|Year : 2013 | Volume
| Issue : 4 | Page : 376-380
Protective effect of Clerodendrum colebrookianum Walp., on acute and chronic inflammation in rats
Lokesh Deb1, Amitabha Dey2, G Sakthivel3, Subrat Kumar Bhattamishra4, Amitsankar Dutta5
1 Pharmacology Laboratory, Medicinal Plants and Horticultural Resources Division, Institute of Bioresources and Sustainable Development, Department of Biotechnology, Government of India, Takyelpat, Imphal, Manipur, India
2 Pharmacology Laboratory, Medicinal Plants and Horticultural Resources Division, Institute of Bioresources and Sustainable Development, Department of Biotechnology, Government of India, Takyelpat, Imphal, Manipur; Department of Pharmacology, Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa, India
3 Department of Nanotechnology, Noorul Islam Centre for Higher Education, Noorul Islam University, Kumaracoil, Thuckalay, Tamilnadu, India
4 Department of Pharmacology, Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa, India
5 Department of Pharmacology, R. K. Pharmacy College, Azamgarh, Uttar Pradesh, India
|Date of Submission||07-Aug-2012|
|Date of Decision||14-Dec-2012|
|Date of Acceptance||23-Apr-2013|
|Date of Web Publication||15-Jul-2013|
Pharmacology Laboratory, Medicinal Plants and Horticultural Resources Division, Institute of Bioresources and Sustainable Development, Department of Biotechnology, Government of India, Takyelpat, Imphal, Manipur
Source of Support: This work supported by Institute of Bioresources & Sustainable Development, Department of Biotechnology, Government of India, Takyelpat, Imphal, Manipur - 795001, India under core research project entitled: "Pharmacological evaluation of active constituents from bioresources used in traditional/folklore medicines of north eastern India and value addition for commercial exploitation" (Code- IBSD/PBD-4), Conflict of Interest: None
Aim: To evaluate antioxidant, anti-inflammatory potential of the aqueous extracts and its aqueous, n-butanol, ethyl-acetate, and chloroform fractions of Clerodendrum colebrookianum Walp. leaves.
Materials and Methods: In this present study, all the test samples were evaluated on in-vivo inflammatory model such as carrageenan and histamine-induced acute-inflammation and cotton pellet induced granuloma formation in albino male rats. Test samples were also employed in in-vitro assays like DPPH* free radical scavenging activity and COX inhibition assay.
Results: The test samples at the dose of 200mg/kg/p.o. were found to cause significant inhibition of carrageenan and histamine-induced inflammation and cotton pallet-induced granuloma formation on acute and chronic inflammation in rats. The test samples, except n-butanol fraction, exhibited inhibitory effect for both COX-1 and COX-2, in in-vitro assay but their percentage of inhibition values differs from each other. The test samples (aqueous extracts, aqueous, n-butanol, ethyl-acetate, and chloroform fractions) at 100 μg concentration exhibits 54.37%, 33.88%, 62.85%, 56.28%, and 57.48% DPPH* radical-scavenging effect respectively in in-vitro antioxidant study.
Conclusion: These observations established the anti-inflammatory effect of C. colebrookianum leaves in acute and chronic stages of inflammation by free radical scavenging and inhibition of COX-1 and COX-2.
Keywords: Antioxidant, Carrageenan, Clerodendrum colebrookianum, COX-1 and COX-2, Histamine, Inflammation
|How to cite this article:|
Deb L, Dey A, Sakthivel G, Bhattamishra SK, Dutta A. Protective effect of Clerodendrum colebrookianum Walp., on acute and chronic inflammation in rats. Indian J Pharmacol 2013;45:376-80
|How to cite this URL:|
Deb L, Dey A, Sakthivel G, Bhattamishra SK, Dutta A. Protective effect of Clerodendrum colebrookianum Walp., on acute and chronic inflammation in rats. Indian J Pharmacol [serial online] 2013 [cited 2019 Nov 16];45:376-80. Available from: http://www.ijp-online.com/text.asp?2013/45/4/376/115021
| » Introduction|| |
Inflammation is the immune system's response to infection and injury and has been implicated in the pathogenesis of diseases such as arthritis, cancer, atherosclerosis, stroke, and epilepsy, as well as neurodegenerative diseases (for example: multiple sclerosis, Alzheimer's and Parkinson's diseases). Two isoforms of cyclooxygenase, COX-1 and COX-2, have been identified. COX-1 is thought to mediate "housekeeping" functions, and is responsible for the production of prostaglandins that are required for normal physiological activities, whereas inducible COX-2 is expressed in immune cells which is a key player in initiating the inflammatory response by converting arachidonic acid (AA, C20:4), an ω-6 polyunsaturated fatty acid (PUFA), into proinflammatory prostaglandins (mainly PGE 2 ) and triggering production of other proinflammatory chemokines and cytokines. Because of this, a therapeutic strategy for inflammatory diseases has involved inhibition of COX-2, though this is now challenged by the finding that COX-2 derived oxidative metabolites of ω-3 PUFAs in activated macrophages. Thus, COX-2 has a dual role in initiation and resolution of inflammation by generating ω-6 and ω-3 PUFA-derived pro- and anti-inflammatory mediators, respectively. 
In globalization and post- GATT scenario, the cost of synthetic medicines is escalating. The side effects associated with various synthetic drugs are also the cause for renewed interest in traditional systems of medicine. In this background, we thought to find out effective safer and cheaper remedy for inflammatory disorders. In this context, a field survey on the use of traditional medicines by native practitioners of Manipur was undertaken. The use of locally available herbs for the purpose was also explored. Several field surveys detected a plant, i.e., Clerodendrum colebrookianum Walp. widely distributed in North-East India, that claimed as highly useful in treating cardiac (hypertension),  hepatic, and inflammatory disorders by the native practitioners of the region. Also there are ethnic reports on the leaves of C. colebrookianum Walp., for use against dizziness, greenish swelling (gland), sore tongue in children, ,[ 4] skin disease, cough, and dysentery  in traditional practice.
| » Materials and Methods|| |
. colebrookianum Walp. leaves were collected from the garden of Institute of Bioresources and Sustainable Development (IBSD), Imphal, Manipur. The plant was identified and authenticated by Dr. Biseswhori Thongam, Scientist - C (Plant Taxonomy), IBSD, Takyelpat, Imphal, Manipur, where a voucher specimen (No.-IBSD/M/1014) was deposited for reference to Plant systematic and conservation Lab, IBSD, Takyelpat, Imphal.
Extraction and sample preparation
The fresh leaves of C. colebrookianum Walp. were cleaned, shade dried, and reduced into coarse powder in a Wiring blender. The powdered material was then subjected to soxhlet extraction with the purified water as solvent in 1:4 (w/v) ratio. Aqueous extract (AECc) was divided in two equal portions. One portion concentrated in vacuum evaporator (Buchi Rotavapor R-210) and dried in vacuum desiccators and the other portion was mixed with equal quantity of petroleum ether in separating funnel to separate aqueous and petroleum ether portions. Same aqueous portion was again mixed with n-butanol, ethyl-acetate, and chloroform one after another and separated out the respective portions to get the aqueous fraction (AFCc), n-butanol fraction (nBFCc), ethyl-acetate fraction (EtFCc), and chloroform fraction (ChFCc), respectively.
The respective fractions were concentrated under reduced pressure in vacuum evaporator (Buchi Rotavapor® R-210) and dried in vacuum desiccators. After drying, all products were stored in refrigerator (8 ± 2°C) and the same was used for in vivo and in vitro studies.
Albino male rats (Wistar) weighing 150 to 200g and Wistar albino male mice weighing 20 to 25g were used. They were procured from Regional Institute of Medical Sciences (RIMS), Imphal. The animals were acclimatized for one week under laboratory conditions. They were housed in polypropylene cages and maintained at 27°C ± 2°C, under 12 hour dark / light cycle and fed with soya bean choke, Gram and water ad libitum. The litter in the cages was renewed daily to ensure hygienic condition and maximum comfort for animals. Ethical clearance for handling the animals was obtained from the Institutional Animals Ethical Committee (IAEC), IBSD, Imphal (approval No.-IBSD/IAEC/Inst./Ph.cology/1) prior to the beginning of the study.
Determination of acute toxicity (ALD50)
The acute toxicity for AECc, AFCc, nBFCc, EtFCc, and ChFCc was determined in albino mice, maintained under standard conditions. The animals (n=3) in each group were fasted overnight prior to the experiment. Fixed dose (OCED Guideline no. 420) method of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) was adopted for toxicity studies. The extract and fractions in suspension were administrated orally. The mortality and abnormality were observed after administering samples at a dose of 2000mg/kg in all animals. ,,
Carrageenan and Histamine Induced Paw inflammation
In two different sets of experiments, the Wister albino rats were divided into seven groups of five animals each for carrageenan-induced inflammation and separate seven groups of five animals each for histamine induced inflammation. Paw inflammation was induced in overnight-fasted rats by injecting 0.1ml of 1% w/v carrageenan sodium salt (Ozone, Mumbai) and 0.1ml of 1% w/v histamine (Ozone, Mumbai) subcutaneously into the sub-plantar region of the rat right hind paw to the respective groups of animals. Animals were pre-treated either with distilled water 2ml/kg body weight p.o. for Control group, diclofenac sodium 8mg/kg p.o. for reference group, and 200 mg/kg, p.o. of AECc, AFCc, nBFCc, EtFCc, & ChFCc to the respective test groups 30 minutes before the carrageenan or histamine injection. The readings of normal paw volume at 0 minutes before injecting inflammatory agents and inflamed paw volume after injecting inflammatory agents was obtained at 1, 2, 3, 4, and 6 hours with the aid of plethysmometer. 
Cotton pellet granuloma method
The effect of test products on chronic or proliferative phase of inflammation was studied in cotton pellet granuloma rat model as described by Deb et al. 2009. The animals were divided into seven groups of five animals each. Autoclaved cotton pellets weighing 50±1mg each was implanted subcutaneously through a small incision made along the axilla or flank region of the anesthetized rats. After implantation, the animals belonging to different test groups received the samples AECc, AFCc, nBFCc, EtFCc, and ChFCc at the dose of 200mg/kg, p.o., once daily for 14 consecutive days. Control group animals received vehicle (distilled water 1 ml/animal, p.o.) and the reference group animals received diclofenac sodium (8mg/kg) orally for the same 14 consecutive days.
On the 14 th day, the cotton pellets covered by the granulomatous tissue were removed from all animals under weak ether anesthesia and dried in hot air oven at 55 ± 5°C till a constant weight was achieved. Granuloma weight was obtained by subtracting the weight of the cotton pellet on 0 day (before start of experiment) from the weight of the cotton pellet on the 14 th day. 
Cyclooxygenase-1 and Cyclooxygenase-2 Inhibition assay
COX-1 and COX-2 assays were performed in separate assay plates (96 flat bottom well plates). COX enzyme activity was determined by using a colorimetric COX inhibitor screening assay kit (Cayman Chemical Company, Ann Arbor, USA) in a free system, according to the manufacturer's instructions. The Colorimetric COX inhibitor Screening Assay measured the peroxidase component of cyclooxygenases. The peroxide activity was assayed colorimetrically by monitoring the appearance of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. All samples were dissolved in DMSO. Briefly, 160μl of assay buffer and 10μl of heme were added to the background well. 150μl of assay buffer, 10μl of heme and 10μl of COX-I & COX - II enzyme were added to the 100% initial activity well. 10μl of samples AECc, AFCc, nBFCc, EtFCc, and ChFCc (the final concentration is 100μM and 50μM) was added to the sample wells and 10μl of DMSO was added to the background wells. The plate was carefully shaken for a few seconds and incubated for 5 minutes at 27°C. 20μl of the colorimetric substrate solution and then 20μl of AA were added to all the wells. The plate was carefully shaken for a few seconds and incubated for 5 minutes at 27°C. The absorbance at 590 nm was read using a microplate reader (Thermo Scientific Multiskan Spectrum) and the inhibition ratio on COX-1 & COX-2 enzymatic activities was calculated according to the manufacturer's instructions. The results were expressed as percentage COX-1 and COX-2 inhibition.
DPPH* radical scavenging activity
The free radical-scavenging activity of all samples (AECc, AFCc, nBFCc, EtFCc, and ChFCc) was measured in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH. Solution of 0.1 mM DPPH (Himedia, Mumbai) in ethanol was prepared and 1.0mL of this solution was added to 3.0mL of all the extracts solution in water at different concentrations (10-100μg/mL). Thirty minutes later, the absorbance was measured at 517nm. , Lower absorbance of the reaction mixture indicates higher free radical-scavenging activity. Rutin (Ozone, Mumbai) was used as a standard antioxidant. The results were expressed as IC 50 (inhibitory concentration 50) value i.e. concentration of samples exhibited 50% inhibition of DPPH* radicals.
Data were expressed as mean ± S.E.M. Differences were considered significant at ***P<0.001, **P < 0.01 or * P<0.05 when the test groups were compared with the control group. For numerical results, one-way analysis of variance (ANOVA) with Dunnett's test (compare rest vs. control) was performed using GraphPad InStat Version 3 (GraphPad Software) and all graphs were made using GraphPad Prism5 software.
| » Results|| |
Determination of acute toxicity (ALD50)
All the test samples (AECc, AFCc, nBFCc, EtFCc and ChFCc) employed in acute toxicity study did not show any sign of abnormality and mortality at the dose of 2000mg/kg in experimental animals for a period of 48 hours initially and then after for a week of observation. Therefore, 2000 mg/kg dose was considered as ALD 50 ; cut off the dose (safe dose) accordingly 1/10 th of that dose was selected (200mg/kg) for in vivo experiments.
Carrageenan and Histamine induced paw oedema
The anti-inflammatory effect of AECc, AFCc, nBFCc, EtFCc, and ChFCc was observed on both acute inflammation models. All the test samples at the dose of 200 mg/kg. p.o. were found to cause significant (**P<0.01) inhibition of carrageenan and histamine-induced inflammation.whereas nBFCc shows highest activity compared to other test group [Table 1]a and b.
Cotton pellet granuloma method
The effect of samples AECc, AFCc, nBFCc, EtFCc, and ChFCc were evaluated on chronic inflammatory model. All the test samples at the dose of 200 mg/kg. p.o. were found to cause significant (***P<0.001) inhibition of granuloma formation, whereas AFCc and nBFCc showed highest activity among them [Table 2].
|Table 2: Effect of C. colebrookianum Walp on cotton pellet-induced granuloma|
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COX 1 and COX-2 inhibition assay
In in-vitro assays, the samples AECc, AFCc, EtFCc and ChFCc inhibited both COX-1 and COX-2 by 38.88%, 44.44%, 33.33%, 27.77% and 51.61%, 32.25%, 16.12%, 74.19% respectively, whereas nBFCc does not show any effect on COX-1 but inhibited COX-2 with a highest 83.14% inhibition [Table 3].
DPPH* radical scavenging activity
In in-vitro assays, the IC 50 values of Rutin, AECc, AFCc, nBFCc, EtFCc, and ChFCc were calculated as 55.24μg, 90.98μg, 92.73μg, 70.81μg, 32.36μg and 83.93μg for DPPH* radical scavenging effect respectively [Table 4].
| » Discussion|| |
In Indian system of medicine, certain herbs are claimed to provide relief of pain and inflammation. The claimed therapeutic reputation has to be verified in a scientific manner. Inflammation is a complex process Reactive Oxygen Species (ROS) plays an important role in the pathogenesis of inflammatory diseases.  In present study, the samples AECc, AFCc, nBFCc, EtFCc, & ChFCc posses DPPH radical scavenging activity.
Carrageenan-induced rat paw edema model is a suitable test for evaluating anti-inflammatory drugs, which has frequently been used to assess the antiedematous effect of the drug. Carrageenan is a strong chemical used for the release of inflammatory and proinflammatory mediators (prostaglandins, leukotrienes, histamine, bradykinin, TNF-α, etc.). The course of acute inflammation is biphasic. First phase starts with the release of histamine, serotonin, and kinins after the injection of phlogistic agent in the first few hours. However, in the second phase prostaglandins like substances are released 2-3 hours after carrageenan injection. Second phase is sensitive to both the clinically useful steroidal and nonsteroidal anti-inflammatory agent. Prostaglandins are the main culprit responsible for acute inflammation.  The intra-plantar injection of Freund's Complete Adjuvant (FCA) produces an acute inflammatory response that peaks at approximately 24 hours, and is maintained for over 30 days post-injection. But, the intra-plantar injection of the polysaccharide carrageenan produces an acute inflammatory response that peaks approximately 3-4 hours post-injection. 
The significant acute anti-inflammatory activity of the samples (AECc, AFCc, nBFCc, EtFCc, & ChFCc) and standard drug observed in the present study may be due to inhibition of the mediators of inflammation such as prostaglandins, histamine and cytokines. In chronic inflammation model (cotton pellet granuloma method) the results indicate that AECc, AFCc, nBFCc, EtFCc, & ChFCc have anti-transudative, anti- exudative and anti-proliferative activity, and this may be due to the inhibition of prostaglandins.
Furthermore, cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) are the two important enzymes which catalyze the formation of mediators involved in the inflammatory process. , In the present study, the samples AECc, AFCc, EtFCc and ChFCc at the dose of 100 μg/ ml, inhibited both COX-1 and COX-2 activity, but their % inhibition differs except nBFCc, which has effect only against COX-2. The report revealed that flavonoids inhibits a wide range of enzymes involved in oxidative stress, such as 5-lipoxygenase, cyclooxygenase, monooxygenase, or xanthine oxidase.  Preliminary phytochemical studies revealed the presence of flavonoid in C. colebrookianum that supports the role of flavonoids in COX-1 and COX-2 inhibition, antioxidant potential and consequent anti-inflammatory effects.  However, detailed studies on isolation, characterization and identification of phytochemical(s) responsible for anti-inflammatory effect through free radical scavenging and COX inhibition is required to understand the exact mechanisms of action.
| » Conclusion|| |
The observations of the present study establish the anti-inflammatory effect of C. colebrookianum leaves in acute and chronic stages of inflammation by free radical scavenging and inhibition of both the COX enzymes. This study also justifies the traditional claim of native practitioners and folklore healers for the usefulness of C. colebrookianum leaves as an alternative source for treatment of different inflammatory ailments.
| » Acknowledgements|| |
The authors thank Dr. N. C. Talukdar, Director and Scientist-'F', Institute of Bioresources and Sustainable Development (IBSD), Department of Biotechnology, Government of India, Imphal, Manipur, and the technical and non-technical staff of Pharmacology Laboratory, IBSD, Imphal, Manipur, for providing the facilities and support for this research work. The authors also thank Dr. D. K. Hore, Dr. Asamanja Chattoraj, and Dr. J. C. Bora for critical discussions and constructive comments.
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[Table 1], [Table 2], [Table 3], [Table 4]