Analgesic and anti-inflammatory activities of aerial parts of Flacourtia sepiaria Roxb.

Sreejith. M¹*, Kannappan. N², Santhiagu. A³, Ajith. P. M⁴

¹Assistant Professor, Department of Pharmaceutical Chemistry, National College of Pharmacy, Calicut, Kerala

²Associate Professor, Department of Pharmacy, Annamalai University, Annamalai Nagar 608002, Tamil Nadu.

³Associate Professor and HOD, Department of Biotechnology, National Institute of Technology, Calicut, Kerala

⁴Assistant Professor, Department of Pharmacology, National College of Pharmacy, Calicut, Kerala

*Corresponding Author E-mail: aksreephd@gmail.com

ABSTRACT:

Aim: To investigate the possible anti-inflammatory and analgesic effects of the crude methanol (MEF) and ethyl acetate (EAEF) extracts of the aerial parts of Flacourtia sepiaria Roxb belonging to the family Flacourtiaceae.

Methods: Anti-inflammatory property of the MEF and EAEF extracts was tested by carrageenan induced acute paw edema in rats. For evaluating analgesic effects, acetic acid induced writhing test in mice and tail immersion tests in rats were conducted. The in vitro cyclooxygenase (COX) inhibitory activity was also conducted using COX inhibitory assay kit.

Result: Both MEF and EAEF of Flacourtia sepiaria were found to have in vitro and in vivo anti-inflammatory effects. The most potent drug was found as diclofenac sodium (67%) in 6 h measurement, while MEF (65%) and EAEF (55.17%) of Flacourtia sepiaria in 400 mg/kg dose were found to have significant ability to reduce inflammation, with respect to control values. The extracts MEF and EAEF showed selective inhibitory activities against in vitro cyclooxygenases (COXs) inhibition assay. The MEF and EAEF decreased the acetic acid induced abdominal contraction and also significantly increased the reaction time of tail immersion test. These results showed that the plant extracts had both central and peripheral analgesic action.

Conclusion: Thus the MEF and EAEF extracts of the aerial parts of Flacourtia sepiaria has significant anti-inflammatory and analgesic activities.

KEYWORDS: Anti-inflammatory, Analgesic, Flacourtia sepiaria, Cyclooxygenase.

1. INTRODUCTION:

Inflammation or phlogosis is a pathophysiological response of living tissues to different factors such as infections, chemicals, thermal and mechanical injuries that leads to the accumulation of plasmatic fluid and blood cells [1]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most clinically important medicine used for the treatment of inflammation-related diseases like arthritis, asthma, and cardiovascular disease [2]. NSAIDs are among the most widely used medications due to their efficacy for a wide range of pain and inflammatory conditions. However, the long-term administration of NSAID may induce various adverse effects like gastro-intestinal ulcers, bleeding and renal disorders [3].

Thus, there has been many efforts to discover newer plant based active principles retaining potent anti-inflammatory efficacy but with very minimum side effects. The research into plants with alleged folkloric use as pain relievers, anti-inflammatory agents, should therefore be viewed as a fruitful and logical research strategy in the search for new analgesic and anti-inflammatory drugs [4].

Flacourtia sepiaria Roxb belonging to the family of Flacourtiaceae is a medium sized tree widely distributed in the dry jungles of Bengal, Bihar, Orissa and all districts of the Madras presidency. Various parts of the plant are widely used in folk medicine, an infusion of the leaves is given in case of snake bites and its bark triturated with sesame oil is used as a liniment in rheumatism and gout. The ashes of root are also given in kidney diseases [5]. It has also been proved to posses antimicrobial [6], xanthine oxidase inhibitory and antioxidant activity [7]. However, no such report is available in the literature regarding anti-inflammatory and analgesic activities.

Thus the purpose of the present study was to investigate the methanol (MEF) and ethyl acetate (EAEF) extract of the aerial parts of Flacourtia sepiaria Roxb for its in vitro and in vivo anti-inflammatory activity using cyclooxygenases (COXs) inhibitory assay and carrageenan-induced rat paw edema model. The antinociceptive properties of the extracts MEF and EAEF was also evaluated and compared with commonly used NSAIDs, using the inhibition of the number of writhes induced by acetic acid in mice and the increase in reaction time in tail immersion test in rats.

2. Materials and Methods:

2.1 Plant material and extracts preparation

The aerial parts of Flacourtia sepiaria Roxb were collected from Tirunalveli district, Tamilnadu, India during the month of March 2011. The plant was identified and authenticated by Mr. Chelladurai, Research Officer- Botany, Central Council for Research in Ayurveda and Siddha, Government of India (Ref No:- NCP/CH/PS02)

The aerial parts of Flacourtia sepiaria were collected, shade dried, powdered mechanically and sieved through No. 20 mesh sieve. About 100g of the powdered aerial part is first defatted with petroleum ether (PEF, 60º-80ºC) and then consecutively extracted with ethyl acetate (EAEF) and methanol (MEF) by soxhlet extraction. The crude extracts were concentrated and dried at room temperature [7].

2.2 HPLC analysis of extracts

The HPLC analyses of the extracts were carried out with chromatographic system (Shimadzu LC-2010CHT) with UV and PDA detector in combination with class LC solution software. The separation was carried on Kromasil C18 (250 x 4.6mm, 5μm) column at temperature 30 ± 1ºC. The mobile phase consists of water and acetonitrile and the separation were performed by gradient elution mode, elution performed at a flow rate of 1.5 ml/min. The sample dissolved in methanol, filtered and injected (20μl) to HPLC and detected at 330nm. Steroids were identified by comparison with chromatographic standards using their retention time, as parameter to identify the compounds [8].

2.3 Animals

Wistar rats (180–220 g) and Swiss albino mice (20–25 g) of either sex, were brought from Central animal house, K. M College of Pharmacy, Madurai. Animals were housed at standard conditions of temperature (22 ± 1◦C) and 12/12 h light/dark cycle. They were fed with standard pellet diet and had free access to water. Six animals are used in each group. Permission for conduct of these experiments was obtained from, Institutional Animal Ethics Committee (Ref no: 661/02/c/CPCSEA)

2.4 Acute Toxicity Study

Toxicity studies were conducted as per internationally accepted protocol drawn under OECD guidelines 423 in Swiss albino mice.

2.5 In vivo anti-inflammatory activity

Wister rats of either sex with a body weight between 180-220 g were used. The animals were fasted for 18 hours prior to the experiment. Animals were divided into six groups of six animals each. Group I received normal saline (0.9%) and served as control. Group II received Diclofenac sodium 50mg/kg b.w i.p and served as standard. Group III, IV, V and VI received the MEF and EAEF extract respectively at dose of 200 mg/kg and 400 mg/kg b.w orally. One hour after oral administration, an injection of 0.1ml of carrageenan (1% carrageenan suspended in 0.9% NaCl) was made into the right hind limb of each rat under the subplantar aponeurosis. Measurement of paw volume was done by means of volume displacement technique using plethysmometer (Ugo Basile Italy) immediately after carrageenan injection and after 6 hour. The results are tabulated by % of inhibition [9].

(vt-vo)Control- (vt-vo)treated

Percentage inhibition = ------------------------------------X 100

(vt-vo)Control

Vt is the average volume for each group after treatment and Vo is the average volume for each before treatment.

2.6 Antinociceptive activity

2.6.1 Writhing test in mice

The abdominal writhing response to the acetic acid (1%, 10 ml/kg i.p) administration involves contractions of the hind limbs. The number of abdominal writhing was recorded for a period of 10 min, starting 5 min after the administration [10]. For the writhing tests, mice received acetic acid injection 30 min after receiving their respective treatment. Group I was pretreated with saline solution (0.9%; 0.1 ml/10 g b.w, i.p). Group II was pretreated with diclofenac (5mglkg b.w, i.p).Group III, IV, V, VI were treated with MEF and EAEF extracts (200 mg/kg b.w o.d; 400 mg/kg b.w, orally) respectively. Antinociception was calculated as percentage of inhibition of writhing constrictions.

(Control group-test group)

Percentage inhibition =----------------------------------- X 100

(Control group)

2.6.2 Tail immersion test in rats

The tail immersion test basically involves the measure of the response latency of rats to a nociceptive stimulus. The procedure was performed following the method of Aydin et al [11]. This involves introducing 3 cm of the rat’s tail in hot water at a temperature of 55 ± 0.5^◦C. Within a few minutes, the rats reacted by withdrawing the tail. The reaction time was recorded with a stopwatch. The animals were treated by MEF and EAEF extracts (200 and 400 mg/kg), or water (vehicle) or standard drug (morphine, 10 mg/kg), 30min before the immersion of the tail. The time reaction is taken at 30, 60, 90 and 120 min after administration of different preparations.

2.7 Cyclooxygenase (COX) inhibitor screening assay

The inhibitory activities against COX 1 and COX 2 were determined using a colorimetric COX (ovine) Inhibitor Screening Assay Kit (cayman chemicals Co.) according to the manufacturer’s protocol [12]. The inhibitory activities of the MEF and EAEF extracts were measured by monitoring the production of oxidized N,N,N’,N’ tetramethyl p phenylenediamine (TMPD) at 590 nm followed by incubation of either COX 1 or COX 2 with arachidonic acid.

The enzymes were preincubated for 5 min at 25ºC with the test compound (5-80 μg/ml) before addition of arachidonic acid (1.1 mM) and TMPD and incubation for 5 min at 25 ºC. The COX inhibiting activity was calculated according to the equation,

(At-Ao)

COX inhibiting activity (%) = [1- --------- ] X 100

(Ac)

Where Ac was the absorbance of the control (without the test compound), At was the absorbance in the presence of the test compound and Ao was the absorbance of sample blank (without TMPD). The IC₅₀ value of the extracts was calculated using a calibration curve (Yang et al., 2009).

2.8 Statistical analysis

All values were expressed as mean ± SEM, and data was analyzed by one way analysis of variance (ANOVA) followed by newmann keuls multiple range tests using GraphPad InStat.

Table 1: Protective effect of MEF and EAEF Extract of Flacourtia sepiaria Roxb (MEFS) on paw edema induced by carrageenan in rat.

Treatment	Dose (mg/kg)	Paw volume(ml) as measured by mercury displacement at 6 hour	Percentage inhibition of paw edema
Group I Normal saline	10ml/kg orally	5.60±0.98	-
Group II Std	50mg/kg I.P.Diclofenac sodium	1.80±0.32	67.85%*a
Group III MEFS	200mg/kg through orally.	2.10±0.52	62.50%*a
Group IV MEFS	400mg/kg through orally.	1.96±0.60	65.00%*a
Group V EAEF	200mg/kg through orally.	2.72±0.32	51.42%*a
Group VI EAEF	400mg/kg through orally.	2.51±0.44	55.17%*a

* Data are expressed as Mean ± S.E.M. *Data were analyzed by one way ANOVA followed by Newman’s keul’s multiple range tests, to determine the significance of the difference between the control group and rats treated with the test compounds. *a Values were significantly different from normal control at P< 0.01.

Table.2: Analgesic activity of MEF and EAEF extract of Flacourtia sepiaria Roxb by acetic acid induced writhing reflux in mice.

Treatment	Dose (mg/kg)	No. of writhing	% reduction in reaction time
Group I Normal saline	Inject 1%v/v acetic acid 1ml/100g(b.w)	38.2±4.6	-
Group II Std	50mg/kg (i.p) Diclofenac sodium	6.4±0.9	83.24%**
Group III MEFS	200mg/kg through orally.	13.2±1.6	65.44%**
Group IV MEFS	400mg/kg through orally.	12.4±1.2	67.53%**
Group V EAEF	200mg/kg through orally.	17.1±1.6	55.23%**
Group VI EAEF	400mg/kg through orally.	15.9±1.4	58.37%**

Values are expressed as mean±SEM. Values were find out by using one-way ANOVA followed by Newman’s keuls multiple range tests. ** Values were considered significant at P< 0.001.

Fig. 1 HPLC profile of Methanol extract (MEF) of Flacourtia sepiaria. (1)- β-sitosterol.

Fig. 2 HPLC profile of Ethyl acetate extract (EAEF) of Flacourtia sepiaria. (1)- β-sitosterol and (2)- Stigmasterol.

Table 3: Protective effect of MEF and EAEF extract of Flacourtia sepiaria Roxb on tail withdrawal reflex induced by tail immersion (reaction time).

Groups	Drug (dose), route	30 min	60min	90 min	120 min
Group I Normal saline	10ml/kg (b.w)	1.3 ± 0.1	1.4 ± 0.2	1.8 ± 0.2	2.5 ± 0.2
Group II Std	10mg/kg (i.p) Morphine	6.4 ± 0.8**	8.5 ± 0.9**	8.6 ± 0.8**	8.9 ± 0.9**
Group III MEFS	200mg/kg through orally.	5.9 ± 0.6 **	6.2 ± 0.7**	6.4 ± 0.6**	6.6 ± 0.8**
Group IV MEFS	400mg/kg through orally.	6.8 ± 0.7**	7.2 ± 0.9**	7.6 ± 1.0**	7.8 ± 1.3**
Group V EAEF	200mg/kg through orally.	5.1 ± 0.8**	5.3 ± 1.4**	5.6 ± 0.6**	5.9 ± 0.2**
Group VI EAEF	400mg/kg through orally.	5.7 ± 0.6**	6.0 ± 0.8**	6.2 ± 0.2**	6.5 ± 1.2**

Values are expressed as mean±SEM. Values were find out by using one-way ANOVA followed by Newman’s keuls multiple range tests. ** Values were considered significant at P< 0.001.

Table 4. Inhibitory effects of MEF and EAEF extract of Flacourtia sepiaria Roxb on ovine COX 1 and COX 2 activity in vitro.

Plant extracts	IC50 (μg/ml)		COXs selectivity*
Plant extracts	COX 1	COX 2	COXs selectivity*
MEF	82.20 ± 0.4	34.22 ± 0.8	2.40 ± 0.6
EAEF	94.80 ± 0.6	78.40 ± 0.2	1.21 ± 0.8
Diclofenac Sodium**	0.39 ± 0.2	0.370 ± 0.6	1.07 ± 0.2

Values are expressed as mean±SEM. *COX selectivity was determined by IC50 COX 1/ IC50 COX 2. **Positive control.

3.4.2 Tail immersion test in mice

Table 3 shows the effects of the methanol and ethyl acetate extract (200 and 400 mg/kg b.w) of Flacourtia sepiaria in the tail immersion test in rats. A significant (p< 0.05) increase in the reaction time was observed in the tail immersion test in rats after 30, 60, 90 and 120 min when compared with the control. Morphine (standard) at dose of 10 mg/kg b.w exhibited more potent activity at 30, 60, 90 and 120 min than the extracts.

3.5 Cyclooxygenase (COX) inhibitor screening assay

As shown in Table 4, MEF and EAEF extracts of Flacourtia sepiaria showed selective inhibitory activity against COXs. MEF extract showed more potent inhibitory activity with a measure of COX 2 selectivity greater than 2.40 ± 0.6.

4. Discussion and conclusion:

Carrageenan induced edema has been commonly used as an experimental animal model for acute inflammation and is believed to be biphasic [14]. The early phase (1-2 h) of the carrageenan model is mainly mediated by histamine, serotonin and increased synthesis of prostaglandins in the damaged tissue surroundings. The late phase is sustained by prostaglandin release and mediated by bradykinin, leukotrienes, polymorphonuclear cells, and prostaglandins produced by tissue macrophages [15]. Since the extract significantly inhibited paw edema induced by carrageenan in the second phase, this finding suggests a possible inhibition of cyclooxygenase synthesis by the extract and this effect is similar to that produced by nonsteroidal anti-inflammatory drugs such as diclofenac sodium, whose mechanism of action is inhibition of the cyclooxygenase enzyme [16]. This has been further confirmed by the in vitro COXs inhibitory assay, where the extracts exhibited potent selective COX 2 inhibitory activity. Thus it shows more selective action towards the inducible COX 2 rather than the COX 1 responsible for housekeeping functions [17]. It is important to point out that the extracts of Flacortia sepiaria are capable of causing lesser side effects compare to other non selective COX inhibitors.

The brain and spinal cord play a major role in central pain mechanisms. The dorsal horn of the spinal cord is endowed with several neurotransmitters and receptors including substance P, somatostatin, neuropeptide Y, inhibitory amino acid, nitric oxide, endogenous opioids, and the monoamines which are the major targets for pain and inflammation [18]. The tail immersion test was considered to be selective to examine compounds acting through opioid receptor. Both the extracts increased pain threshold which means basal latency, which indicates that it may act via centrally mediated analgesic mechanism.

On the other hand, acetic acid-induced writhing model represents pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from the tissue phospholipid[19]. The acetic acid induced writhing response is a sensitive procedure to evaluate peripherally acting analgesics. The response is thought to be mediated by peritoneal mast cells [20], acid sensing ion channels [21], and the prostaglandin pathways [22]. The MEF and EAEF extract inhibited acetic acid induced writhing indicating its peripheral analgesic activity.

Moreover, MEF extract showed highest analgesic activity in all the experimental model which may be due to its high flavonoid contents which are responsible for free radical scavenging activity, as these free radicals are involved during pain stimulation, and antioxidants showed reduction in such pain [23]. The results of the present study have shown that the crude extract of the investigated plant exhibited very high anti-inflammatory and analgesic activities. These activities may be linked with the presence of polyphenolic compounds present in the extract.

Flavonoids, steroids and saponins are well known for their ability to inhibit pain perception as well as anti-inflammatory properties due to their inhibitory effects on enzymes involved in the production of the chemical mediator of inflammation [24]. The ability of flavonoids and sterols like β-sitosterol to inhibit eicosanoid biosynthesis has been documented [25, 26]. Eicosanoids, such as prostaglandins, are involved in various immunological responses and are the end products of the cyclooxygenase and lipoxygenase pathways [27]. Further, flavonoids are able to inhibit neutrophils degranulation and thereby decrease the release of arachidonic acid [28].

Thus, the presence of polyphenolics like flavonoids and identified steroids like β-sitosterol in the extracts of Flacourtia sepiaria Roxb (MEF and EAEF) might be responsible for the anti-inflammatory and analgesic activity. Furthermore, these compounds might be helpful in treating or slowing the progress of inflammation and pain. But, further investigations on the isolation of active compounds present in the extracts are necessary to identify a potential chemical entity for clinical use in the treatment of inflammation and pain.

5. Acknowledgement:

We are grateful to Dr. N. Chidambaranathan, Head, Department of Pharmacology, K.M. College of Pharmacy, Madurai for providing lab facilities for this research work.

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Received on 31.01.2015 Accepted on 25.02.2015

Asian J. Res. Pharm. Sci. 5(1): Jan.-March 2015; Page 12-17

DOI: 10.5958/2231-5659.2015.00003.X