Anti-inflammatory, antioxidant and anticancer activity

International Journal of Research in Pharmaceutical and Biomedical Sciences

ISSN: 2229-3701

__________________________________________________________________________Research Paper

Anti-inflammatory, antioxidant and anticancer activity of Quercetin and its analogues Urmila J.Joshi, aAmol S.Gadge, aPriscilla D’Mello, Srivastava and bGirjesh Govil a

aPrin

b

Ragini Sinha,

Sudha

b*

K M Kundanani College of Pharmacy, Cuffe Parade, Mumbai 400005, India

bNational

Facility for High Field NMR, Tata Institute of Fundamental Research, Mumbai-5, India

ABSTRACT Quercetin has been reported to be effective in inflammation, arteriosclerosis, bleeding, allergy and swellings. It is also known to be associated with reduced risk of certain types of cancers. However, the major problem associated with the use of quercetin, is the very low bioavailability. Based on our molecular dynamics simulation results, structurally modified analogues of quercetin (Q) were synthesized to get Q–Cl and Q–OCH3 by modifying phenolic–OH groups of ring A and ring B. The compounds were tested for antioxidant activity by TBARS assay, anti-inflammatory activity by carragenan induced rat paw edema. A possible anticancer activity against breast cancer (MCF7), hepatic cancer (HepG2), prostate cancer (PC3) and colon carcinoma (HCT15) cell lines is also tested. It is observed that the structural modifications result in significant reduction in the antiinflammatory activity and antioxidant activity of these compounds. The anticancer activity of Q–Cl is comparable to quercetin in HepG2 cell lines and to a lesser extent in other cell lines. It is proposed that these compounds may have better bioavailablity and need to be explored by further structural modifications for a better activity. Based on these results and by correlating the bioactivity of quercetin and its analogues, a possible structure activity relationship has been proposed. Keywords: Quercetin, MD simulations, Antioxidant, Anticancer, Anti-inflammatory INTRODUCTION Quercetin (3,5,7,3´,4´-pentahydroxy flavone) is one of the most abundant bioflavonoids. It is present in edible fruits and vegetables [1]. It consists of two aromatic rings A and B, linked by an oxygencontaining heterocyclic ring C (Fig.1a). Because of its potent antioxidant and metal ion chelating capacity, quercetin has been reported to be effective in inflammation, arteriosclerosis, bleeding, allergy and swellings [2, 3]. In addition, epidemiological data suggests that quercetin is associated with reduced risk of certain types of cancers [4, 5]. It is considered to be one of the most potent flavonoid which is capable of interacting with and modulating activity of a variety of enzyme systems including cyclooxygenase, lipooxygenase, phosphodiesterase and tyrosine kinase. However, the major problem associated with the use of quercetin, is the very low bioavailability [6]. In natural form, quercetin is present in the form of glycosides. The glycosides are too polar to penetrate the intestinal membranes and hence, are not easily absorbed. The release of the aglycone by the action of the microfloral enzymes is needed for the compound to become absorbable. Although _______________________________________ *Address for correspondence: E-mail: [email protected]

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aglycones are permeable, the bioavailability is not high due to poor solubility and a greater extent of conjugation. Thus, the bioavailability of quercetin is an important challenge [7]. Factors such as solubility, permeation across membrane and metabolism tend to affect its bioavailability. The number of hydroxyl groups, presence of a methoxy group in the B ring and lipophilicity are known to influence the bioavailability of flavonoids [8]. We have modified the structure of quercetin to get two analogues Q–Cl and Q–OCH3 by modifying phenolic–OH groups of ring A and ring B. In order to enhance the lipophilicity, one of the phenolic– OH group in ring B is replaced by a –Cl and a – OCH3 group to get Q-Cl and Q-OCH3, respectively (Fig. 1 b, c). Using molecular dynamics (MD) simulation, we have attempted to understand the effect of these groups on the binding of quercetin, Q–Cl and Q–OCH3 to the lipid bilayer. The two compounds were tested for their antioxidant, antiinflammatory and possible anticancer activity. Based on these results and by correlating the bioactivity of quercetin and its analogues, we have deduced structure activity relationship.

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MATERIALS AND METHODS Animals and preparations: Albino Wistar rats of either sex weight between 120-150 g were obtained from a registered breeder; Bharat Serum and Vaccines, and maintained under standard conditions of temperature (22 ± 1◦C), relative humidity (65 ± 10%), and 12 h/12 h light/dark cycle. They were fed with standard pellet food and water ad libitum. The rats were maintained in hygienic conditions in groups of 6 in clean polypropylene cages with wire mesh top. The experimental protocol was approved by the institutional animal ethics committee as per CPCSEA guidelines (Ref. No.25/1999/CPCSEA) after reviewing the research project. Goat testes were procured from a slaughter house and used within 1 hour of the sacrifice. One set of experiments was performed on cells taken from two testes of the same goat. Cells from the cauda region of epididymis were prepared by gentle mincing and tweezing in Dulbecco buffer. Tissue pieces were removed and cells were washed, made into a pellet by centrifugation, and then suspended in an appropriate quantity of buffer to maintain a concentration of 1x106 cells/ml. Cell count and motility was checked with a cytometer. Cells exhibiting motility greater than 50% were used. Synthesis of Quercetin analogues: Quercetin analogues Q–Cl (2–(4–chlorophenyl)–3– hydroxy–chromen–4–one) and Q–OCH3 (2–(4– methoxyphenyl)–3–hydroxy–chromen–4–one) were synthesized from 2–hydroxyacetophenone and 4–substituted benzaldehydes in two steps. The first step involves Claisen Schimdt reaction giving rise to chalcone, followed by treatment with H2O2 resulting in the synthesis of the above flavonols. Typical procedure for the synthesis has been described in Scheme 1. A suspension of 4–chlorobenzaldehyde (1.36 g) (for Q–Cl)/4–methoxybenzaldehyde (for Q–OCH3 ) and 2–hydroxyacetophenone (1.405 g) in ethanol (25 ml) was cooled to 10 0C and 7.5 ml of 40 % w/v KOH solution was added drop wise. The reaction mixture was stirred for 18 h at room temperature. Dichloromethane (125 ml) was added and the organic layer was washed with H2O (3x50 ml), dried over sodium sulphate and concentrated under vacuum. The oily residue was dissolved in ethanol and 5.4% (w/v) NaOH solution followed by drop wise addition of 4 ml of 35% H2O2. The reaction mixture was stirred in an ice bath for 3 h and subsequently at room temperature for 12 h resulting in a yellow suspension. After acidification with 2M HCl, the precipitate was filtered and dried. The crude product was purified by passing it through silica gel column using chloroform as eluent, to give pure Q–Cl and Q–OCH3. The analogues thus prepared were characterized by elemental analysis and by NMR spectral data. The

Vol. 2(4) Oct - Dec 2011

ISSN: 2229-3701

1

H and 13C NMR chemical shifts of the quercetin, Q–Cl and Q–OCH3 in DMSO–d6 at 323 K are given in Table 1. Q–Cl: (found: C, 66.157; H, 3.181. C15H9O3 Cl requires C, 66.150; H, 3.302 ); IR O–H stretch : 3284, C–H stretch: 2812-2875, –C=O stretch : 1614. Q–OCH3 : (found: C, 66.157; H, 3.181. C15H9O3Cl requires C, 66.150; H, 3.302 ); IR O–H stretch : 3204, C-H stretch: 2812–2892, –C=O stretch : 1612; Molecular Dynamics (MD) simulations: MD simulations were carried out using Desmond 2.2 running on CentOS 5.4 Linux workstation using OPLS force field, 24 DPPC and 723 water molecules. The pressure coupling constant of 1.0 ps and the temperature coupling constant of 0.1 ps was used. The temperature and the pressure were set to 323K and 1.01325 bar respectively which are commonly applied conditions for the study of DPPC bilayers [9]. The system was coupled to a Berendsen thermostat and barostat. Periodic boundary conditions were enforced. The non– bonded cut–off was set to 9Ǻ. All the bond lengths involving hydrogens were constrained with SHAKE algorithm. The simulated system was constructed by inserting the molecule under consideration (quercetin/Q– Cl/Q–OCH3) into the middle of the lipid bilayer where there is a relatively large free volume available and therefore the insert position results in least perturbation. The net charge on the system was adjusted to zero. To start simulation, the system was energy minimized with gradually decreasing harmonic restraints followed by a 0.5 ns relaxation to remove bad contacts by initial relaxation of the lipid and the solvent molecules followed by relaxation of the molecules–lipid complex. The energy of intermolecular interaction was monitored and was found to equilibrate in this time. This was followed by a 20 ns MD simulation. The trajectories were recorded every 4.8 ps. Antioxidant activity: Thiobarbituric acid (TBA), trichloroacetic acid (TCA) and PNMC (3-methy-4-nitrophenol) were purchased from Aldrich Chemical Co. (USA). Dulbecco’s medium (pH=7.2) was used as buffer along with 0.1% glucose (w/v) which served as fuel to maintain cellular metabolism. For antioxidant testing, mammalian sperm cells have been used from goat. These cells are highly susceptible and undergo peroxidation under mild environmental conditions during storage such as UV radiation, cold, or mild peroxidizing agents. Lipid peroxidation in these cells was induced by exposure of the spermatozoa to UV radiation in one


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set of experiments and using oxidizing agent PNMC in the other set. For UV radiation, 600 l of cell samples previously incubated with different concentration (0.6 mM – 5 mM) of drugs for 30 minutes were used. Samples were placed in a glass vial and exposed to UV radiation from a 30W Philips lamp at a distance of 15 cm from the source for 60 minutes For peroxidation, 5mM solution of PNMC in dimethyl sulphoxide is used under identical drug incubation conditions as above. Production of malonaldialdehyde (MDA) was used as an index of spontaneous lipid peroxidation by sperm cell sample using TBA assay [10]. Incubation of sperm suspensions in the two experiments were terminated at fixed time intervals by chilling the cells in liquid nitrogen for 15 minutes and then 2 ml of TBA reagent (15% w/v trichloroacetic acid and 0.25 N HCI) was added. The mixture was heated in a boiling water bath for 15 minutes. After cooling, the suspension was centrifuged at 1,500 rpm for 10 minutes. The absorbance of supernatant was measured at 532 nm on Specord 205, Analytikjena, UV–Vis Spectrometer. The amount of MDA measured has been normalized with respect to control sample (in absence of drug/analogues) taken as 100 and expressed as % MDA. Anti-inflammatory activity: For anti-inflammatory test [11], the animals were starved overnight and given free access to water. Drug suspensions were prepared by using 1% (w/v) carboxymethyl cellulose (CMC). The suspension was administered orally to each rat in the experimental group of six rats, in the dose level of 50 mg/kg, using an oral galvage. The dose determination was done according to standard procedure. The standard reference group was treated orally with 10 mg/kg body weight of diclofenac aqueous solution while one control group was given normal saline while the other group was given CMC solution. After one hour, the rats were challenged by a subcutaneous injection of 0.05 ml of 1% solution of carrageenan into the plantar side of the left hind paw. The paw was marked with ink at the level of the lateral malleous and immersed in mercury up to this mark. The paw volume was measured plethysmographically immediately after the injection, again at 1 h, 2 h, 3 h, 4 h, 6 h, and eventually 24 h after the challenge. Inhibition of paw edema was calculated as:

where, Vt is the rat paw volume at time ‘t’, Vo is the initial rat paw volume i.e. before carrageenan injection, (Vt–Vo)control is edema produced in

Vol. 2(4) Oct - Dec 2011

ISSN: 2229-3701

control group and (Vt-Vo)treated is edema produced in treatment groups. The results were calculated by comparing the positive control and the drug groups with their corresponding negative control. with control group. Anticancer activity: Anticancer activity was determined by sulphorhodamine B (SRB) assay [12, 13]. The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mM Lglutamine. For screening experiment, cells were inoculated into 96 well microtiter plates in 100 µl. After inoculation, the microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to the addition of experimental drugs. After 24 h, one 96 well plate containing 5x103 cells/well was fixed in situ with TCA to represent a measurement of the cell population (Tz) at the time of drug addition. Experimental drugs were initially solubilized in dimethyl sulfoxide at 100 mg/ml and diluted to 1 mg/ml using water and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml with complete medium. Aliquots of 10 µl of these different drug dilutions were added to the appropriate microtiter wells already containing 90 µl of medium, resulting in the required final drug concentrations i.e.10 μg/ml, 20 μg/ml, 40 μg/ml, 80 μg/ml. Plates were incubated at standard conditions for 48 hours and assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 30 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded; plates were washed and air dried. SRB solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing with 1 % acetic acid and air dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was read on a plate reader at a wavelength of 540 nm with 690 nm reference wavelength. Percent growth has been calculated on a plate-byplate basis for test wells relative to control wells. Percent growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells * 100. Six absorbance measurements [at time zero (Tz), control growth (C), and test growth in the presence of drug at the four concentration levels (Ti)] were used to calculate the percentage growth inhibition. Percentage growth inhibition at each of the drug concentration was calculated as: [(Ti–Tz)/(C–Tz)] x 100 for concentrations for which Ti ≥ Tz (Ti–Tz) positive or zero


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[(Ti–Tz)/Tz] x 100 for concentrations for which Ti < Tz. (Ti–Tz) negative. Statistical methods: The data obtained were computed using GraphPad Prism software and later analyzed using ANOVA followed by Dunnett’s statistical method of analysis. The P values