Evaluation of Anti-inflammatory Activity of leaf extract of Morinda citrifolia L. (Noni)

 

Khuntia Tapas Kumar1*, Panda D. S. 2, Khuntia S.3

1Department of Pharmacology, K.L.R. Pharmacy College, Paloncha, A.P., India

2Department of Pharmaceutics, Institute of Pharmacy and Technology, Salipur, Orissa

3College of Pharmaceutical Sciences, Puri, Orissa

 

ABSTRACT:

Morinda citrifolia L. (Noni) (Rubiaceae) has been used in folk remedies by Polynesians, Indians for over 2000 years, and is reported to have a broad range of therapeutic effects, including antibacterial, antiviral, antifungal, antitumor, analgesic, hypotensive, and immune enhancing effects. Petroleum ether extract of leaves of Morinda citrifolia L. (Noni) was studied for its in-vivo anti-inflammatory potential using carageenen induced rat paw edema and cotton pellet induced granuloma methods. The results of the study indicate that the petroleum ether extract possess significant anti-inflammatory activity at doses 150mg/kg and 300mg/kg.

 

KEYWORDS: Immune enhancing, antiviral, antifungal, antitumor, granuloma

 

INTRODUCTION:

Inflammation is defined as a local response of living mammalian tissue to injury due to any agent. It is a body defense reaction in order to eliminate or limit the spread of injurious agent as well as to remove the consequent necrosed cells and tissues. [1]

A large number of Indian medicinal plants are attributed with various pharmacological activities because they contain a diversified class of Phytochemicals. It is believed that current analgesia-inducing drugs such as opoids and non-steroidal anti-inflammatory drugs are not useful in all cases, because of their side effects and potency. [2] Several plants have been used in folklore medicine as anti-inflammatory agents and they play an important role in health services around the globe. [3] Morinda citrifolia L. (Noni) (Rubiaceae) is reported to have a broad range of therapeutic effects, including antibacterial, antiviral, antifungal, antitumor and immune enhancing effects. [4] Lack of scientific data with respect to the pharmacological properties of the leaf extract of Morinda citrifolia L. (Noni) (Rubiaceae) encouraged for the evaluation of its anti-inflammatory potential.

 

MATERIALS AND METHODS:

Plant Material:

Leaves of Morinda citrifolia L (Noni) were collected from the coconut gardens of Salipur, Orissa in the month of December. The plant was identified, authenticated and a voucher specimen was kept in the herbarium of Department of Pharmacognosy, Institute of Pharmacy and Technology, Salipur, Orissa.

 

Extract Preparation:

The leaves were collected and washed thoroughly in water, chopped, air dried for a week at 35-400 C and pulverized in electric grinder. 150 gm. of the powder subjected to Soxhlet apparatus using petroleum ether as solvent.


The solvent was then removed under reduced pressure, which obtained a greenish- black colored residue. The yield was 11.9 %. The prepared extract was used for phytochemical screening [5] and evaluation of anti-inflammatory activity.

 

Experimental Animals:

Animal care and handling was carried out according to Institutional Animal Ethics Committee (IAEC). Eight to ten weeks old Wistar rats of either sex, weighing (160±20 g) were selected from the in-bred colony maintained under controlled conditions of temperature (22±3oC) humidity (40-70%) and light (12 and 12h of light and dark, respectively). They were feed with standard rodent chew, Nutrilab Rodent (Tetragon Chemie Pvt. Ltd., Bangalore) and purified drinking water. Before each test, the animals were fasted for at least 12 hours. All the experiments were performed in the morning according to current guidelines for investigations of experimental pain in conscious animals. [6]

 

Chemicals:

Anesthetic ether from Kabra Drugs Limited, INDORE, Absolute alcohol, Hayman, ENGLAND and Sterillium disinfectant solution was procured from Bode Chemie Hamburg, GERMANY.

 

EXPERIMENTAL DESIGN:

(a) Carageenen Induced Rat Paw Edema: [7]

In each group six Wistar rats were taken. The animals were kept fasted throughout the experimental period, but were provided with water ad libitum. After 30 minutes 0.1 ml carageenen (1%) was injected into the planter region of hind paw of rats. Measurement of paw volume (ml) was made by mercury displacement technique using Plethysmometer immediately before and 3hr after carageenen injection.

 

Group-I: Control [Animals received Carboxy methyl cellulose (100mg/kg P.O.)]

Group-II: Standard [Indomethacin treated Animals (10mg/kg I.P.)]

Group-III: Test-1 [Petroleum ether extract of M. citrifolia (150mg/kg)]

 Group-IV: Test-2 [Petroleum ether extract of M. citrifolia (300mg/kg)]

 

(b) Cotton Pellet induced Granuloma: [8]

The animals were divided in to four groups (n=6). The animals were anaesthetized with ether; the back skin was shaved and disinfected with 70% ethanol. An incision was made in lumbar region. Using a blunted forceps, subcutaneous tunnels were formed and sterilized cotton pellets weighing 20±0.5 mg were implanted on either sides of the scapular region of each rat. Group-I served as control and received the vehicle.

 

The Petroleum ether extract of M. citrifolia at concentrations of 150mg/kg and 300mg/kg were administered orally to Group-II and Group-III animals respectively for 7 days. Group-IV animals received Indomethacin at a dose of 10-mg/kg p.o. for the same period. On the 8th day, the animals were sacrificed and the pellets together with the granuloma tissue were carefully removed, dried in an oven at 600 C, weighed and compared with the control.

The percentage activity of anti-inflammatory effect of petroleum ether extract of Morinda citrifolia L. (Noni) was calculated by the following formula:

 

               Percentage inhibition= (C-T)/C´ 100

Whereas,

T- Increase in paw volume after administration of test extract

C- Increase in paw volume of control group

 

(c) Egg albumin induced paw edema:

The experiment was conducted by inducing egg- albumin to healthy rats. [9] All four groups were pretreated with vehicle or Indomethacin or the extract at both doses (150mg/kg, 300mg/kg) respectively. After 30 min, each group was injected with 0.5ml raw egg albumin sub-plantar to the left hind paw. The paw volume compared to that of the control animals was recorded at 0, 1, 3, 6, 12hr and considered as anti-inflammatory response.

 

Statistical Evaluation:

The data were statistically analyzed by student’s t- test and all the values were expressed as mean± SEM. The data were also analyzed by one way ANOVA followed by Dunnet’s t- test and values P<0.05 were considered significant. [10]

 

RESULTS AND DISCUSSION:

Administration of Carrageenan in paw edema of rats produced a short inflammatory response indicated by increase in paw volume. Oral administration of Pet. Ether extract of Morinda citrifolia showed a significant (P<0.05) inhibition of Carrageenan induced paw inflammation at dose 150mg/kg (34% inhibition) and at 300mg/kg (42% inhibition). (Table-1)

 

Table-1 Effect of Petroleum ether extract of M.citrifolia on carageenen induced paw edema in Rats.

 

Group No.

(n=6)

 

Design of Treatment

 

Dose (mg/kg)

Increase in paw edema at the end of 3hr.

 

Percentage of Inhibition

I

Control

-

0.49±0.02

-

II

Indomethacin

10

0.21±0.01**

57

III

Test-1

M.citrifolia

150

0.32±0.05**

34

IV

Test-2

M.citrifolia

300

0.28±0.01**

42

**P<0.01 Vs control. The data were statistically analyzed by Student’s t-test and all values were expressed as Mean± SEM. The data were also analyzed by one way ANOVA followed by Dunnet’s t-test and values P< 0.05 were considered significant.

 

 

Table-2 Effect of extract of M.citrifolia in cotton pellet induced Granuloma model.

 

Group No.

(n=6)

 

Design of Treatment

 

Dose (mg/kg)

Weight of cotton pellet

(mg)

 

Percentage of Inhibition

I

Control

-

70.2±0.7

-

II

Test-1

M.citrifolia

150

48.9±0.6*

32

III

Test-2

M.citrifolia

300

30.5±0.9*

44

IV

Indomethacin

10

26.3±0.7*

55

*P<0.05 Vs control. The data were statistically analyzed by Student’s t-test and all values were expressed as Mean± SEM. The data were also analyzed by one way ANOVA followed by Dunnet’s t-test and values P< 0.05 were considered significant.

Table-3 Effect of Petroleum ether extract of M. citrifolia on egg-albumin induced rat paw edema.

Groups

(n=6)

Dose (mg/kg)

Paw Volume (ml)

0hr

1 hr

3 hr

6hr

12 hr

Control

1% CMC

1.27±0.01

1.82±0.02

2.41±0.01

1.50±0.02

1.42±0.01

Indomethacin

10

1.27±0.01

1.74±0.01**

1.39±0.01***

1.19±0.00***

1.15±0.01***

Test-1 M. citrifolia

150

1.25±0.01

1.83±0.01

1.64±0.02***

1.48±0.01

1.40±0.00

Test-2 M.citrifolia

300

1.26±0.01

1.78±0.02

1.50±0.02***

1.3±0.01***

1.28±0.01***

**P<0.01, *** P<0.001 Vs control. The data were statistically analyzed by Student’s t-test and all values were expressed as Mean± SEM. The data were also analyzed by one way ANOVA followed by Dunnet’s t-test and values P< 0.05 were considered significant.

 

 

Table-4. Qualitative Phytochemical Analysis of petroleum ether extract of Morinda citrifolia L (Noni).

Plant constituents

Seed oil of Morinda citrifolia L (Noni)

Tests/Reagents used

 

1

Triterpenoids

 

(i) Tin and Thionyl chloride

+++

     2

Saponins

 

(i)   Foam test

+

 

(ii)  Haemolysis test

+

3

Tannins and phenolics                 

 

(i) Ferric chloride solution

++

 

(ii) Lead acetate solution

+

 

(iii) Potassium dichromate sol. 

+

 

(iv) Gelatin solution 

+

4

Flavonoids

 

(i)   Magnesium and HCl

+++

 

(ii)  Lead acetate solution 

++

 

(iii) NaOH and H2SO4

+

5

Alkaloids

 

(i)   Mayer’s test

+

 

(ii)  Wagner’s test

+

 

(iv) Dragendorff’s test

+

+++  Phyto constituent in high concentration, ++ Medium conc., + Low conc.

 

 

In cotton Pellet induced granuloma the percentage inhibition (44%) at 300 mg/kg was found significant and comparable to Indomethacin at 10 mg/kg. (Table-2)

 

The effect of petroleum ether extract of M. citrifolia was evaluated on egg-albumin induced rat paw edema. It was observed that the test compound at dose 300mg/kg produced a significant reduction in the paw volume at 3, 6 and 12hr, which is comparable with the standard drug Indomethacin (10mg/kg). (Table-3)

 

The phytochemical studies on the petroleum ether extract of M. citrifolia revealed the presence of various constituents like triterpinoids, saponins, tannins and phenolics, flavonoids and alkaloids. (Table-4)

 

CONCLUSION:

The petroleum ether extract of Morinda citrifolia L. (Noni) showed significant anti-inflammatory activity against carrageenan and egg-albumin induced paw edema and cotton pellet induced granuloma models in rats. The anti-inflammatory activity may be attributed to the presence of different Phyto-constituents present in the plant leaf extract, especially flavonoids, which are found to act by reducing the release of inflammatory substance like prostaglandin there by reducing tissue exaggeration. [11] Further detailed investigation needs to be underway to determine the exact Phytoconstituents, which are responsible for the anti-inflammatory activity and may provide deeper insight to the discovery of a potent drug for the treatment of

 

 

inflammation. The inhibitory activity of the extract justified the use of the plant as a non-specific anti-inflammatory activity in folk medicine.

 

ACKNOWLEDGEMENT:

The authors are thankful to the staff, K.L.R. Pharmacy College, Paloncha, A.P. and Institute of Pharmacy and Technology, Salipur, Orissa, for providing the necessary facilities and support to carry out this work.

 

REFERENCE:

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2.          Adhmadiani A. et al., Antinociceptive and anti-inflammatory effects of Sambucus ebulus rhizome extract in rats. J Ethanopharmacol. 1998; 61: p229

3.          Premanathan M et al., A survey of some Indian medicinal plants for anti-human immunodeficiency virus (HIV) activity. Indian J. Med. Res. 2000; 112: p73-77

4.          WANG Mian-Ying et al., Morinda citrifolia (Noni): A literature review and recent advances in Noni research. Acta Pharmacol Sin 2002; 23: p1127 -1141

5.          Odebiyi O.O. et al., Phytochemical screening of Nigerian Medicinal plants, ll., Lloydia, 1978; 41: p234-246.

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8.          Meier R, Schuller W. Anti-inflammatory activity of Ennryngium maritimum L. rhizome extracts in intact rats. Experientia. 1950; 6: p469

9.          Akah P A, Nwambie A L. Evaluation of Nigerian traditional medicines 1: Plants used for rheumatic (inflammatory) disorders. J. Ethnopharmacol. 1994; 42: p179-182

10.       Bennet C A, Franklin N L. “Statistical Analysis in Chemistry and chemical Industry”. John Wiley Sons, New York, 1967; p133

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Received on 05.01.2010

Accepted on 20.02.2010 

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics 2(2): March –April 2010: 195-197