ANALGESIC ACTIVITY OF CINNAMALDEHYDE PER SE AND IT’S INTERACTION WITH DICLOFENAC SODIUM AND PENTAZOCINE IN SWISS ALBINO MICE.

ANALGESIC ACTIVITY OF CINNAMALDEHYDE PER SE AND IT'S INTERACTION WITH DICLOFENAC SODIUM AND PENTAZOCINE IN SWISS ALBINO MICE.

Ritesh Churihar ^*, Pooja Solanki, Savita Vyas, Hemant Tanwani and Shubham Atal

Department of Pharmacology, M.G.M. Medical College, A.B. Road, Indore - 452001, Madhya Pradesh, India.

ABSTRACT: Cinnamon is one of the best-known spices used as herbal medicine. Cinnamaldehyde is the most important constituents of cinnamon. The present study was aimed to evaluate the analgesic activity of Cinnamaldehyde per se and its interaction with diclofenac sodium and pentazocine in Swiss albino mice. Healthy mice of either sex weighing 20-30 grams were divided into 6 groups of 6 animals each. Peripheral analgesic activity was evaluated by acetic acid induced writhing test, and central analgesic activity was studied using Eddy's hot plate method. Cinnamaldehyde (100 and 200 mg/kg) and its combination (Cinnamaldehyde 100 + 2.5 mg/kg standard drug), diclofenac sodium (2.5 and 5 mg/kg) and pentazocine (2.5 and 5 mg/kg) were given orally. Acetic acid induced writhing model showed that diclofenac sodium at doses of 2.5 and 5 mg/kg reduces writhing 44% and 66% respectively as compared to control when administered alone. Cinnamaldehyde at 100 and 200 mg/kg showed dose-dependent decrease in writhes 54% and 81%, but when Cinnamaldehyde (100 mg/kg) was co-administered with diclofenac sodium (2.5mg/kg) showed a significant decrease in writhes 84.43% concerning control. While in Eddy's hot plate method Cinnamaldehyde not only showed hyperalgesia when given alone as compared to control but also decreases the analgesic effect of pentazocine when combined with pentazocine in comparison with pentazocine alone and control group. The findings suggest that the Cinnamaldehyde significantly increases the analgesic activity of diclofenac sodium, but decreases the analgesic activity of pentazocine.

Cinnamaldehyde, Acetic acid, Writhing, Eddy's hot plate, Hyperalgesia

INTRODUCTION: Pain is one of the most common and frequent complaints of a human being. It can be either defined to one specific area, or it can be generalized to the whole body. Perception of pain is a standard physiologic response of healthy nervous system ¹ and is mediated by various mediators like prostaglandins, serotonin, substance P, etc.

Currently, there are various medications available for pain. Depending on the type of pain, either NSAIDs or opioids can be used. But there are various adverse effects related to these medications.

NSAIDs has a high risk of gastric ulcers ² similarly opioids has a wide range of CNS adverse effects like tolerance, dependence, CNS depression ³ etc. As an alternative to NSAIDs and opiates, newer analgesic drugs with lesser or negligible adverse effects along with high efficacy are being searched all over the world. According to WHO, about 80% of the world population still rely mainly on plant-based drugs ⁴. Cinnamon is one of the oldest and best-known spices in the world and is used as herbal medicine ⁵. It belongs to family Laurence and is found in South India, Srilanka, Indonesia, Vietnam, Bangladesh, and Nepal and commonly known as dal-chini, darchini or dhall cheene in Hindi. The active component of commercial cinnamon is the dried inner stem-bark of aromatic evergreen tree 10-15 meters tall. The most important constituents of cinnamon are Cinnamaldehyde and eugenol, which are present in the essential oil of the bark thus contributing to the fragrance and the various biological activities observed with cinnamon ⁶.

Cinnamon has been investigated for antioxidant property ⁷, inhibition of tau aggregation ⁸, anti-inflammatory activity ⁹, anti-nociceptive activity ¹⁰, peptic ulcer protection effects ^{11, 12} effect on cardiovascular system ^{11, 13, 14} and hepato-protective effects ¹⁵, antihyperlipidemic activity ¹⁶ and antidiabetic ¹⁷. The present study was planned to investigate whether Cinnamaldehyde has any analgesic activity per se and how does it interact with diclofenac sodium and pentazocine?

MATERIALS AND METHODS:

Drugs and Chemicals: Cinnamaldehyde (CNM) was obtained from the Science center (Sunchem Pharma), Indore, Madhya Pradesh, India, having 98% purity. Tween twenty was purchased from the same and used as a vehicle. Acetic acid- Ranbaxy lab limited, Mumbai, diclofenac sodium (DICLO) (Voveron injection, 25 mg/ml- 3 ml ampoule- Novartis Pharmaceuticals) and pentazocine (PTZ) (Fortwin injection, 30 mg/ml – 1 ml ampoule, Ranbaxy Pharmaceuticals Ltd.) were purchased from their authorized representatives.

Animals: Swiss albino mice (20-30 gm) of either sex were used for the study. The animals were housed in polypropylene cages in the central animal house. The rooms were maintained at the temperature of 25 ± 5 ºC with 12 h light/dark cycles. All the animal experiments were carried out according to the Committee for Control and Supervision of Experiments on Animals (CPCSEA), India guidelines. The animals were fasted overnight before the experiment and were given only water ad libitum. The study was approved by the Institutional Animal Ethics Committee (IAEC), M.G.M. Medical College, Indore, India (IAEC-709/20/2010, dated 25/03/2015) and the work was conducted in the Department of Pharmacology.

Preparation of Drugs for the Animal Experiment: CNM and other drugs were dissolved in tween twenty 20% to maintain uniformity of the solvent.

Experimental Design: Animals were grouped in 6 groups comprising of 6 animals each. (N=6, n=6). All the drugs were administered orally as per the grouping is done to all animals at the stipulated doses. The grouping done as follows:- Group I - Tween-20 20% (10 ml/kg), Group II - Diclofenac sodium for peripheral activity and pentazocine for central activity (2.5 mg/kg), Group III - diclofenac sodium or pentazocine 5 mg/kg (as per the peripheral or central activity), Group IV- CNM (100mg/kg), Group V  - CNM (200 mg/kg), and GroupVI – CNM + diclofenac sodium or pentazocine (100 + 2.5 mg/kg)

Procedure and Experiment: Two different models of nociception assessed antinociceptive activity.

Acetic Acid Induced Writhing Method to Demonstrate Peripheral Analgesic Activity:

Principle: Writhing method was used for the evaluation of the peripheral analgesic activity. Painful reactions can be produced in experimental animals by applying noxious stimuli using chemical irritants such as acetic acid and bradykinin. Acetic acid induces writhing after intraperitoneal administration in mice.

Procedure: Abdominal constrictions were induced, by 1% v/v glacial acetic acid solution (10 ml/kg, i.p.) ¹⁸, after 1 hour of group-wise drug treatments. The number of abdominal writhes was measured over 10 min after the intraperitoneal injection of acetic acid. Results are expressed as percentage inhibition of abdominal constrictions concerning control, the total number of writhes and onset time of writhes were recorded. Abdominal constriction followed by extension of at least one hind limb is considered as one writhe.

Eddy’s Hot Plate Model to Demonstrate Analgesic Activity:

Principle: Painful reactions can be produced in experimental animals by applying noxious stimuli such as thermal – using radiant heat as a source of pain, chemical – using irritants such as acetic acid and bradykinin and physical pressure – using tail compression.

In the Eddy’s hot plate model, the animals are placed on Eddy’s hot plate which consists of an electrically heated surface. The responses are jumping, withdrawal of the paws and licking of the paws. The time until these responses occur is prolonged after administration of centrally acting analgesics (opioid analgesics), whereas peripheral analgesics do not generally affect these responses.¹⁹

Procedure: Animals were weighed, dosed according to grouping and placed on the hot plate maintained at a temperature of 55 ± 1 ºC. Responses such as jumping, withdrawal, and licking of the paws were seen. The period (latency period), from when the animals were placed and until the responses occurred, was recorded using a stopwatch. To avoid tissue damage of the animals 10 sec was kept as a cut off time ^{20, 21}.

The time obtained in all the untreated groups of animals was considered as basal reaction time. Increase in the basal reaction time was the index of analgesia. All the animals were screened at least three times initially in this way and the animals were showing a broad range of variation in the basal reaction time were excluded from the study.

After selecting the animals, the drugs were administered to all animals as per grouping. The reaction times of the animals were then noted at 0.5, 1, and 2 h interval after drug administration.

Statistical Analysis: SPSS-20.00 statistical computer software was used to evaluate the results. Results are expressed as mean ± SEM. One way ANOVA followed by Tukey's test was applied for multiple comparisons amongst different groups. p<0.05 was regarded as statistically significant.

RESULTS:

Analgesic Activity of Individual Drugs:

(a) Cinnamaldehyde: In acetic acid induced writhing method, CNM at the dose of 100 and 200 mg/kg showed a significant decrease in number and delayed onset of writhes in a dose-dependent manner (p<0.05) Table 1.

TABLE 1: EFFECT OF CINNAMALDEHYDE (CNM) IN ACETIC ACID INDUCED WRITHING IN MICE

One way ANOVA followed by multiple Tukey's comparison tests.

Values are mean ± SEM, n= 6 in each group, df = 5, 30

* p<0.05, as compared to control.

^# p<0.05 as compared to diclofenac 2.5 mg/kg.

^†p<0.05 as compared to diclofenac 5 mg/kg.

Note: Percent reduction = [1 – (mean of writhes in test or standard group / mean of writhes in control group)] × 100

While on Eddy's hot plate it showed a hyperalgesic effect at the dose of  200 mg/kg as compared control and pentazocine during all time points of study (p<0.05). But with 100 mg/kg dose hyperalgesia was observed only at 0.5 hr as compared to control (p<0.05) and at all time point when compared with pentazocine (5mg/kg) Table 2.

(b) Diclofenac Sodium: Diclofenac sodium at a dose of 2.5 and 5 mg/kg produced a significant decrease in number and delayed the onset of writhes as compared to control (p<0.05) Table 1.

(c) Pentazocine: In Eddy's hot plate method, with a dose of 5 mg/kg of pentazocine, the significant antinociceptive effect was observed at 0.5 h after the treatment and persisted for the entire test period (p<0.05). With the sub-therepeutic dose of pentazocine (2.5 mg/kg) the antinociceptive effect was observed only at 2 h (p<0.05) Table 2.

 Analgesic Activity of Combinations:

(a) CNM and Diclofenac Sodium: Diclofenac sodium (2.5mg/kg) in combination with CNM (100mg/kg) produced a significant decrease in some writhes when compared to control value or either of the treatment alone (p<0.05) Table 1.

(b) CNM and pentazocine: When CNM (100mg/kg) given in combination with pentazocine (2.5mg/kg) decreases the reaction time as compared to control and pentazocine 2.5 mg/kg significantly (p<0.05) Table 2.

No toxicity or mortality was observed during an observation period of seven days after the completion of the experiment.

TABLE 2: EFFECT OF CINNAMALDEHYDE (CNM) ON EDDY'S HOT PLATE METHOD IN MICE

One way ANOVA followed by multiple turkey's comparison tests.

Values are mean ±SEM, n= 6 in each group, df = 5, 30

* p<0.05 as compared to control

# p<0.05 as compared to pentazocine 5 mg/kg

† p<0.05 as compared to CNM 100 and 200 mg/kg

DISCUSSION: Thus in the present study, Cinnamaldehyde (CNM) was studied for its analgesic potential in both peripheral (non-narcotic) and central (narcotic) type of pain models. Diclofenac sodium (2.5 and 5 mg/kg, p.o.) and pentazocine (2.5 and 5 mg/kg, p.o.) were used as standard drugs for comparing analgesic effects at peripheral and central levels, respectively.

The study on peripheral analgesic activity using the glacial acetic acid (1%) induced writhing method showed that CNM has peripheral analgesic potential. CNM increases pain threshold in Swiss albino mice in a dose-dependent manner, i.e. 100 mg/kg and 200 mg/kg. There was a significant decrease in some writhes (p<0.05) and also delay in the onset time of writhes (p<0.05) with CNM 200 mg/kg concerning control and diclofenac 2.5 mg/kg and diclofenac 5 mg/kg.

The combination group (i.e., CNM 100 mg/kg and diclofenac sodium 2.5mg/kg), showed significantly better analgesic activity as compared to control and standard drug groups (i.e., half and a full dose of diclofenac sodium) (p<0.05).

On the contrary study for central analgesic activity reveals that CNM has a hyperalgesic effect in a dose-dependent manner and when it was given in combination with pentazocine it markedly decreased the analgesic effects of pentazocine. Table 2

CNM has the hyperalgesic effect, at both doses, i.e. 100mg/kg and 200 mg/kg and it remained for whole study period, i.e. 0 h, 1 h, 2 h, as compared to control and standard drug groups. (p<0.05) but there is no significant difference between the hyperalgesic effect of both CNM doses (p>0.05).

When CNM (100 mg/kg) was given in combination with pentazocine (2.5 mg/kg), we found a significant decrease in analgesic activity as compared to control and pentazocine (5 mg/kg) alone (p<0.05). The decreased analgesia, compared to the standard group pentazocine (5 mg/kg), was evident as early as 0.5 h and remained for the entire duration of the study.

If we analyze the above findings, we can say that the CNM showed hyperalgesic activity not only when given alone but also showed the same effects when it was combined with even subtherapeutic dose of the standard drug. The lowering of analgesic effect response of pentazocine probably can’t be explained by peripheral pharmacokinetic interactions like lowered absorption of pentazocine by CNM or increased metabolism of pentazocine by microsomal enzyme system because hyperalgesic activity was seen with incremental dose of CNM per se also, which could be due to antagonism of endogenous narcotic endopeptidases like endorphins, enkephalins, and dynorphins which are acting on various opiate receptors like mu, kappa, and delta. This activity is similar to narcotic antagonist drugs like naloxone and naltrexone. The hyperalgesic activity of CNM could be due to the stimulation of nociceptive receptors (NOP) or through agonistic action on Transient Receptor Potential Ankyrin 1 (TRPA1) which are involved in the process of nociception and hyperalgesia ^{22, 23}.

Pain is a complex process mediated by many physiological mediators, e.g. prostaglandins, bradykinin, substance P, etc. In the acetic acid-induced writhing model the constrictions induced by acetic acid in mice results from an acute inflammatory reaction with the production of PGE2 and PGF2α in the peritoneal fluid ^{24, 25}. Therefore, it is likely that CNM might suppress the formation of these substances or antagonize their action for exerting analgesic activity. Nonsteroidal anti-inflammatory drugs inhibit COX and thereby inhibits production of prostaglandins ²⁶. The similar analgesic activity has been reported with CNM by Atta & Alkofahi in 1998 ¹⁰ and Annegowda HV in 2012 ⁷. Against gastric ulceration ¹² if we consider this as a principle mechanism of CNM, then like prostaglandin synthesis inhibitors it must impair the mucosal defense of GIT by increasing gastric acid secretion, reducing mucus formation and increasing mucosal permeability. However, the study of Alqasoumi S et al., showed that CNM is protective against gastric ulceration ¹¹. Thus, it appears that the analgesic activity of CNM is due to some other mechanism rather than inhibition of prostaglandin synthesis.

The pharmacokinetic reasons may be responsible for the enhancement of analgesia of a low dose of diclofenac. This could be due to increased blood flow due to GIT irritation with consequently enhanced absorption. CNM may improve the absorption of drugs, perhaps by increasing GI blood flow by vasodialatation ^{11, 13}.

Further studies are needed to reveal the exact mechanism of action responsible for the enhanced activity of diclofenac sodium and decreased activity of pentazocine. However, the study adds to our concept that CNM can improve the activity of diclofenac and inhibit the activity of pentazocine. So, the addition of CNM may reduce the required dose of diclofenac that may help in the reduction of toxicity. Contrastingly co-administration with pentazocine may cause the requirement of higher dose to lead to increased toxicity. Hence, further studies are required to acknowledge these facts.

ACKNOWLEDGEMENT: The author is grateful to the Department of Pharmacology MGM Medical College Indore for their support and help.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Fields HL and Martin JB: Pain: pathophysiology and management. In: Dr. Faucis, editor. Harrison’s Principles of internal medicine, 18^th New York: McGraw-Hill 2012.
2. Grosser T, Smyth EM and Fitzgerald GA: Anti-inflammatory, antipyretic and analgesic agents; pharmacotherapy of gout. In: Brunton LL, Chabner BA, Knollmann BC, editor. Goodman and Gilman’s The pharmacological basis of therapeutics, 12^th New York: McGraw-Hill 2011: 973-6.
3. O'Brien CP: Drug Addiction. In: Brunton LL, Chabner BA, Knollmann BC, editor. Goodman and Gilman’s The pharmacological basis of therapeutics, 12th ed. New York: McGraw-Hill 2011: 658-63.
4. Kumara NK: Identification of strategies to improve research on medicinal plants used in Sri Lanka. In: WHO Symposium, University of Ruhuna, Galle, Sri Lanka, 2001.
5. Senanayake UM and Wijesekera ROB: In: Ravindran PN, Nirmal-Babu K, Shylaja M (Ed.). Cinnamon and Cassia: The Genus Cinnamomum, CRC Press 2003: 80-120.
6. Yeh HF, Luo CY and Lin CY: Methods for thermal stability enhancement of leaf essential oils and their main Constituents rom Indigenous Cinnamon (Cinnamomum osmophloeum). J Agric Food Chem 2013; 61(26): 6293-8.
7. Annegowda HV, Gooi TS and Awang SHH: Evaluation of Analgesic and Antioxidant Potency of Various Extracts of Cinnamomum iners Int J Pharmacol 2012; 8(3): 198-203.
8. George RC, Lew J and Graves DJ: Interaction of cinnamaldehyde and epicatechin with tau: implications of beneficial effects in modulating Alzheimer's disease pathogenesis. J Alzheimers Dis 2013; 36(1): 21-40.
9. Vetal S, Subhash LB and Vishwaraman M: Anti-inflammatory and anti-arthritic activity of type-A procyanidins polyphenols from the bark of Cinnamomum zeylanicum in rats. Food Sci Hum Wellness 2013; 2: 59-67.
10. Atta AH and Alkofahi A: Anti-nociceptive and anti-inflammatory effects of some Jordanian medicinal plant extracts. J Ethnopharmacol 1998; 60: 117-24.
11. Harada M and Yano S: Pharmacological studies on Chinese cinnamon. II. Effects of cinnamaldehyde on the cardiovascular and digestive systems. Chem Pharm Bull (Tokyo) 1975; 23(5): 941-7.
12. Tankam JM, Sawada Y and Ito M: Regular ingestion of cinnamomi cortex pulveratus offers gastroprotective activity in mice. J Nat Med 2013; 67(2): 289-95.
13. Xue YL, Shi HX and Murad F: Vasodilatory effects of cinnamaldehyde and its mechanism of action in the rat aorta Vasc Health Risk Manag 2011; 7: 273-80.
14. Raffai G, Kim B and Park S: Cinnamaldehyde and cinnamaldehyde-containing micelles induce relaxation of isolated porcine coronary arteries: the role of nitric oxide and calcium. Int J Nanomedicine. 2014; 21(9): 2557-66.
15. Eidi A, Mortazavi P and Bazargan M: Hepatoprotective activity of cinnamon ethanolic extract against CCl₄-induced liver injury in rats. EXCLI J 2012; 11: 495-507.
16. Javed I, Faisal I and Rahman Z: Lipid-lowering effect of Cinnamomum zeylanicum in hyperlipidaemic albino rabbits. Pak J Pharm Sci 2012; 25(1): 141-7.
17. Hassan SA, Barthwal R and Nair MS: Aqueous bark extract of Cinnamomum zeylanicum: a potential therapeutic agent for streptozotocin-induced type 1 diabetes mellitus (T1DM) rats. Trop J Pharm Res 2012; 11: 429-35.
18. De S, Maroo N and Saha P: Ethanolic extract of Piper betle leaves reduces nociception via modulation of the arachidonic acid pathway. Indian J Pharmacol 2013; 45(5): 479-82.
19. Gupta SK, Velpandian T, Sengupta S: Influence of piperine on nimesulide induced antinociception. Phytother Research 1998; 12(4): 266-9.
20. Vogel H: Drug discovery and evaluation Pharmacological 2^nd ed. New York: Springer-Verlag Berlin Heidelberg 2002: 696. ISBN 3-540-42396-6
21. Simpi CC, Nagathan CV and Karajgi SR: Evaluation of marine brown algae Sargassum ilicifolium extract for analgesic and anti-inflammatory activity Pharmacognosy Res 2013; 5(3): 146-9.
22. Moura JC de, Noroes MM, Rachetti V and de PS: The blockade of transient receptor potential ankirin 1 (TRPA1) signaling mediates antidepressant- and anxiolytic-like actions in mice. Br J Pharmacol 2014; 171: 4289-99.
23. Tsagareli MG, Tsiklauri N and Zanotto KL: Behavioral evidence of thermal hyperalgesia and mechanical allodynia induced by intradermal CNM in rats. Neurosci Lett 2010; 473(3): 233-6.
24. Deraedt R, Jouquey S and Benzoni J: Inhibition of prostaglandin biosynthesis by non-narcotic analgesic drugs. Archives Internationales de Pharmacodynamic et de Thérapie 1976; 224: 30-42.
25. Deraedt R, Jouquey S and Delevalee F: Release of prostaglandins-E and prostaglandins-F in an algogenic reaction and its inhibition. Eur J Pharmacol 1980; 61(1): 17-24.
26. Grosser T, Smyth EM and Fitzgerald GA: Anti-inflammatory, antipyretic and analgesic agents; pharmacotherapy of Gout. In: Brunton LL, Chabner BA, Knollmann BC, editor. Goodman and Gilman’s. The pharmacological basis of therapeutics, 12^th New York: McGraw-Hill 2011: 920-3.
    

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    How to cite this article:

    Churihar R, Solanki P, Vyas S, Tanwani H and Atal S: Analgesic activity of cinnamaldehyde Per se and it's interaction with diclofenac sodium and pentazocine in Swiss Albino mice. Int J Pharmacognosy 2016; 3(2): 97-02. doi: 10.13040/IJPSR.0975-8232.3(2).97-02.

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