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PRELIMINARY SCREENING OF PHARMACOLOGICAL ACTIVITIES OF E-3 ARYLIDENE FLAVANONES.

Byline: Lincy Joseph and Mathew George - Email: mathewlincg@yahoo.com

Abstract

Objective of the present study was to synthesize E-3 -arylidene flavanones by one pot method and screen their analgesic, anti-oxidant and anti-bacterial activities. A set of four E-3 arylidene flavanones were synthesized by simple base catalysed condensation of appropriate aryl aldehydes and 2'4'dihydroxy acetophenone. Screened analgesic activity by hot plate method,anti-oxidant activity by spectrophotometric method and anti-bacterial activity by cup-plate method. Result showed all four synthesized compounds were found to exhibit reliable degree of analgesic activity and anti-oxidant action produced by the synthesized compounds were in reverse order to that of analgesic activity. A few of synthesized compounds showed activity against some organisms. Even though all synthesized compounds have same basic nucleus, biological activities expressed by different compounds are not same.

Due to structural similarity with those of natural flavanones, all the synthesized compounds were expected to exhibit analgesic activity, but only three were found to exhibit analgesic action. But all showed a reliable degree of anti- oxidant activity. In anti-bacterial studies unsubstituted compound didn't showed any kind of antibacterial activity against any of the tested organisms.

Keywords: Analgesic activity, Anti-oxidant activity, E-3arylidene flavanone, 2'4'dihydroxy acetophenone.

INTRODUCTION

Flavonoids are a group of polyphenolic compounds which are widely distributed through out the plant kingdom (Kuhna, 1976). Flavonoids can be classified into flavonols, flavones, flavanones and dihydroflavonols (Hyun Koo et al., 2002). 3-arylidene flavanones are also known as flavindogenides. Basic structure of E- 3 arylidene flavanones have four rings.

In addition to basic structure of flavanone,E-3 -arylidene flavanone have an extended conjugation at C-3 with carbonyl group. The special feature of long conjugation with the keto groups of flavanone moiety is expected to impart very significant biological activity of this type of compounds. Krishnamurthy et al. (1989) suggests E-3 arylidene flavanones and their heterocyclic analogues have poor solubility in aqueous medium starting either from 2-hydroxy chalcone or from o-hydroxy acetophenone. The reactivity of condensing aldehyde is an important factor in the synthesis. Presence of alcohol enhances the formation of products which have been utilized for alkali catalyzed synthesis of many arylidene flavanones been reported by them. Seiket et al. (1962) and Shah et al. (1964) reported the formation of 3-arylidene flavanone in alkaline medium.

Chawla et al. (1987) reported the synthesis of seven 3-arylidene flavanone by condensing 2-Hydroxy acetophenones with aromatic aldehydes in aqueous alkaline medium, along with corresponding chalcones. According to these authors arylidene flavanones were accessible only by a lowyielding circuitous route. Knishnamurthy et al. (1989) concluded that 3-arylidene flavanones are obtained by acid catalyzed condensation between flavanone and aryl halide.

Their first representatives were synthesized by Katshalowsky and Kostanecky in 1904 (Kastanecki et al., 1904). For a long time, E-3 Arylidene flavanones (E-34) were synthesized solely by the acid catalyzed condensation of flavanone (Lorand et al., 1971) and aromatic aldehydes (Ryan et al., 1929, Algar et al., 1930, Diesbach et al., 1945, Szell et al., 1968, Reichel et al., 1968, Hempel et al., 1966 and Dhara et al., 1996). The reaction usually has been performed in alcoholic solution saturated with anhydrous hydrochloric acid at various temperatures and for different time. Albert Levai et al. (1978) introduced a very simple base-catalyzed condensation for the synthesis of E3-arylidene flavanones. A mixture of equimolar amounts of flavanones and aromatic aldehyde and a few drops of piperidine was allowed to react at 150oC and E-3 arylidene flavanone was obtained in good yield without any purification. On the basis of 1H NMR spectra Keane et al. (1970) explained the stereochemistry of synthetic E and Z-3-arylidene flavanones.

So here is an attempt made to synthesize a few E-3-arylidene flavanones by alkaline condensation between 2',4'-dihydroxy acetophenone and aromatic aldehydes,in presence of alcohol,then to screen the synthesized compounds for the analgesic, anti-oxidant and anti-bacterial activities.

EXPERIMENTAL

For the synthesis of proposed compounds, 2',4'-dihydroxy acetophenone have been purchased from Sigma Aldrich Chemical Company Inc. U.S.A., Benzaldehyde, Furfuraldehyde, P-Chloro benzaldehyde, and 3-Nitrobenzaldehyde have been purchased from S.D. Fine Chemicals, Mumbai.

Synthetic protocol

The each proposed compounds were synthesized as per the following procedure (Dhara et al., 1996). To a mixture of 2'4'-dihydroxy acetophenone (1 m mole) and substituted aromatic aldehyde (2.5 m mole), a warm solution of KOH (15%) in aqueous methanol (1:1) was added with constant stirring until to get clear solution. Then the reaction mixture was cooled to room temperature by adding methanol dropwise. The reaction mixture is kept for 96 hrs at room temperature with stoppered condition. The solid separated out was filtered and washed with cold aqueous methanol (1:1) and crystallized from chloroform-light petroleum ether (1:3) to give TLC pure crystals.

Biological Experimental Protocol for Analgesic activity (S.K.Kulkarni, 1993 and R.A Turner 1965)

Hotplate method

To study the analgesic activities of the synthesized compounds, albino mice of either sex were used. All mice were screened by exposure to thermal stimulus. Mice weighing between 20-25 g selected and made into six groups having six animals in each group. One group served as control which received 2% gum acacia suspension. Second group received Diclofenac sodium as standard drug orally at a dose of 75 mcg/Kg body weight of animal (suspension) of test compound (150 mcg/ml) prepared in 2% gum acacia) . Animals were placed on perspax cylinder on heated surface and the time to produce discomfort reaction (licking paws or jumping) was considered as reaction time with the cut off time being 60 seconds. The first reading was taken immediately after administration of compounds and afterwards at the intervals of 30 minutes. The results were recorded.

MATERIALS AND METHOD

1. Albino mice each weighing between 20-25g

2. Standard drug-Diclofenac Sodium

3. Hot plate (model 7280 ugo Brazil, Italy)

Suspension of standard drugs and test compounds

1. Solution of Diclofenac Sodium containing 75 mcg/ml of drug were prepared

2. Suspension of test compounds (150 mcg/ml) were prepared in 2% gum acacia.

Experimental protocol for Anti-oxidant activity (Jayaprakash et al., 2004)

Mixture of 1,1-diphenyl-2-picrylhydrazyl(3.9 mg in 10 ml ethanol) and test compounds (10 mg /10 ml ethanol) mixed and kept for 20 minutes at room temperature. Then absorbance measured at 517 n.m. Curcumin used as standard drug to compare the activity.

Experimental protocol for Anti-bacterial activity

The antibacterial activity of arylidene flavanones were studied by cup-plate method with ciprofloxacin at a concentration of 10 ug/0.1 ml in DMF (dimethyl formamide) as reference standard using two gram +ve organisms used were Staphylococcus aureus and Bacillus subtilis and two gram -ve organisms namely Escherichia coli and Pseudomonas aeruginosa.

Materials and Methods for antibacterial studies

1. Medium have been prepared as described in Indian Pharmacopoeia.

2. Sterilized petridishes, pipettes, boiling tubes and beakers.

3. 8 to 24hrs. old growth cultures in nutrient broth

4. Sterilized test tubes

5. Sterile cork borer.

6. Sterile inoculation loops

7. Sterilized fine pointed forceps

8. Nutrient agar

9. Tuberculin syringes.

Preparation of solutions of test compounds

10mg of each test compound was dissolved in 10ml of DMF (dimethyl formamide) in serially and suitably labeled sterile test tubes, thus giving a final concentration of 10ug/1ml.

Preparation of Media

Media mentioned in Indian pharmacopoeia was prepared by dissolving bacteriological peptone (6g), pancreatic digest of casein (4g), yeast extract (3g), beef extract (1.5g), dextrose (1.0g) and agar (15.0g) in distilled water to produce one litre of medium.

The pH of the solution was adjusted to 6.5-6.6 by using 1M sodium hydroxide and 1M hydrochloric acid. Then it was sterilized for 30 minutes at 15 lbs pressure.

10mg of each test compound was dissolved in 10 ml of DMF (dimethyl formamide) in serially and suitably labelled sterile test tubes, thus giving a final concentration of 10ug/1ml. Using sterile pipettes the standard and the sample solutions (0.1ml) of known concentrations were fed into the bored cups. As Cup-1: Standard (ciprofloxacin).Cup-2: solvent control (DMF).Cup3-: Test compound.

Table 1. Analgesic activity of synthesized compounds by Hot plate method

Compound###Dose(orally)###Average reaction time in seconds a###

I.D###mcg/kg###0###30###60###90###

1###200###3.00###3.10###3.10###3.10###

2###200###2.55###3.25###4.50###4.50###

3###200###3.00###4.00###4.00###4.00###

4###200###3.50###5.00###5.20###5.20###

Std.###200###3.00###5.25###8.25###8.28###

Control###-----###3.00###3.00###3.00###3.00###

a = Average reaction time taken as mean (+-S.D)of a group of six animals. Table 2. Anti-oxidant activity by in vitro method

Compound###Absorbance at 517 n.m###Relative % activity considering that of standard as 100%###

Curcumin###2.596###100%###

1###0.764###29.4%###

2###2.482###95.6%###

3###2.264###87.2%###

4###2.168###83.5%###

Table 3 Antibacterial activity cup-plate method###

Compound###Zone of inhibition in m.m###

###E.coli###P.aerugenosa Staphylococcus###Bacillus subtilis###

###aureus

1###-###-###-###-###

2###17###10###16###15###

3###13###11###15###12###

4###18###15###16###24###

Ciprofloxacin###28###26###24###32

Assessment of Synthesized Compounds

Physical datas of tested compounds are as follows. Methanol had been used as solvent to find out ? -max by U.V spectroscopy. KBr pellets used to measure I.R spectrum and CDCl3 used for 1 H NMR spectrum. Using NMR and IR spectrum structures of proposed compounds elucidated.

Compound 1:

? -max-248 n.m,

IR (KBr):1600 cm -1 (C=O),1260and 1111 cm -1 (C- 0-C), 3070 and 2935 cm -1 (C-H), 834 and 745 cm -1 (C-H def), 1461and 1577 cm -1 (C=C), 3548 cm -1(O-H).

1H NMR(CDCl3 ppm):6.6-6.68(H- 2), 6.9-7.1 (H-6,H-8),7.22(CHCl3),7.8-8 (H-3),7.22-7.4 (H-B).

Compound 2:

? -max - 243 n.m,

IR (KBr): 1654 cm -1 (C=O), 1254 and 1110 cm -1 (C-O-C), 865 cm -1 (C-H ), 748cm -1 (Cl),1595 and 1570-1457 cm -1 (C=C).

1H NMR(CDCl3 ppm) 6.12-6.2(H-3',H-4'), 6.48-6.54(H-4"), 6.94-6.98(H-.9-8(H-5)8),

7-7.2(H-6),7.2-7.28(H-2),7.3 CHCl3, 7.4-7.5 (H-7), 7.54-7.6(H-5"),7.8-8(H-5)

Compound 3:

?-max-256 n.m.

IR(KBr):1671 cm -1(C=O), 1172 and 1260 cm -1(C-O-C),832 and 743 cm -1(C-H def), 1574 and 1462 cm - 1(C=C).

1H NMR(CDCl3ppm)6.52-6.58(H-2),6.9-7.2(H-6,H-8),7.88-7.94(H-5),8.06-8.08(H-B).

Compound 4:

?-max-254 n.m.

IR(KBr):1674 cm -1(C=O), 1220 and 1109 cm -1(C-O-C), 842 and 816 cm -1(C-H def), 1603 and 1489 cm -1(C=C) 1408 cm -1(N--O).

1H NMR(CDCl3ppm)6.52-6.56(H-2),6.8-7(H-6,H-8),7.8-7.96(H-5),8.2-8.5(H-B).

RESULTS

As per the synthetic protocol four E-3 arylidene flavanones have been synthesized and performed their analgesic activity by hot plate method. Anti-oxidant activity tested by spectrophotometric method. Observations for analgesic activity are shown in Table 1, that of anti-oxidant activity in Table 2 and anti-bacterial activity in Table 3.

The basic structure of the synthesized compounds can be drawn out as follows:

Compound 1; R= H, Compound 2; R= Cl, Compound 3; R=OCH3, Compound 4;R=NO2

DISCUSSION

The results of the assessment of synthesized compounds have good agreement with the datas given in the literature for compounds of same category.

Due to structural similarity with those of natural flavanones, all synthesized compounds were expected to exhibit analgesic activity, as per the studies three were found to exhibit analgesic action. The results shows less analgesic activity for all tested compounds than the standard drug namely diclofenac sodium. The compound-4 showed maximum analgesic activity. Compound-1 showed least analgesic activity. Only three compounds exhibited analgesic activity,the compound- 4 showed more activity than others, probably due to the presence of nitro group.

Considering anti- oxidant activity, compound-2 showed maximum activity and compound-1 showed least activity. More anti-oxidant activity of compound-2 may be due to the presence of chlorine and hydroxyl groups.

In anti- bacterial studies only compound -4 showed good activity against all organisms. Compound -1didn't showed any activity against any organisms. Compound -2 also showed good activity except against Pseudomonas auregenosa.

From the present study can be concluded that nitrated flavanones are more potent than unsubstituted flavanones in producing antianalgesia.In the case of antibacterial activity, maximum efficacy shown by nitrated compounds followed by halogenated compounds. Among the synthesized compounds halogenated compound showed maximum antioxidant activity than unsubstituted/ nitrated/methoxyl compounds.

ACKNOWLEDGEMENTS

The authors are thankful for those who gave suggestions and comments during the research which made more progress. Also wish to thank Sigma Aldrich and S.D Fine chemicals for providing the chemicals.

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Lincy Joseph* and Mathew George Jaipur National University, Rajasthan, India
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Publication:Journal of Basic & Applied Sciences
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Geographic Code:9INDI
Date:Dec 31, 2010
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