Synthesis and Gastroprotective Evaluation of New Chalcone Derivatives

Imran Khan Pathan*, Narendra Babu K., K.K. Ashok Kumar, Ishwarya K., J. Hima Kiran

Department of Pharmacology, The Erode College of Pharmacy, Erode- 638112, Tamilnadu, India.

ABSTRACT:

The etiology of gastrodeodenal ulcers is influenced by various aggressive and defensive factors such as acid pepsin secretion, parietal cells, mucosal barrier, mucous secretion, blood flow, cellular regeneration, endogenous productive agents (PGS and epidermic growth factors), and Helicobacter pylori (H. Pylori). However, it has been suggested that free radicals are closely related with peptic ulcer and gastritis. Oxygen free radicals are detrimental to the integrity of biological tissues and mediate their injury. The mechanism of damage involves lipidperoxidation, which destroys cell membranes with the release of intracellular components, such lysosomal enzymes, leading to further tissue damage. The radicals also promote mucosal damage by causing degradation of the epithelial basement membrane components, complete alteration of the cell metabolism and DNA damage. Therefore, by scavenging free radicals, the reactive oxygen metabolites might be useful by protecting the gastric mucosa from oxidative damage or by accelerating healing of gastric ulcer. Antioxidants act as scavengers inhibit lipidperoxidation and other free radicals mediated process, and therefore they protect the human body from several diseases attributed to the reactions of radicals. In this study, the gastroprotective effect of orally administrable newly synthesized chalcone derivatives on gastric lesions by pylorus ligation induced gastric ulcer in rats and In vitro free radical scavenging activity were studied.

KEYWORDS: Gastrodeodenal ulcers, Helicobacter pylori, Oxygen free radicles, Antioxidants, Chalcone.

INTRODUCTION

In recent years there has been is growing interests in polyphenolic compounds and their presumed role in the prevention of various diseases, such as gastric ulcer, atherosclerosis, cancer, chronic inflammation and diabetes mellitus. Chalcones are flavonoid lacking heterosycling C ring. Among this category, flavonoids have been identified as an interesting compounds that is associated with several biological activities¹. Chalcones are readily synthesized by the condensation of base-catalyzed Claisen-Schmidt condensation of an aldehyde and an appropriate ketone in polar solvent like methanol. This method is versatile, convenient, although yields may be variable. The potent antioxidant activity of hydroxyl chalcones was evaluated for their ability to scavenge various exogenous radicals¹. Chalcones are continue to attract considerable scientific significant and attention because of their association with their various biological activities. Skilful structural manipulation of the chalcone framework may yet narrow its range of biological activity and enhance its potency for a targeted pharmacological profile. Chalcones are important intermediates in the metabolic pathway converting of tyrosine to the flavonoids, and have a general chemical structure consisting of two phenyl groups, both with hydroxyl groups (s), connected by a C₃ bridge [-C=C-(CO)-]. Therefore, the chalcones are polyphenols and can be expected to act as antioxidants.

MATERIALS AND METHODS:

General Procedure for Chalcone Preparation:

Compounds Ia to Ig were obtain by reactions of appropriate acetophenone(0.01M) and benzaldehyde (0.01M) in presence of 10% NaOH and ethanol and the following procedure was adapted to synthesis individual compounds. The mixture was stirred for 2 hrs until entire mixture become very cloud. Then the mixture was poured slowly in to 400ml of water with constant stirring and kept in refrigerator for 24 hrs. The products obtained were filtered, washed and recrystallised from ethanol. The reaction was manipulated by TLC (Benzene: Ethyl acetate).

Melting points were determined with Lab line melting point (Science house, Chennai) apparatus and are uncorrected. Infra-Red spectra were recorded on a Shimadzu 8400-s spectrophotometer using KBr pellets. The reactions were monitored by thin layer chromatography (TLC) using silica gel-G (benzene: ethylacetate, 9:1).

Scavenging Activity against DPPH Radical

Scavenging activity on DPPH (1,1-diphenyl-2-picryl-hydrazyl radicals) of chalcone derivatives was measured². Each sample stock solution was diluted to final concentrations of 10-640 μg/ml, and 0.2 ml of methanol and 0.3 ml of various concentrations of the samples in methanol were mixed in a 10 ml test tube. To this was added 2.5 ml of 75 μM DPPH (1,1-diphenyl-2-picryl-hydrazyl) in methanol to achieve a final volume of 3 ml. The solution was kept at room temperature for 90 min, and the absorbance at 517 nm was measured. Butylated hydroxy toluene (BHT) was used as a reference compound. The DPPH (1,1- diphenyl-2-picryl-hydrazyl) scavenging effect and IC₅₀ were calculated using linear regression method.

Control - samples

% Radical inhibition = -------------------------- × 100

Control

Nitric Oxide Radical Scavenging

At physiological pH, nitric oxide generated from aqueous sodium nitroprusside (SNP) solution interacts with oxygen to produce nitrite ions, which may be quantified by the Griess reaction³. The reaction mixture contained 10 mM SNP, phosphate buffered saline (pH 7.4) and various concentrations (10-640 μg/ml) of the test solution in a final volume of 3 ml. After incubation for 150 min at 25°C, 1 ml sulfanilamide (0.33% in 20% glacial acetic acid) was added to 0.5 ml of the incubated solution and allowed to stand for 5 min. Then 1 ml of napthylethylenediamine dihydrochloride (NED) (0.1% w/v) was added and the mixture was incubated for 30 min at 25°C. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and subsequent coupling with NED was measured spectrophotometrically at 540 nm against a blank sample. All tests were performed six times. Ascorbic acid was used as a standard.

Control - samples

% Radical inhibition = ---------------------------- × 100

Control

Gastro-Protective Effect-Pylorus Ligation Induced Ulcer

The screening of antiulcer activity was conducted at Pharmacology laboratory. Experiments were performed complied with the rulings of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) New Delhi, India, and the study was permitted by the institutional ethical committee of the Erode College of Pharmacy, Erode India.

Albino rats (Wistar strain) of either sex weighing 150-200g were kept at room temperature (25-30 ^°C) and fasted for overnight before performing experiments. The pyloric ligation assay was performed using the method of Shay et al⁴ with few modifications. All the compounds were given orally thirty minutes prior to pylorus ligation. The animals were divided into five groups (n = 6). After 24 h of fasting, the animals were anesthetized with thiopental sodium (10 mg/kg, i.p.), the abdomen was incised and the pylorus was ligated. Vehicle (2 ml/kg), compounds I_b, I_c and I_f were orally administered at the dose of 100 mg/kg, Ranitidine 50 mg/kg to the respective group. The pyloric portion of stomach was identified, slightly lifted outside and ligated, avoiding traction to the pylorus or damage to the blood supply. The stomach was then replaced carefully and the abdominal wall closed by interrupted sutures. Animals were kept deprived of food and water during post operative period and were scarified at the end of 4 hr after the operation. Four hours later, the animals were sacrificed by cervical dislocation; the abdomen was opened and another ligature was placed at the oesophageal end. The stomach was separated as a whole and the whole content were brought out. The stomachs were removed and the gastric content collected and centrifuged at 3000 rpm (1000 rpm, 25^°C, 10 min). The amount of gastric acid (ml) and the pH values were determined.

The total acid secretion in the gastric content was determined in the supernatant volume by titration to pH 7.0 using a 0.01M NaOH solution, and phenolphthalein as indicator. Mean ulcer score for each group of animals was expressed as ulcer index^5,6. The number of ulcer per stomach was also recorded and the percent of ulcer incidence of each group was compared with the control.

Acidity was expressed as below,

Volume of NaOH × Normality × 100

Acidity (mEq/L) =--------------------------------------------------------

0.1

The ulcers were scored as below,

0=Normal coloured stomach

0.5=Red colouration

1.0=Spot ulcers

1.5=Hemorrhagic streaks

2.0=Ulcers ≥ 3 ≤ 5

3.0=Ulcer > 5

Table.no.1. Scheme and Physiochemical Properties of New Chalcone Derivatives

Compound	x (Ketone)	y (Aldehyde)	% Theoretical yield	Melting point (C)	Solubility	IR data (kbr)v(cm^-1)
I_a	Acetophenone	4-hydroxy 3-methoxy benzaldehyde	63.17	192-197	alcohol	1844.19(c=o), 1636.05(c=c), 29819(c-H), 2885.6(-oCH₃)
I_b	Acetophenone	2-nitro benzaldehyde	56.37	223-228	alcohol	1733.93(c=o), 1624.77(c=c)
I_c	Acetophenone	2-hydroxy benzaldehyde	58.13	155-160	alcohol	1844.21(c=o), 1540.51(c=c), 2907.74(C-H)
I_d	2-nitro acetophenone	2-nitro benzaldehyde	56.40	172-177	alcohol	1772.03(c=o), 1653.61(c=c) 2856.08(C_H)
I_e	2-nitroAceto phenone	2,5-di methoxy benzaldehyde	60.26	183-188	alcohol	1793.54(c=o), 1659.93(c=c), 2996.3(C-H)
I_f	2-nitro acetophenone	3,4-dimethyoxy benzaldehyde	52.69	202-207	alcohol	1733.97(c=o), 1636.1(c=c), 2944.64(C-H)
I_g	2-nitro acetophenone	4-hydroxy 3-methoxy benzaldehyde	52.34	148-153	alcohol	1867.28(c=o), 1636,03(c=c), 2929.88(C-H)

Table 2. Gastroprotective effect of new Chalcone compounds on pylorus ligated Shay ulcer rats

Treatment	Gastric volume (mL)	pH	Acidity (mEq/L)	Ulcer Index	Percentage protection from Ulcer (%)
Control ( 2ml/kg, p.o) 2 % CMC	4.48 ±0.27	1.60±0.49	18.17±2.17	6.50±1.18	...............
Compound I_b(100 mg/kg, p.o)	2.45±0.67	3.63±0.26	11.67±1.91	4.59±0.69	29.53
Compound I_c(100 mg/kg, p.o)	2.47±0.42	3.50±0.50	15.2±1.99	4.83±0.919	25.69
Compound I_f(100 mg/kg, p.o)	3.07±2.45	2.25± 0.45	17.17±1.40	3.83±0.17	41.07
Ranitidine (50 mg/kg, p.o)	1.02±0.20	6.67±0.45	5.67±1.67	2.50±1.10	61.53

Values are expressed as mean ± SEM of six animals (n=6). Statistical analysis was performed using one way analysis of variance (ANOVA) using Dunnett’s t test by Graphpad Instat software (USA). P value .Significant when compared with standard group and Shay ulcer control rats.

Statistical Analysis

The difference between the groups were determined using the one-way analysis of variance (ANOVA) followed by Dunnett’s test using Graphpad Instat Software, USA.

RESULTS AND DISCUSSIONS:

Chemistry

The scheme Figure.1 shows the general synthesis procedure employed for the chalcone derivatives. The compounds were generally obtained in good yields (50-60 %). The compounds have low solubility in common solvents. All the synthesized compounds were characterized by TLC, melting point and IR and the results were shown in Table.1

Pharmacology

The antioxidant activity of several hydroxyl chalcones were evaluated for their ability to scavenge various free radicals. The free radical scavenging activity of new chalcone compounds were studied for their in-vitro radical scavenging activity in the presence of DPPH and NO^- radicals and the results were shown in Figure 2 and 3 respectively. All the synthesized chalcone derivatives were evaluated for their free radical scavenging activity using 1,1-diphenyl-2-picryl hydroxyl (DPPH) and Nitric oxide radical assay systems. Among them, except compound I_d, all other compounds exhibited marked radical scavenging activity against DPPH and NO^- radicals. The antioxidant nature of these new chalcone compounds may be due to their polyphenolic nature and since polyphenols are important compounds because of their antioxidant nature and these compounds can be explored as antioxidant agents.

Compounds showed higher in-vitro antioxidant activity were subjected to gastroprotective effect using pylorus ligation induced gastric ulcer in rats. Volume of gastric juice pH, total acidity and ulcer index were shown in Table.2 and Figure.4 Compounds I_b (29.53 %) and I_f (41.07 %) showed good percentage of gastroprotective effect as comparable to standard (61.63 %) drug (Ranitidine 50 mg/kg) at the dose of 100 mg/kg. The capacity to increase the pH and reduce the acidity, gastric juice content, ulcer index was significant (P value) for compound I_f. All the screened compounds exhibited moderate ulcer index values as compared to Shay ulcer control rats. Among them, compound I_f shared better antiulcer potential. The increase in the gastroprotective activity could be attributed to their antioxidant and phenolic nature of chalcones. Among seven, three synthesized chalcone compounds were found to have antioxidant and gastro-protective activities at the doses employed. Further studies are in progress to optimize these lead compounds and to characterize the mode of action.

CONCLUSION:

The chalcone derivatives possessed a marked cytoprotective activity against experimentally induced gastric ulcer. Among them, morphological analysis of the stomach and ulcer index of compound I_ftreated animal results shown that the cytoprotective effect observed in this study were based on the reduced gastric secretion (anti-secretory) and acidity in the stomach, All the synthesized chaconne derivative shown low anti-oxidant activity as determined by DPPH and NO^- scavenging assays. However, for clinical studies, further pharmacological tests need to be carried out to determine appropriate mode of action and advanced investigation is required to uncover any side effects caused by these chalcone compounds.

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Received on 25.09.2013

Modified on 28.10.2013

Accepted on 05.11.2013

Research J. Pharmacology and Pharmacodynamics. 5(6): November –December 2013, 325-330