETP-induced antinociception was prevented by the pretreatment of mice with

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naloxone (5 mg/kg, non-selective opioid receptor antagonist, s.c.) in acetic acid-induced writhing test. A two way ANOVA revealed significant differences of naloxone pretreatment [F(1,20)=47.49, P<0.0001], AETP treatment [F(1,20)=18.80,P<0.0001] and naloxone pretreatment × AETP treatment interaction

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[F(1,20)=666.80,P<0.0001]. In contrast, the pretreatment of mice prazosin (1 mg/kg, an α1-adrenoceptor antagonist, i.p.) or yohimbine (1 mg/kg, an α2-adrenoceptor antagonist, i.p.) failed to reverse AETP-

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induced antinociception. Two way ANOVA revealed non-significant differences of prazosin or yohimbine pretreatment [F(2,30)=2.72,P=0.08], extract treatment [F(1,30)=790.00,P<0.0001] and prazosin or yohimbine pretreatment × extract treatment interaction [F(2,30)=2.23,P=0.12] (Fig. 3B). Fig. 3C also shows that the pretreatment of mice with ondansetron (0.5 mg/kg, 5-HT3 receptor antagonist,

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i.p.) or glibenclamide (10 mg/kg, AETP-sensitive K+ channels blocker) failed to prevent the AETPinduced antinociceptive effect in mice. A two way ANOVA revealed significant differences of ondansetron

or

glibenclamide

pretreatment

[F(2,30)=3.73,P=0.08],

treatment

× extract treatment

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[F(1,30)=2621,P<0.0001] and ondansetron or glibenclamide pretreatment

extract

interaction [F(2,30)=13.35,P<0.01]. ). Interestingly, post hoc analysis showed that the pretreatment of

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mice L-arginine (150 mg/kg, i.p.; nitric oxide precursor) did not affect AETP-induced antinociception but the pretreatment of mice with L-NNA (10 mg/kg, i.p., neuronal nitric synthase inhibitor) prevented AETP-induced antinociception. In addition, two way ANOVA revealed significant effect of treatment [F(1,30)=587.70,P<0.0001], L-arginine or L-NNA pretreatment F(2,30)=16.91,P<0.0001], and Larginine or L-NNA pretreatment × AETP treatment interaction [F(2,30)=20.70,P>0.05] (Fig. 3D). In contrast, two way ANOVA revealed non-significant differences of methylene blue (MB) (2 mg/kg, a

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non-specific inhibitor of NO/soluble guanylyl cyclase inhibitor) pretreatment [F(1,20)=0.05,P=0.82], extract treatment [F(1,20)=435.60,P<0.0001] and MB pretreatment

× extract treatment interaction

[F(1,20)=0.75,P=0.40] (Fig. 3E). These results suggest that the inhibition of the NO-cGMP signal

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pathway at the soluble guanylyl cyclase level is not involved in AETP-induced antinociception.

Anti-inflammatory activity

3.4.1

Effect of AETP on carrageenan-induced paw oedema in rats

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Intraplantar injection of carrageenan into the right hind paw of rats induced a time course increase in paw circumference that peaked at 3 h (1.65±0.16 mm) compared to vehicle treated. However, oral

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administration of AETP (250 mg/kg) produced time course inhibition of oedema formation with peak effect 50.60% inhibition at 6 h post carrageenan injection. As expected, ibuprofen (100 mg/kg) significantly reduced paw oedema by 58.22% at 6 h post carrageenan injection (Table 2). Effect of AETP on CFA-induced arthritis in rats

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3.4.2

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As shown in the table 3, injection of CFA into the right hind paw of the animals produced an increase in paw diameter which peaked on day 12 and thereafter gradually decreased till day 28. However, AETP 250 mg/kg produced significant decrease in paw diameter on day 3 (0.82±0.15; 46.75% inhibition), with peak effect on day 28 (0.82±0.19; 58.56% inhibition). The standard reference drug, celecoxib 50 mg/kg, significantly reduced (p<0.01) paw size on day 12 (1.55±0.16; 49.51% inhibition), with peak effect on day 27 (0.78±0.09; 60.61% inhibition) (Table 3). 3.4.3 Effect of AETP on arthritic index and body weight 15

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To evaluate the effect of AETP on CFA-induced arthritis, we assessed the arthritic index. The severity of arthritis was evaluated using a macroscopic scoring system ranging from 0 to 4. CFA-vehicle treated rats showed a gradual increase in mean macroscopic scores with a peak score 4 on day 12 post CFA

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injection, which reduced to score 3.88±0.13 on day 28. Two way ANOVA revealed significant effect of treatment [F(4,125)=7.79,P<0.05]. Post hoc analysis showed that the pretreatment of rats with AETP or celecoxib caused reduction in arthritic index from day 24 (Fig. 4). On the other hand, the growth of

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arthritic rats was found to be impeded as they showed a significant reduction in body weight throughout the experiment when compared to normal rats, indicating the severity of arthritis disease. Post hoc

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analysis showed that the intraplantar injection of CFA caused a significant decrease (p<0.05) in body weight in CFA-vehicle treated group as compared to vehicle-control normal (Fig. 5). Two way ANOVA revealed significant effect of treatment [F(3,120)=32.92,P<0.0001]. However, post hoc analysis showed that the pretreatment of rats with AETP or celecoxib prevented the decrease in body weight when

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compared to vehicle-CFA treated control group.

3.4.4. Effect of AETP on CFA-induced motor deficits

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Post hoc analysis showed that intraplantar injection of CFA induced time course deficit in locomotor activity from day 8 in OFT indicative of loss of function. Two way ANOVA revealed significant effect

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of treatment [F(5,90)=25.95,P<0.0001] (Fig. 6A). The decrease in spontaneous motor activity induced by CFA was prevented with pretreatment of rats with AETP or celecoxib. To corroborate this finding CFA reduced staircase climbing activity of the animal which was ameliorated with the pretreatment of rats with AETP. . Two way ANOVA revealed significant effect of treatment [F(5,90)=67.92,P<0.0001] (Fig. 6B). 3.4.5

Effect of AETP on haematological parameters in arthritic rats

16

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Intraplantar injection of CFA into the right hind paw of rats caused significant increase in WBC (×106/µL), granulocyte count and erythrocyte sedimentation rate (ESR) with decrease in level of haematocrit. However, the pretreatment of rats with AETP (10, 50 or 250 mg/kg) or celecoxib caused

7). Effect of AETP on oxidative stress in arthritic rats

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3.4.6

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significant decrease in WBC (×109/L), granulocyte count and erythrocyte sedimentation rate (ESR) (Fig.

As shown in Fig. 8A, intra-plantar injection of CFA increased MDA generation by 2.46, 1.68 and 1.90

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folds in the spleen, liver and limbs, respectively, which was attenuated by AETP administration. CFA induced deficits antioxidant enzymes activity: GSH level (2.25 folds) in spleen (Fig. 8B), SOD activity (1.75 folds) in spleen and limbs (Fig. 8C) as well as catalase activity (2.20 folds) in spleen and limbs (Fig. 8D), in comparison to vehicle treated control. Post hoc analysis showed that the decrease in

3.7.4

Histological analysis

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antioxidant enzymes activity in the spleen and limbs were ameliorated by AETP administration.

The vehicle control showed normal joint architecture with a preserved synovium, presence of intact

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cartilage and absence of inflammation (Fig. 9A). However, CFA-vehicle treated rat showed severe

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synovial hyperplasia and cellular infiltration with pannus formation (Fig. 9B). The pretreatment of rats with AETP (10, 50 and 250 mg/kg) reduced the severity of synovial hyperplasia and cellular infiltration to a moderate level. AETP treated rats showed reduction in inflammation and protection of tibiotarsal with presence of lesser oedema. (Fig, 9C-E). Celecoxib (50 mg/kg) CFA-treated rats restored joint space as well as reduction in cellular infiltration (Fig. 9F). 4.0

Discussion

17

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Findings from this study showed that the aqueous whole plant extract of Trianthema portulacastrum possesses antinociceptive activity in chemical-/thermal-induced pain possibly through opioidergic and nitrergic mechanisms as well as anti-inflammatory effect on carrageenan-induced acute inflammation

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and CFA-induced arthritis via attenuation of oxidative and nitrosative stress. To estimate the antinociceptive property of new drugs, it is essential to employ different tests which differ in stimulus quality, intensity, and duration (Tjolsen and Hole, 1997; Ishola et al., 2011). To assess the peripheral

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antinociceptive effect of T. portulacastrum, acetic acid-induced nociceptive assay was carried out. Intraperitoneal injection of acetic acid induced abdominal writhing due to significant increase in the

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levels of PGE2 and PGF2α in the peritoneal cavity (Ayoub and Botting, 2010; Oliveira-Fusaro et al., 2012). Interestingly, the pretreatment of mice with T. portulacastrum elicits a dose dependent inhibition of the acetic acid-induced visceral nociceptive response in mice indicative of its peripheral antinociceptive action. To investigate the putative effect of the extract in centrally mediated forms of

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pain, the hot plate test (thermal threshold test) was carried out. It is a complex pain model, producing two behavioural components (paw licking and jumping) considered to be spinal and supraspinally integrated responses (Ishola et al., 2011, 2015). In this study, T. portulacastrum produced time course

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increase in pain threshold in hot plate test possibly due to its effect on spinal dorsal horn and supraspinal processing indicative of centrally mediated antinociceptive activity (Millan, 2002). Moreover, T.

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portulacastrum-induced antinociceptive actions did not affect motor output of the spinal reflex arc, evidenced in absence of motor impairment in rotarod test (data not shown). In this study, an attempt was made to characterise some of the mechanisms through which T. portulacastrum exerts its antinociceptive action in acetic acid-induced nociception model in mice at a dose that did not affect motor function. The acetic acid-induced writhing test is a typical model for inflammatory pain, with peripheral polymodal receptors around small vessels that signal to the CNS via 18

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sensory afferent C-fibres entering the dorsal horn being its major transmission pathway (Kumazawa et al., 1996; DalBó et al., 2006). Findings from this study showed that the pretreatment of mice with naloxone (non-selective opioid receptor antagonist) reversed T. portulacastrum-induced antinociceptive

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action corroborating its central antinoceptive action as well as opioidergic involvement in its mechanism of action (Adeyemi et al., 2018). In another experiment, the role of monoaminergic pathways in T. portulacastrum-induced antinociception was assayed. The modulatory actions of descending brainstem

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monoaminergic pathways have been shown to dampen pain behaviours, possibly to aid facilitation of escape and survival in response to threatening stimuli (Martin et al., 1999; Millan, 2002). In this study,

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the pretreatment of mice with prazosin (α1-adrenoceptor antagonist), yohimbine (α2-adrenoceptor antagonist) or ondansetron (5-HT3 receptor antagonist) alone did not change the response to acetic acidinduced writhing. Similarly, combination of T. portulacastrum and ondansetron did not affect the nociceptive response, thus, ruling out involvement of monoaminergic receptors. In another study, the

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role of L-arginine-nitric oxide pathway in T. portulacastrum-induced antinociceptive action was evaluated. The pretreatment of rats with the inducible nitric oxide synthase inhibitor, L-NNA, at a dose that produced no significant effect on acetic acid-induced nociception, significantly reversed the

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antinociceptive action of T. portulacastrum (Ishola et al., 2014a). Thus, this findings showed the role of nitrergic pathway in antinociceptive effect of T. portulacastrum (Alves et al., 2004). Our results also

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showed that the pretreatment of mice with methylene blue (soluble guanylate cyclase inhibitor) or glibenclamide (an AETP-sensitive K+ channel inhibitor) did not affect T. portulacastrum-induced antinociception. In summary, T. portulacastrum-induced antinociception is produced through opioidergic and nitrergic signaling (Ishola et al., 2014b). In the anti-inflammatory models, the extracts showed significant inhibition of inflammation in both the carrageenan-induced rat paw swelling and CFA induced arthritis models. Carrageenan induced 19

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oedema is a biphasic event in which a primary acute phase (up to 2h) involves generation and step-wise release of the inflammatory mediators such as histamine, serotonin and bradykinin whereas secondary chronic phase (2 to 5 h) is regulated by neutrophil infiltration in sustained production of arachidonic

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metabolites or nitric oxide (Romero et al., 2005; Queiroz et al., 2013). In the study, T. portulacastrum decreased inflammation in the secondary chronic phase possibly due to inhibition of the neutrophil

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infiltration or release of nitric oxide.

The effect of the extract on chronic inflammation was investigated in CFA-induced arthritic model. CFA

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induces an inflammatory cascade causing swelling and pain lasting 24 h to 12 days. The classical symptoms of this model are swelling of the CFA-injected paw (oedema), redness, allodynia and hyperalgesia. It is well reported that the injection of CFA into the rat hind paw results in marked joint inflammation, characterised by release of various inflammatory mediators such as prostaglandins, cytokines, kinnins, substance P and ROS (Shah et al., 2012; McCarson, 2015). In this study, intraplantar

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injection of CFA produced time course increase in arthritic index and erythrocyte sedimentation rate and oxidative stress (Ishola et al., 2015b). However, the pretreatment of rats with T. portulacastrum produced significant and dose dependent inhibition of oedema, alleviate loss of motor function, reduced

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erythrocyte sedimentation rate, and decreased arthritic index.

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The neutrophil and macrophages infiltration leads to increased generation of reactive oxygen species (ROS) (e.g., superoxide, hydrogen peroxide and hydroxyl radicals) and reactive nitrogen species (e.g., nitric oxide) (Bogdan, 2001). ROS induce an array of tissue damage, destructive oxidation of membrane lipid (lipid peroxidation) and nitric oxide is involved in the development of joint destruction in RA (Del Carlo and Loeser, 2002). Nitric oxide and superoxide anion may react together to produce significant amounts of peroxynitrite anion, which is a potent oxidizing agent that can cause DNA fragmentation and lipid peroxidation (Valko et al., 2007). T. portulacastrum extract significantly attenuated CFA-induced 20

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increase in lipid peroxidation, with a concomitant decrease in glutathione, superoxide dismutase and catalase activities in the spleen, liver and limbs. Thus, the anti-arthritic effect of the extract involved enhancement of antioxidant defense systems. Interestingly, the preliminary phytochemical analysis of T.

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portulacastrum revealed the presence of flavonoids, saponins, alkaloids, terpenoids, phenols and cardiac glycoside. Saponins have previously been reported to display anti-nociceptive, anti-inflammatory and antipyretic activities while terpenoids, phenols and flavonoids possess antioxidant, antinociceptive, and

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anti-inflammatory activities (Mohammad et al., 2004; Aquila et al., 2009). HPLC profiling showed that that the extract contain rutin and quercetin. Moreso, the GC-MS assay demonstrated the presence of

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epoxyδ-lactone ring and cyclohexane moieties. Sesquiterpene lactones are a large group of secondary plant metabolites, exert a broad variety of different biological activities. Therapeutic relevance of sesquiterpene lactones and epoxide derivatives in inflammation, especially arthritis have been reported (Zarpelon et al., 2017; Choodej et al., 2018). Additionally, the epoxide derivative 6A: might represent a

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lead compound for further anti-TNF-α therapies, owing to its potent activity and reduced toxicity (Choodej et al., 2018). The antinociceptive and anti-inflammatory effects of AETP could be attributed to the presence of these phyto-active components. However, further study will be carried out to isolate,

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characterize and elucidate the active principles in AETP responsible for the observed pharmacologic

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effect. Conclusion

Findings from this study suggest that the aqueous whole plant extract of T. portulacastrum possesses both peripheral and central anti-nociceptive effects through interaction with nitrergic and opioidergic systems as well as anti-inflammatory action through inhibition of inflammatory cells release and enhancement of antioxidant defense system. Thus, confirms its use in the folklore for the treatment of inflammatory conditions. 21

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Acknowledgement The authors are grateful to Mr. S.O. Adenekan (Department of Biochemistry), and Mr M.C Chijioke of

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the Department of Pharmacology, Toxicology and Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria, for technical assistance provided. References

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Cytokines. Inflammation, 40(6):2020-2032

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List of tables Table 1: time course effect of ATP on hot plate-induced nociception in mice Treatment(mg/kg)

0

30

60

90

120

150

180

2.44±0.28 2.34±0.11

1.98±0.25

1.94±0.14

1.88±0.13 1.66±0.15 1.54±0.14

ATP 10

2.00±0.25 3.0±0.38

2.92±0.44

2.36±0.21

2.14±0.18 1.94±0.14 1.20±0.03

11.72

5.21

3.02±0.30

2.84±0.30

12.97

11.17

2.86+0.20

2.72+0.14

10.97

9.68

5.67

7.43

4.73

4.6 ± 0.30 6.0 ± 0.70b 6.2 ± 0.70b

4.5 ± 1.00

2.5 ± 0.40

2.0±0.35

26.70

3.90

3.10

2.42±0.24 3.62±0.12 0.26

ATP 250

2.18±0.28 4.10+0.29 3.44

Morphine 10

16.71

2.0 ± 0.20 5.82

a

22.98

3.2

3.36

20.60 42.00

4.02

2.28±0.12 1.56±0.14 1.22±0.08 17.54

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ATP 50

8.62

4.93

3.78

2.34+0.16 2.28+0.15 1.94+0.22

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5.82

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Vehicle

44.10

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Values are expressed as mean ± S.E.M. (n=5). ap < 0.05, bp<0.01, cp<0.001 versus vehicle-control treated. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test. [Note: values in bold represent maximum possible effect (MPE)]

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2h

3h

6h

24 h

Vehicle

0.90±0.13

1.64±0.2

1.65±0.16

1.58±0.17

0.70±0.18

ATP 10

0.98±0.09 8.89

1.71±0.04 4.27

1.31±0.09 20.61

1.43±0.19 9.49

0.51±0.07 27.14

ATP 50

0.91±0.09

1.45±0.23

1.29±0.10

1.17±0.08

1.11

11.59

21.82

ATP 250

0.84±0.06 6.67

1.20±0.09 26.83

1.07±0.09a 35.15

Ibuprofen 100

0.74±0.09

1.22±0.08

0.92±0.06b

17.78

25.61

44.24

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1h

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0.56±0.13

25.95

20.00

0.78±0.16 b 50.63

0.39±0.13a 44.28

0.66±0.12a

0.35±0.07

58.22

50.00

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Treatment (mg/kg)

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Table 2: time course effect of ATP on carrageenan-induced paw oedema in rats

AC C

EP

Values are expressed as mean ± S.E.M. (n=5). ap < 0.05, bp<0.01, cp<0.001 versus Vehicle + CFA. Statistical level of significance analysis by twoway ANOVA followed by Bonferroni post hoc multiple comparison test. [Note: values in Bold represent percentage inhibition of oedema].

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Table 3: time course effect of ATP on CFA-induced Arthritis in rats Change in paw circumference (mm) Days 6 0.09±0.02

9 0.11±0.02

12 0.14±0.02

15 0.16±0.02

CFA

0.64±0.07

1.54±0.18

2.29±0.20

2.3±0.16

3.07±0.31

2.41±0.14

ATP 10

0.58±0.06

1.33±0.11

2.07±0.21

2.33±0.22

2.25±0.30b

9.38

13.64

6.94

1.3

26.71

0.44±0.11

0.93±0.20

1.55±0.31

1.96±0.28

31.25

39.61

32.31

0.33±0.06

0.82±0.15a

48.44 CEL 50

21 0.24±0.05

24 0.36±0.04

27 0.37±0.04

2.30±0.17

2.26±0.13

2.15±0.12

1.98±0.11

1.94±0.21

1.87±0.21

1.77±0.21

1.58±0.17

1.12±0.27b

19.5

18.7

21.68

26.51

43.43

2.08±0.40c

1.95±0.28

1.61±0.44a

1.41±0.18b

1.35±0.18a

1.09±0.12b

14.78

32.25

19.09

30.43

38.88

37.21

43.4

1.48±0.34

1.77±0.40

2.00±0.28c

1.78±0.37

1.49±0.21b

1.26±0.26c

1.05±0.26c

0.82±0.19c

46.75

35.37

23.04

34.85

26.14

35.22

44.25

51.16

58.56

0.30±0.04

0.88±0.09

1.45±0.12

1.74±0.16

1.55±0.16c

1.47±0.14b

1.32±0.13c

1.26±0.13c

1.18±0.14b

0.78±0.09c

53.13

42.86

36.68

24.35

49.51

39.04

42.61

44.23

45.12

60.61

SC

M AN U

ATP 250

18 0.21±0.03

RI PT

0.29±0.03

3 0.02±0.03

ATP 50

1

TE D

Treatment (mg/kg) Vehicle

AC C

EP

Values are expressed as mean ± S.E.M. (n=8). ap < 0.05, bp<0.01, cp<0.001 versus Vehicle + CFA. Statistical level of significance analysis by twoway ANOVA followed by Bonferroni post hoc multiple comparison test. Values in Bold represent percentage inhibition of oedema.

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M AN U

SC

RI PT

List of figures

AC C

EP

TE D

Figure 1A: The chromatogram of mixture 100 µg/mL of rutin and quercetin standard

Figure 1B: The chromatogram of plant extract (name of plant) indicating presence of rutin and quercetin.

M AN U

SC

RI PT

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AC C

EP

TE D

Figure 2: effect of AETP on acetic acid-induced mouse writhing test. Values are expressed as mean ± S.E.M. (n=6). *p < 0.05, **p<0.01, ***p<0.001 versus vehicle-control treated. Statistical level of significance analysis by one way ANOVA followed by Tukey post hoc multiple comparison test.

AC C

SC

EP

TE D

M AN U

Mean number of writhes in 30 min

RI PT

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Fig. 3A-E: effect of (a) naloxone (NAL) (5mg/kg, s.c.), (b) prazosin (PRAZ) (1mg/kg, i.p.) or yohimbine (YOH) (1 mg/kg, i.p.), (c) ondansetron (ONDA) (0.5mg/kg, i.p.) or glibenclamide (GLIB) (10mg/kg, i.p.), (d) L-arginine (L-ARG) (150mg/kg, i.p.) or L-nitro-arginine (L-NNA) (10mg/kg, i.p.), and (e) methylene blue (MB) (2 mg/kg, i.p.) on AETP-induced antinociception in mice. Values are expressed as mean ± S.E.M. (n=6). # p < 0.05 versus AETP (250 mg/kg), ***p<0.001 versus vehicle-control treated. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test.

SC

RI PT

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AC C

EP

TE D

M AN U

Fig. 4: effect of AETP on arthritic index in rats. Values are expressed as mean ± S.E.M. (n=8). *p < 0.05 versus Vehicle + CFA. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test.

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body weight (g)

b

180

b

b a

a

160

a a

140 120

28 ay

SC

D

D

D

ay

ay

21

14

7 ay D

D

ay

1

100

CEL 50 AETP 250 AETP 50 AETP 10 CFA vehicle 10 ml/kg

RI PT

200

AC C

EP

TE D

M AN U

Fig. 5: effect of CFA and AETP on body weight in rats. Values are expressed as mean ± S.E.M. (n=8). ap < 0.05, bp < 0.01 versus Vehicle + CFA; αp < 0.05; βp < 0.01 versus vehicle control. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test.

TE D

M AN U

SC

RI PT

Number of line crosses

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AC C

EP

Fig. 6A-B: effect of AETP on (a) spontaneous motor activity (b) number of stairs climbed in CFA-induced motor deficits. Values are expressed as mean ± S.E.M. (n=6). #p < 0.05 versus vehicle-control treated, *p<0.05, ** p<0.01 versus CFA-vehicle treated. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test

SC

RI PT

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AC C

EP

TE D

M AN U

Fig 7: effect of AETP on leukocyte count (WBC) and erythrocyte sedimentation rate in CFA-induced arthritic rats. Values are expressed as mean ± S.E.M. (n=6). *p < 0.05, **p < 0.01 versus Vehicle + CFA; ##p < 0.01, ###p < 0.001 versus vehicle control. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test.

D

Vehicle CFA AETP 10 AETP 50 AETP 250 CEL 50

M AN U

25

SC

RI PT

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20 15 10

cc # cc

#

TE D

5 0

Spleen

Liver

Limb

AC C

EP

Fig. 8A-D: effect of AETP on (a) MDA level, (b) GSH level, (c) SOD activity, (d) Catalase activity in the liver, spleen and limbs of rats. Values are expressed as mean ± S.E.M. (n=6). #P<0.05 versus vehicle control; ap< 0.05, bp<0.01, cp<0.001 versus vehicle + CFA. Statistical level of significance analysis by two way ANOVA followed by Tukey post hoc multiple comparison test.

SC

RI PT

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AC C

EP

TE D

M AN U

Figure 9A-F: Micrograph showing right hind limb histological section of soft tissue overlying the skin and skeletal muscle tissues in treated rats. A: vehicle control [no area of inflammation seen]; B: CFA induced arthritic rat [Intense mixed inflammatory infiltrates seen]; C: AETP 10 mg/kg treated [Moderate mixed inflammation seen]; D: AETP 50 mg/kg treated [Mild to moderate inflammation seen]; E: AETP 250 mg/kg treated [reduced infilterates seen], and F: Celecoxib 50 mg/kg treated [Mild inflammation seen] ×100.

AC C

EP

TE D

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SC

RI PT

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Supplementary 1: GC-MS chromatogram of AETP