Pharmacological Evaluation of Trichilia connaroides Bark for Analgesic and Anti-inflammatory activity in Experimental Animal Models

Muhammed Shakkeel K.V¹*, Anjan Kumar¹, Veeresh Babu. D¹, Narayana Swamy V.B²

¹Department of Pharmacology, Karavali College of Pharmacy, Mangalore.

²Department of Pharmacognosy, Karavali College of Pharmacy, Mangalore

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

ABSTRACT:

The present study was designed to evaluate the analgesic and anti-inflammatory activity of “Trichilia connaroides” bark using different animal models. Analgesic activity was evaluated by using various two animal models namely, Writing Test using acetic acid in mice and Hot Plate Method. The degree of analgesic activity was determined by the number of wriths was recorded for each animal in Writing Test using acetic acid in mice model and the delay in reaction time for of each animal when place in hot plate maintained at 55±0.10c is recorded in Hot Plate Method. Anti-inflammatory activity was evaluated using Formalin induced paw edema model, Croton oil ear edema in rats. Trichilia connaroides bark ethanolic extracts produced significant analgesic activity in both Hot plate and acetic acid induced writhing models in mice. In hot plate method percentage increase in reaction time was determined where as in acetic acid induced writhing model percentage decrease in writhings was determined. Evaluation of anti inflammatory activity was done by Formalin induced paw edema model, Croton oil ear edema model. In Formalin induced paw edema model mean change in paw volume and percentage protection were calculated. In croton oil ear edema model the difference between untreated ear and treated ear were determined which indicated degree of inflammatory edema. The study revealed that the “Trichilia connaroides”bark possess a significant analgesic and anti-inflammatory activity.

KEY WORDS: “Trichilia connaroides” bark, Writing Test using acetic acid, Hot Plate Method, Formalin induced paw edema model, Croton oil ear edema in rats.

INTRODUCTION:

Trichilia is a flowering plant genus in the family Meliaceae. These plants are particularly diverse in sub-Saharan Africa and tropical South America. The genus name “Trichilia” is derived from Greek “tricho” referring to the 3-lobed fruits. Several species of Trichilia were used in folk medicine and shamanism – e.g. T. rubescens against malaria,¹ T. catigua was used as aphrodisiac and body stimulant,² T.emetica as an anticonvulsant³ and T.connaroides as an analgesic.⁴

Trichilia is a genus of trees, widely distributed in south east of Asia. Trichilia connaroides (W. & A.) Bentilizen, belonging to family Meliaceae well known as karai, karavilangu found in Kerala, Tamilnadu and Andhra pradesh. T.connaroides are distributed in Western ghats and in Central Sahyadris. Phytochemical screening of leaves have been reported by several authors like isolation of two tetracyclic triterpenoids having a 9,19-cyclopropane ring and two new tetranortriterpenoids-trijugin A and B from chloroform extract of T. connaroides.⁵

A new limonoid, trichiliton B was isolated from the twigs and leaves of T. connaroides. Preliminary phytochemical screening of chloroform extract of T. connaroides revealed presence of flavonoid compounds. Flavonoids are known to target prostaglandins, which are involved in the late phase of acute inflammation and pain perception. ⁶ Some of the activities attributed to flavonoids include: anti-allergic, anti-tumour, antibacterial, spasmolytic and estrogenic effects.^7-12 They act in plants as photoreceptors, visual attractors, feeding repellants. Many studies have suggested that flavonoids exhibit analgesic, anti-inflammatory, anti-viral, anti-microbial, anti-cancer activities.^13-18 Flavonoids are formed in plants from aromatic amino acids phenylalanine, tyrosine and malonate.But till now there is no study has been carried out which indicates the effect of Trichilia connaroides on analgesic and anti-inflammatory activity. So the present study has been designed to evaluate the analgesic and anti-inflammatory activity of Trichilia connaroides using different experimental models.

MATERIALS AND METHOD

The Trichilia connaroides bark belonging to the family Meliaceae were collected from Kesavans Plantations (Dealers in Ayurvedic Pharmaceuticals) Wayanad, Kerala It is preserved in the departmental library for future reference.

Animal selection

Swiss albino mice weighing 18-30 gm, and albino rats of wistar strain weighing 200-250g were used for the study. The mice and rats were inbred in the central animal house of the Department of Pharmacology, Karavali College of Pharmacy, Mangalore, under suitable conditions of housing, temperature, ventilation and nutrition were used for antidepressant activity. They were kept in clean dry cages week before the beginning of the experiment to acclimatize with the experimental conditions. The animals were fed with standard pelleted diet (Lipton India Ltd., Mumbai) and distilled water and libitum was maintained at 21⁰C-23⁰C under a constant 12hrs light and dark cycle. The animal care and experimental protocols were in accordance with CPCSEA /IAEC.

Preparation of test solution

Naturally available wood of Trichilia connaroides will be collected and the bark collected will be shade dried followed by powdering the bark to obtain the bark in coarse form which will be defatted with hexane followed by soxhlet extraction with ethanol and the the drug extract will be concentrated using rotary evaporator. The extract will be suspended in 1% tween 80 and administered orally.

A. ANALGESIC ACTIVITY

1. Hot plate test using mice:^19-22

Albino mice were randomly grouped into six groups of six animals each. Group I served as control. BVE and BVA 200 and 400 mg/kg body weight, p.o., and the standard tramadol at 5 mg/kg body weight i.p., was administered to the animals of group II to group VI respectively. The delay in response time (Jumping and hind paw licking response) of animals when placed on the hot plate which was maintained at 55 ± 1ºC was recorded at 0,30,45,60,90,120 and 180 min. the percentage increase in reaction time was calculated.

Percentage protection against thermal pain was calculated by applying the formula:

% Protection against thermal pain = (Ta– T_b) x 100/ T_b

Where, Ta – Mean reaction time of test and Tb – Mean reaction time of control.

2. Abdominal writhing test using acetic acid in mice:^19-22

Albino mice were used for the study and were divided into six groups of six animals in each. Group I served as control. The II, III, IV and V group animals received BVE (Trichilia connaroides. bark ethanolic extract) and BVA (Trichilia connaroides. aqueous extract) 200 and 400 mg/kg body weight respectively by oral route. Group VI animals received the standard drug indomethacin 10 mg/kg body weight by oral route.

Writhings were induced 30 min later by i.p. injection of 0.1 ml of 0.6 % acetic acid to all animals of the various groups. The numbers of writhes were counted for 20 min, starting immediately after acetic acid injection. Percentage protection was calculated for all the groups by applying the formula:

Writhings in control – Writhings in test

Percentage protection =____________________________________

Writhings in control

Statistical Analysis:

The data were expressed as mean ± SD. Results were analysed statistically by one-way analysis of variance (ANOVA) followed by Dunnet and Tukey’s test. P-value <0.05 was regarded as statistically significant.

ANOVA (Analysis of variance)

In statistics, analysis of variance is a collection of statistical models and their associated procedures, in which the observed variance is partitioned into components due to different explanatory variables. In its simplest form ANOVA gives a statistical test of whether the means of several groups are all equal and therefore generalize Dunnett’s multiple comparison tests to more than two groups.

B. ANTI-INFLAMMATORY ACTIVITY

1. Formalin induced paw edema model^19-22

Animal Used: Albino Rats of Wistar strain

Chemical agent used to induce inflammation: Formalin (0.1ml injected intra peritoneally to subplanatar region of left hind paw)

Route Of Administration: Oral^*

The method²⁰was used for this study. Animals were divided into five groups denoted as Control group, Positive control group (Standard-Ibuprofen 100mg/kg) group), Test group I (TCE-100), Test group II (TCE-200) and Test group III (TCE-400). Each group consisting of 6 albino wistar rats.

Control group received orally 0.1ml of 1% suspension in sodium CMC at the dose of 10 ml/kg body weight and Positive control group received orally at the dose of 100mg/kg body weight. Test group I and Test group II, Test group III were treated with test Sample orally at the dose of 100,200 and 500 mg/kg body weight. 0.2 ml of 3 % formalin was injected into the dorsal surface of the left hind paw of rats 1 h after oral administration of the extracts. The time spent by each animal in licking the injected paw was observed for 5 min. (from 0- 5min post formalin injection) and 10 min (from 20-30 min post formalin injection). The mean of the licking time was determined and compared with the mean for the control group.

2. Croton oil ear edema^19-22

Albino rats were divided into five groups of six animals each. Animals were treated orally with the extract (TCEE 100, TCEE 200 and TCEE 400 mg/kg), Ibuprofen (100 mg/kg) and distilled water (3 ml/kg). Thirty minutes later, edema was induced in each rats group by applying a drop of croton oil to the inner surface of the right ear. After 15 min, the animals were sacrificed under ether anesthesia and both ears cut off, sized and weighed. The anti inflammatory activity was expressed as the difference in (weight of untreated ear – weight treated ear) of edema in the treated mice in comparison with the control rats.

RESULTS:

A. ANALGESIC ACTIVITY OF TRICHILIA CONNAROIDES BARK

The ethanolic extracts of bark of Trichilia connaroides were evaluated for analgesic activity by hot plate and acetic acid induced writhing models, the results obtained are as follows,

1. Hot plate method:

The ethanolic extracts significantly and dose dependently protected the mice against thermally induced pain stimulus. All the extracts at various time intervals at which they were tested produced increase in reaction time. The comparison of analgesic activity with the standard drug Tramadol at various time intervals is as follows. At 30 min, only TCEE 400 produced analgesic activity comparable (P<0.05) to that of standard. The percentage protection against thermally induced pain stimulus by TCEE 400 and the standard drug, tramadol was 85.34±5.22 and 69.84±6.74 respectively. At 45 min TCEE 400 produced analgesic activity comparable (P<0.05) to that of tramadol, the percentage protection was 75.92±7.21 and 81.42±5.30 respectively. At 60 min TCEE 200 and 400 produced analgesic activity comparable (P<0.05) to that of tramadol. At 90, 120 and 180 min, all extracts at all doses produced analgesic activity better (P<0.01) than tramadol.

Table 1:Analgesic effect of Trichilia connaroides ethanolic extract (TCEE) and tramadol in mice by hot plate method.

Treatment	Dose (mg/kg)	Percentage increase in reaction time
Treatment	Dose (mg/kg)	30 min	45 min	60 min	90 min	120 min	180 min
Standard (STD)(Tramadol)	5	69.84±6.74	81.42±5.30	78.74±6.40	71.77±7.00	66.45±7.47	31.69±8.07
TCEE 100	100	41.75±11.26	64.44±8.73	81.49±16.81	83.44±7.00^**	80.30±6.67^**	66.87±12.79^**
TCEE 200	200	46.97±15.05	70.30±19.31	83.08±6.06^*	80.87±6.67^**	80.87±8.38^**	66.67±14.91^**
TCEE 400	400	85.34±5.22^*	79.92±7.21	78.64±5.66^*	85.35±5.21^**	86.29±5.19^**	47.12±18.48^**

n=6, values represent mean ±SD

Where, TCEE 100, TCEE 200 and TCEE 400 indicates Trichila connaroides ethanolic extracts at doses 100, 200 and 400 mg/kg body weight respectively.

*Symbols represent statistical significance.** P < 0.01., *P < 0.05. as compared to tramadol.

Figure 1: Effect of Trichilia connaroides ethanolic extract in mice by hot plate method

Table 2: Effect of Trichilia connaroides ethanolic extracts and Aspirin on acetic acid induced writhes in rats.

Treatment	Dose(mg/kg)	Number of writhes in 20 (min)	Percentage inhibition
Control	-	39.67± 3.18	-
TCEE 100	100	21.83 ±2.92^**a	44.25 ±7.46
TCEE 200	200	36.3± 1.36	7.23 ±3.48
TCEE 400	400	7.33 ±1.96^{a, b}	81.28 ±5.01^{c, d}
Aspirin	10	9.16 ±1.47^**a	76.60± 3.76

Values represent mean ±SD.

Where, TCEE 100, TCEE 200 and TCEE 400 indicate Trichila connaroides ethanolic extracts at doses 100, 200 and 400 mg/kg body weight respectively.

*Symbols represent statistical significance. ** P < 0.01., *P < 0.05. as compared to aspirin. . ‘a’ as compared with control, ‘b’ is comparision of TCEE 400 with other treatment groups, ‘c’ as compared with Aspirin and ‘d’ is comparision of TCEE 400 with other treatment groups.

Figure 2.1: Analgesic activity of Trichila connaroides on acetic acid induced writhes.

Where n=6, values are mean ±SEM,

TCEE 100, TCEE 200, 400, indicates Trichila connaroides ethanolic extracts at doses 100, 200 and 400 mg/kg body weight respectively.

Figure 2.2: Analgesic activity of Trichila connaroides on acetic acid induced writhes.

Where, n=6, values are mean ±SEM, TCEE 100, TCEE 200, 400 indicates Trichila connaroides aqueous and alcoholic extracts at doses 200 and 400 mg/kg body weight respectively.

B. ANTI-INFLAMMATORY ACTIVITY OF TRICHILIA CONNAROIDES ETHANOLIC EXTRACT (TCEE)

1. FORMALIN INDUCED PAW EDEMA MODEL

Table 3: Acute anti-inflammatory activity of the TCEE (Trichilia connaroides ethanolic extract) and Ibuprofen (reference drug) on formalin induced paw edema in Wistar rats.

Group	N	30 min		60 min	120 min	180 min	240 min	300 min
Control	6	1.25 ±0.014	1.30 ±0.01		1.34±0.03	1.32 ±0.056	1.26 ±0.084	1.24 ±0.070
TCEE 100	6	1.09 ±0.013	1.15 ±0.049		1.20 ±0.021	1.14 ±0.049	1.12* ±0.070	1.06* ±0.021
TCEE 200	6	1.08 ±0.056	1.17 ±0.014		1.25 ±0.042	1.23±0.035	1.10* ±0.014	1.00* ±0.028
TCEE 400	6	1.10±0.056	1.20±0.014		1.27±0.042	1.24±0.035	1.11* ±0.014	1.00* ±0.028
Ibuprofen (100mg/kg)	6	1.25 ±0.007	1.31 ±0.028		1.33 ±0.028	1.20 ±0.007	1.02 ±0.014	0.93* ±0.035

Data are the mean ± SEM values for six rats in each group.

*p < 0.05, **p < 0.01 as compared to the control.

At 400mg/kg dose (1.00±0.028), the activity of the extract showed almost similar activity compare to standard drugs

2. CROTON OIL EAR EDEMA MODEL

Table 4: Effect of TCEE on Croton oil ear edema in rats

Group	Dose (mg/kg)	N	Weight of Untreated ear (Right ear) (mg)	Weight of treated ear (Left ear) (mg)	Difference
Control	1ml/kg	6	37.53 ±1.08	25.02 ±1.17	13.17 ±1.24
TCEE 100	100mg/kg	6	37.49 ±0.37	28.14 ±0.28	9.35±0.09
TCEE 200	200mg/kg	6	37.02 ±0.51	29.04 ±1.20	7.98* ±0.85
TCEE 400	400mg/kg	6	36.82 ±0.44	30.66 ±0.63	6.16**±0.69
Ibuprofen	100mg/kg	6	37.43 ±0.64	32.47 ±0.57	4.95* ±0.11

Data are the mean ± SEM values for six rats in each group.

*p < 0.05, **p < 0.01 as compared to the control.

DISCUSSION:

Analgesic activity:^19-22

Antinociceptive or analgesic activity of Trichilia connaroides was evaluated using both chemical and thermal models of nociception in mice. These models are used to detect central and peripheral analgesics respectively. Acetic acid induced writhing test is used for detecting both central and peripheral analgesics, where as hot plate model is more sensitive to centrally active analgesics.

1.Acetic acid induced writhing test:^19-22

Acetic acid induced writhing test is very sensitive and able to detect anti-nociceptive effects of compounds at dose levels that may appear inactive in other methods like tail flick test. However the test is not specific as it does not indicate whether activity is central and/or peripheral. The intraperitonial administration of acetic acid produces abdominal writhing response due to sensitization of chemosensitive nociceptors by prostaglandins. Acetic acid releases PGE₂ and PGF₂α as well as lipooxygenase product into the peritoneal fluid.

TCEE (Trichilia connaroides ethanolic extract) produced decrease in number of writhes at doses TCEE 200, TCEE 400. The percentage decrease in writhing ± SEM by TCEE 400 and aspirin were found to be 81.28±2.04, and 76.60±1.53 respectively. The abdominal constrictions produced after administration of acetic acid is related to sensitization of nociceptors to prostaglandins. It is therefore possible that the extracts exert their analgesic effect probably by inhibiting the synthesis or action of prostaglandins. The analgesic effect of the extracts may therefore be due to either its action on visceral receptors sensitive to acetic acid, or due to the inhibition of the production of algogenic substances or the inhibition at the central level of the transmission of painful impulses.

2. Hot plate test:^19-22

Thermal induced nociception indicates narcotic involvement²³. The ability of the extracts to prolong the reaction latency to thermally induced pain (Hot plate test) in mice further suggests central analgesic activity. Thermal nociceptive tests are sensitive to opioid μ receptors²³. The ethanolic extracts significantly and dose dependently increased the reaction time at the various time intervals at which they were tested. At higher doses the extracts showed activity which was comparable to that of Aspirin (30, 45 and 60 min) and was better than aspirin at 90, 120 and 180 min. This indicates that the extracts exhibit analgesic effect by central action.

From the results obtained by both the models it can be concluded that the extracts may be showing analgesic activity both by peripheral and central mechanisms. Flavonoids, alkaloids and saponins are reported to have analgesic effect. The analgesic effect of the extract may be due to the presence of flavonoids, tannins, alkaloids and saponins either singly or in combinations which were found to be present in the extracts during phytochemical tests.

Anti inflammatory activity^19-22

1. Formalin induced paw edema model

In the case of Formalin induced paw TCEE (Trichilia connaroides ethanolic extract) (TCEE 400) (1.27±0.042) exhibited almost similar anti-inflammatory activity compared to standard ibuprofen at (1.33 ±0.028).Anti-inflammatory activity exhibited by (Trichilia connaroides ethanolic extract)was found to be in a dose dependent manner i.e at lower dose less activity and higher dose more activity. So the order of exhibition of activity was observed as: TCEE 400>TCEE 200 >TCEE 100. Decline in anti inflammatory activity was observed after 120 minutes.

2. Croton oil ear edema model

In the case of Croton oil ear edema model (Trichilia connaroides ethanolic extract) (TCEE 400) (6.16±0.69 ) exhibited almost similar anti-inflammatory activity compared to standard ibuprofen at (4.95±0.11).Anti-inflammatory activity exhibited by (Trichilia connaroides ethanolic extract) was found to be in a dose dependent manner i.e at lower dose less activity and higher dose more activity. So the order of exhibition of activity was observed as: TCEE 400>TCEE 200 >TCEE 100. Decline in anti inflammatory activity was observed after 120 minutes.

CONCLUSION:

The study was taken up to evaluate ethanolic extracts of Trichilia connaroides bark for analgesic, and antiinflammatory activities. The acute toxicity study conducted for ethanolic extracts indicated that they are safe up to 2000 mg/kg body weight.

Trichilia connaroides bark ethanolic extracts produced significant analgesic activity in both Hot plate and acetic acid induced writhing models in mice. In hot plate method percentage increase in reaction time was determined where as in acetic acid induced writhing model percentage decrease in writhings was determined.

Evaluation of anti inflammatory activity was done by Formalin induced paw edema model and Croton oil ear edema model. In Formalin induced paw edema model mean change in paw volume and percentage protection were calculated. In croton oil ear edema model the difference between untreated ear and treated ear were determined which indicated degree of inflammatory edema. It was evident from the results that Trichilia connaroides bark ethanolic extracts produced significant anti inflammatory activity in the two models. From the results obtained it can be concluded that Trichilia connaroides bark has analgesic, anti inflammatory activity.

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Received on 16.07.2015 Accepted on 29.09.2015

Asian J. Pharm. Res. 5(3): July- Sept., 2015; Page 138-144

DOI: 10.5958/2231-5691.2015.00021.0