1. INTRODUCTION:

Coriander sativum (Umbelliferae) is an annual herb 1-3 feet with small, white or pinkish flower born on compound terminel Umbels¹. It is cultivated throughout India and also widely seen in Syria, Palestine, Mesopotamia and Greece. The leaves are hypnotic, analgesic, and useful in hiccough, suppuration, piles, inflammation, and pain in the eyes. It is used in the treatment of jaundice, stomatitis, and toothache, bleeding of gums, scabies and tuberculosis. The seed is hot, aphrodisiac used in syphilis, dyspepsia, and headache, biliousness, applied to ulcers on the penis. Coriander fruits are used as a spice and flavoring agent in medicine throughout the east. They have carminative and corrective properties. The roots and leaves are powdered and macerated with alcohol are used to touch the eruption of measles in children ². Due to the limitations of opioid and NSAID therapy, there is continuing search for new analgesics. In view of this and on account of the alleged usefulness of this plant in the traditional treatment of some painful and inflammatory conditions which has not yet been scientifically verified. This current study was aimed at investigating possible anti nociceptive and anti inflammatory activities on chloroform extract of the leaves of Coriander sativum in mice and rats.

2. MATERIALS AND METHODS:

2.1 Preparation of plant material and extracts:

Coriander sativum leaves (identified by Dr. Sasikala Ethirajulu, Research officer in Siddha Hospital and Research in Chennai, Tamil Nadu) were collected in Medicinal Garden in Chennai, Tamil Nadu. A Voucher specimen is in our laboratory for future reference. The leaves of the plant was dried, pulverized by mechanical grinder into a dried powder and extracted in soxhlet apparatus using petroleum ether, chloroform and methanol. These extracts were concentrated for further studies at reduced temperature and pressure in a rotary evaporator. The percentage yield of the extracts was calculated to be 1.5005, 3.021 and 10.51 respectively.

2.2. Phytochemical Screening:^3-6

Preliminary phytochemical tests were performed to observe the presence of steroids, flavonoids, tannins, and terpenoids. The solvents used were of AR grade and procured from S.D. Fine Chemicals, Mumbai, India.

2.3. Animals:

Male and female Swiss Albino rats weighing between 180 and 220 gm and Swiss Albino mice of either sex between 18 and 20 gm were utilized in various experiments. The animals were kept in plastic cages at room temperature (25 ± 4⁰C) with free access to diet and water Ad labium. Both animals and diet purchased from Pasteur Institute, Coonur, India. Experiments were performed according to the current guiding principles of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India) and under institutional approval.

2.4. Acute Toxicity studies: LD:₅₀

Toxicity study of the extract of Coriander sativum was performed to get the information about the safety of extract and for their therapeutic usefulness. LD₅₀ values of chloroform and methanol extracts of this plant were determined by the ‘Acute Toxicity Test’. These extracts were dissolved separately in 0.025% of Carboxy Methyl Cellulose (CMC) at different concentrations. The LD₅₀ values of the above extracts of the leaves of Coriander sativum were derived by the method of Litchfield and Wilcoxa^7,8

2.5. Antinociceptive study:

Pentazocine injection i.p. (Fortwin injection, Tablets India Ltd, Chennai, India) was used as a standard drug. Chloroform extract of the leaves of Coriander sativum and Pentazocine were dissolved in 0.025% Carboxy Methyl Cellulose (CMC) which was purchased from S.D. Fine Chemicals, Mumbai, India at definite concentrations separately for the present study.

2.6. Acetic acid induced Writhing:⁹

The method of was adapted. Mice (n=6-19 per group) were injected i.p. with 0.6% acetic acid (10 ml/kg) body weight and after 10 minutes, the number of constrictions were registered for 20 minutes. Animals were pretreated i.p. with chloroform extract of leaves of Coriander sativum (100, 150, 200 mg/kg, dissolved in 0.025% CMC) 30 minutes before acetic acid administration. Control animals were injected with a similar volume of CMC. Pentazocine (5 mg/kg, i.p.) was used as a reference drug.

2.7. Hot Plate Test:

This test was carried out using an UGO Basile (Italy) hot plate apparatus (Socrel Model D –S 37), maintained at 53 ± 0.5⁰ C ¹⁰ was employed. Only mice that showed initial nociceptive responses within 20 seconds were selected for the experiment. The reaction time of the mice to the thermal stimulus, taken to be the interval between the instant, the animal reached the hot plate to the time it licked its hind paw, was measured before and at 30, 60, 90 minutes after the administration of the chloroform extract at dose level of 100, 150, 200 mg/kg i.p. Licking of the paws and jumping were the parameters evaluated as the thermal stimulus. The control group was administered 0.025% CMC at similar volumes to the test groups. Cut off time for the response was set at 60 seconds to avoid tissue damage to the mice paws and percentage inhibition in mice was calculated. Pentazocine (5 mg/kg, i.p.) was used as a reference drug.

2.8. Anti-inflammatory effect:

The following drugs and chemical were used: Diclofenac sodium (Diclomax, Torrent Pharmaceutical Pvt. Ltd, Ahmedabad, India) and Carrageenan (Sigma Chemicals Company, St.Louis, MO, USA) 0.025% Carboxy Methyl Cellulose (S.D Fine Chemicals, Mumbai, India). The plant extract and the standard drug were suspended in 0.025% of Carboxy Methyl Cellulose.

2.8.1. Carrageenan induced paw edema in rats:

The chloroform extract of Coriander sativum leaves at the dose levels of 100, 150, 200 mg/kg and Diclofenac sodium at the dose level of 10 mg/kg were administered orally to different groups of the rats. Acute inflammation was induced half an hour after above treatment by sub planter injection of 0.1 ml of freshly prepared 1% suspension of carrageenan in right hind paw of rats ¹¹. The paw volume was measured initially and then at 1, 2, 3 and 4 hrs after carrageenan injection by using plethysmographic method ¹²

2.9. Statistical analysis:¹³

Data were expressed as mean ± SEM from 6 rats in each group. The biochemical parameters were analyzed statistically using one-way ANOVA followed by Dunnett’s multiple comparison test (DMRT). The minimum level of significance was fixed at P<0.05 significant.

3. RESULTS:

3.1. Phytochemical Screening:

On phytochemical screening, the extracts of leaves of Coriander sativum revealed the presence of flavonoids, tannins, terpenoids and steroids.

3.2. Acute toxicity studies:

The LD₅₀ of chloroform extract was estimated to be 250 mg/kg (i.p.) and > 2500 mg/kg (p.o) and the LD₅₀ of methanol extract was found to be 200 mg/kg (i.p.) and 1500 mg/kg (p.o) in mice. During observations the animals exhibited decreased mobility without loss of righting reflex prior to death.

Table-1: Antinociceptive effect of Coriander sativum leaf chloroform extract on acetic acid Induced writhing in mice

Group/Dose	No of abdominal constrictions (20 min)	Inhibition %
Control (19) 0.025% of CMC (10ml)	13.2 ± 2.4	-
Pentazocine 5 mg/Kg (8)	6.0 ± 1.0^a	52.5
EXTRACT
100 mg/kg (6)	4.1 ± 0.8^a	66.7
150 mg/kg (16)	2.0 ± 0.4^a	84.0
200 mg/kg (14)	0.06 ± 0.06^a	98.4

Values are reported as mean ± SEM for the number of animals shown in parentheses, ^a (p<0.05) significantly different from control.

3.3. Antinociceptive studies:

The chloroform extracts doses of 150 and 200 mg/kg showed antinociceptive activity in two different models for nociception, which are used to investigate the anti nociceptive effects of the extract in this study. The effect of chloroform extract on acetic acid induced writhing test and hot plate test are shown in tables 1, 2 respectively. In acetic acid induced writhing method Coriander sativum showed a significant dose – dependent antinociceptive effect upon the abdominal constrictions induced by acetic acid in mice. Inhibition rates (p<0.0001) were observed at the doses of 100 (66.7%), 150 (84.0%), 200 (98.4%) mg/kg compared to controls (Table – 1) pentazocine inhibited abdominal constrictions by (52.5%). In hot plate method the extract considerably increased the animal reaction time to the heat stimulus, the values were found to be significant (p<0.05) at 60 minutes after the treatment with 150 mg/kg while the values for 200 mg/kg were significant (p<0.001) at 90 minutes.

Table -2: Effect of Coriander sativum leaf chloroform extract in hot plate test

Groups	Dose (mg/kg)	Paw licking time in seconds				Paw jumping time in seconds
Groups	Dose (mg/kg)	0 min	30 min	60 min	90 min	0 min	30 min	60 min	90 min
Control	0.025 % CMC (10ml)	3.0±0.1	2.8±0.3	2.7±0.21	2.8±0.1	3.0±0.1	2.7±0.5	2.8±0.7	2.7±0.4
Penta- zocine	5 mg/kg (i.p.)	3.5±0.1	4.4±0.2 (36.36 %)	5.4±0.12 (50 %)	6.2±0.12 (54.83 %)	3.2±0.1	6.7±0.3 (59.70%)	7.4±0.6 (62.16%)	8.0±0.21 (66.24 %)
Extract	100 mg/kg	3.6±0.21	3.6±0.25 (22.22%)^a	4.4± 0.1 (38.63%)^a	5.1±0.10 (45.09%)^a	3.4±0.3	6.0±0.1 (55 %)	6.4±0.10 (56.25%)	6.5±0.4 (58.46 %)
Extract	150 mg/kg	3.4±0.25	4.5±0.10 (37.77 %)	4.6±0.10 (41.30%)^a	5.5±0.12 (49.09%)^a	3.6±0.3	6.5±0.4 (58.46%)	6.8±0.4 (58.82%)	6.9±0.6 (60.86%)^b
Extract	200 mg/kg	3.7±0.1	4.6±0.2 (39.1 %)	4.7± 0.8 (42.55 %)	5±0.5 (44 %)	4.2±0.5	6.3±0.7 (57.14%)	6.7±0.12 (58.20%)	7.0±0.4 (61.42%)^b

Values are mean ± SEM ^a p < 0.05,^b p < 0.001 significantly different from control; Figures in the parenthesis indicate percentage inhibition of nociception of hot plate test in mice

Table – 3: Effect of chloroform extract of Coriander sativum leaves on carrageenan induced paw edema in rats

Treatment		Percentage inflammation after carrageenan injection
Groups	Dose	1 hr	2 hr	3hr	4hr
Control	0.025 % CMC (10 ml)	43.40 ± 2.20	97.70 ± 3.60	128.02 ± 2.50	130.65 ± 5.30
Extract	100 mg/kg	29.75 ± 2.70	71.80 ± 9.60	98.71 ± 8.60 ^a	112.50 ± 10.30 ^a
	150 mg/kg	26.40 ± 3.50	60.50 ± 8.50	80.60 ± 7.20 ^a	90.60 ± 9.30
	200 mg/kg	25.70 ± 4.20^b	44.32 ± 1.01	72.65 ± 7.80 ^b	81.04 ± 4.55 ^b
Diclofenac	10 mg/kg	13.25 ± 1.90^b	36.30 ± 4.05^b	43.22 ± 4.06 ^b	43.03 ± 5.16 ^b

Values are mean ± SEM, ^a p < 0.01, ^b p < 0.001 significantly different from control, Paired t- test (n=5)

3.4. Anti-inflammatory studies:

The effect of chloroform extract on carrageenan induced paw edema in rats is shown in table – 3. There was dose dependent significant reduction in carrageenan induced rat paw oedema at the dose level of 150, 200 mg/kg of extract as of 10 mg/kg Diclofenac sodium over a period of 4 hrs. The values are found to be significant (p<0.01) after treatment with 100, 150 mg/kg at 3 and 4 hrs while the values for 200 mg/kg were found to be significant (p<0.001) at 3 and 4 hrs respectively.

4. DISCUSSION AND CONCLUSION:

In this study we present evidence that the chloroform extract of Coriander sativum leaves contains potent analgesic and anti-inflammatory activities, validating the medicinal folk use of this plant in inflammatory diseases. Since the analgesics and anti-inflammatory activities are mostly associated with particular compound, this lead us to investigate the anti-inflammatory and antinociceptive activity of the extract. The analgesic effect of the chloroform extract Coriander sativum leaves was demonstrated using a novel and objective behavioural test in which quantization is independent of the observer and is sensitive to all classes of analgesics ¹⁴. It detects the anti nociceptive activity of cyclo-oxygenase inhibitors, sympatholytics, as well as peripheral direct acting analgesics such as dipyrone, methyl morphine and methyl nalorphine. This method is also able to detect centrally acting analgesics such as morphine; pentazocine¹⁵ the chloroform extract of Coriander sativum leaves significantly decreased the acetic acid induced writhing effect in mice at 200 mg/kg. Acetic acid acts indirectly by inducing the release of endogenous mediators of pain sensitive to non steroidal anti- inflammatory drugs and opioids¹⁶. Besides, the induction of the acetic acid writhing in mice is an effect of the acute inflammatory reaction related to increase in levels of prostaglandins E₂and F_2α in the peritoneal fluid¹⁷. The hot plate is a specific central antinociceptive test in which opioid agents exert their analgesic effects via supra spinal and spinal receptors ¹⁸. In the hot plate test the chloroform extract of Coriander sativum leaves showed a significant action at 90 minutes after the administration of 200 mg/kg of the extract.

The chloroform leaf extract of Coriander sativum possess significant anti-inflammatory effect in carrageenan induced paw edema method. Diclofenac, a non steroidal anti-inflammatory drug (NSAID) is commonly employed in the treatment and or management of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis ^19-21for its anti-inflammatory and analgesic effects²². Diclofenac has also been reported to suppress inflammation induced by various phlogestic agents in experimental animal models^23,24

The present experimental findings suggest that Coriander sativum leaf chloroform extract may probably exert its antinociceptive and anti-inflammatory effects by inhibiting the release, synthesis and or production inflammatory cytokines and mediators, including prostaglandins, histamines and so on.

Although the exact chemical compounds responsible for the antinociceptive, anti- inflammatory effects of Coriander sativum leaf chloroform extract still remain speculative, experimental evidence obtained in the present laboratory animal study indicate that chloroform leaf extract of the herb possess antinociceptive and anti-inflammatory properties.

ACKNOWLEDGEMENTS:

Authors are thankful to the authorities of Kamalakshi Pandurangan College of Pharmacy for providing all facilities.

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Received on 07.11.2009 Modified on 14.01.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 744-747