Research Article

ANTI-NOCICEPTIVE ACTIVITIES OF COMPLEXES OF NARINGIN WITH CO (II) METAL IONS

SUDHANSHU SEKHAR ROUTa, RUDRA NARAYAN SAHOOa, SOVAN PATTANAIKa, ABHISEK PALa, SUDAM CHANDRA SIa, PRAKASH MOHANTYb

aSchool of Pharmaceutical Sciences, Siksha‘O’AnusandhanUniversity, Kalinganagar, Ghatikia, Bhubaneswar-751003, Odisha, India, bDepartment of Chemistry, Utkal University, Vani Vihar, Bhubaneswar-751004, Odisha, India.

Received: 20 Apr 2013, Revised and Accepted: 28 May 2013

ABSTRACT

Objective: The activities of flavonoid are believed to increase when they are coordinated with transition metal ions. An attempt was made to understand the contribution of the metal coordination and the type of interaction between a flavonoid and the metal ion & evaluation of its biological activity.

Methods: In this study a new metal complex of Co(II) with naringin was synthesized and characterized by FT-IR, UV-VIS spectrophotometer. Groups of six mice were treated with Cobalt Naringin Complex (CNC) (10 mg/kg body weight), administered intraperitonealy. Different tests were used for evaluating the anti-nociceptive effect or the influence on behavior of the tested substances on thermal nociception (hot plate test, tail flick test), chemical nociception (writhing test) and spontaneous behavior (activity cage assay).

Results: In the current study it has been evaluated that the complex has a Co(II) ion coordinated via 4 and 5 position of the flavonoid. The metal binds with the flavonoid in the ratio 1:2, which was confirmed by Jobs method. Our preliminary data for response latencies for hot-plate and tail-flick tests suggest that systemically administered CNC produces a significant analgesic effect under thermal nociceptive stimulation. The spontaneous behavior assay also shows a significant decrease of activity in the tested animals. Pain inhibition is even more significant in conditions of chemical nociceptive stimulation, in a model of visceral pain.

Conclusion: The mechanism through which CNC exerts its analgesic effect is still unclear, and will require more investigations, including dose-effect analysis, though it may be related to the influence of haemoxygenase-1 on the inflammatory pain pathways .

Keywords: Cobalt Naringin Complex (CNC), Flavonoid, Anti-nociceptive effect, Haem oxygenase-1

INTRODUCTION

A number of new bio active ligands involving natural product as reported to function properly upon co-ordination with metal ions [1]. As inorganic elements play crucial role in biological and biological medical processes and it is evident that many organic compounds used in medicine don’t have a purely organic mode of action, some are activated or biotransformed by metal ion metabolism [2]. Some therapeutic agents are proved to be more effective upon co-ordination. Because of those metal based drugs are now-a-days emerged as a promising alternative for possible replacement for some of the current drugs.

Flavonoids consist of a large group of polyphenolic phytochemicals with great therapeutic properties. This class of compounds has received much attention because of their pharmacological activities in the treatment of diseases such as allergy, Diabetes mellitus, cancer, viral infection, inflammations and others. Most of these effects are related to enzymatic inhibition, anticancer and antioxidant activity and interference with reactions such as the formations of free radicals [3].

Naringin (4’,5,7-trihydroxy flavanone) is major flavanone glycoside in Grape fruit and other citrus fruits(Fig.1). Like most flavonoids naringin also has metal chelating, antioxidant, and anti-inflammatory properties [4-6].Thus the aim of the present work comprise of the studies on the synthesis of a Co(II) transition metal complex bearing a naringin ligand and it is characterized by UV-Visible and FTIR spectroscopy. As Co(II) is an essential divalent trace element well known for its influence on the anti-nociceptive processes, the analgesic activity of CNC was studied.

Fig. 1: Structure of naringin

MATERIALS AND METHODS

Instrumentation and material:[7]

Prior to the synthesis of metal complex, the metal: drug ratio was determined by continuous variation method (Jobs plot).For Jobs plot the UV-Vis spectra of the drug and metal solutions (in different combination ratios) were recorded on UV-Vis spectrophotometer (using the JASCO V-630 spectrophotometer coupled to a PC and loaded with spectra manager software). Thin layer chromatography (TLC) was performed on silica gel GF 254TLC plates and the sample was visualized under UV lamp. Melting point of the metal complex was recorded on an ELICO melting point apparatus. The characterization of naringin metal complex was carried out by UV-Vis spectrophotometer (JASCO V-630) and Fourier Transform Infrared spectrophotometer (JASCO FT/IR-4100 coupled to a PC and loaded with spectra manager software). The disc was placed in the holder directly in the IR laser beam. Spectra were recorded at a resolution of 2cm-1, and 80nos of scans were accumulated.

Naringin was purchased from sigma and the product (compound purity >90% from the citrus fruits) was identified by spectral methods. The solvent and metal salt used was of analytical grade. All the glassware was washed with chromic acid followed by thorough washing with double distilled water which was freshly prepared in the laboratory [8].

Synthesis of metal complex:

Synthesis of metal complex of naringin was carried out according to their calculated mole ratios [8]. For this purpose naringin along with Co(Ac)2. 4H2O were purified by recrystallization. Solvents used for the synthesis of this complex were double distilled water and methanol.

The Co(II) complex was synthesized by dissolving naringin ( 0.146 g,2.5x 10-4mol) in methanol (10 mL) and a solution of Cu(II) acetate ( 0.0311 g, 1.25x10-4mol) in distilled water (2mL) was slowly added to it. pH was adjusted to 6.2 with ammonia solution. The mixture was stirred for 30 min at room temperature. Complex was filtered in vacuum system, washed with water and ethanol and air-dried for 2 days.

International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 5, Issue 3, 2013

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Anti-nociceptive study

Antinociceptive activity of metal complexes was investigated by Hot plate (Thermal) method and acetic acid induced twitching method in mice model of algesia.

a) Hot plate (Thermal) method[9-10]

The mice were first treated with Cobalt chloride 3.75 mg/kg, naringin 25mg/kg and CNC 5mg/kg after 1 h of administration they were placed on a hot plate maintained at 55±1.0°C. A cut-off period of 60 s was considered as maximal latency to avoid injury to the paws. The time taken by the animals to lick the fore or hind paw or jump out of the place was taken as the reaction time. Pentazocine (5 mg/kg s.c.) was used as a reference drug.

b) Acetic acid-induced writhing test[11-12]

The mice were first treated with Cobalt chloride 3.75 mg/kg, naringin 25mg/kg and CNC 5mg/kg. The abdominal stretch, or writhing assay, was performed by injecting 0.1 ml of 1.0% acetic acid intraperitoneal, in manually restrained mice. Immediately after injection, animals were placed in a large glass cylinder. Indomethacin (10 mg/kg) was administered 30 min before the acetic acid injection as standard drug.

RESULTS AND DISCUSSION

Naringin has a favorable solubility in methanol. The CNC was soluble in methanol, DMSO and sparingly soluble in water. The complex was found to be stable at room temperature for two days [13].The stability was checked by taking melting points of the complex at interval of 24 hours and 48 hours. The complex was stored in room temperature (25°C). No appreciable change in the melting point was observed. Melting point of the metal complex (230°C)when compared with the reference drug (166°C)differs considerably from

naringin.Jobs plot reveal that the metal:drug complex is in the ratio 1:2 [7].

The Co(II) complex was synthesized by aqueous solution of Co(Ac)2.4H2O and methanol solution of naringin in 1:2 molar ratio (metal salt/ligand ratio). Ligands are deprotonated by ammonia solution. The structure of Co(II) complex was characterized by UV-VIS and FTIR measurement. The UV-VIS experiment was carried in pH-6.2 solution. Free naringin exhibits an absorption maximum in methanol solution at 283 nm corresponding to the A ring portion and a very weak band at 327nm corresponding to the B ring portion. Upon binding with the Co(II) ion forming the complex the maximum absorption band shifted to 288 nm and the weak band shifted to 376nm with respect to the free flavonoid (Fig. 2) suggesting an interaction of the Co(II) ion with the condensed ring of the flavanone in position 4 and 5. These results are in agreement with the result of others [14-18] who indicates that the bond shift is caused by the binding of the metal ion in this position.

FTIR spectrum data in KBr of free naringin and CNC showed bonds at 1645cm-1(νco). Upon binding to Co(II) and forming complex the bond shifted to 1601cm-1suggesting an interaction of the Co(II) ion with the corresponding ring via carbonyl ring in position 4 and by the oxygen of the hydroxyl group in position 5 (Fig.3) in agreement with UV-Vis data. A broad diffuse band of medium intensity in the region (3100-3500 cm-1)is assign to the OH stretching vibration of the water molecule.

Our studies suggest that naringin acts as a monoanionic bidentate ligand and interacts with the metal centre through 5-hydroxy and 4-oxygen atom. From the result obtained it is proposed that the Co(II) complex is probably six coordinate with two molecules of naringin chelating the central metal atom from four sides and two molecules of water at the vertices of an octahedron(Fig. 3).

Fig. 2: Overlay spectra of naringin and naringin-Co(II) complex

Fig. 3: Tentative structure of Co(II) complex of naringin

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The CNC increased the reaction time in (p<0.05) to the thermal stimulus. The Cobalt chloride (3.75 mg/kg), naringin (25mg/kg) shows increased the reaction time but the significant nociperception of thermal stimulus was exhibited at a dose (5 mg/kg) of, CNC which was comparable to that of pentazocine(Fig. 4).

Maximum number of inhibition of writhing responses (p<0.05) exhibited by the CNC (5mg/kg).The Cobalt chloride (3.75 mg/kg), naringin (25mg/kg) shows reduction in acetic acid induced writhing responses but the significant nociperception of chemical stimulus was exhibited at a dose (5 mg/kg) of, CNC which was comparable to that of standard Indomethacin (10 mg/kg) that caused pain reduction(Fig.5).

Fig. 4: Effect of Co-naringin complex in hot plate reaction time in thermal induced algesia in hot plate method.

The values are expressed as mean ±SEM. Comparisons are made between: Group I with group II and group V

Statistical significant test for comparison was done by one way ANOVA followed by Dunnet’s t-test.*p<0.05

Fig. 5: Effect of CNC in steching number in chemo induced algesia in acetic acid induced method.

The values are expressed as mean ±SEM. Comparisons are made between: Group I with group II and group V

Statistical significant test for comparison was done by one way ANOVA followed by Dunnet’s t-test.*p<0.05

The flavonoid (naringin) is a potent analgesic by act on nociceptive receptor and also cobalt having a effective anti-nociceptive activity

by blocking Ca2+ ion. The metal complex of flavonoid enhances the potency of both compounds. The 3- or 5-hydroxy group and the 4-

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carbonyl group in the C ring are main metal complexion domains of naringin which interact with metal ions [19-21].

Since acetic acid induced writhing can be considered a model of prostaglandin synthesis sensitive response[22],the enhanced analgesic effect of CNC may be due to inhibition of the synthesis of arachidonic acid metabolites via inhibiting COX-2.The enhanced analgesic effect of CNC in the hot plate test might again be due to the inhibitory action on prostaglandin synthesis.

CONCLUSION

Many drugs possess modified toxicological and pharmacological properties when in the form of metal complexes [23]. The use of transition metal complexes as therapeutic compounds has become more and more pronounced. The complexes offer a greater diversity in their action; they don’t have only anticancer proprieties but also has been used as anti-inflammatory, anti-infective and anti-diabetic compounds [24]. Present work deals with the synthesis of naringin metal complex and the evaluation of pharmacological efficacy in compare to the parent, through the difference in their biological activity. The data obtained from the UV-VIS and FTIR studies suggested that the coordination of Co(II) is in position 4 and 5 of the condensed ring of naringin and the increased biological activity of the synthesized complex could be due to the coordination of Co(II) in this position. The analgesic activity potentiated with CNC, in which the metal toxicity of cobalt is minimized and the efficacy, potency of two molecules enhanced, which indicate a new light of therapeutic applicability of the natural molecule.

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