In-vitro anti-viral screening and cytotoxicity evaluation of copper-curcumin complex

Abstract

Multiple therapeutic activities attributed to curcumin deliver a challenge to explore its hidden qualities. Structural features set an opportunity to chelate metal ions and enhance the therapeutics in a specified direction. A metallo-herbal complex (MHco) of curcumin with copper (Cu²⁺) ions was synthesized and characterized by various spectroscopy techniques. It was screened for its antiviral activity and cytotoxicity. Studies revealed that the synthesized compound has good microbicidal activity and would be utilized for the development of vaginal microbicidal gel against viral infections.

Keywords::

• antiviral
• copper
• curcumin
• cytotoxicity
• metallo-herbal complex

Introduction

Curcumin (Cur), commonly called diferuloyl methane, is a hydrophobic polyphenol derived from the rhizome (turmeric) of the herb Curcuma longa. Turmeric has been used traditionally for many ailments because of its wide spectrum of pharmacological activities. Cur, a yellow pigment present in the Indian spice turmeric (associated with curry powder), has been linked with suppression of a number of diseases. Cur has been shown to exhibit antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic activities. It also has hepatoprotective and nephroprotective activities, suppresses thrombosis, protects against myocardial infarction, and has hypoglycemic and antirheumatic properties. Moreover, Cur has been shown in various animal models and human studies to be extremely safe even at very high doses (Aggarwal et al. 2003, Jagetia and Aggarwal 2007, Aggarwal et al. 2007, Suzuki et al. 2005, Shishodia et al. 2007, Shishodia et al. 2005, Goel et al. 2008a, 2008b, Anand et al. 2007, Aggarwal and Harikumar 2009, Kunnumakkara et al. 2008a, 2008b, Anand et al. 2008).

Various metallic chelates of Cur are prepared and evaluated for their therapeutic potential. The manganese complexes of Cur and its diacetyl derivative were found to show greater superoxide dismutase (SOD) activity (Shim et al. 2002), HO radical scavenging activity (Shim et al. 2002), and nitric oxide radical-scavenging activity (Sumanont 2004) than the parent molecules. The copper complex of Cur also has been found to exhibit antioxidant, superoxide-scavenging, and SOD enzyme-mimicking activities superior to those of Cur itself (Barik 2005).

As above, there are many reports about Cur and its metal ion complexes, and their therapeutic profile. In this study we synthesized and investigated the structure of Cur -copper chelate (Cur-Cu) and investigated its antiviral potential and cell cytotoxicity against different viruses.

Materials and method

Materials

The materials used in the study are Cur ≥ 95% purity (Himedia), cupric acetate (Sigma), DMSO (Himedia), methanol (CDH).

Synthesis of copper- Cur complex

25 ml methanolic solution of cupric acetate (0. 220 g, 4 mmol) was added into the 13.5 ml of methanolic solution of Cur (0.185 g, 2 mmol). Dark reddish brown precipitates were produced immediately. The mixture was refluxed for 2 hour under a nitrogen atmosphere. The solid product was then filtered, washed with cold methanol and water to remove the residual reactants, and then the product was dried in vacuum overnight (Barik 2005, Krishnankutty and Venugopalan 1998).

Spectroscopy

Ultraviolet-visible spectroscopy

UV absorption spectra of Cur and Cur-Cu complex in DMSO were recorded in UV-VIS Spectrophotometer (1700 UV Shimadzu, Japan). Cur and its metallic complex were scanned between 200 and 600 nm and λ_max was determined.

FT-IR spectroscopy

Study was performed for identification and conformation of the structural aspects of Cur and Cur-Cu complex using FTIR (Fourier transformed infrared) Spectrophotometer (Nicolet −380, Thermo, USA). The KBr disk technique was employed using 1 mg of Cur and Cur-Cu powder in 100 mg of spectroscopic grade dried KBr. Mixture was ground into a fine powder using an agate mortar/pestle and compressed into KBr disc under a hydraulic press at 10,000 psi. Each KBr disc was scanned 32 times at 4 mm s^‐1 at a resolution of 2 cm^‐1 over a wave number region of 4000–400 cm^‐1 and characteristic bands were recorded.

FT NMR Spectra

FT NMR (H-1) of Cur and Cur-Cu complex using DMSO solvent was performed at PU Chandigarh (Bruker avance II 400 NMR) for identification and structural analysis of Cur and Cur-Cu. This spectral data was required to compare and authenticate the complex formation.

In-vitro antiviral and cell cytotoxicity screening

This experiment was performed in Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute, Belgium. Five different cell cultures Hela, Vero, HEL (Human embryonic lung), MDCK: (Madin Darby canine kidney cells), CRFK: (Crandell-Rees Feline Kidney cells) are used for the antiviral activity determination against herpes simplex virus strains, vaccinia virus, vesicular stomatitis virus, coxsackie virus b4, respiratory syncytial virus, para-influenza-3 virus, reovirus-1, sindbis virus, punta toro virus, feline corona virus (fipv), feline herpes virus and various influenza strains. Brivudin, cidofovir, acyclovir, ganciclovir, DS-5000, (s)-DHPA, ribavirin, HHA, UDA, oseltamivir carboxylate, amantadine, rimantadine were taken to establish the comparison of the activity and cytotoxicity. 50% effective concentration, or concentration producing 50% inhibition (EC₅₀^c) is determined by cell viability with the colorimetric formazan-based MTS assay and in some cases of anti-influenza activity, (EC₅₀^c) is determined by MTS and visual scoring of the of virus-induced cytopathic effect (CPE) assays. For toxicity determinations, Cur-Cu and reference drugs were added in the absence of viral challenge concentration and 50% cytotoxic concentration (CC₅₀), is determined.

Result and discussion

Spectroscopy

UV spectroscopy

UV spectra of curcumin and Cur-Cu are shown in Figure 1. Spectra show a single peak for Cur at 428 nm and two peaks for Cur-Cu complex at 425 and 343 nm.

Figure 1. UV-Spectral comparison of curcumin (cur) and curcumin-copper (Cur-Cu) complex.

IR spectroscopy

IR spectra of Cur, cupric acetate and Cur-cu are shown in Figure 2. The 1627 and 1602 cm^‐1 bands correspond to the mixtures of stretching vibrations of t(C = C) and t(C = O) in Cur (Krishnankutty and Venugopalan 1998) and they are red shifted to 1617 and 1588 cm^‐1 in the Cur-Cu complex. A comparison with the spectrum of complex with reactants (Cur and cupric acetate) reveals that the new bands at 535 and 477 cm^‐1 in Cur-Cu spectra indicates the interaction between Cu(II) and oxygen (O) atom of Cur (Kolev 2005) as shown in Figure 3.

Figure 2. IR spectra of (a) curcumin, (b) cupric acetate and (c) Cur-Cu.

Figure 3. Attachment of Cu(II) on curcumin.

NMR spectroscopy

1H NMR spectrum of Cur is as same as that published from a database (Bong 2000) and the spectra of Cur-Cu complexes are shown in Figure 4. Characteristic peaks of Cur from 6–8 ppm disappear in the spectra of the metal complex, indicating Cur chelation with the metallic ion (Zhou et al. 2010).

Figure 4. NMR spectra of curcumin and Cur-Cu.

In-vitro antiviral and cell cytotoxicity screening

Antiviral activity studies against herpes simplex virus strains, vaccine virus, vesicular stomatitis virus, coxsackie virus b4, respiratory syncytial virus, para-influenza-3 virus, reovirus-1, sindbis virus, punta toro virus are done in HEL, HeLa and VeRo cell culture. Antiviral activity and cytotoxicity details are mentioned in Table I. Antiviral activity and cytotoxicity studies against feline corona virus (fipv), feline herpes virus and various influenza strains are performed in CRFK and MDCK cell cultures, data are mentioned in Table II.

Table I. Antiviral activity and cell cytotoxicity profile.

HEL cell cultures
Compound	Minimum cytotoxic concentration^a (μg ml^‐1)	EC50^b (μg ml^‐1)
		Herpes simplex virus-1 (KOS)	Herpes simplex virus-2 (G)	Vaccinia virus	Vesicular stomatitis virus	Herpes simplex virus-1 TK^‐ KOS ACV^r
Cur-Cu	>4	> 4	> 4	> 4	> 4	> 4
Brivudin (μM)	> 250	0.03	96	4	> 250	22
Cidofovir (μM)	> 250	2	0.9	22	> 250	3
Acyclovir (μM)	> 250	0.08	0.2	> 250	> 250	29
Ganciclovir (μM)	> 100	0.03	0.03	> 100	> 100	4
HeLa cell cultures
Compound	Minimum cytotoxic concentration^a (μg ml^‐1)	EC50^b (μg ml^‐1)
		Vesicular stomatitis virus	Coxsackie virus B4	Respiratory syncytial virus
Cur-Cu	4	> 0.8	> 0.8	> 0.8
DS-5000	> 100	2	> 100	0.8
(S)-DHPA (μM)	> 250	> 250	> 250	> 250
Ribavirin (μM)	> 250	6	112	4
VeRo cell culture
Compound	Minimum cytotoxic concentration^a (μg ml^‐1)	Para-influenza-3 virus	EC50^b (μg ml^‐1)
			Reovirus-1	Sindbis virus	Coxsackie virus B4	Punta Toro virus
Cur-Cu	>4	≥ 4	> 4	> 4	> 4	> 4
DS-5000	> 100	> 100	> 100	> 100	> 100	> 100
(S)-DHPA (μM)	> 250	> 250	> 250	> 250	> 250	> 250
Ribavirin (μM)	> 250	50	146	> 250	50	50

^aCytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay.

^bMinimum compound concentration that causes a microscopically detectable alteration of normal cell morphology.

Table II. Antiviral activity and Cell cytotoxicity profile.

CRFK cell cultures
Compound	CC50^a (μg ml^‐1)	EC50^b (μg ml^‐1)
		Feline corona virus (FIPV)			Feline herpes virus
BC-1	11.4	> 4			> 4
HHA	> 100	40.3			17.6
UDA	61.8	9.7			8.2
Ganciclovir (μM)	> 100	> 100			3.1
MDCK cell cultures
Compound	CC50^a	Minimum cytotoxic concentration^b	EC50^c (μg ml^‐1)
			Influenza A H1N1 subtype		Influenza A H3N2 subtype		Influenza B
			Visual CPE score	MTS	Visual CPE score	MTS	Visual CPE score	MTS
BC-1 μg ml^‐1	10.6	≥ 4	> 4	> 4	> 4	> 4	> 4	> 4
Oseltamivir carboxylate μM	> 100	> 100	0.8	2.0	9	8.6	34	20.2
Ribavirin μM	> 100	> 100	4	4.7	2	2.9	2	1.8
Amantadine μM	> 200	> 200	5	2.2	0.7	2.3	> 200	> 200
Rimantadine μM	> 200	> 200	> 200	> 200	0.4	0.6	> 200	> 200

^a50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay.

^bMinimum compound concentration that causes a microscopically detectable alteration of normal cell morphology.

^c50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by measuring the cell viability with the colorimetric formazan-based MTS assay.

Discussion

Spectral data of synthesized Cur-Cu complex confirmed the synthesis of metallo-curcumin chelate when compared with Cur spectral data (Zhou et al. 2010). Comparison of the activity against standard drugs clearly showed its antiviral potential. But on the other side cytotoxicity profiles in various cell cultures revealed very low safety margin. Antiviral EC⁵⁰ value is same (4 μg/ml) against all the viral strains except vesicular stomatitis virus, coxsackie virus B4, respiratory syncytial virus (0.08 μg ml^‐1 minimum cytotoxicity value of > 4 μg ml^‐1) in all cell cultures.

Cur-Cu possesses good antiviral activity against vesicular stomatitis virus, Coxsackie virus B4, Respiratory syncytial virus. But cytotoxicity may create hurdles in its therapeutic use. Exploring the methodology to control the toxicity parameter surely presents a good approach of using Cur-Cu as a broad-spectrum antiviral agent.

Acknowledgement

Authors want to acknowledge the Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute, BELGIUM for conducting in-vitro antiviral screening of developed compound.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.