Chander Hass1, Parveen Kumar2, Dheeraj Rajak1, S.K. Jain1 and Manish M. Wanjari2*
1Institute of Pharmacy, Bundelkhand University, Jhansi – 284128 2Central Research Institute (Ayurveda), Gwalior - 474009
*Corresponding Author E-mail: wmanish@yahoo.com
ABSTRACT:
In view of ethnobotanical and traditional use of Neolamarckia cadamba in pain and inflammatory conditions, the present study evaluated the analgesic and anti-inflammatory activity of methanolic extract of its bark in rodents. Analgesic effect was studied in acetic acid induced writhing and hot plate analgesic model of pain while anti-inflammatory activity was investigated using carrageenan induced paw edema (acute) and cotton pellet granuloma and grass pith granuloma (sub-acute) models of inflammation. The methanolic extract of bark of Neolamarckia cadamba was administered orally in the doses of 100, 200, 400 and 800 mg/kg/day of body weight. The extract showed significant peripheral and central analgesic effect and anti-inflammatory activity which were comparable to standard drugs. The observed effects were attributed to the various phytochemicals present in bark of N. cadamba. These investigations provide the scientific rationale for the traditional claim of Neolamarckia cadamba as analgesic and anti-inflammatory agent.
KEYWORDS: Neolamarckia cadamba, analgesic, anti-inflammatory, traditional, bark.
INTRODUCTION:
Herbal medicines derived from plants/extracts are being increasingly utilized to treat wide variety of clinical diseases. A large number of plants are being used in pain and inflammatory conditions.1,2 The preference to the herbal agents is more compared to conventional non-steroidal anti-inflammatory drugs (NSAIDs) because NSAIDs pose adverse effect like gastric ulceration, thrombocytopenia, skin rashes, etc. There is growing interest in the pharmacological evaluation of various plants used in the Indian traditional system of medicine.
Neolamarckia cadamba (Roxb.) Bosser, syn. Anthocephalus cadamba var A. chinensis (Family: Rubiaceae) commonly known as Kadam is a large tree up to 45 m high, frequently found in moist deciduous evergreen forests and widely distributed throughout the greater part of India. The bark is gray, smooth in young trees, rough and longitudinally fissured in old trees.3,4 In traditional system of medicine, warm aqueous extract of Neolamarckia cadamba leaves is used to alleviate pain, swelling and for cleansing and better healing of wounds as well as for the treatment of menorrhagia.5
The dried stem bark is used as folk medicine in the treatment of fever, inflammation, anemia, uterine complaints, blood and skin diseases, diarrhoea, dysentery, colitis, stomatitis and for improvement of semen quality.6,7 The bark and leaves of the plants are also reported to possess various medicinal uses such as astringent, mucolytic, analgesic, anti-inflammatory, febrifuge, diuretic, laxative, antiseptic, antimicrobial, wound healing, antioxidant,5,8-11 anthelmintic,12 hypoglycaemic, lipid lowering activities and is given in cough.10,13,14 Chlorogenic acid isolated from the leaves has been reported to possess hepatoprotective activity in vitro and antioxidant activity in liver microsomes in vivo.15 The root extract has been found useful in urinary ailments dysurea, calculi and glycosuria.16 Despite the traditional medicinal use of the bark of Neolamarckia cadamba in pain and inflammatory conditions, it has not been scientifically evaluated fully in various animal models.
In the present study, attempts were made to examine the analgesic activity of Neolamarckia cadamba in the peripheral and central models of pain and the anti-inflammatory activity in both acute and subacute models of inflammation.
MATERIALS AND METHODS:
Preliminary phytochemical screening:
NCBE was subjected to various preliminary phytochemical screening as per the standard procedures.17
Animals:
Male Swiss albino mice (25-30 g) were used for assessment of analgesic activity and male Wistar albino rats (220-280 g) were used for assessment of anti-inflammatory activity. Animals procured from Central Drug Research Institute, Lucknow were kept at departmental animal house at 25±2°C, relative humidity 55-65% and light and dark cycles of 12 h respectively. Animals were provided with standard pellet diet and water ad libitum. Principles of laboratory animal care guidelines were followed and prior permission was sought from the Institute Animal Ethics Committee for conducting the study (Regd. No. 716/02/a/CPCSEA).
Aspirin (Arora Chemicals, New Delhi) and Indomethacin (Alfa Remedies, Ambala) were obtained as gift samples. Diethyl ether (Merck, Mumbai) and Sodium carboxymethylcellulose (Himedia, Mumbai) were procured from local market. Carrageenan was procured from Sigma Aldrich, USA. Pentazocin (Ranbaxy Laboratories Ltd., Ahmedabad) and Thiopentone sodium (Neon Laboratories, Mumbai) were purchased in injectable form. Drug solutions were freshly prepared and their doses are expressed in terms of their free bases.
Doses and Treatments:
Mice and rats were divided into six different groups (n=5). NCBE was suspended in 0.5% sodium carboxymethylcellulose in normal saline for administration purpose and administered in graded doses of 100, 200, 400, 800 mg/kg, p.o. Pentazocin (10 mg/kg, i.p.) was administered as standard drug in Eddy’s hot plate analgesia method while aspirin (150 mg/kg, p.o.) was administered as standard in cotton pellet and grass pith granuloma models.18 Indomethacin (10mg/kg, p.o.) was used as standard drug in acetic acid induced writhing19 and carrageenan induced paw oedema.20 Carrageenan (0.1 ml of 1% w/v freshly prepared suspension in normal saline) was administered in the sub-plantar region in hind paw of rats for causing paw oedema and acetic acid (10 ml/kg of 0.6 % acetic acid, i.p.) was injected for inducing writhing episodes. Thiopentone sodium (25mg/kg, i.p.)21 was used an anaesthetic agent in surgery of animals in granuloma models. All above doses were given 10 ml/kg, p.o. and 5 ml/kg, i.p. All rodends model were fasted for 12 h before starting the test.22
Acute toxicity Study:
The study was carried out according to OECD guidelines 423.23 NCBE in dose of 2000 mg/kg, (p.o.) was administered in group of three rats and the rats were observed for a period of 72 hours and thereafter up to 14 days for behavioral, neurological, autonomic profiles and for any lethality, moribund state or death.
Assessment of analgesic activity:
Assessment of peripheral analgesic effect of NCBE:
The peripheral analgesic effect of NCBE was evaluated using acetic acid induced writhing test model.24 Writhing is defined as stretch, torsion to one side and retraction of the abdomen so that the belly of the mouse touches to the floor. Abdominal writhing episodes were induced in mice by i.p. injection of acetic acid. Mice were pretreated with vehicle or NCBE or indomethacin 1 h prior to acetic acid injection and thereafter the observations were made. The number of stretching occurred immediately within 30 min. after the acetic acid injection was recorded. The results were evaluated by calculating the mean number of stretching per group and represented as % inhibition of stretching movement compared to control group as below.
% Analgesic activity = (n′-n/n) x 100
Where, n is the average no of stretching of control group and n′ is the average no of stretching in test group.
Assessment of central analgesic effect of NCBE:
The central analgesic effect of NCBE was studied using Eddy’s hot plate test.25 The mice were placed on hot plate maintained at 55±1°C and time in seconds for the paw licking or jumping was recorded as the reaction time. Mice which have shown basal reaction time period below 6 seconds were selected and used for the screening. Mice were pretreated with vehicle or NCBE or pentazocin. The mouse activity on hot plate was closely observed and the reaction time (latency period) was recorded in seconds as the time taken for the animal to react to the thermal pain by licking its paw or attempting to jump out.
Assessment of Anti-inflammatory Activity:
Acute paw oedema (acute inflammation) was induced by sub-plantar injection of carrageenan suspension in normal saline into the right hind paw of each rat. The left hind paw was injected with same volume of 0.1 ml of normal saline. Rats were pretreated with Vehicle or NCBE or aspirin 1 h prior to carrageenan administration. The paw size was measured in mm using Vernier Caliper before (0 min) and at an interval of 30, 60, 120, 150, 180, 240 min. after carrageenan administration27. The percent inhibition was calculated by following formula.
Percentage Inhibition = (Ct -Co) control — (Ct - Co) treated / (Ct - Co) Control
Where, Ct = Linear circumference of paw after carrageenan injection and
Co = Linear Circumference of paw before carrageenan injection
Cotton pellet-induced granuloma model:
Under thiopentone sodium anesthesia, sterile cottons pellets weighing 10±1 mg were implanted subcutaneously in both axilla region of each rat through a single needle incision, on each side. Rats were administered vehicle or NCBE or aspirin once daily up to 7 days from the day of cotton pellet implantation. On day 8, rats were sacrificed under overdose of ether. The granuloma was dissected out and dried at 60° C for 24 h to constant weight. The weight of the cotton pellet before implantation was subtracted from the weight of the dried dissected weight. The increase in dry weight of pellet was taken as the increase in granuloma formation.28
Grass pith granuloma model:29
This method is used for the study of sub-acute inflammation. Under thiopentone sodium anesthesia with semi aseptic measures in the rats, subcutaneous dead space wounds were inflicted in the region of the groin by making a pouch through a small nick in the skin. Granuloma formation was induced by implanting grass piths in groin regions. Cylindrical grass pith measuring 2.5 cm in length and 0.2 cm in diameter was introduced into the pouch. The wounds were sutured and mopped with alcoholic swabs. Rats were placed into their individual cages after recovery from anesthesia. These animals received vehicle, NCBE or aspirin treatment once daily up to 7 days. On day 8, rats were sacrificed under overdose of ether. Granulomas surrounding the grass piths were excised and slit open and preserved in 10% formalin. The sections (5 µm) were cut and slides were prepared and studied for histopathology. The parameters like infiltration of mononuclear cells, vascularization and granuloma formation were observed in the slides under microscope.
Statistical Analysis:
The data were analyzed by one-way ANOVA and one-way repeat measure ANOVA, wherever applicable followed by Dunnett’s post hoc test. The difference of P<0.05 was considered significant in all the cases.
RESULTS:
Phytochemical Screening:
The phytochemical screening of NCBE revealed the presence of alkaloid, carbohydrates, sterols, glycosides and flavonoids.
Acute Toxicity Study:
The limit test for acute toxicity showed that NCBE at 2000 mg/kg, p.o. in rats exhibited normal behavioral, neurological and autonomic profiles. No mortality or toxicity of any nature was observed during 72 hours and thereafter up to 14 days observation. This indicated that approximate LD50 value of NCBE is more than 2000 mg/kg, p.o. and it is relatively safe and non-toxic to rats.
Assessment of Analgesic Activity:
Peripheral analgesic effect of NCBE in acetic acid induced writhing test in mice:
One-way ANOVA revealed a significant (P<0.0001) influence of NCBE on acetic acid-induced writhing compared to control. Post hoc Dunnett’s test showed that NCBE at 200, 400 and 800 mg/kg caused significant inhibition (P<0.05-0.001) of writhing but NCBE 100 mg/kg did not produce inhibition of writhing (P>0.05). The maximum inhibition (64.20%) was noted for NCBE 800 mg/kg while NCBE 200 and 400mg/kg produced 33.00 and 56.70% inhibition, respectively. The effects were comparable with that of reference standard, indomethacin that showed 36.00% inhibition of writhing (P<0.01) (Table.1).
Table. 1
Treatments (mg/kg) |
No. of Writhing episodes/30 min |
% Inhibition of writhing |
Vehicle NCBE 100 NCBE 200 NCBE 400 NCBE 800 Indomethacin 10 |
66.60 ±6.84 54.00 ±5.59 44.60 ±4.58* 28.80 ±4.08** 23.80 ±2.52** 42.60 ±4.26** |
… 18.91 % 33.00 % 56.70 % 64.20 % 36.00 % |
Table. 1 Effect of NCBE on acetic acid induced writhing in mice. Mice were pretreated with vehicle or NCBE (100, 200, 400 and 800 mg/kg, p.o.) or indomethacin (10 mg/kg, p.o.) 1 h prior to acetic acid injection. The number of stretchings occured immediately within 30 min. after the acetic acid injection, was recorded. The results are represented as % inhibition of stretching movement. Results are expressed as mean±S.E.M. (n=5) *P<0.05 or **P<0.01 Vs control (One-way ANOVA followed by Post hoc Dunnett’s test).
Central analgesic effect of NCBE in hot plate test in mice:
One-way repeat measure ANOVA exhibited significant (P<0.0001) influence of NCBE on hot plate reaction time. Dunnett’s test showed that NCBE at 200, 400 and 800 mg/kg showed significant (P<0.001) increase in reaction time but NCBE 100 mg/kg did not show any significant effect compared to control (P>0.05). NCBE 800 mg/kg produced maximum effect and increased reaction time from 3.80 to 6.26 s at the end of 150 min. The effects were comparable to pentazocin that showed significant increase (P<0.001) in reaction time from 3.51 to 7.88 s at the end of 150 min (Table. 2).
Assessment of Anti-inflammatory Activity:
Effect of NCBE on carrageenan-induced rat paw edema:
One-way repeat measure ANOVA showed significant (P<0.0001) influence of NCBE on carrageenan-induced inflammation. Dunnett’s test indicated NCBE 200, 400 and 800 mg/kg caused significant (P<0.01) decrease in paw oedema compared to vehicle while NCBE 100 mg/kg did not show any effect. NCBE 800 mg/kg produced a reduction in paw oedema 58.28 and 52.15 % where indomethacin10 mg/kg shows significant maximum reduction 65.64% and 65.64% in comparison of the control
Table. 2
Treatment mg/kg |
Hot plate reaction time (sec) |
|||||
0 min |
30 min |
60 min |
90 min |
120 min |
150 min |
|
Vehicle NCBE 100 NCBE 200 NCBE 400 NCBE 800 Pentazocin 10 |
3.80 ±0.29 3.80 ±0.51 4.40*** ±0.79 3.96*** ±0.49 3.80*** ±0.45 3.51*** ±0.15 |
3.27 ±0.13 3.82 ±0.48 4.90*** ±0.53 4.90*** ±0.54 4.88*** ±0.61 4.33*** ±0.13 |
3.25 ±0.43 3.96 ±0.70 5.66*** ±0.77 5.82*** ±2.82 5.88*** ±0.86 5.62*** ±0.06 |
3.54 ±0.34 4.40 ±0.85 5.89*** ±1.00 6.12*** ±0.57 6.36*** ±0.65 6.23*** ±0.10 |
3.46 ± 0. 23 5.09 ±0.72 6.26*** ±1.06 6.42*** ±0.83 6.98*** ±0.64 7.14*** ±0.13 |
4.05 ±0.91 4.93 ±0.54 5.49*** ±0.48 5.95*** ±0.83 6.26*** ±0.60 7.88*** ±0.08 |
Table. 2 Effect of NCBE on hot plate test in mice. Mice were pretreated with vehicle or NCBE (100, 200, 400 and 800 mg/kg, p.o.) or pentazocin (10 mg/kg, i.p.). The reaction time (latency period) was recorded in seconds as the time taken for the animal to react to the thermal pain by licking its paw or attempting to jump out. Results are expressed as mean±S.E.M. (n=5) ***P<0.001 Vs control (One-way repeat measure ANOVA followed by Dunnett’s test).
Table. 3
Treatment (mg/kg) |
Change in Paw oedema in mm |
||||||
Time (minutes) |
|||||||
30 60 90 120 150 180 240 |
|||||||
Vehicle
NCBE 100
NCBE 200
NCBE 400
NCBE 800
Indomethacin |
1.04±0.41 ------ 0.86 ±0.22 (17.31%) 0.62 ±0.71 (40.39%)** 0.56±0.18 (46.15%)** 0.32 ±0.17 (69.23%)** 0.32 ±0.30 (69.23%)** |
1.70 ±0.36 ------ 1.45 ±0.18 (14.70%) 1.24 ±0.07 (27.05%)** 0.90 ±0.16 (47.05%)** 0.98 ±0.27 (42.35%)** 0.72 ±0.31 (57.65%)** |
3.02±0.05 ------ 2.58 ±0.16 (14.57%) 1.94 ±0.12 (35.76%)** 1.70±0.15 (43.71%)** 1.26 ±0.34 (58.28%)** 1.08±0.65 (65.64%)** |
3.62 ±0.49 ------ 2.90 ±0. 37 (11.04%) 2.54±0.18 (22.08%)** 2.36 ±0.33 (27.60%)** 1.56 ±0.48 (52.15%)** 1.12 ±0.37 (65.64%)** |
3.58 ±0.36 ------ 3.22 ±0.44 (7.26%) 3.02 ±0.14 (15.64%)** 2.30 ±0.43 (32.96%)** 2.08 ±0.40 (41.90%)** 1.66 ±0.57 (53.63%)** |
3.98 ±0.37 ------ 3.56 ±0.15 (10.55%) 3.06 ±0.16 (23.12%)** 2.30 ±0.23 (42.21%)** 2.30 ±0.38 (42.21%)** 1.90 ±0.47 (52.26%)** |
4.28 ±0.33 ------ 3.28 ±0.16 ((23.36%) 3.46±0.21 (19.15%)** 2.94 ±0.21 (31.13%)** 3.24±0.37 (24.29%)** 2.72±0.46 (36.44% )** |
Table. 3 Effect of NCBE on carrageenan-induced rat paw edema. Rats were pretreated with vehicle or NCBE (100, 200, 400 and 800 mg/kg, p.o.) or indomethacin (10 mg/kg, p.o.), 1 h prior to carrageenan administration. The paw size was measured in mm after carrageenan administration and the percent inhibition was calculated. Results are expressed as mean±S.E.M. (n=5) **P<0.001 Vs control (One-way repeat measure ANOVA followed by Dunnett’s test). Value in parenthesis shows percentage inhibition.
group at the end of 90 and 120 min respectively. Except NCBE 100 mg/kg all treatment indicating significantly anti-inflammatory activity from 30 min. onwards when compared to control (Table. 3).
Effect of NCBE on cotton pellet granuloma:
One-way ANOVA showed significant (P<0.0001) influence of NCBE on on cotton pellet granuloma compared to control group. Post hoc Dunnett’s test showed that NCBE at 200, 400 and 800 mg/kg caused significant inhibition (P<0.05-0.01) of granuloma formation but NCBE 100 mg/kg did not produce any significant effect (P>0.05). The maximum inhibition (51.75%) was noted for NCBE 800 mg/kg while NCBE 200 and 400 mg/kg produced 32.24% and 38.24% inhibition respectively. The effects were comparable with that of reference standard, aspirin that showed 44.38% inhibition of granuloma formation (P<0.01) (Table. 4).
Effect of NCBE on grass pith granuloma:
The slides were observed under the microscope for presence of aggregation of macrophages, lymphocytes, plasma cells, fibroblasts and collagen and granuloma formation and necrosis. Representative photomicrographs are presented. Photomicrograph from vehicle treated rat (Fig. 1 A) revealed the presence of nonspecific fully developed granulomatous features with marked mononuclear cells infiltration and neovasularization. This is considered as positive control against which the effect of other treatments was compared. Photomicrographs from aspirin treated rats (Fig. 1 B) highlight the marked inhibition of inflammatory response with lesser degree of mononuclear cells infiltration and neovasularization compared to vehicle treated rats. Photomicrograph from NCBE (400 and 800 mg/kg) treated rats (Fig. 1 C and D) indicates the marked dose dependent inhibition of inflammatory response with lesser degree of mononuclear cells infiltration and neovasularization compared to vehicle treated rats. The inhibition obtained with NCBE (800 mg/kg) is comparable to that of aspirin.
Figure. 1 Effect of NCBE on grass pith granuloma. Rats were administered vehicle, NCBE (100, 200, 400 and 800 mg/kg, p.o.) or aspirin (150 mg/kg, p.o.) once daily up to 7 days. On day 8, slides of granulomatous were observed under the microscope for presence of aggregation of macrophages, lymphocytes, plasma cells, fibroblasts and collagen and granuloma formation and necrosis. Representative photomicrographs are presented.
Table. 4
Treatment (mg/kg) |
Mean dry wt. of granuloma (mg)/100gm body wt. |
% Inhibition |
Vehicle NCBE 100 NCBE 200 NCBE 400 NCBE 800 Aspirin 150 |
27.69±0.95 20.40±2.46 18.58±1.16* 17.10±1.26** 13.36±0.92** 15.40±1.41** |
… 26.32% 32.24% 38.24% 51.75% 44.38% |
Table. 4 Effect of NCBE on cotton pellet granuloma. Rats were administered vehicle or NCBE (100, 200, 400 and 800 mg/kg, p.o.) or aspirin (150 mg/kg, p.o.) once daily up to 7 days from the day of cotton pellet implantation. On day 8, the increase in dry weight of pellet was taken as the increase in granuloma formation. Results are expressed as mean±S.E.M. (n=5) *P<0.05 or **P<0.01 Vs control (One-way ANOVA followed by Post hoc Dunnett’s test).
DISCUSSION:
The results of the present study showed that NCBE exhibited significant analgesic and anti-inflammatory activities. The results of the analgesic test experiments suggest that NCBE possesses both peripheral and central analgesic properties. Acetic acid-induced writhing and hot plate test methods are useful techniques for the evaluation of peripherally and centrally acting analgesic drugs, respectively.30-32 NCBE exhibited significant dose dependent inhibition of acetic acid-induced writhing in mice, comparable to indomethacin while delayed the reaction times of mice in the ‘hot-plate’ analgesia in a dose dependent fashion comparable to pentazocin. The peripheral analgesic effect is mediated through inhibition of cyclooxygenase and/or lipoxygenase (and other inflammatory mediators) or inhibition of pain responses mediated by nociceptors peripherally33,34 while the central analgesic action is mediated via inhibition of central pain receptors.35,36 Therefore, it is possible that the NCBE may be showing its analgesic effect through these mechanisms.
The carrageenan-induced rat paw edema is a suitable test for evaluating anti-inflammatory drugs37,38. Development of the edema in the paw of the rat after injection of carrageenan is a biphasic event. The initial phase observed during the first hour is attributed to the release of histamine and serotonin while the second phase of edema is owed to the release of prostaglandins, protease and lysosomes.34,39-48 In carrageenan paw edema model of acute inflammation, NCBE showed significant dose dependent reduction in paw size, comparable to indomethacin. This might be due to the inhibition of the biphasic response induced by the carrageenan. Acute anti-inflammatory effect is the result of inhibition of either cyclo-oxygenase and/or lypo-oxygenase enzyme or inhibition of synthesis, release and action of above inflammatory mediators. In subacute inflammation study, NCBE significantly reduced the granuloma formation (both cotton pellet and grass pith granuloma) in rats. Multiplications of small blood vessels as well as proliferation of fibroblast are the characteristic features of the repair phase of inflammation. NCBE showed significant dose dependent reduction in weight of granuloma in cotton pellet granuloma model while decreased the aggregation of macrophages, lymphocytes, plasma cells, fibroblasts and collagen, and reduced granuloma formation and necrosis in grass pith granuloma model. The effect of NCBE was comparable to aspirin in both cases. From the above results, it appears that NCBE might be showing anti-inflammatory action through cyclo-oxygenase and/or lypo-oxygenase enzyme inhibition or inhibition of synthesis, release and action of above inflammatory mediators and decrease in the aggregation of macrophages, lymphocytes, plasma cells, fibroblasts and collagen.
Several plant extracts showed analgesic and anti-inflammatory effect in animal models and their effects have been attributed to the presence of triterpenoids,49 alkaloids,50,51 glycosides,22,52 flavonoids53,54 and sterols.55,56 The analgesic and anti-inflammatory activity of some flavonoids,53,57-61 terpenoids and saponins has been already reported. Saponins isolated from about 50 plants have been shown to possess anti-inflammatory activities against several experimental models of inflammation in mice and rats.61 The phytochemical screening of NCBE revealed the presence of triterpenes, glycosides, saponins, indole alkaloids; cadambin, 3′α-dihydrocadambine, cadamine, isocadamine and isodihydrocadambine,62-65 secoiridoid glucoside and two phenolic apioglucoside kelampayaosides,63 two triterpenoid glycosides, glycosides A and B and two novel triterpenoid saponins, phelasin A (1) and phelasin B (2)66. Therefore, it is possible that the analgesic and anti-inflammatory activity of NCBE can be due to the presence of these phytochemical constituents. Further bioactivity guided studies are required to determine the exact constituent responsible for the analgesic and anti-inflammatory effect of NCBE.
It can be concluded that NCBE has potential analgesic and anti-inflammatory activities and this study provides a rationale for the use of Neolamarckia cadamba in traditional medicine as a pain reliever and in the treatment of inflammatory diseases.
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Received on 06.05.2010 Modified on 20.05.2010
Accepted on 31.05.2010 © RJPT All right reserved
Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1178-1184