Antinociceptive activity of acute and chronic administration of Murraya koenigii L. leaves in experimental animal models.

AIM: To evaluate the antinociceptive activity of acute and chronic administration of petroleum ether extract of Murraya koenigii L. leaves (PMK) and total alkaloids separated from petroleum ether extract of Murraya koenigii leaves (AMK) in mice.
MATERIALS AND METHODS: PMK was subjected for isolation of total alkaloid fraction AMK. The antinociceptive activity of PMK (100 and 300 mg/kg, p.o.) and AMK (100 and 300 mg/kg, p.o.), after acute and chronic administration (for 15 days), was evaluated using peripheral model like acetic acid-induced writhing method and central model like hot plate method and tail immersion method. Statistical analysis was carried out by one-way ANOVA followed by Dunnett's test. RESULT: In acute studies, PMK and AMK significantly and dose-dependently reduced the number of acetic acid-induced writhing, significantly increased the latency of paw licking in hot plate method, and significantly increased the basal reaction time in tail immersion method. With chronic administration of PMK and AMK, highest activity was observed on day 9 in acetic acid-induced writhing model. In hot plate and tail immersion method, chronic administration of PMK and AMK initially showed fluctuating responses but produced highest degree of antinociception on day 9 of the study.
CONCLUSION: The degree of antinociception produced by PMK and AMK at the end of 15 days study suggest that Murraya koenigii has potential to use as an analgesic.

Introduction

Pain is a direct response to an untoward event associated with tissue damage, such as injury, inflammation or cancer.[1] Analgesics like nonsteroidal anti-inflammatory agents (NSAIDs) act peripherally to exhibit an inhibitory action on the cyclooxygenase that catalyzes the biosynthesis of prostaglandins and thromboxane from arachidonic acid, while opioids interfere with central appreciation of pain. Analgesics relieve pain as a symptom without affecting its cause.[2] Most of the drugs used at present for analgesic effect are synthetic in nature, prolonged use of which causes several side and toxic effects including respiratory depression, constipation, kidney damage, physical dependence as well as gastric irritation.[3] In this context, there arise new scopes for evaluation of herbs and herbal formulation in treatment of pain.

Murraya koenigii L. (curry tree), belonging to family Rutaceae, is a tropical to sub-tropical tree native to India. Traditionally, the plant is used as tonic, stomachic, and carminative.[4] Fresh juice of the root is taken to relieve pain associated with kidney.[5] Methanolic extract of Murraya koenigii leaves possess antinociceptive and anti-inflammatory activity.[6] The plant is reported to contain carbazole alkaloids[7] having antioxidant,[8-10] anti-inflammatory, and anti-tumor activities.[4] No research on analgesic activity has been carried out. Hence, the present research aimed to evaluate the antinociceptive activity of acute and chronic administration of pet ether extract of Murraya koenigii leaves (PMK) and total alkaloids separated from pet ether extract of Murraya koenigii leaves (AMK). Peripheral and central antinociceptive activity was evaluated using acetic acid-induced writhing method,[11] hot plate method,[12] and tail immersion method.[13]

Materials and Methods

Animals

Adult Swiss albino mice (20 ± 2 g) were used for this study. The animals were housed at 24 ± 2°C and relative humidity 55 ± 5 with 12:12 h light and dark cycle. They had free access to food and water ad libitum. The animals were acclimatizated for a period of seven days before the study. The experimental protocol was approved by the Institutional Animals Ethics Committee (IAEC) of MGV's Pharmacy College, Nasik.

Drugs and Treatment Schedule

Aspirin (Research Lab, Mumbai), pentazocine (Fortwin, Ranbaxy) were used for this study. All other chemicals were of analytical grade. Mice (20 ± 2 g) of both the sexes were randomly divided into six groups, each containing five animals. On the day of the experiment, control group received 0.1% carboxy-methyl cellulose, p.o., 1 h before the experiment. PMK (100 and 300 mg/kg) was suspended in 0.1% CMC and (AMK) (100 and 300 mg/kg) was dissolved in water and administered orally to animals 1 h before the test.

Plant Material and Extraction

Leaves of Murraya koenigii L. were purchased from the local market and were identified by Dr. P.G. Diwakar Jt. Director, Botanical Survey of India, Pune, where a voucher specimen (MUKKID 1) has been retained. The leaves were dried in shade and powdered mechanically. Then the powdered leaves of Murraya koenigii were defatted with the petroleum ether (60-80°C). The filtrate was concentrated to get PMK (yield=12.6%w/w). The extract was further subjected to isolation of alkaloids according to the method of Cordell.[14] PMK was suspended in tartaric acid (pH=5) and partitioned with ethyl acetate presaturated with water to produce aqueous-acid phase. The phase was made alkaline with Na2CO3 solution (pH=11) and again partitioned with ethyl acetate to give aqueous portion containing alkaloids. The aqueous portion was dried to get AMK (yield=48.33%).

Phytochemical Analysis

Phytochemical analysis of PMK was carried out according to methods described earlier.[15]

Identification

UV-visible spectra: AMK revealed peak at 295.0, 256.50 and 246.50 nm when spectra was run using Shimadzu-2450.

FTIR: AMK depicted presence of functional groups like N-H stretch (3394.83 to 3556.85 ϋ) Aliphatic C-H stretch superimposed on N-H stretch (2974.33 ϋ), N-H bend (1610.61), C-N vibration (aromatic, secondary) (1276.92 ϋ).

Acetic Acid-Induced Writhing Method

Antinociceptive activity of PMK and AMK was assessed by counting the number of writhes induced by 0.6% acetic acid (10 ml/kg, i.p.)[11] in the following 20 min. Aspirin (100 mg/kg, p.o.) was used as a reference standard. Percentage protection against writhing was taken as an index of analgesia.

% inhibition= (Number of writhing in control group – Number of writhing in treated) × 100 (Number of writhing in control group)

Hot Plate Method

Pentazocine (17.5 mg/kg, i.p.) was used as reference standard. Animals were placed individually on hot plate maintained at a temperature of 55 ± 0.5°C.[12] The latency to lick the paw was the reaction time. The reaction time was noted at 0, 15, 30, 45, 60, 90, and 120 min. The cut off time was set at 20 sec to avoid damage to the skin.

Tail Immersion Method

Pentazocine (17.5 mg/kg, i.p.) was used as a reference standard. The distal 2-3 cm portion of the mouse-tail was immersed in hot water maintained at 55 ± 0.5°C.[13] The time taken by the animal to withdraw the tail from hot water was noted as reaction time at 0, 15, 30, 45, 60, 90, and 120 min.

Antinociceptive Activity after Chronic Administration

Further, the study was carried out for 15 days to evaluate the effect of PMK and AMK in chronic pain. The animals were treated with PMK and AMK for 15 days and each of above test was performed on 1st, 3rd, 5th day and so on with interval of one day in between.[16]

Statistical Analysis

All data were expressed as mean ± SEM. Statistical analysis was carried out by one-way ANOVA followed by Dunnett's test. The values considered significant at P<0.05 when compared with the control group.

Results

Phytochemical analysis of PMK revealed the presence of alkaloids, triterpenoids, flavonoids, tannins, and phenols.

PMK and AMK significantly (P<0.05) and dose-dependently reduced the number of acetic acid-induced writhing, when compared to vehicle treated group indicating significant peripheral antinociceptive activity. The percentage inhibition on single administration of test substance was found to be increased upto 53.52 with PMK (300 mg/kg, p.o.) and 61.03 with AMK (300 mg/kg, p.o.). Aspirin, used as reference standard, produced maximum inhibition (73%) [Table 1]. Chronic administration of PMK and AMK increased the degree of inhibition. In acetic acid-induced writhing model, a highest activity was observed after day 9 [Table 2].

Table 1

Effect of Murraya koenigii on acetic acid-induced writhing in mice

Table 2

Effect of Murraya koenigii on acetic acid induced writhing after chronic administration

In hot plate model, pretreatment with PMK and AMK significantly (P<0.05) increased the latency of paw licking after acute administration, but a similar dose of PMK exhibited greater activity than AMK [Table 3]. Further, the studies involving chronic administration of PMK and AMK revealed that AMK produced greater degree of antinociception at 30 and 60 min interval than PMK [Tables 4 and 5]. After single dose, the basal reaction time in tail immersion method was significantly (P<0.05) increased by PMK and AMK. The highest reaction time was observed at 60 min interval [Table 3]. The chronic administration of PMK and AMK further enhanced tail withdrawing time with greater activity shown by AMK [Tables 6–8]. Chronic administration of PMK and AMK during hot plate model and tail immersion method initially showed fluctuating responses, but produced highest degree of antinociception after day 9 of the study AMK [Tables 4, 7, 8].

Table 3

Effect of Murraya koenigii on latency of paw licking in hot plate method

Table 4

Effect of Murraya koenigii on paw licking in hot plate method after chronic administration in mice at 30 min

Table 5

Effect of Murraya koenigii on paw licking in Hot plate method after chronic administration at 60 min

Table 6

Effect of Murraya koenigii on tail immersion method in mice

Table 8

Effect of Murraya koenigii on Tail immersion method after chronic administration at 60 min

Table 7

Effect of Murraya koenigii on Tail immersion method after chronic administration at 30 min

Discussion

The present study was carried out to study the possible peripheral and central antinociceptive activity of PMK and AMK in acute as well as chronic pain. The analgesic agents used currently in pharmacotherapy of pain has very potent side effects. Acute and chronic administration of drugs like aspirin induces anemia associated with leukocytosis.[17] Therefore, naturally originated agents with very little side-effects are required to substitute chemical therapeutics of acute and chronic pain.

The constriction response of abdomen produced by acetic acid is a sensitive procedure for peripheral analgesic agents. Acetic acid causes an increase in peritoneal fluid levels of prostaglandins (PGE2 and PGF2α), involving in part peritoneal receptors[18] and inflammatory pain by inducing capillary permeability.[19] PMK and AMK exhibited significant reduction in number of writhing, suggesting involvement of prostaglandins.

The hot plate and tail immersion are the most common tests to evaluate the central analgesic effects of drugs. The nociception experience is short lasting and it is well accepted that agonist of μ-opioid receptors produced antinociception in acute pain models. Therefore, it is believed that substances that are effective in tail immersion exert their effects predominantly through μ-opioid receptors. The hot plate test is considered to be selective for opioid like compounds, which are centrally acting analgesics in several animal species.[20] PMK and AMK had antinociceptive activity in hot plate test that may in part be mediated by opioid receptors. Present study indicates that PMK and AMK significantly increased basal reaction time in tail immersion test. These findings indicate that PMK and AMK act as analgesic probably through central mechanism or as already known for NSAIDs, it inhibited the pain receptors at the site of noxious stimuli.

The degree of antinociception produced by PMK and AMK at the end of 15 days study suggest that Murraya koenigii can be effectively used in the management of acute and chronic pain.

The infra red (IR) spectrum of AMK showed characteristic absorption bands in the range 3394.83 to 3556.85 ϋ (N-H), 2974.33 ϋ (C-methyls), 1610.61 ϋ (aromatic system) suggesting the presence of pyranocarbazole nucleus. The UV spectrum of AMK showing absorption peaks at 295.0, 256.50, and 246.50 nm also supports the presence of pyranocarbazole nucleus.[21-23]

The presence of alkaloids, triterpenoids, and flavonoids in Murraya koenigii may be responsible for antinociceptive activity as all the three constituents have been reported to posses analgesic and anti-inflammatory activity.[2425]