Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524112EnglishN-0001November30HealthcareSYNTHESIS AND BIOLOGICAL SCREENING OF NOVEL ARYLOXYACETIC ACID ANALOGS
English0311Ramninder KaurEnglishKomalpreet KaurEnglishA series of 5-Bromo- 2-formyl phenoxyacetyl amino acids and peptides have been synthesized by coupling of the 5-Bromo- 2-formyl phenoxyacetic acid with amino acid/methyl esters/dipeptides/ tripeptides using DCCas coupling agent and NMM as base. The structures were elucidated FTIR and 1HNMR .The newly synthesized compounds were evaluated for their antibacterial, antifungal and anthelminitic activities. The compounds (2, 6, 11 and 13) were found to exihibit potent antibacterial activity against Bacillus subtilis, Staphylococcus aureus (gram positive bacteria) and Escherichia coli (gram negative) bacterias.Thecompounds (5, 6 and 13) were found to exihibit potent antifungal activity against Candida albicans and Aspergillus niger. The moderate to good anthelminitic activity was shown by the synthesized compounds (7 and 13) against Eudrilus spieces.
EnglishPhenoxyacetic acid, amino acids, antibacterial, antifungal and anthelminitic.Phenoxyacetic acid is among the most vital moieties which are associate with potent antidiabetic (Rival et al 2004), antimycobacterial (Yar et al 2007),diuretic (Lebedev et al 1985, Woltersdorf et al 1976, Bicking et al 1976), anti-inflammatory (Kunsch et al 2005, Shokol et al 2005), antibiotic (Grardin et al 1995), anti-obesity (Kiso et al 1999), diagnostic (Ohmomo et al 1989), inhibition of platelet aggregation (Meanwell et al 1993, Seiler et al 1994) activities. The review of literature has suggested that incorporation of amino acids and peptides into aromatic and heterocyclic congeners have resulted in compounds with potent bioactivities.
Introducing an amino acid or peptide into aromatic compounds can increase the potency, decrease the toxicity and prolong its action. Among aromatics, phenolic compounds have wide range of activities. Further phenoxylation the resulting compound phenoxyacetic acid is obtained, which is well known for their biological potential. Thus keeping in view the biological potency of phenoxyacetic acids as well as taking advantage of biodegradability and biocompatibility of a novel series of substituted phenoxyacetic acid derivatives of amino acids and peptides have been synthesized with an anticipation to get potent agents with
good therapeutic efficacy with negligible side effects. The study of IR and 1HNMR spectrum gives us most of the required information of certain vibrational bands or characteristic groups present in the molecule. The IR spectrum of newly synthesized compounds showed characteristic bands in the region 3329- 3318, 1654-1642, 1537-1532 cm-1 which can be assigned as N-H stretching, C=O stretching and N-H bend respectively. In their 1HNMR spectra, a singlet appeared in the range 8.33-8.36 ppm corresponding to the CO-NH proton. The compounds were found to exhibit potent antimicrobial and moderate anthelmintic activity in comparison to standard drugs against the same concentration.
Results and discussion
Antibacterial Activity:
The synthesized peptide derivatives were screened for antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis using modified Kirby-Bauer disc diffusion method (DMF as a solvent).The test samples were tested at the concentrations 25, 50, 100 µg/ml. The petri plates inoculated with bacterial cultures were incubated at 37 C for 18 hrs. The diameters obtained for the test sample were compared with that produced by the standard drug ciprofloxacin. The results are shown in Table 2
Antifungal Activity:
The synthesized peptide derivatives were screened for antifungal activity against Candida albicans and Asperigillus niger. DMSO is used as negative control. The test samples were tested at the concentrations 25, 50, 100 pg/ml. The petri plates inoculated with fungal cultures were incubated at 25 C for 48 hrs. Diameters of the zone of inhibition were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced by the standard drug griseofulvin. The results are shown in Table 2
Anthelmintic Activity:
The anthelminitic activity was carried out against earthworms Eudrilus species by Garg and Atal method at 2 mg/ ml concentration. Suspension of samples was prepared by triturating synthesized cyclic peptide (200 mg) with Tween 80 (0.5 %) and distilled water. Suspension of the standard drug albendazole was prepared with the same concentration in a similar way. The paralyzing and death times were noted and their mean was calculated for triplicate sets. The death time was ascertained by placing the earthworms in warm water (50 C) which stimulated the movement. The results were shown in Table 3.
The results of biological activities revealed that newly synthesized peptide derivative 73 at 50pg/ml concentration exhibited highest zone of inhibition against Staphylococcus aureus and 75 at 50pg/ml concentration exhibited highest zone of inhibition against Candida albicans. Morover, other compounds showed moderate antimicrobial activities against tested organisms. Comparison of anthelmintic activity data revealed that peptide derivative 75 was found to exhibit potent anthelmintic activity and other peptide derivatives showed good to moderate activity.
Experimental
Melting points were determined and uncorrected. The amino acids, di-tertbutyl pyrocarbonate (Boc2O), 5- Bromosalicyaldehyde, DCC and NMM were obtained from Spectrochem Limited, Himedia laboratories Limited mumbai and Sd-fine-chem Limited, Mumbai, India. The IR spectra were recorded on a Perkin Elmer Fourier transform infrared spectrophotometer using KBr pellets. The 1HNMR spectra were recorded on the Bruker Avance II- 400 NMR spectrometer using CDCl3 as the solvent. The purity of all the compounds was controlled by TLC on silica gel G plates. Chloroform:Methanol (9:1 v/v) was used as developing solvent system and dark brown spots were detected on exposure to iodine vapours in a tightly closed chamber. The physical data of synthesized compounds is listed in Table 1. The scheme of synthesis is given in Scheme 1.
Synthesis of Boc amino acids (1-3):
L-Leucine (1.31gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L-1 ) and 10 ml of i-propanol. ditert.butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of i-propanol was added followed by 10 ml of sodium hydroxide (1 mol L-1 ) to the resulting solution. The solution was stirred at room temperature for 2 hr, washed with 10 ml of light petroleum ether (b.p. 40-60 C), acidified to p H 3.0 with 1 mol L-1 sulphuric acid and finally extracted with chloroform (3 x 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was purified by recrystallization from methanol and ether at 0 C to get pure Boc-Leucine (1). Similarly, Boc-Serine (2) and Boc-Alanine (3) were prepared by stirring di-tert.butylpyrocarbonate (3 ml, 13 mmol) with Boc-Serine (1.05gm, 10 mmol) and Boc-Alanine (0.89gm, 10 mmol) respectively.
Synthesis of L-amino acid methyl ester hydrochlorides (4-6) :
Thionyl chloride (0.73mL, 10 mmol) was slowly added to methanol (50 mL) at 0 C and 1.15 gm of L- Proline (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 C. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude product was purified by recrystallization from methanol and ether at 0 C to get Lproline methyl ester hydrochloride (4). Similarly, L-leucine methyl ester hydrochloride (5) and L-tryptophan methyl ester hydrochloride (6) was prepared by refluxing 1.31 gm of Lleucine (10 mmol) and 2.04 gm of Ltryptophan with 50 ml methanol in the presence of 0.73 ml of thionyl chloride (10 mmol).
Synthesis of Boc-dipeptide methyl esters (7-9) :
To a mixture of 1.65 gm compound 4 (10 mmol) in 20 ml of chloroform, 2.3 ml of N- methylmorpholine (21mmol) was added at 0 C. The reaction mixture was stirred for 15 min. 2.31gm compound 1 (10mmol) in 20 ml chloroform and 2.1gm of DCC (10mmol) were added under stirring to the above mixture. After 36 hrs, the reaction mixture was filtered and the residue was washed with 30 ml of chloroform and added to the filterate. The filterate was washed with 5% sodium hydrogen carbonate and saturated sodium chloride solution (25 ml each). The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated in vacuum. The crude product was recrystallized from mixture of chloroform and petroleum ether (b.p. 40-60 C) followed by cooling at 0 C to get Boc-Leu-Pro-OMe (7). Similarly Boc-Ser-Leu-OMe (8) and Boc-Ala-ProOMe (9) were prepared by stirring compounds 2 and 3 with amino acid methyl ester hydrochlorides 5 and 4, respectively in the presence of DCC and NMM.
Deprotection of dipeptides at carboxyl end (8a, 9a) :
To a solution of 3.32 gm of compound 8 (10 mmol) in 36 ml of THF/H2O (1:1), 0.36 gm lithium hydroxide (15mmol) was added at 0 C. The mixture was stirred at room temperature for 1 hr, and acidified to 1 pH 3.5 with 0.5 mol LH2SO4. The aqueous layer was extracted with diethyl ether (3 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from methanol and ether to get Boc-SerLeu-OH (8a). Similarly compound 9 was hydrolyzed under alkaline conditions to obtain Boc-Ala-Pro-OH (9a).
Deprotection of dipeptide at amino end (7a):
Compound 7 (3.42 gm, 10mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of trifluoroacetic acid (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by recrystallization from mixture of chloroform and light petroleum ether (b.p. 40-60 C) to get pure Leu-Pro-OMe (7a).
Synthesis of Boc-tripeptide methyl esters (10, 11) :
To synthesize Boc-Ser-Leu-Trp-OMe (10), 3.18 gm of dipeptide unit 8a (10 mmol) was coupled with 2.54 gm of amino acid methyl ester hydrochloride 6 (10 mmol) in the presence of DCC and NMM following the same procedure as adopted for the synthesis of Bocdipeptide methyl esters 7-9. Similarly Boc-Ala-Pro-Pro-OMe (11) was prepared by coupling 2.86 gm of deprotected dipeptide unit 9a and 1.65 gm of amino acid methyl ester hydrochloride 4 using DCC as the coupling agent and NMM as the base.
Deprotection of tripeptides at amino end (10a, 11a):
Compound 10 (5.18 gm, 10mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of trifluoroacetic acid (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by recrystallization from mixture of chloroform and light petroleum ether (b.p. 40-60 C) to get pure Ser-Leu-TrpOMe (10a). Similarly Ala-Pro-Pro-OMe (11a) was prepared by stirring compound 11 with 2.28 gm of trifluoroacetic acid (20 mmol).
Synthesis of free acid
Sodium hydroxide (0.89gm, 22.4mmol) in 25 ml water was slowly added with stirring to 2.01gm of 5-bromo-2- hydroxyaldehyde (10mmol) and 0.94gm of chloroacetic acid (10mmol). The mixture was heated on heating mantle to remove all the liquid and the residue was treated with 30 ml water. The mixture was cooled and filtered and clear solution was acidified with dilute hydrochloric acid. The aqueous layer was extracted with diethyl ether (2 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate. The crude product was recrystallized from ethanol and purified by recrystallization from ethanol-water (1:1) to get 5-Bromo-2-formylphenoxyacetic acid (12).
Synthesis of 5-Bromo-2-formylphenoxyacetyl amino acid and peptide methyl esters
L-Proline methyl ester hydrochloride (1.65 gm, 10mmol) was dissolved in Tetrahydrofuran (75 mL). To this, 2.3 ml of N-methylmorpholine (21mmol) was added at 0 C and the reaction mixture was stirred for 15 min. 2.01gm of compound 12 (10mmol) in tetrahydrofuran and 2.1gm dicyclohexylcarbodiimide (10mmol) were added under stirring to the above mixture. After 36 hrs, the reaction mixture was filtered and the residue was washed with 30 ml of tetrahydrofuran and added to the filterate. The filterate was washed with 5% NaHCO3 and saturated NaCl solution (25 ml each). The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated in vacuum. The crude product was recrystallized from mixture of chloroform and n-hexane followed by cooling at 0 C to get 5-Bromo-2-formylphenoxyacetyl-proline methyl ester (13). Similarly, 5-Bromo-2-formylphenoxyacetyl-leucyl-proline methyl ester (14), 5-Bromo-2-formylphenoxyacetyl-seryl-leucyl-tryptophan methyl ester (15) and 5-Bromo-2- formyl-phenoxyacetyl-alanyl-prolylproline methyl ester (16) were prepared by stirring compound 7a, 10a and 11a with compound 12 respectively in the presence of DCC and NMM.
Deprotection of 5-Bromo-2-formylphenoxyacetyl-alanyl-prolyl-proline methyl ester at carboxyl end (16a) :
To a solution of 4.39 gm of compound 16 (10 mmol) in 36 ml of THF/H2O (1:1), 0.36 gm lithium hydroxide (15mmol) was added at 0 C. The mixture was stirred at room temperature for 1 hr, and acidified to 1 pH 3.5 with 0.5 mol L-H2SO4. The aqueous layer was extracted with diethyl ether (3 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from methanol and ether to get 5- Bromo-2-formyl-phenoxyacetyl-alanylprolyl-proline (16a).
ACKNOWLEDGEMENTS
The authors are thankful to the Head, Department of Chemistry for providing research facilities, SAIF Department, Punjab University, Chandigarh (India), for providing spectral details in time.
R1 = Br; R2 = H; R3 = H; R4 =CHO (12-16a)
l = ClCH2COOH, NaOH, RT 1hr,
ll = DCC, NMM, RT 36hrs,
lll = LiOH, THF: H2O (1:1) RT1hr X = Pro (13), Leu -Pro (14), Ser-Leu-Trp (15), Ala-Pro-Pro (16, 16a)
Englishhttp://ijcrr.com/abstract.php?article_id=2270http://ijcrr.com/article_html.php?did=22701. Y. Rival., A. Stennvin., L. Puech, A.Rouquete, C.Cathala, F. Lestienna,. and D.Junquero, J. Pharmacol. Exp. Therap., 2004, 311, 2, 467-475.
2. M.S Yar., A.A. Siddiqui and M.A. Ali, Bioorg. Med. Chem., 2007, 14, 4571-4574. 3. A.A. Lebedev, V.A. Smirnoc, M. Bazhmina and Karpacheva T.A., Pharm. Chem. J., 1985, 19,
3, 171- 173.
4. C. Kunsch, J. Luchoomul, G.L. Dodd, K.S. Karu, J.D. Piper and C.L. Sundell, J. Pharmcol. Exp. Therap., 2005 313, 2, 492-501.
5. P. Gerardin, M. Ahrach, R. Schneider, F. Houillon, B. Loubinoux, M. Sprenge and J.L. Colin, Bioorg. Med. Chem. Lett., 1995, 5, 14, 1467-1470.
6. T. Kiso, T. Kakita, T. Shoqaki and Y. Ohtsubo, Bio. Pharm. Bull., 1999, 22, 10, 1073-1078.
7. Y. Ohmomo, S. Okuyama, Y. Magata, Y. Ueno, C. Tanaka and A. Yokoyama, Chem. Pharm. Bull., 1989, 37, 9, 2276-2281.
8. N.A Meanwell, M.J. Rosenfeld, J.J. Kim Wright, C.L. Brassard, J.O. Buchanan M.E. Federici, and S.M. Seiler, J. Med. Chem., 1993 36, 3871-3883.
9. R. Dahiya, D. Pathak and S. Bhatt, J. Saudi. Chem. Soc., 2006, 10, 1, 165-176.
10. A.W. Bauer, W.M. Kirby and M. Turck, Amer. J. Clin. Path., 1966, 45, 493-496.
11. L.C. Garg and C.K Atal, Indian J. Pharm. Sci., 1963, 59, 240-245.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524112EnglishN-0001November30HealthcareSYNTHESIS AND BIOLOGICAL SCREENING OF CYCLIC HEPTAPEPTIDE
English1222Komalpreet KaurRamninder KaurEnglishA new bioactive cyclic heptapeptide cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) was synthesized using the solution phase technique by cyclization of the linear peptide Boc- Gly-Tyr-Val-Pro-Leu-Trp-Pro after proper deprotection at carboxyl and amino terminals. All the coupling reactions were performed at room temperature utilizing dicyclohexylcarbodiimide (DCC) as the coupling agent and N-methylmorpholine (NMM) as the base. Structures of all new compounds were characterized by IR and 1HNMR. The synthesized cyclopeptide was
screened for antimicrobial and anthelminitic activities and found to exhibit good antibacterial activity against Bacillus subtilis and moderate antifungal activity against Candida albicans and Asperigillus niger. In
addition the cyclic peptide was found to exhibit good anthelminitic activity against earthworms Eudrilus species.
Englishcyclic heptapeptide, antimicrobial activity, anthelminitic activity.Cyclic congeners possess unusual or modified amino acid residues and exhibit there bioactivities through binding to corresponding enzyme. This characteristic feature can allow bioactive cyclopeptides to act as therapeutic agents in this resistant world. Cyclopepetides having multiple peptide bonds are concerned with a wide spectrum of biological activities such as antimicrobial, anti-inflammatory, antimalarial, cyctotoxic, and antifungal activities. Cyclic peptides are more important compounds for medicinal purposes and represent an important class of natural products. Since only minute quantities are obtained from natural resources many of these compounds were attempted to synthesize in the laboratory. Keeping in view the biological potential of cyclic peptide as well as to obtain a bioactive compound in a good yield, the present investigation aimed at synthesis of cyclic heptapeptide cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) in a convenient and economical manner. Synthesized cyclic heptapeptide was evaluated for pharmacological activities. The antibacterial and antifungal activities were carried out against variety of pathogenic microorganism like Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, Asperigillus niger. The anthelminitic activity was carried out against Eudrilus species of earthworms.
MATERIALS AND METHODS
Melting points were determined and uncorrected. The amino acids, di-tert-butyl pyrocarbonate (Boc2O), p-nitrophenol (pnp), DCC and NMM were obtained from Spectrochem Limited and Sd-fine-chem Limited, Mumbai, India. The IR spectra were recorded on a Perkin Elmer Fourier transform infrared spectrophotometer using KBr pellets. The 1HNMR spectra were recorded on the Bruker Avance II-400 NMR spectrometer using CDCl3 as the solvent. The purity of all the compounds was controlled by TLC on silica gel G plates. Chloroform:Methanol (9:1 v/v) was used as developing solvent system and dark brown spots were detected on exposure to iodine vapours in a tightly closed chamber. The physical data of synthesized compounds is listed in Table 1. The scheme of synthesis is given in Scheme 1.
Synthesis of Boc amino acids (1-3) :
L-tyrosine (1.81gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L-1 ) and 10 ml of i-propanol. ditert.butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of i-propanol was added followed by 10 ml of sodium hydroxide (1 mol L-1 ) to the resulting solution. The solution was stirred at room temperature for 2 hr, washed with 10 ml of light petroleum ether (b.p. 40-60 C), acidified to pH 3.0 with 1 mol L-1 sulphuric acid and finally extracted with chloroform (3 x 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was purified by recrystallization from methanol and ether at 0 C to get pure Boc-Tyrosine (1). Similarly, Boc-leucine (2) and Boc-proline (3) were prepared by stirring ditert.butylpyrocarbonate (3 ml, 13 mmol) with Boc-proline (1.15 gm, 10 mmol) and Boc-leucine (1.31gm, 10 mmol) respectively.
Synthesis of L-amino acid methyl ester hydrochlorides (4-7) :
Thionyl chloride (0.73mL, 10 mmol) was slowly added to methanol (50 mL) at 0 C and 1.15gm of L- proline (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 C. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude product was purified by recrystallization from methanol and ether at 0 C to get L-proline methyl ester hydrochloride (4). Similarly, L-valine methyl ester hydrochloride (5), L-tryptophan methyl ester hydrochloride (6) and glycine methyl ester hydrochloride (7) were prepared by refluxing 1.17 gm of L-valine (10 mmol), 2.04 gm of L-tryptophan (10 mmol) and 0.75 gm of glycine (10 mmol) with 50 ml methanol in the presence of 0.73 ml of thionyl chloride (10 mmol).
Synthesis of Boc-dipeptide methyl esters (8-10):
To a mixture of 1.67gm compound 5 (10 mmol) in 20 ml of chloroform, 2.3 ml of N- methylmorpholine (21mmol) was added at 0 C. The reaction mixture was stirred for 15 min. 2.81gm compound 1 (10mmol) in 20 ml chloroform and 2.1gm of DCC (10mmol) were added under stirring to the above mixture. After 36 hrs, the reaction mixture was filtered and the residue was washed with 30 ml of chloroform and added to the filterate. The filterate was washed with 5% sodium hydrogen carbonate and saturated sodium chloride solution (25 ml each). The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated in vacuum. The crude product was recrystallized from mixture of chloroform and petroleum ether (b.p. 40-60 C) followed by cooling at 0 C to get Boc-Tyr-Val-OMe (8). Similarly Boc-Leu-Trp-OMe (9) and Boc-Pro-Gly-OMe (10) were prepared by stirring compounds 2 and 3 with amino acid methyl ester hydrochlorides 6 and 7, respectively in the presence of DCC and NMM.
Deprotection of dipeptides at carboxyl end (8a, 9a) :
To a solution of 3.94 gm of compound 8 (10 mmol) in 36 ml of THF/H2O (1:1), 0.36 gm lithium hydroxide (15mmol) was added at 0 C. The mixture was stirred at room temperature for 1 hr, and acidified to 1 pH 3.5 with 0.5 mol L-H2SO4. The aqueous layer was extracted with diethyl ether (3 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from methanol and ether to get Boc-Tyr-Val-OH (8a). Similarly compound 9 was hydrolyzed under alkaline conditions to obtain Boc-LeuTrp-OH (9a).
Deprotection of dipeptide at amino end (10a):
Compound 10 (2.86 gm, 10mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of trifluoroacetic acid (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by recrystallization from mixture of chloroform and light petroleum ether (b.p. 40-60 C) to get pure Pro-Gly-OMe (10a)
Synthesis of Boc-tri/tetrapeptide methyl esters (11, 12) :
To synthesize Boc-Tyr-Val-Pro-OMe (11), 3.80 gm of dipeptide unit 8a (10 mmol) was coupled with 1.66 gm of amino acid methyl ester hydrochloride 4 (10 mmol) in the presence of DCC and NMM following the same procedure as adopted for the synthesis of Boc-dipeptide methyl esters 8- 10. Similarly Boc-Leu-Trp-Pro-Gly-OMe (12) was prepared by coupling 3.36 gm of deprotected dipeptide unit 9a and 1.86 gm of 10a using DCC as the coupling agent and NMM as the base.
Synthesis of Boc-heptapeptide methyl ester (13):
To synthesize Boc-Tyr-Val-Pro-LeuTrp-Pro-Gly-OMe (13), 4.77 gm of tripeptide unit 11 mmol was deprotected at carboxyl end to get Boc-Tyr-Val-Pro-OH (11a) following the same procedure as adopted for the synthesis of compounds 8a and 9a from compounds 9 and 10, respectively. Tetrapeptide unit (12) (5.85 gm, 10 mmol) was deprotected at amino end to get Leu-Trp-Pro-Gly-OMe (12a) following the same procedure as adopted for the synthesis of compounds 10a from compound 10. The deprotected tripeptide unit 11a (4.77 gm, 10 mmol) and 4.85 gm of tetrapeptide unit (10 mmol) were coupled in the presence of DCC and NMM to get linear heptapeptide unit 13 under the same experimental conditions as adopted for the synthesis of Boc-dipeptide methyl esters (8- 10).
Synthesis of cyclic heptapeptide, cyclo(Gly-Tyr-Val-Pro-Leu-Trp-Pro) (14):
To synthesize cyclo(Gly-Tyr-Val-ProLeu-Trp-Pro) (14), 9.44 gm of linear heptapeptide unit(10 mmol) (13) was deprotected at carboxyl end using 0.36 gm of LiOH (15 mmol) to get Boc-Tyr-Val-ProLeu-Trp-Pro-Gly-OH (13a) following the same procedure as adopted for the synthesis of compounds 8a and 9a from compounds 8 and 9 respectively. The deprotected heptapeptide unit 13a (4.65 gm, 5 mmol) was dissolved in 50 ml of CHCl3 at 0 C. To the above solution, 0.94 gm of pnp (6.7 mmol) was added and stirred at room temperature for 12 hrs. The reaction mixture was filtered and the filtrate was washed with 10% NaHCO3 solution (3 x 15 ml) until excess of pnp was removed and finally washed with 5% HCl (2 x 10 ml) to get the corresponding p-nitrophenyl ester Boc-Tyr-Val-Pro-Leu-Trp-Pro-Gly-O-pnp (13b). To compound 13b (4.20 gm, 4 mmol) dissolved in 35 ml of CHCl3, 0.91 gm of TFA (8 mmol) was added, stirred at room temperature for 1 hr and washed with 10% NaHCO3 solution (2 x 25 ml). The organic layer was dried over anhydrous sodium sulphate to get Tyr-Val-Pro-Leu-Trp-ProGly-O-pnp (13c), which was dissolved in 25 ml of CHCl3 and 2.3 ml of NMM (21 mmol) was added. Then all the contents were kept at 0 C for 7 days. The reaction mixture was washed with 10% NaHCO3 until the byproduct p-nitrophenol was removed completely and finally washed with 5% HCl (3 x 15 ml). The organic layer was dried over anhydrous sodium sulphate. Finally, chloroform was distilled off and the crude cyclized product was crystallized from CHCl3 and n-hexane to get the pure compound 14.
Antibacterial Activity:
The synthesized cyclic peptide was screened for antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis using modified Kirby-Bauer disc diffusion method. A spore suspension in sterile distilled water was prepared from five days old culture of the test bacteria growing on nutrient broth media. About 20 ml of the growth was transferred into sterilized petri plates and inoculated with 1.5 ml of the spore suspension. Each petri plate was divided into five equal portions along the diameter to place one disc. Three discs of test sample were placed on three portions together with one disc reference drug ciprofloxacin (50pg/ml) and a disc impregnated with the solvent DMF as negative control. The test samples were tested at the concentrations 25, 50, 100 pg/ml. The petri plates inoculated with bacterial cultures were incubated at 37 C for 18 hrs. Diameters of the zone of inhibition were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced by the standard drug ciprofloxacin. The results are shown in Table 2.
Antifungal Activity:
The synthesized cyclic peptide was screened for antifungal activity against Candida albicans and Asperigillus niger. A spore suspension in normal saline was prepared from culture of the test fungi on sabouraud?s broth media. After transferring growth media, petri plates were inoculated with spore suspension. After drying, wells were made using agar punch and the test samples, reference drug (griseofulvin) (50pg/ml) and negative control (DMSO) were placed in labeled wells in each petri plate. The test samples were tested at the concentrations 25, 50, 100 µg/ml. The petri plates inoculated with fungal cultures were incubated at 25 C for 48 hrs. Diameters of the zone of inhibition were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced by the standard drug griseofulvin. The results are shown in Table 2.
Anthelmintic Activity:
The anthelminitic activity was carried out against earthworms Eudrilus species by Garg and Atal method at 2 mg/ ml concentration. Suspension of samples was prepared by triturating synthesized cyclic peptide (200 mg) with Tween 80 (0.5 %) and distilled water. The resulting mixture was stirred using mechanical stirrer for 30 min. The suspensions were diluted to contain 0.4 % w/v of the test samples. Suspension of the standard drug albendazole was prepared with the same concentration in a similar way. Three sets of five earthworms of almost similar sizes were placed in petri plates containing 50 ml suspension of Tween 80 (0.5 %) and distilled water. The paralyzing and death times were noted and their mean was calculated for triplicate sets. The death time was ascertained by placing the earthworms in warm water (50 C) which stimulated the movement. The results were shown in Table 3.
RESULTS AND DISCUSSION
The results revealed that newly synthesized cyclic peptide 14 at 50 pg ml-1 concentration exhibited highest zone of inhibition against B. subtilis. The cyclic peptide 14 at 50 pg ml-1 concentration exhibited moderate antifungal activity against A. niger and C. albicans. Anthelmintic activity data revealed that cyclic peptide 14 possessed moderate anthelmintic activity against Eudrilus sp. comparable to that of reference drug. Cyclization of linear heptapeptide fragment 13 was indicated by disappearance of absorption bands at 1736 cm-1 (C=O stretching of ester) and 1392, 1367 cm-1 (CH band of t butyl group) and presence of additional Amide I and Amide II bands at 1659 and 1539 cm-1 in IR spectrum of synthesized cyclic peptide 14. Formation of cyclic peptide was further confirmed by disappearance of singlets at 3.62 and 1.60 ppm corresponding to three protons of methyl ester group and nine protons of t butyl group of di-tert.butylpyrocarbonate in 1HNMR spectrum of the cyclic heptapeptide 14 showed characteristic peaks confirming the presence of all seven amino acid moieties.
ACKNOWLEDGEMENTS
The authors are thankful to the Head, Department of Chemistry for providing research facilities, SAIF Department, Punjab University, Chandigarh (India), for providing spectral details in time.
TABLE 1 : PHYSICAL DATA OF THE SYNTHESIZED COMPOUNDS
Table 2 : ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF COMPOUND 14:
TABLE 3 : ANTHELMINTIC ACTIVITY OF COMPOUND14 :
TABLE 4: SPECTRAL DATA OF SYNTHESIZED COMPOUNDS
Scheme (1)
Synthesis of Cyclo(glycyl-tyrosinyl-valyl-prolyl-leucyl-tryptophanyl-prolyl)
Englishhttp://ijcrr.com/abstract.php?article_id=2271http://ijcrr.com/article_html.php?did=22711. Poojary B. and Belagali S.L. Synthetic studies on cyclic octapeptides, Yunnanin F and Hymenistatin. Eur. J. Med. Chem., 2005; 40 : 407-412.
2. Dahiya R. and Pathak D. Synthetic studies on a natural cyclic tetrapeptide Halolitoralin C. J. Pharm. Res., 2006; 5, 3 : 69-73.
3. Morita H., Yun Y.S., Takeya K., Itokawa H. and Shirota O., A cyclic heptapeptide from Vaccaria Segetalis. Phytochemistry, 1996; 42, 2 : 439-441.
4. Dahiya R., Pathak D., Himaja M. and Bhatt S. First total synthesis and biological screening of hymenamide E. Acta Pharm., 2006; 56 : 399-415.
5. Bauer A.W., Kirby W.M. and Turck M. Amer. J. Clin. Path., 1966; 45 : 493-496.
6. Garg L.C. and Atal C.K. Indian J. Pharm. Sci., 1963; 59 : 240- 245.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524112EnglishN-0001November30HealthcareGARLIC: A WONDER NUTRACEUTICAL
English4451Ajay G. PiseEnglish Shilpa PiseEnglish D. SreedharEnglish J. ManthanEnglish Virendra S. LigadeEnglish N. UdupaEnglishEnglishHistory
Thousand years ago garlic (Allium sativum) has been used as a food product and known for its medicinal values by civilizations around the world. Old existing corpus can supports its use in Chinese, Egyptian, French and Ayurvedic medicine. Garlic is indigenous to central Asia, where people live the longest, and the occurrence of cancer is the lowest known is not a seer coincidence. Garlic was included in the diet of the slaves for strength and endurance who built the pyramids in Egypt.13 Evidence shows that Egyptians worshiped garlic, they placed clay models of the bulb in Tutankhamen's tomb. It is said that Hippocrates himself used garlic vapors to treat certain cancers.11 It was included as one of the first medicines in the Codex Ebers, a 1550 B.C. papyrus that consider the oldest medical text, and was mentioned even earlier in clay, cuneiform tablets from the library of Nineveh in Mesopotamia. Pliny, in his Natural History listed garlic as a remedy for sixty-one ailments.16 Egyptian medical papers dating back to 1550 B.C. mentioned about eight hundred formulas for the therapeutic uses of garlic.19 Gravediggers in early eighteenth-century in France drank a concoction of crushed garlic in wine to protect themselves from getting the plague that killed many people in Europe. During World Wars I and II, soldiers were given garlic to prevent gangrene.2 Regulatory Status22 US: generally recognized as sage UK: included in general sales list Canada: over-the-counter drug status France: traditional medicinal use Germany: commission E approved as over-the counter drug.
Medicinal value
It is found that garlic (pulp) contains more than 200 chemical compounds including volatile oil with sulphur-containing compounds like allicin, alliin, and ajoene. It also contains enzymes such as allinase, peroxidase and myrosinase. It is considered that Allicin is responsible for antibiotic properties and strong odor; it also shows fibrinolytic activity which reduces platelet aggregation by inhibiting prostaglandin E2. Ajoene also contributes to the anticoagulant action of garlic. Apart from this garlic also contains citral, geraniol, linalool, Aphellandrene and B phellandrene. The allyl contained in garlic is also found in several members of the onion family and is considered a very valuable therapeutic compound.22 Alliin and diallyl dysulphur are highly unstable substances and melt easily into liquids and gases. When transported by the blood, they infuse all tissues and organs of the body, thus they act on the whole body.13
Allium sativum pulp contains vitamins especially B-1, vitamin C, vitamin A, flavonoids, ascorbic acid, phosphorous, potassium, sulphur, selenium, calcium, magnesium, germanium, sodium, iron, manganese and trace iodine. Seventeen amino acids are found in it, including eight essential ones.22
Garlic as nutraceutical
It is evident that garlic has been used as a food product across the globe. Today it became inseparable part of our diet. Scholars around the world have proved garlic for its medicinal use in treatment and prevention of certain diseases. It possesses both curative and preventative properties; new focus is on its use in prevention of heart attack and cancer. By studying these properties of garlic it can be categorised as nutraceuticals.
Clinical applications
From centuries garlic is being used in the treatment of bronchitis, respiratory problems, gastrointestinal problems, flatulence, leprosy, menstrual cramps, high blood pressure, diabetes and externally for warts, corns, arthritis, muscle pain, neuralgia and sciatica. In Ayurvedic medicine garlic is considered heating, diuretic, diaphoretic (enduces sweating), expectorant, carminative, anti-coagulant, anthelmintic and immune-enhancing. Homeopathically, garlic is used to treat upper respiratory tract inflammation, rheumatism and digestive problems.16 Researchers are studying the use of garlic in prevention and treatment of several diseases.
In cardiovascular diseases
Dioscorides was a well-known first century physician who wrote that garlic "clears the arteries and opens the mouths of veins."11 Today, studies around the globe have suggested that garlic consumption can reduce the risk of heart disease caused by the hardening of the arteries.14 Garlic is considered good for the health of heart because it lowers the cholesterol and blood fats called triglycerides in the bloodstream. According to Yu-Yan Yeh, Ph.D., professor of nutrition science at Pennsylvania State University in University Park, many of garlic protective effects take place in the liver, where cholesterol is produced. In laboratory studies, rats given garlic extract produced 87% less cholesterol and 64% fewer triglycerides. In a review of 16 studies involving 952 people, British researchers found that eating garlic, whether fresh or in powdered form lowered total cholesterol an average of 12 to 13%.21Thirty years of research has shown garlic to be affective in reducing cholesterol levels. It is known to reduce systolic blood pressure and lower the blood sugar.20 Sulfur compounds of garlic, including diallyldisulfide (DADS) which seem to help smooth blood flow by preventing platelets from sticking together and clotting. In a Brown University study, researchers gave 45 men with high cholesterol aged garlic extract (the equivalent of about 5-6 cloves of fresh garlic). When they examined the men's blood, they saw that the rate at which platelets clumped and stuck together had dropped anywhere from 10 - 58%.21 It decreases the level of LDL cholesterol in the blood, because it makes its absorption by the intestine more difficult. It has been proven that in the hours following a breakfast of toast with butter, the level of cholesterol increases 20%, however when the bread is rubbed with garlic, even if it has butter, this increase does not take place.13 Garlic also has been shown to protect blood vessels from the deleterious effects of free radicals. This antioxidative activity has also been linked to its blood cholesterol-lowering action and its ability to decrease deposits of cholesterol on the walls of blood vessels.5 A garlic-supplementation trial on 432 patients over a three-year period done by Arun Bordia, M.D. a cardiologist at India's Tagore Medical College, shows that 10- percent drop in blood cholesterol and in blood pressure, and less expressions of angina in the population who ate garlic daily. While the non-garlic eaters saw no cardiovascular changes.5
As an anticancer
Recent researches have supported the fact that garlic shows excellent potential in the prevention of cancer. Laboratory tests on animal studies suggest that garlic may have some anti-cancer activity. Population-based observation studies suggest that people who have more raw or cooked garlic in their diet are less likely prone to colon, stomach breast, prostate, and laryngeal cancers.2 Researchers have found that certain enzymes contained in some cancers are totally inhibited by alliinase and other compounds contained in garlic. Several Japanese experiments suggest that injecting garlic into rats with certain types of sarcoma blocked tumor cell reproduction and caused mutations in the cancer cells themselves. Garlic?s role in simulating the body?s immune defenses may also be linked to cancer control and prevention, in laboratory experiment, the natural killer cells of garlic-eating subjects destroyed 159 percent more tumor cells than those who had not consumed garlic. 22 A 1994 study done on 41,000 women who consumed a weekly serving of garlic demonstrated a 35% decrease in the risk of colon cancer.20 In animal studies by Weisberger and Pensky of Western Reserve University, as reported in Science, mice injected with cancer cells died within 16 days. When cancer cells were treated with Garlic extract and injected into the animals, no deaths occurred for a period of 6 months. In other studies, feeding fresh Garlic to female mice completely inhibited the development of mammary tumors. It is considered that the high germanium content of garlic may also play a role in cancer treatment and prevention. 22 The ajoene in garlic is showing some promise in the treatment of skin cancer.18
According to John Milner, Ph.D., professor and head of the department of nutrition at the Pennsylvania State University, s-allycysteine of garlic appears to stop the metabolic action that causes a healthy cell to become cancerous. The substance called DADS chokes cancer cells until their numbers are reduced and they start dying. Another substance in garlic is diallyl trisulfide (DATS), which is 10 times more powerful than DADS at killing human lung cancer cells. Its effectiveness is comparable to that of 5 flluorouracil, a widely used chemotherapy agent. In his view, garlic contains compounds that help prevent nitrites, common substances found in some foods and pollutants from transforming themselves into nitrosamines, harmful compounds that can trigger cancerous changes in the body21
As an antibiotic
Louis Pasteur first verified garlic's antibacterial properties scientifically in 1858, and showed how it killed bacteria under laboratory conditions.16 Several modern extensive studies confirm that garlic has definite antibiotic properties and is effective against many bacteria, fungi and viruses. According to Wright State University, garlic is approximately one per cent as potent an antibiotic as penicillin. The significant advantage of using garlic as antibiotic is that, the body does not seem to build up a resistance to it as it does to many modern antibiotics.1 Garlic is effective against diverse types of fungi, yeasts, and some viruses, including herpes. The active principles of garlic are supposed to interact with the nucleic acids of the virus, thus limiting its proliferation.13 Researchers around the globe are extensively studying the role of Garlic in enhancing the immune system, as antidiabetic, and anti oxidant ability to neutralize damaging free radicals before they can harm healthy cells.
As an antidiabetic
Few studies suggest that garlic may have some mild blood-sugar-lowering properties. It is believed that garlic lower blood sugar levels by decreasing the rate at which insulin is inactivated and degraded by the body, effectively increasing quantities of circulating insulin and decreasing blood glucose levels. Overall, these effects do not appear to be strong enough to warrant use of garlic as a bloodsugar-lowering agent.45 In general, sulfur compounds in garlic are believed to exert hypoglycemic activity by competing with insulin.44
Safety
Garlic may increase bleeding, especially in patients already taking certain anti-clotting medications. Rarely, gastrointestinal upset symptoms are considered as possible side effects.32 It is advised that people taking warfarin, regular doses of aspirin, or other blood thinners should not use garlic for anything other than seasoning.35 At the National Institutes of Health?s (NIH) HIV clinic, one woman who was on ritonavir (Norvir) treatment and then started garlic supplements was developed severe nausea and vomiting, which resolved after stopping the garlic. It is considered that the garlic may have increased the levels of ritonavir. It was also supported in one more case study. Still it is unclear, if garlic was increasing the risks of ritonavir related side effects or if it was the actual cause of them. Subsequently, small single-dose studies of ritonavir and garlic do not suggest a serious herb-drug interaction.36 Garlic may also increase the risk of side effects associated with other anti-HIV drugs. It is assumed that garlic has an effect on p450 enzyme. Garlic supplements should not be used while taking saquinavir as the sole protease inhibitor due to the risk of decreased saquinavir plasma concentrations.30 Moreover, people using the supplement with anti HIV drugs who experience serious stomach problems might consider stopping it to see if these symptoms lessen. 36 Garlic may intensify the effects of drugs that decrease blood sugar levels (hypoglycemic drugs, such as insulin and glipizide) causing an excessive decrease in blood sugar levels (hypoglycemia).25
Dosage and dosage form
Garlic supplement preparations are available in oil, extract, powder, capsules and tablet forms. It is observed that chemical composition of these preparations may not mirror the composition of fresh garlic clove. Hence it is always advisable to take daily dose equivalent to 4 g41 of fresh garlic cloves, which is about the size of one average clove of garlic. Average daily dosage: fully-dried powder, 400-1200 mg; fresh (air-dried) bulb, 2-5 g; garlic oil, 2-5 mg. Fully-dried powder, 400-1200 mg, fresh (air-dried) bulb, 2-5 g; garlic oil, 2-5 mg.28
Conclusion
Garlic is being used from thousand of years for its medicinal properties. Numerous researches have proved its beneficial role in cardiovascular condition. Indeed, garlic does indeed have cardioprotective properties. Researches also proved its active role as anticancer, natural immunity booster, antioxidant, antibiotic and antidiabetic product. On other hand studies also report some side effects of garlic if it is used with blood-thinners, anti HIV, or hypoglycemic drugs. This observation suggests that more research is needed in safety and active use of garlic as a Nutraceutical.
Englishhttp://ijcrr.com/abstract.php?article_id=2273http://ijcrr.com/article_html.php?did=22731 "Garlic as an Antibiotic" cited from http://www.garlic-central.com/garlichealth.html accessed on 19th Feb 2007
2 "Garlic" cited from http://www.umm.edu/altmed/ConsLookup s/Herbs.html accessed on 20th Feb 2007
3 "Garlic: Do its medicinal properties match its gastronomic attributes?" cited from http://www.healthandage.com/Home/gm= 0!gc=30 accessed on 24th Feb 2007
4 Information provided by An International Information Service on the Medicinal Benefits of Garlic? on http://www.garlic.mistral.co.uk/cholest.ht m. Accessed on 19th Feb 2007
5 Foster, Steven "Garlic the spice of life" cited from http://www.accessmylibrary.com/coms2/br owse_JJ_B044. Accessed on 20th Feb 2007
6 Carol Geck "The medicinal and healing properties of garlic" cited from http://www.proliberty.com/observer/index. htm. Accessed on 19th Feb 2007
7 "Medicinal properties and a bit more chemistry" cited from http://www.chm.bris.ac.uk/webprojects200 1/gray/morechemistry.htm. Accessed on 19th Feb 2007
8 "Herbal plants and medicinal properties" cited from www.ayurvedam.com. Accessed on 19th Feb 2007
9 "Herb facts and tips- garlic" cited from http://www.glenbrookfarm.com/herbs/herb facts.htm. Accessed on 20th Feb 2007
10 "Garlic Gold" cited from http://www.garlicgold.com. Accessed on 24th Feb 2007
11 Pat Kendall "Garlic: Medicinal Plant of The Mediterranean" cited from http://www.ext.colostate.edu/index.html. Accessed on 19th Feb 2007
12 "All about garlic Medicinal properties" cited from http://www.theworldwidegourmet.com/?ac tion=ingredient_showand id=5and lg=en. Accessed on 20th Feb 2007
13 Naveena "The medicinal properties of garlic" cited from http://foodrelatedhub.blogspot.com/index. html. Accessed on 19th Feb 2007
14 "Garlic's Medicinal Properties" cited from http://www.sacredorganics.com/index.html . accessed on 20th Feb 2007
15 "Exploring the medicinal properties of onion and garlic" cited from http://www.dummies.com/WileyCDA/Sect ion/id-100009.html. Accessed on 19th Feb 2007
16 "Garlic: An Herb Society of America Guide" cited from http://www.herbsociety.org/garlic/ggarden. php. Accessed on 24 Feb 2007
17 "Garlic Health Benefits" cited from http://www.garlic-central.com/garlichealth.html. Accessed on 19th Feb 2007
18 "Garlic Miracles: The Health and Medicinal Benefits of Garlic" cited from http://www.cookingnook.com/index.html. Accessed on 19th Feb 2007
19 "The Ancient Bulb with 21st Century Medicinal Properties" cited from http://www.dentalplans.com/articles/categ ory.aspx?CatID=4. Accessed on 22 Feb 2007
20 Hwaa Irfan "The Advantages and Disadvantages of the Medicinal Properties of Garlic" cited from http://www.islamonline.net/servlet/Satellit e?pagename=Zone-EnglishHealthScience/HSEZone. Accessed on 22 Feb 2007
21 "GARLIC" cited from http://www.spoonstruck.com/med.php. Accessed on 19th Feb 2007
22 "Garlic-nature?s amazing nutritional and medicinal wonder food" published by Woodland Publishing Inc. P.O. Box 160 Pleasant Grove, UT 84062 cited from http://www.nutraceutical.com/educate/pdf/ garlic.pdf. Accessed on 19th Feb 2007
23 "Drug interaction" cited from http://www.rxlist.com/cgi/generic/warfarin _wcp.htm. Accessed on 19th Feb 2007
24 Chris Woolston "Alternative health: Herb-drug interactions" cited from http://healthresources.caremark.com/topic/ alternative. Accessed on 19th Feb 2007
25 "Medicinal herbs and nutraceuticals"cited from http://www.merck.com/mmhe/sec02/ch01 9/ch019a.html. Accessed on 19th Feb 2007
26 Volker Schulz and Andrea Johne "Side effects and drug interactions" cited from http://www.springerlink.com/content/xx1u 78. Accessed on 19th Feb 2007
27 "What's the Story? Drug-Supplement Interaction published by American Council on Science and Health. cited from cited from http://www.acsh.org/publications/view.,rec No.11/pub_listing.asp. Accessed on 20th Feb 2007
28 "Herbal reference guide" published by American Botanical Council. Cited from http://www.herbalgram.org/default.asp?c= herb_info. Accessed on 21st Feb 2007
29 "Herb-Drug Interactions cited from http://www.cancer.prostatehelp.org/contents.htm. Accessed on 20th Feb 2007
30 Side effects" cited from http://www.rxlist.com/cgi/generic/saquin_ wcp.htm#P. Accessed on 20th Feb 2007
31 Shari Margolese "Drug Interactions" cited from http://www.thewellproject.org/en_US/Trea tment_and_Trials/Things_to_Consider/ind ex.jsp. Accessed on 20th Feb 2007
32 Subhuti Dharmananda, "Checking for Possible Herb-Drug Interactions" published on http://www.itmonline.org. Accessed on 19th Feb 2007
33 Melanie Johns Cupp, West Virginia University School of Pharmacy Mo0rgantown, West Virginia "Herbal Remedies: Adverse Effects and Drug Interactions" published by American Academy of Family physicians. Vol 59 / no. 5 (March 1, 1999)
34 "Herbs, Supplements and HIV" cited from http://www.projinf.org/fs/herbs.html. Accessed on 19th Feb 2007
35 Drug Interactions" cited from https://www.wellpointrx.com/wps. Accessed on 20th Feb 2007
36 "Herbs, Supplements and HIV" cited from http://www.projinf.org/fs/herbs.html. Accessed on 20th Feb 2007
37 Budoff MJ, Takasu J, Flores FR, "Aged Garlic Extract (AGE) Effects on Coronary Calcification" published on http://www.herbalgram.org/herbclip/defaul t.asp. Accessed on 19th Feb 2007
38 Antoine Al-Achi, PhD Campbell University School of Pharmacy Buies Creek, North Carolina. "Herbs That Affect Blood Glucose Levels" published in US Pharmacist 2005; 4: HS-16-HS-18.
39 "Concentration of Chemicals in Plants with Antidiabetic Activity" cited from www.garynull.com. Accessed on 21st Feb 2007
40 Steven Foster "Garlic: the spice of life" published in Better Nutrition, Feb, 1997 cited from http://www.findarticles.com. Accessed on 20th Feb 2007
41 Steven Foster "Garlic cited from http://www.stevenfoster.com/index.html. Accessed on 19th Feb 2007
42 L. Pari, R. Saravanan "Antidiabetic effect of diasulin, a herbal drug, on blood glucose, plasma insulin and hepatic enzymes of glucose metabolism in hyperglycaemic rats"Published in ,Diabetes, Obesity and Metabolism? 6 (4), 286-292. July 2004
43 Sheela CG, Augusti KT. "Antidiabetic effects of S-allyl cysteine sulphoxide isolated from garlic Allium sativum Linn." Published in Indian J Exp Biol. 1992 Jun;30(6):523-6.
44 "Plants with Antidiabetic Activity' HerbalGram. 1997;40:21 American Botanical Council.
45 Belinda O?Connell, "Herbal Supplements in Diabetes Management" December 17, 2006. cited from http://www.nutritionupdate.info/store/stop diabetes.asp?ppc=1and at=889
46 R. C. Jain "Effect of garlic on serum lipids, coagulability and fibrynolytic activity of blood" published in American Jouranal of Clinical Nutrition cited from http://www.ajcn.org/
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524112EnglishN-0001November30HealthcareASSESSMENT OF THE TREATMENT PATTERN, CLINICAL OUTCOME, AND QUALITY OF LIFE IN PATIENTS WITH BLADDER OUTLET OBSTRUCTION IN A TERTIARY CARE TEACHING HOSPITAL
English2337R.RajeshEnglish Nitha.VEnglish Sureshwar PandeyEnglish Arun chawlaEnglishAim: This study provides an insight into the assessment of the treatment pattern, clinical outcome and quality of life in patients with Bladder outlet obstruction and to assess the safety and efficacy of (0.2 mg) and (0.4 mg) tamsulosin in female patients with Bladder outlet obstruction in a tertiary care teachinghospital.
Method: Institutional ethics committee approval was obtained from kasturba hospital, Manipal for conducting this study.This was a randomised controlled study conducted for eight months at Dept. of Urology, Kasturba Hospital, Manipal. Inclusion criteria were all male bpatients in age group greater than 45years with bladder outlet obstruction symptoms due to BPH and all the female patients aged greater than 35 years with bladder outlet obstruction were included for the study. Exclusion criteria were allMale patients with Stricture urethra, acute cystitis, Carcinoma of prostate, Neurogenic voiding dysfunction and all female patients with Pelvic masses, previous pelvic surgery, and acute cystitis were excluded from the study. The American Urological Association Symptom Score (AUASS) was adopted to assess the severity of urinary symptoms and to check the effectiveness of the treatment. The percentage of improvement of treatment was also assessed in terms of both objective and subjective parameters like Maximum
Flow Rate (MFR), Post Voidal Residual Volume (PVR) and bladder thickness. Female patients were randomised into two groups? first group of patients receiving (0.2 mg of tamsulosin) while second group of patients receiving (0.4 mg of tamsulosin) daily for a period of months with periodic follow up at 2nd , 4 th and 8 th week, assessed with IPSS (International Prostate Symptom Score) and uroflowmetry andultrasonography at 8 th week. The quality of life in patients with bladder outlet obstruction was evaluated by using the quality of life questionnaire.
Results: A total number of 177 patients diagnosed with bladder outletobstruction were aged between 55 to 65 years and males (n=152) predominated over females (n=25). Weak stream urinary symptoms were found to be the highest complained symptom. The co morbidities associated with bladder outlet obstruction was Hypertension. Surgery was the main line of treatment in patients with bladder outlet obstruction. The drug tamsulosin was the most implicated drug in the medical group. The most common surgical procedure performed was transurethral resection of the prostate. The safety and efficacy of tamsulosin in female patients showed that patients were having AUASS score between 8-19 (moderate)
before treatment and most of them showed an improvement in the score from moderate to mild (1-7) aftertreatment. The subjective parameters showed that the female patients had an MFR value of < 15ml/s before treatment and have improved after treatment from < 15ml/s to >15ml/s. The PVR values were >50ml in all patients before treatment and < 50ml after treatment. The bladder thickness showed a reduction from > 5mm to < 2.5mm. Our results show tamsulosin to be very effective in the management of bladder outlet obstruction in female patient if detected early.
Conclusions: In female patients? use of tamsulosin was found to be a very safe, well tolerated showing significant improvement in urinary outflow symptoms, reducing post void urinevolume and decreasing IPSS with minimal tolerable adverse events. The correct and timely diagnosis of bladder outlet obstruction in women was difficult, since clinical features are similar to those of other voidingdisorders and diagnostic modalities are often inconclusive or even misleading.
Englishbladder outlet obstruction, Medical management, tamsulosin, urodynamics.Introduction
Bladder outlet obstruction (BOO) is a blockage at the base of the bladder that reduces or prevents the flow of urine into the urethra, the tube that carries urine out of the body1 . Numerous gender-specific etiologies are responsible for bladder outlet obstruction. BOO may be induced by specific functional and anatomic causes. The resulting obstruction frequently produces lower urinary tract symptoms (LUTS), although the degree of bother by LUTS is highly variable and not predictable on the basis of the specific inciting etiology2 . Induced LUTS symptoms may be predominantly obstructive, irritative, or often a combination of both. Typically, obstructive symptoms include hesitancy, sensation of incomplete bladder emptying, diminished urinary stream, and post voiding urinary dribbling.Irritative complaints include urinary urgency, frequency of urination, occasional dysuria, and nocturia. Rarely are symptoms related to BOO isolated; often the individual experiencing LUTS presents with a variety of mixed symptoms of obstruction and irritation. BOO may also occur in the complete absence of symptoms and be first identified in the scenario of urinary retention or decompensation of the upper urinary tracts. BOO often occur in both male and females; it is more common in aging men. urodynamic evaluation and pressure flow evaluation is the gold standard diagnostic tool, other modalities may also be used, including post void residual analysis, urinary flow rates, cystoscopy, and selected radiologic ones. Patient self-appraisal of symptoms using various inventories such as the American Urologic Association Symptom Index3 or the International Prostate Symptom Score is relevant to the initial assessment and subsequent follow up. Analysis of secondary symptoms of obstruction in women is often performed using a subjective symptom appraisal and is determined urodynamically, assessing the pressureflow relation during voiding. The complete assessment of LUTS arising from BOO often includes several of these modalities to fully define the obstructive impact on the individual's urinary function and quality of life4 . There are a number of treatment options in both male and female patients with bladder outlet obstruction. The treatment options in male patients includes watchful waiting5 (A strategy of management in which the patient is monitored but receives no active treatment), medical treatment which includes alpha blocker therapy which inhibit contraction of prostatic smooth muscle and finasteride therapy, an enzyme inhibitor that lowers prostatic androgen levels and decreases prostate size, surgical treatment which includes balloon dilation (A catheter with a balloon at the end is inserted through the urethra and into the prostatic urethra. The balloon is then inflated to stretch the urethra narrowed by the prostate), Transurethral Incision of the Prostate (TUIP) an endoscopic surgical procedure in which patients with smaller prostates (25ml/s), bladder wall thickness (15ml/s. The PVR values were >50ml in all patients before treatment and < 50ml after treatment. The bladder thickness showed a reduction from > 5mm to < 2.5mm. No suspected adverse drug reactions were reported during the study period. The results of the quality of life questionnaire showed that most of the patients were unhappy by their disease state before treatment and mostly satisfied after treatment. There was a significant improvement in the urinary symptoms after treatment in both groups as shown in the figure 1.
Discussion
Bladder outlet obstruction (BOO) is becoming an increasingly recognized entity over the past several years. The goal of treatment of bladder outlet obstruction is primarily to provide a rapid and sustained improvement in lower urinary tract symptoms (LUTS) and to reduce the long term complications. LUTS associated with BOO are a common disorder in urology. Surgical removal of prostate tissue or pharmacological prostate/bladder neck smooth muscle relaxation with alpha1AAR antagonists relieves outlet obstruction but does not improve irritative symptoms.9 Tamsulosin and nonselective subtype antagonists have the ability to relieve obstructive and irritative symptoms in men, while in women there is also some evidence for this. Although, recent studies suggest that it is an under diagnosed cause of female lower urinary tract symptoms.10 our study showed that the male patients were having Benign Prostatic Hyperplasia (BPH) as the main risk factor for BOO. Symptomatic BPH is thought to be due to bladder outflow obstruction and is often referred to as lower urinary tract symptoms suggestive of BOO11. This study systemically explored the treatment pattern and clinical outcome of treatment in patients with BOO in the Urology Department of Kasturba Hospital, Manipal. A total of 177 patients with BOO were evaluated during the eight month study period. On studying the overall gender wise distribution of the 177 patients with BOO, it was observed that there was male (n=152) predominance over female (n=25). This was because of under diagnosis and failure of the female patients to visit the Urology Department. In males, the occurrence of BOO was more common because gonadal androgens play a major role. The hormone testosterone is converted to dihydrotestosterone (DHT) in males as the age progresses and this DHT has a pivotal role in the development of BPH. This observation was consistent with observation made by McConnell12 which revealed about a relationship between DHT and BPH. The male to female ratio was 63:1 with a mean age of 62.16 + 11.23 years for males and 53.76 + 9.41 years for females. A study by Nunzio et al5 showed similar results with males having mean age 64.5 + 7.6 years and a study by Pummangura and Kochakarn13showed that mean age of females affected by bladder outlet obstruction is 45.3 + 12.9. In this study, majority (42%) of the patients with BOO were in the age group of 55-65 years. This was because the chance of occurrence of this disease increases with the progression of age. Verhamme et al14 has observed that BPH is one of the most common condition associated with ageing men and approximately 40% of men are affected at the age fifties. The study patients were evaluated for the distribution of obstructive and irritative urinary symptoms with BOO. However, in our study these symptoms, which were mainly of the obstructive type (weak urinary stream), were reported. This finding is in accordance with the study carried out by Lee et al15 which showed a similar observation with weak stream as the most commonly observed obstructive urinary symptom. The result of our study further supports the co morbidity between Hypertension and bladder outlet obstruction. The prevalence of BPH and arterial Hypertension increases with age and hence both are frequent disease state in the elderly people with bladder outlet obstruction. This study result was consistent with a study carried out by Nicolas et al16 which confirms that there is a statistically significant relationship between Hypertension and LUTS secondary to BPH and correlation between mean blood pressure and prostate size. In our study majority of the patients had undergone surgery for bladder outlet obstruction because their urinary symptoms were moderate to severe in AUA index score and also patients presented with reoccurrence of urinary symptoms which suggestive of clear indication for surgery. This may be due to the fact that the treatment pattern followed was similar to the AUA guidelines in the management of Bladder outlet obstruction and also the European Urology guidelines on BPH. As per the guidelines, watchful waiting is recommended for patients with mild symptoms, medical treatment for patients with mild to moderate symptoms and surgery for patients who failed medication or conservative management and who have moderate to severe symptoms and/or complications of BPH. Although in our study men with LUTS due to BPH are generally managed with watchful waiting or medical therapy, some of the patients presented with progressive disease that can result in symptom deterioration, acute urinary retention, urinary tract infection, renal failure, and thereby indicated for surgery. Our study results showed that majority of the patients were prescribed with Tamsulosin, in the treatment of BOO and several studies have shown its superiority for treatment of lower urinary tract symptoms associated with Bladder outlet obstruction. Recently tamsulosin was found to be effective in patients with neurogenic lower urinary tract dysfunction; the drug decreased the maximal urethral pressure significantly in the long term follow up and improves cystometric parameters related to bladder storage and emptying17 . Interestingly, in our study it is preferred by the physicians because of the propensity to cause adverse drug reaction with alpha blocker is comparatively less with that of other classes of drugs. Transurethral Resection of Prostate (TURP) is the gold standard of treatment for BPH. In our study most of the patients with BOO in whom BPH was the risk factor had bothersome BPH symptoms refractory to medical treatment and because of the symptom severity and reoccurrence of symptoms there was a clear indication for transurethral Resection of Prostate surgery. This finding is consistent with the European Urology guidelines where it is clearly indicated that TURP is the most frequently performed surgical procedure for those patients who have bothersome symptoms.18 Most of the female patients in our study were presented with urethral and meatal narrowing, which causes obstruction in them and in these patients cystoscopy and urethral dilatation has been preferred as an empirical treatment. Bladder neck resection surgery has been preferred because of reoccurrence of symptoms. Critical analysis of our result data very clearly illustrates the safety and efficacy of tamsulosin in medical management of bladder outlet obstruction. Being an uroselective antagonist it controls both irritative and obstructive symptoms thereby giving a total therapeutic effect in relieving urinary symptoms. The result of our study in comparable to many international studies conducted. Abrams et al 19 in their randomized placebo control clinical trial on 296 patients found tamsulosin to be well tolerated drug showing significant improvement in UFR and symptoms score. Lee et al 20 from Korea found tamsulosin to be a safe drug showing statistically significant improvement in urine flow and symptom score as compared to placebo. The Japanese study conducted by Kawabe et al21 and the Chinese study conducted by Yan Jun et al22 showed results similar to our result. Our study showed that AUA score decreased from 23.9 to 16.1 after treatment with tamsulosin, mean MFR was 11 ml/s before treatment and 22 ml/s after treatment with tamsulosin. Similarly, there was an improvement in PVR and bladder thickness as early as 2 nd week and improvement was maintained throughout the treatment period. The improvement was seen both in 0.2 mg and 0.4 mg tamsulosin after treatment. The improvement was seen both in IPSS and urodynamic parameters. Tamsulosin was found to be very effective, safe and well tolerated drug with no significant differences with 0.2 mg and 0.4 mg as far as the vital signs and adverse events were concerned. Quality of life improved among the patient prescribed with tamsulosin due to improved flow rate and decreased symptom score. Many of the patients in our hospital were prescribed tamsulosin for management of their features of prostatism. In our study about 70% of patients taking Tamsulosin showed significant overall improvement in both objective and subjective parameters.
Conclusions
The AUA symptom index score may be useful as a bothersomeness index in women with bladder outlet obstruction. A complete urodynamic evaluation is essential for making the diagnosis, although standard urodynamic definitions are still lacking. Further epidemiological and pathophysiological investigations are needed to evaluate the causes and risk factors for bladder outlet obstruction in women. Better understanding the pathophysiological mechanisms associated with bladder outlet obstruction in women may provide the possibility of using appropriate diagnostic and treatment modalities, thus, avoiding any unnecessary intervention.
Acknowledgements
The author?s wishing to thank staff of urology department and administrative staff of Kasturba Medical College, Manipal University, Manipal for their technical support and encouragement.
Table.1.Age group and gender wise distribution of Bladder outlet obstruction.
Table.2.The distribution of obstructive and irritative urinary symptoms associated with bladder outlet obstruction.
Table.3.Co morbidities associated with bladder outlet obstruction.
Table.4.Treatment pattern associated with bladder outlet obstruction.
.5. Drug Class most commonly prescribed in patients with bladder outlet
Table.6.Drugs implicated in the medical treatment with bladder outlet obstruction.
Table.7.Surgical procedures performed in bladder outlet obstruction.
Table.8. Objective and subjective improvement for all types of treatment in bladder outlet obstruction.
Figure 1 Safety and efficacy of tamsulosin in female patients with bladder outlet obstruction.
? AUA - American Urological Association symptom score
? MFR – Maximum Flow Rate
? PVR – Post Void Residual Volume
? BT – Bladder Thickness
Englishhttp://ijcrr.com/abstract.php?article_id=2295http://ijcrr.com/article_html.php?did=22951. Bladder outlet obstruction. Medline plus Medical Encyclopaedia. [Online]. 2008 [cited 2009 Jun 2]; Available from: http://www.nlm.nih.gov/medlineplus/en cy/article/002238.htm
2. Zderic, S. A., Chacko, S., DiSanto, M. and Wein, A. J.: Voiding function: relevant anatomy, physiology, pharmacology, and molecular aspects. In: Adult and Edited by J. Gillenwater, J. Grayhack, S. Howard and M. Mitchell. Philadelphia: Lippincott Williams and Wilkin. Pediatric Urology, 4th ed. 1061– 1114, 2002
3. Barry, M. J., Fowler, F. J., Jr., O?Leary, M. P. et al: The AmericanUrological Association symptom index for benign prostatic hyperplasia. Measurement Committee of the American Urological Association. J Urol, 148: 1549, 1992
4. Kupelian V, Wei JT, O?Leary MP, Kusek JW, McKinley JB. Prevalence of Lower Urinary Tract Symptoms and effect on quality of life in a racially and ethnically diverse random sample: The Boston Area Community Health Survey. Archieves of Internal Medicine 2006; 166 (21): 2381- 2387.
5. Nunzio C, Franco G, Rocchegiani A, Iori F, Leonardo C, Laurenti C. The evolution of detrusor over activity after watchful waiting, medical therapy and surgery in patients with bladder outlet obstruction. The Journal of Urology 2003; 169: 535-39.
6. Dunn, Christopher J, Matheson, Anna, Diana M. Tamsulosin: a review of pharmacology and therapeutic efficacy in the management of lower urinary tract symptoms: Drug evaluation. Drugs and aging 2002; 19(2):135-161.
7. Arnold EP. Tamsulosin in men with confirmed Bladder Outlet Obstructive Syndrome: A clinical and urodynamic analysis from a single centre in New Zealand. BJU International 2001; 87(1): 24-30.
8. Rahardjo D, Soebadi DM, Sugandi S, Birowo P, Djati W, Wahyudi I. Efficacy and safety of Tamsulosin hydrochloride compared wiyh doxazozin in the treatment of Indonesian patients with Lower Urinary Tract Symptoms due to Benign Prostrate Hyperplasia. International Journal of Urology 2006; 13(11): 1405-9.
9. Blue, D., Zinner, N., Grino, P., Crager, M. and Ford, A.:RO700004, a selective alpha1A-adrenoceptor antagonist, does not improve lower urinary tract symptoms in men with benign prostatic hyperplasia. J Urol, suppl., 167: 265, abstract 1044, 2002
10. Athanasopaulose A, Gyftopaulose K, Giannitsas K, Fisfis J, Perimenis P. Combination treatment with an alpha blocker plus an anticholinergic for bladder outlet obstruction: A prospective, randomized controlled study. Journal of Urology 2008; 169: 2253 - 56.
11. Speakman MJ. Benign Prostatic Hyperplasia. European Urology 2001; 39 (3): 13-19.
12. McConnell JD. Prostatic growth: new insights into hormonal regulations. British Journal of Urology 1995; 76 (1): 5-10.
13. Pummangura N and Kochakarn W. Efficacy of Tamsulosin in the Treatment of Lower Urinary Tract Symptoms (LUTS) in Women. Asian Journal of Surgery (2007); 30 (2): 131-7.
14. Verhamme KMC, Dieleman JP, Bluemink GS, Sturkerboom MCJM. Incidence and prevalence of lower urinary tract symptoms suggestive of benign prostatic hyperplasia in primary care-The triumph project. European Urology 2002; 42: 323-28.
15. Lee KS, Choo MS, Yoo TK, Park HJ, Jeong H. Efficacy and safety of tamsulosin for the treatment of nonneurogenic voiding dysfunction in female: an 8 week prospective study. Urotoday ICS 2006; 1: 379-380.
16. Nicolas TJ, Tornero RJ, Banon PV. Relation between hypertension and clinical cases of benign prostatic hyperplasia. Archivos Espanoles Urologia 2003; 56 (4): 355-8.
17. Morant SV, Reilly K, Bloomfield GA, Chappel C. Diagnosis and treatment of lower urinary tract symptoms suggestive of overactive bladder and bladder outlet obstruction among men in general practice in the UK. International Journal of Clinical Practice 2008; 62: 668-9.
18. Jean JCMH, Alivizatos G, Maderbacher S, Perachino M. EAU Guidelines on Benign Prostatic Hyperplasia. European Urology 2001; 40: 256-63.
19. Abrams P, Schulman CC. Vaage S and the European Tamsulosin Study Group. Tamsulosin, a selective α1c, - adrenoreceptor antagonist: a randomized, controlled trail in patients with benign prostatic `obstruction' (symptomatic BPH). Br J Urol 1995; 76; 325-36.
20. Lee E, Lee C. Clinical comparison of selective and nonselective α1A adrenoreceptor antagonists in benign prostatic hypetplasia. Studies on tamsulosin in a fixed dose and terazocin in an escalating doses in Korean patients. Br J Urol 1997: 32; 210-213.
21. Kawabe K, Nijima T, Ueno T, Takimoto Y, Aso Y, Kato H. Use of an α1 blocker, YN617 in the treatment of benign prostatic hypertrophy. J Urol 1990; 144: 908-911
22. Yan-Jun N, Ying Lu G, Fang Liu G et al. Clinical comparison of selective and non selective α1A adrenoreceptor antagonists for bladder outlet obstruction associated with benign prostatic hyperplasia. 3rd Asian Congress Urology 1996: 22: 301-7.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524112EnglishN-0001November30HealthcareSYNTHESIS AND ANTITUBERCULAR ACTIVITY OF SOME NEW BENZOPYRONE DERIVATIVES
English3843Shashikant R.PattanEnglish Nachiket s DigheEnglish Jayshri S PattanEnglish Santosh R ButleEnglish Santosh G JadhavEnglish Deepak S Musmade Suwarna H KaleEnglishA series of 5-Bromo- 2-formyl phenoxyacetyl amino acids and peptides have been synthesized by coupling of the 5-Bromo- 2-formyl phenoxyacetic acid with amino acid/methyl esters/dipeptides/ tripeptides using DCC as coupling agent and NMM as base. The structures were elucidated FTIR and 1HNMR .The newly synthesized compounds were evaluated for their antibacterial, antifungal and anthelminitic activities. The compounds (2, 6, 11 and 13) were found to exihibit potent antibacterial activity against Bacillus subtilis, Staphylococcus aureus (gram positive bacteria) and Escherichia coli (gram negative) bacterias.The compounds (5, 6 and 13) were found to exihibit potent antifungal activity against Candida albicans and Aspergillus niger. The moderate to good anthelminitic activity was shown by the synthesized compounds (7 and 13) against Eudrilus spieces.
EnglishPhenoxyacetic acid, amino acids, antibacterial, antifungal and anthelminitic.Phenoxyacetic acid is among the most vital moieties which are associate with potent antidiabetic (Rival et al 2004), antimycobacterial (Yar et al 2007),diuretic (Lebedev et al 1985, Woltersdorf et al 1976, Bicking et al 1976), anti-inflammatory (Kunsch et al 2005, Shokol et al 2005), antibiotic (Grardin et al 1995), anti-obesity (Kiso et al 1999), diagnostic (Ohmomo et al 1989), inhibition of platelet aggregation (Meanwell et al 1993, Seiler et al 1994) activities. The review of literature has suggested that incorporation of amino acids and peptides into aromatic and heterocyclic congeners have resulted in compounds with potent bioactivities.
Introducing an amino acid or peptide into aromatic compounds can increase the potency, decrease the toxicity and prolong its action. Among aromatics, phenolic compounds have wide range of activities. Further phenoxylation the resulting compound phenoxyacetic acid is obtained, which is well known for their biological potential. Thus keeping in view the biological potency of phenoxyacetic acids as well as taking advantage of biodegradability and biocompatibility of a novel series of substituted phenoxyacetic acid derivatives of amino acids and peptides have been synthesized with an anticipation to get potent agents with good therapeutic efficacy with negligible side effects. The study of IR and 1HNMR spectrum gives us most of the required information of certain vibrational bands or characteristic groups present in the molecule. The IR spectrum of newly synthesized compounds showed characteristic bands in the region 3329- 3318, 1654-1642, 1537-1532 cm-1 which can be assigned as N-H stretching, C=O stretching and N-H bend respectively. In their 1HNMR spectra, a singlet appeared in the range 8.33-8.36 ppm corresponding to the CO-NH proton. The compounds were found to exhibit potent antimicrobial and moderate anthelmintic activity in comparison to standard drugs against the same concentration.
Results and discussion
Antibacterial Activity:
The synthesized peptide derivatives were screened for antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis using modified Kirby-Bauer disc diffusion method (DMF as a solvent).The test samples were tested at the concentrations 25, 50, 100 pg/ml. The petri plates inoculated with bacterial cultures were incubated at 37C for 18 hrs. The diameters obtained for the test sample were compared with that produced by the standard drug ciprofloxacin. The results are shown in Table 2.
Antifungal Activity:
The synthesized peptide derivatives were screened for antifungal activity against Candida albicans and Asperigillus niger. DMSO is used as negative control. The test samples were tested at the concentrations 25, 50, 100 pg/ml. The petri plates inoculated with fungal cultures were incubated at 25 C for 48 hrs. Diameters of the zone of inhibition were calculated in triplicate sets. The diameters obtained for the test sample were compared with that produced by the standard drug griseofulvin. The results are shown in Table 2.
Anthelmintic Activity:
The anthelminitic activity was carried out against earthworms Eudrilus species by Garg and Atal method at 2 mg/ ml concentration. Suspension of samples was prepared by triturating synthesized cyclic peptide (200 mg) with Tween 80 (0.5 %) and distilled water. Suspension of the standard drug albendazole was prepared with the same concentration in a similar way. The paralyzing and death times were noted and their mean was calculated for triplicate sets. The death time was ascertained by placing the earthworms in warm water (50C) which stimulated the movement. The results were shown in Table 3. The results of biological activities revealed that newly synthesized peptide derivative 73 at 50pg/ml concentration exhibited highest zone of inhibition against Staphylococcus aureus and 75 at 50pg/ml concentration exhibited highest zone of inhibition against Candida albicans. Morover, other compounds showed moderate antimicrobial activities against tested organisms. Comparison of anthelmintic activity data revealed that peptide derivative 75 was found to exhibit potent anthelmintic activity and other peptide derivatives showed good to moderate activity.
Experimental
Melting points were determined and uncorrected. The amino acids, di-tertbutyl pyrocarbonate (Boc2O), 5- Bromosalicyaldehyde, DCC and NMM were obtained from Spectrochem Limited, Himedia laboratories Limited mumbai and Sd-fine-chem Limited, Mumbai, India. The IR spectra were recorded on a Perkin Elmer Fourier transform infrared spectrophotometer using KBr pellets. The 1HNMR spectra were recorded on the Bruker Avance II- 400 NMR spectrometer using CDCl3 as the solvent. The purity of all the compounds was controlled by TLC on silica gel G plates. Chloroform:Methanol (9:1 v/v) was used as developing solvent system and dark brown spots were detected on exposure to iodine vapours in a tightly closed chamber. The physical data of synthesized compounds is listed in Table 1. The scheme of synthesis is given in Scheme 1.
Synthesis of Boc amino acids (1-3):
L-Leucine (1.31gm, 10mmol) was dissolved in 10 ml of sodium hydroxide (1 mol L-1 ) and 10 ml of i-propanol. ditert.butylpyrocarbonate (3 ml, 13 mmol) in 5 ml of i-propanol was added followed by 10 ml of sodium hydroxide (1 mol L-1 ) to the resulting solution. The solution was stirred at room temperature for 2 hr, washed with 10 ml of light petroleum ether (b.p. 40-60 C), acidified to p H 3.0 with 1 mol L-1 sulphuric acid and finally extracted with chloroform (3 x 20 ml). The organic layer was dried over anhydrous sodium sulphate and evaporated under reduced pressure to give crude product. The crude product was purified by recrystallization from methanol and ether at 0 C to get pure Boc-Leucine (1). Similarly, Boc-Serine (2) and Boc-Alanine (3) were prepared by stirring di-tert.butylpyrocarbonate (3 ml, 13 mmol) with Boc-Serine (1.05gm, 10 mmol) and Boc-Alanine (0.89gm, 10 mmol) respectively.
Synthesis of L-amino acid methyl ester hydrochlorides (4-6) :
Thionyl chloride (0.73mL, 10 mmol) was slowly added to methanol (50 mL) at 0 C and 1.15 gm of L- Proline (10 mmol) was added to the above solution. The resulting mixture was refluxed for 9 hrs at 110 C. Methanol was evaporated and the residue was triturated with ether at 0 C until excess dimethyl sulphite was removed. The crude product was purified by recrystallization from methanol and ether at 0 C to get Lproline methyl ester hydrochloride (4). Similarly, L-leucine methyl ester hydrochloride (5) and L-tryptophan methyl ester hydrochloride (6) was prepared by refluxing 1.31 gm of Lleucine (10 mmol) and 2.04 gm of Ltryptophan with 50 ml methanol in the presence of 0.73 ml of thionyl chloride (10 mmol).
Synthesis of Boc-dipeptide methyl esters (7-9) :
To a mixture of 1.65 gm compound 4 (10 mmol) in 20 ml of chloroform, 2.3 ml of N- methylmorpholine (21mmol) was added at 0 C. The reaction mixture was stirred for 15 min. 2.31gm compound 1 (10mmol) in 20 ml chloroform and 2.1gm of DCC (10mmol) were added under stirring to the above mixture. After 36 hrs, the reaction mixture was filtered and the residue was washed with 30 ml of chloroform and added to the filterate. The filterate was washed with 5% sodium hydrogen carbonate and saturated sodium chloride solution (25 ml each). The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated in vacuum. The crude product was recrystallized from mixture of chloroform and petroleum ether (b.p. 40-60 C) followed by cooling at 0 C to get Boc-Leu-Pro-OMe (7). Similarly Boc-Ser-Leu-OMe (8) and Boc-Ala-ProOMe (9) were prepared by stirring compounds 2 and 3 with amino acid methyl ester hydrochlorides 5 and 4, respectively in the presence of DCC and NMM.
Deprotection of dipeptides at carboxyl end (8a, 9a) :
To a solution of 3.32 gm of compound 8 (10 mmol) in 36 ml of THF/H2O (1:1), 0.36 gm lithium hydroxide (15mmol) was added at 0 C. The mixture was stirred at room temperature for 1 hr, and acidified to 1 pH 3.5 with 0.5 mol LH2SO4. The aqueous layer was extracted with diethyl ether (3 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from methanol and ether to get Boc-SerLeu-OH (8a). Similarly compound 9 was hydrolyzed under alkaline conditions to obtain Boc-Ala-Pro-OH (9a).
Deprotection of dipeptide at amino end (7a):
Compound 7 (3.42 gm, 10mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of trifluoroacetic acid (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by recrystallization from mixture of chloroform and light petroleum ether (b.p. 40-60 C) to get pure Leu-Pro-OMe (7a).
Synthesis of Boc-tripeptide methyl esters (10, 11) :
To synthesize Boc-Ser-Leu-Trp-OMe (10), 3.18 gm of dipeptide unit 8a (10 mmol) was coupled with 2.54 gm of amino acid methyl ester hydrochloride 6 (10 mmol) in the presence of DCC and NMM following the same procedure as adopted for the synthesis of Bocdipeptide methyl esters 7-9. Similarly Boc-Ala-Pro-Pro-OMe (11) was prepared by coupling 2.86 gm of deprotected dipeptide unit 9a and 1.65 gm of amino acid methyl ester hydrochloride 4 using DCC as the coupling agent and NMM as the base.
Deprotection of tripeptides at amino end (10a, 11a):
Compound 10 (5.18 gm, 10mmol) was dissolved in 15 ml of chloroform and treated with 2.28 gm of trifluoroacetic acid (20 mmol). The resulting solution was stirred at room temperature for 1 hr and washed with 25 ml of saturated sodium hydrogen carbonate solution. The organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by recrystallization from mixture of chloroform and light petroleum ether (b.p. 40-60 C) to get pure Ser-Leu-TrpOMe (10a). Similarly Ala-Pro-Pro-OMe (11a) was prepared by stirring compound 11 with 2.28 gm of trifluoroacetic acid (20 mmol).
Synthesis of free acid
Sodium hydroxide (0.89gm, 22.4mmol) in 25 ml water was slowly added with stirring to 2.01gm of 5-bromo-2- hydroxyaldehyde (10mmol) and 0.94gm of chloroacetic acid (10mmol). The mixture was heated on heating mantle to remove all the liquid and the residue was treated with 30 ml water. The mixture was cooled and filtered and clear solution was acidified with dilute hydrochloric acid. The aqueous layer was extracted with diethyl ether (2 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate.The crude product was recrystallized from ethanol and purified by recrystallization from ethanol-water (1:1) to get 5-Bromo-2-formylphenoxyacetic acid (12).
Synthesis of 5-Bromo-2-formylphenoxyacetyl amino acid and peptide methyl esters
L-Proline methyl ester hydrochloride (1.65 gm, 10mmol) was dissolved in Tetrahydrofuran (75 mL). To this, 2.3 ml of N-methylmorpholine (21mmol) was added at 0 C and the reaction mixture was stirred for 15 min. 2.01gm of compound 12 (10mmol) in tetrahydrofuran and 2.1gm dicyclohexylcarbodiimide (10mmol) were added under stirring to the above mixture. After 36 hrs, the reaction mixture was filtered and the residue was washed with 30 ml of tetrahydrofuran and added to the filterate. The filterate was washed with 5% NaHCO3 and saturated NaCl solution (25 ml each). The organic layer was dried over anhydrous sodium sulphate, filtered and evaporated in vacuum. The crude product was recrystallized from mixture of chloroform and n-hexane followed by cooling at 0 C to get 5-Bromo-2-formylphenoxyacetyl-proline methyl ester (13). Similarly, 5-Bromo-2-formylphenoxyacetyl-leucyl-proline methyl ester (14), 5-Bromo-2-formylphenoxyacetyl-seryl-leucyl-tryptophan methyl ester (15) and 5-Bromo-2- formyl-phenoxyacetyl-alanyl-prolylproline methyl ester (16) were prepared by stirring compound 7a, 10a and 11a with compound 12 respectively in the presence of DCC and NMM.
Deprotection of 5-Bromo-2-formylphenoxyacetyl-alanyl-prolyl-proline methyl ester at carboxyl end (16a) :
To a solution of 4.39 gm of compound 16 (10 mmol) in 36 ml of THF/H2O (1:1), 0.36 gm lithium hydroxide (15mmol) was added at 0 C. The mixture was stirred at room temperature for 1 hr, and acidified to 1 pH 3.5 with 0.5 mol L-H2SO4. The aqueous layer was extracted with diethyl ether (3 x 25 ml). Combined organic extracts were dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was recrystallized from methanol and ether to get 5- Bromo-2-formyl-phenoxyacetyl-alanylprolyl-proline (16a).
ACKNOWLEDGEMENTS
The authors are thankful to the Head, Department of Chemistry for providing research facilities, SAIF Department, Punjab University, Chandigarh (India), for providing spectral details in time.
Table 1 : Physical data of the synthesized compounds
Table 2: Antimicrobial activity data of synthesized compounds
Table 3: Anthelmintic activity data of synthesized compounds
Table 4: Spectral data of synthesized compounds
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