|Year : 2014 | Volume
| Issue : 3 | Page : 66-69
Study of anti-inflammatory effect of neem seed oil (Azadirachta indica) on infected albino rats
Manas Ranjan Naik1, Ayon Bhattacharya1, Rasmirekha Behera1, Divya Agrawal2, Suhasini Dehury3, Sanjay Kumar1
1 Department of Pharmacology, Institute of Medical Sciences and SUM Hospital, Siksha O Anusandhan University, Bhubaneshwar, India
2 Department of Anatomy, Institute of Medical Sciences and SUM Hospital, Siksha O Anusandhan University, Bhubaneshwar, India
3 Department of Pharmacology, Shri Rama Chandra Bhanja Medical College, Cuttack, Odisha, India
|Date of Web Publication||24-Mar-2015|
Manas Ranjan Naik
Department of Pharmacology, Institute of Medical Sciences and SUM Hospital, K-8, Kalinga Nagar, Ghatikia BO, Bhubaneshwar - 751 003, Odisha
Source of Support: None, Conflict of Interest: None
Objective: To study the anti-inflammatory effect of neem seed oil (NSO) on albino rats using carrageenan-induced hind paw edema. Materials and Methods: The study sample was divided into six groups with each group consisting of six rats as follows: Group I: Control (distilled water 0.5 ml/rat); Group II: Standard (aspirin 200 mg/kg body weight orally); Groups III, IV, V, and VI (NSO 0.25 ml, 0.5 ml, 1 ml, and 2 ml/kg body weight, respectively, i.p.). The inflammation induced in the form of hind paw edema was produced by injection of 0.1 ml of 1% suspension of carrageenan in normal saline below the plantar aponeurosis of right hind paw of rats; the left hind paw served as control. The volume of edema was measured by water displacement method in microburette. Volumes of both the hind paws were measured before and after 1, 2, 3, 4, 6, 12, and 24 h of carrageenan injection. Results: NSO in the dose of 0.25 ml/kg body weight did not show any significant anti-inflammatory activity. NSO showed increased inhibition of paw edema with the progressive increase in dose from 0.25 ml to 2 ml/kg body weight. At the dose of 2 ml/kg body weight, NSO showed maximum (53.14%) inhibition of edema at 4 th hour of carrageenan injection. Aspirin in the dose of 200 mg/kg body weight showed maximum inhibition of hind paw edema. Conclusion: The present study concludes that NSO exhibits significant anti-inflammatory action.
Keywords: Anti-inflammatory, carrageenan, edema, neem seed oil, paw edema
|How to cite this article:|
Naik MR, Bhattacharya A, Behera R, Agrawal D, Dehury S, Kumar S. Study of anti-inflammatory effect of neem seed oil (Azadirachta indica) on infected albino rats. J Health Res Rev 2014;1:66-9
|How to cite this URL:|
Naik MR, Bhattacharya A, Behera R, Agrawal D, Dehury S, Kumar S. Study of anti-inflammatory effect of neem seed oil (Azadirachta indica) on infected albino rats. J Health Res Rev [serial online] 2014 [cited 2019 Nov 21];1:66-9. Available from: http://www.jhrr.org/text.asp?2014/1/3/66/153880
| Introduction|| |
Neem (Azadirachta indica) is a well-known plant grown in the semi-arid regions throughout Asia and Africa. It belongs to the family of Mahogany known as Meliaceae.  It is a medium to large-sized tree which grows to an average height of 30 cm.  In Sanskrit, it is known as "arishtha" meaning "perfect, complete, imperishable," "reliever of sickness," hence it has been known by many names like "Sarbarogaribarini," and "wonder tree."  Neem is known since prehistoric times and has a number of folklore claims. The earliest writings of neem were in Sanskrit about the health benefits of neem's fruits, seeds, oil, leaves, roots, and bark.  The neem seed oil (NSO) has been reported of having analgesic and antiarthritic activity.  Neem has a multitude of medicinal properties like antipyretic, antimalarial, antitumor, antiulcer, antidiabetic, and antifertility effects.  The anti-inflammatory principles of neem discovered by phytochemical analysis are triterpenes, flavonoids, tannins, saponins, nimbidin, sodium nimbidate, gallic acid, catechin, and polysachharides. , The modern-day nonsteroidal anti-inflammatory drugs (NSAIDs) have their own side effects, hence the need for herbal NSAIDs has become a priority of the society.
Aims and objectives
The present study was done to find out the anti-inflammatory action of NSO on carrageenan-induced infection in rats.
| Materials and methods|| |
The study was conducted from August 2013 to July 2014. This study is a randomized placebo-controlled trial conducted on albino rats.
The ethical consent for the study was obtained from IAEC, School of Pharmaceutical Sciences, SOA University.
Collection of plant extract
NSO extract was procured from Indian Herbs Research Supply Co. Ltd., Saharanpur, India.
Aspirin (Burgoyne Burbidges and Co, Mumbai, India), carrageenan Sd Fine-Chem Ltd, Mumbai, India)
Albino rats of either sex, weighing 150-200 g, were selected for the study. These animals were obtained from the central animal house of IMS and SUM Hospital, SOA University under the Department of Pharmacology. The animals were kept at ambient temperature of 22 ± 1°C in a 12 h light and dark cycle. Food and water were given ad libitum. The animals were acclimatized to laboratory conditions for 7 days before starting the experiments.
Carrageenan-induced rat paw edema
The animals were divided into six groups with each group consisting of six rats as follows:
- Group I: Control (distilled water 0.5 ml/rat)
- Group II: Standard (Aspirin 200 mg/kg body weight orally)
- Groups III, IV, V, and VI: (NSO 0.25 ml, 0.5 ml, 1 ml, and 2 ml/kg body weight, respectively, administered intraperitoneally).
The total volume of the oral dose was kept constant at 1 ml/rat. For all intraperitoneal injections, the volume was kept constant at 0.5 ml/rat. It is a randomized control study.
The inflammation induced in the form of hind paw edema was produced by injection of 0.1 ml of 1% suspension of carrageenan in normal saline below the plantar aponeurosis of right hind paw of rats. Normal saline was injected in the left hind paw of rats in the control group. A mark was made on both the hind limbs at the tibio-tarsal joint.  Volume of paw edema was measured by water displacement method and the displaced water was collected in the microburette. A glass tube with side outlet was fixed inside the microburette of 2 ml capacity. The glass tube and the microburette were fixed on different stands. During the experiment, the hind limb was dipped inside the tube up to the given mark at the tibio-tarsal joint causing water to overflow from the side outlet to the microburette. Volume of water displaced was read in the microburette which had micro-graduations. The volume of displaced water was equal to the volume of paw. Aspirin and intraperitoneal NSO were given in all groups except the control group, 1 h before carrageenan injection. Standard and the test drugs were given 1 h before carrageenan injection. Volumes of both the hind paws were measured before and after 1, 2, 3, 4, 6, 12, and 24 h of carrageenan injection. The ability of the drug to inhibit paw edema was found and compared with control.
The percentage inhibition of paw edema was calculated by the formula: 
where Vc = Mean volume of paw edema in the control group of animals and Vt = Mean volume of paw edema in the drug treated group of animals.
| Results|| |
Results were tabulated using unpaired "t" test. Level of significance was taken at P < 0.05. More the inhibition of edema, lesser volume of water is displaced and lesser the inhibition of edema, more volume of water is displaced. If the edema in hind paw is more, it means more volume of water is displaced by the paws of rats and more volume of water is measured in the microburette. In the groups having NSO, there was more inhibition of edema and, thus, lesser volume of water displaced as compared to the control group. NSO in the dose of 0.25 ml/kg body weight did not show any significant anti-inflammatory activity. However, 1 ml/kg of NSO showed significant inhibition of edema at 3 rd and 4 th hour of carrageenan injection, whereas 2 ml/kg body weight of NSO showed significant reduction of edema from 1 to 6 h of carrageenan injection [Table 1]. NSO in the dose of 0.5 ml/kg body weight inhibited the edema significantly only at 4 th hour of carrageenan injection. NSO in the dose of 0.5 ml/kg body weight showed 16.3% inhibition of edema at 4 th hour of carrageenan injection, whereas 1 ml/kg body weight of NSO caused 18.8% inhibition of edema at 3 rd hour and 17.8% inhibition at 4 th hour. NSO in the dose of 2 ml/kg body weight showed maximum (53.14%) inhibition of edema at 4 th hour of carrageenan injection. Aspirin in the dose of 200 mg/kg body weight caused maximum inhibition of hind paw edema [Table 2], [Figure 2].
|Table 2: Percentage inhibition of edema at various doses of NSO at different hours|
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| Discussion|| |
Inflammation occurs in phases: The first phase is characterized by vascular permeability, exudation of plasma, release of mediators; the second phase is due to migration of leukocytes; and the final phase is by granuloma formation. 
The study is a randomized control experimental study. Carrageenan is used here and acts as a phlogistic agent. The paw edema induced by carrageenan in rats is biphasic: The first phase (0-2 h) is due to release of 5-HT, histamine, and bradykinin from the mast cells, the plateau phase (3 h) is maintained by kinins, and the second phase (4 h) is due to prostaglandins, protease, and lysosomes. ,
A similar dose-dependent anti-inflammatory effect of nimbidin was also observed by Pillai et al. at the doses of 20 mg, 40 mg, and 80 mg/kg body weight. Nimibidin is an active compound obtained from NSO.  Jagadeesh et al. had studied the anti-inflammatory effect of neem leaf oil at 1, 2, 4, and 8 ml/kg body weight on albino rats, which showed 45.2%, 50%, and 48.2% reduction of paw edema at 1, 2, and 4 ml/kg, respectively, at 3 h in carrageenan-induced rat paw edema test. In the present study, NSO at 2 ml/kg showed 52.99% reduction in paw edema at 3 h. 
The phytochemical ingredients like triterpenes, flavonoids, tannins, saponins, nimbidin, sodium nimbidate, gallic acid, catechin, and polysachharides could also be responsible for the anti-inflammatory effect of NSO. Flavonoids play a major role here as they not only inhibit prostaglandin biosynthesis, but also inhibit endoperoxidases and the enzymes like protein kinases and phosphodiesterases that are involved in the inflammation process. ,
In [Figure 1], we can appreciate a clear visual impact of NSO at 0.5, 1, and 2 ml/kg in comparison to control. However, aspirin showed the maximum inhibition of paw edema as seen at the bottom line in the line diagram [Figure 1]. NSO at 0.25 ml/kg did not show an appreciable difference with respect to control. NSO in the dose of 2 ml/kg body weight showed maximum (53.14%) inhibition of edema at 4 th hour of carrageenan injection. In the present study, the edema suppression was maximum at 3 rd and 4 th hour, indicating that NSO could act by inhibiting prostaglandin-like substances by inhibiting the cyclooxygenase (COX) enzyme.
|Figure 1: Line diagram showing the effect of NSO and aspirin on carrageenan-induced hind paw edema at various time intervals|
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|Figure 2: Bar diagram showing the percentage (%) inhibition of paw edema of NSO and aspirin on carrageenan-induced hind paw edema|
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The future prospects of this study can be extended to unravel the effect of this plant on long-term dosing schedules. One among the strengths of this study was the cost-effectiveness. The sample size taken for this study was small; hence, on increasing the sample size, the results could be analyzed with parametric tests. In this study, only one model of inflammation was used; it could be extended using more models of inflammation. In this study, we postulated that the mechanism of action of this plant could be due to its COX inhibitory action. The mechanism of action of the plant can be studied using in vitro models. The ability of the test compound to inhibit COX-1 and COX-2 (IC 50 values, μM) can be determined using enzyme immunoassay kit. Isolation of individual phytochemical ingredients will yield fruitful results as neem contains various bioactive compounds; so further studies need to be conducted in the isolation of phytoconstituents by column chromatographic techniques. This can be followed by the relevant toxicity studies.
| Conclusion|| |
Thus, it can be concluded from the present study that NSO exhibits anti-inflammatory action with increase in dose. The results of the present study could help in forming an effective and inexpensive anti-inflammatory health intervention for low socio-economic communities. However, further research is needed to clarify and confirm the exact mechanism of action.
| Acknowledgment|| |
The authors are highly indebted to Professor Dr. Indira Devi and Professor Dr. Shantilata Patnaik for giving their ideas toward this research.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]