Universal Journal of Pharmacy Take Research to ew Heights Research Article ISS 2320-303X SYTHESIS, ATI-IFLAMMATRY, ATIMICRBIAL AD ATHELMITIC EVALUATI F AXIASTATI-1 *Corresponding author: Dr. (Mrs.) M. Himaja Pharmaceutical Chemistry Division, School of Advanced Sciences, VIT University, Vellore 632014, Tamil adu, India Mobile: +91-9944796228, Malipeddi Himaja * * Pharmaceutical Chemistry Division, School of Advanced Sciences, VIT University, Vellore - 632014, Tamil adu, India Received 29-08-2015; Revised 27-09-2015; Accepted 25-10-2015 ABSTRACT Axinastatin 1, a cyclic heptapeptide, was synthesized by solution phase peptide synthesis using -(Benzotriazol-1-yl)-,,, -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent and triethylamine as the base. The structure of the compound was confirmed by FTIR, 1 H MR, FABMASS and elemental analysis. The synthesized cyclic peptide was evaluated in vitro anti-inflammatory activity by membrane stabilizing method and antimicrobial activity against four strains of bacteria and two strains of fungi. It was also evaluated for anthelmintic activity against earthworms. The compound showed moderate anti-inflammatory activity as compared to the standard drug, ibuprofen. It possessed potent antibacterial activity only against Gram positive bacteria and moderate anthelmintic activity as compared to the standard drug, mebendazole. Keywords: Axinastatin 1, Solution phase peptide synthesis, Anti-inflammatory activity, Antimicrobial activity, Anthelmintic activity. ITRDUCTI Peptides belongs to one of the most important classes of organic compounds possessing many biological activities like antioxidant, anthelmintic, antitubercular, anti-inflammatory 1-3, antifungal and antibacterial activities 4,5. The objective in peptide synthesis is to design peptide ligands with specific physical, chemical and biological activities. Peptides ligands generally act by interaction with acceptor or receptor molecules (enzymes, hormones, neurotransmitters, etc.) 6,7. Axinastatin 1 is a cyclic peptide, possessing relatively simple chemical structure. It is a cyclic heptapeptide, produced by the Western Pacific marine sponge Axinella species. It was first isolated by George Pettit et al. in the year 1991 8. The sponge was collected in Palau and extracted with a mixture of methylene chloride and 2-propanol (1:1). Axinastatin 1 was crystallized from methylene chloride. Axinastatin 1 exhibited cytotoxicity against the PS leukemia cell line. The structure of Axinastatin 1 consists of three L-Valine, one L- Phenylalanine, two L-Proline and one L-Asparagine units (Figure 1). In order to carry out the synthesis, the cyclic heptapeptide was disconnected into three dipeptide units and a single amino acid unit. The dipeptides were prepared from the respective protected amino acids. The amino group was protected with tertiary Butyloxycarbonyl (Boc-) group and the carboxyl group was protected by converting it into methyl ester. The Boc-amino acids were coupled with the amino acid methyl ester hydrochlorides by using -(Benzotriazol-1-yl)-,,, -tetramethyluronium tetrafluoroborate (TBTU) as the coupling agent and triethylamine (TEA) as the base. Universal Journal of Pharmacy, 04(05), September-ctober 2015 33 H 3 C C H 3 H 2 CH 3 H CH H 3 C 3 CH 3 Figure 1: Structure of Axinastatin 1
MATERIALS AD METHDS All the reactions requiring anhydrous conditions were conducted in flame dried apparatus. All the reactions were magnetically stirred unless otherwise stated. rganic extracts were dried over anhydrous sodium sulphate. Melting points were determined by capillary method and were uncorrected. Amino acids, di-tertbutylpyrocarbonate, trifluoroacetic acid and triethylamine were obtained from Spectrochem Ltd. Mumbai. TBTU, DCM, diethyl ether, methanol and chloroform was obtained from AVRA. IR spectra were recorded on Perkin-Elmer IR spectrometer using a thin film supported on KBr pellets for solids and chloroform as a solvent for semisolids. The values are reported as ν max (cm 1 ). 1 H MR spectra were recorded on Bruker JEL (400MHz) MR spectrometer. The spectra were obtained in CDCl 3 and the chemical shift values are reported as values in ppm relative to TMS (δ = 0) as internal standard. FABMASS spectra were recorded on a Joel Sx 102/DA-6000 mass spectrometer using xenon as the carrier gas. The spectra were recorded at room temperature, m-nitrobenzyl alcohol was used as the matrix. The protection of amino group, carboxyl group and side chain and their deprotection were done by standard procedures 9,10. Preparation of Dipeptides 10 mmol of amino acid methyl ester hydrochloride was dissolved in 20 ml of dichloromethane (DCM). To this, triethylamine (TEA) (4 ml, 28.7 mmol) was added at 0 C and the reaction mixture was stirred for 15 mins. Boc-amino acid (10 mmol) in DCM (20 ml) and TBTU (10 mmol) were added with stirring. After 1 h, the reaction mixture was filtered and the residue was washed with DCM (30 ml). The filtrate was washed with 5% ahc 3 (20 ml), 5% HCl (20 ml) and distilled H 2 (20 ml). The organic layer was dried over anhydrous a 2 S 4, filtered and evaporated in a vacuum. The residue was purified by recrystallization from DCM. Boc-Pro-Phe-Me, Boc- Val-Val-Me and Boc-Pro-Val-Me was prepared in this manner. Protection of the Side chain function The carboxamide side function of Boc-Asparagine was protected by introducing the benzhydryl group (bzh) by Bodanszky 10 method (Scheme 1). Preparation of Axinastatin 1 The ester group of Boc-Val-Val-Me was removed with LiH and the Boc- group of Boc-Pro-Val-Me 2 was removed by trifluoroacetic acid (TFA). The deprotected dipeptides were coupled using TBTU and TEA to get the tetrapeptide Boc-Val-Val-Pro- Val-Me. The Boc- group of the tetrapeptide and the ester group of the dipeptide Boc-Pro-Phe-Me were removed coupled to give the hexapeptide Boc-Pro-Phe-Val-Val-Pro-Val-Me. After the removal of the Boc- group of the hexapeptide unit, it was coupled with Boc-Asn(bzh)-H to obtain the heptapeptide Boc-Asn(bzh)-Pro-Phe-Val-Val-Pro- Val-Me. The ester group of the heptapeptide was removed and p-nitrophenyl group (pnp) was introduced. The Boc- and the bzh- groups were removed by TFA and the linear fragment was cyclized by adding pyridine and keeping the whole contents at 0 o C for ten days to get the Axinastatin 1 (Scheme 2). In vitro Anti-inflammatory Activity In vitro Anti-inflammatory Activity of Axinastatin 1 was carried out by the below mentioned procedures 11. Preparation of erythrocyte suspension 10 ml of blood was collected and centrifuged at 3000 rpm for 10 mins in a centrifuge. Plasma was discarded and the pellet was washed with 0.9% saline. The volume of the blood was reconstituted as 20% v/v suspension with 10 mm sodium phosphate buffer solution (ph 7.4). Heat induced haemolysis The membrane stabilizing activity of Axinastatin 1 was measured by heat induced haemolysis of human blood. The reaction mixture contained solutions of Axinastatin 1 (50-200 µg/ml) in different tubes. 20% RBCs suspensions (1 ml) was added to all the tubes. Instead of the compound only 10 mm sodium phosphate buffer solution was added to the control tube. The tubes containing the mixture were incubated at 56 o C for 30 minutes in a water bath. The reaction mixture was centrifuged at 3000 rpm for 10 minutes and the absorbance of the supernatants was measured at 560 nm using Hitachi U-2910 Spectrophotometer. Ibuprofen (50-200 µg/ml) was used as the reference standard. Membrane stabilizing activity (in %) was calculated by the following formula: Inhibition (%) = 100 Where, A t = Absorbance of test sample and A c = Absorbance of control. (bzh) Boc-H CH 3 CH, benzhydrol conc. H 2 S 4, RT, 12 h Boc-H H H Boc-Asn(bzh) Scheme 1: Protection of side chain of Asparagine Universal Journal of Pharmacy, 04(05), September-ctober 2015 34
Boc-Pro + Phe-Me Boc-Val + Val-Me Boc-Pro + Val-Me a, b Boc-Pro-Phe + a, b Boc-Val-Val + Val-Val-Pro-Val-Me Pro-Val-Me Pro-Phe-Val-Val-Pro-Val-Me a Boc-Asn(bzh) Boc-Asn(bzh)-Pro-Phe-Val-Val-Pro-Val-Me a = TBTU, TEA, DCM, RT, 1 h b = LiH, THF:H 2 (1:1), RT, 1 h b, d, c, e c = TFA, CHCl 3, RT, 1 h d = p-nitrophenol, CHCl 3, RT, 12 h Axinastatin 1 e = Pyridine, CHCl 3, 10 days, 0 o C Scheme 2: Synthetic scheme of Axinastatin 1 Antimicrobial Activity For antimicrobial activity study four strains of bacteria and two strain of fungi were used. The bacterial isolates included two Gram negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and two Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis). The fungal cultures were Candida albicans and Aspergillus niger. The antimicrobial activity of Axinastatin 1 was tested at 50 µg/ml, using agar well diffusion assay method 12. The bacterial test organisms were grown in nutrient broth at 37 o C for 24 h and then spread on Muller Hinton Agar (MHA) plates. For the fungal cultures, spore suspensions was grown in Sabor Dextrose Broth (SDB) at 25 o C for 48 h and then spread on Potato Dextrose Agar (PDA) plates. Dimethyl formamide was used as negative control and disks containing penicillin (10µg/disk), streptomycin (10µg/disk) and griseofulvin (25µg/disk) as positive controls. All the tests were performed in triplicates. The antimicrobial activity was evaluated based on the diameter of zone of inhibition. Anthelmintic Activity Anthelmintic activity study of Axinastatin 1 was carried out against earthworms (Eudrilus eugeniae 13 ). Suspension of Axinastatin 1 was prepared by triturating it with 15% Tween 80 and distilled water and the mixture was stirred for 30 mins using a magnetic stirrer. The suspension was diluted to contain 0.1% and 0.2% w/v in 20 ml of the test sample. The standard drug, mebendazole was also prepared in a similar way. Earthworms were placed in three petri dishes containing 20 ml of each sample, control (20 ml suspension of distilled water and 15% Tween 80) and standard drug, respectively at room temperature. The time needed for the paralysis and death of the earthworms were noted. The death time was determined by placing the earthworms in warm water at 50 o C, which stimulated the movement if the earthworms were alive. RESULTS AD DISCUSSI Physical Data and Spectral Analysis Axinastatin 1: Yield 69%; pale white solid; m.p. 280-283 o C; IR spectrum (ν/cm -1 ): 3300 (br. s), 2930 (s), 2965 (s), 1705 (s), 1690 (s), 1670 (br. s), 1645 (br. s), 1620 (s), 1520 (s), 1470 (s), 1450 (s), 1280 (s), 1165 (s), 1110 (s), 1040 (s), 895 (s) cm -1 ; 1 H MR spectrum (δ, ppm): 8.5(3H, br. s), 7.5 (4H, br. s),, 7.3-7.1 (5H, m), 4.7-4.6 (1H, m), 4.5-4.3 (2H, m), 4.2-3.9(4H, m), 3.5-3.2(6H, m), 2.2-1.5 (10H, m), 1.4-1.1 (3H, m), 1.0 (18H, d, J = 6.0 Hz); FABMASS: m/z (M + H) + = 753.4; Elemental Analysis: M. F. = C 38 H 56 8 8, M. W. = 752, Found (Cal) %C: 60.61 (60.64), %H: 7.46 (7.45), %: 14.90 (14.89). Anti-inflammatory Activity Anti-inflammatory activity of Axinastatin 1 are reported in Table 1. From the obtained results it was concluded that Axinastatin 1 has a significant membrane stabilizing activity which was comparable to the standard ibuprofen. The maximum inhibition of 71.3±0.69% was observed with 200 µg/ml of Axinastatin 3. Whereas, diclofenac sodium, showed the maximum inhibition of 89.4±0.63% at 200 µg/ml. Universal Journal of Pharmacy, 04(05), September-ctober 2015 35
Table 1: In vitro anti-inflammatory activity of Axinastatin 1 Concentration (µg/ml) Standard (ibuprofen) Axinastatin 1 50 71.9±0.86 53.4±1.1 100 82.4±0.59 62.1±0.59 200 89.4±0.63 71.3±0.69 Values are expressed as mean ± S. E of the three replicates. Antimicrobial Activity Evaluation of antibacterial and antifungal activities was done for Axinastatin 1 (Table 1) and it was found to possess antibacterial activity against Gram positive (B. subtilis and S. aureus) bacteria. However, it did not show antibacterial activity against Gram negative (P. aeruginosa and E. coli) bacteria and antifungal activity against C. albicans and A. niger. Table 2: Antimicrobial activity of Axinastatin 1 Diameter of zone of inhibition (in mm). P. aer E. coli B. sub S. aur C. alb A. niger Sl. o. Compound 1 Axinastatin 1 - - 8.1±0.45 9.0±0.25 - - 2 Penicillin 11.2±1.2 12.3±0.5 17.9±0.6 18.2±1 - - 3 Streptomycin 16.2±0.2 16.1±0.5 12.0±1 12.2±0.8 - - 4 Griseofulvin - - - - 20.1±0.7 18.2±1 Values are expressed as mean ± S. E of the three replicates. Zone of inhibition not include the diameter of the well (7 mm) Anthelmintic activity The synthesized cyclic peptide, Axinastatin 1 have shown moderate anthelmintic activity as compared to CCLUSI the standard drug, mebendazole. Table 2 illustrates the anthelmintic activity of sample and standards. Table 3: Anthelmintic activity of Axinastatin 1 Sl. o. Compound Conc. of the Mean paralysing time Mean death time compound (mg) (mins) ± S.E (mins) ± S.E 1 Control 100 200 2 Mebendazole 100 17.44 ± 0.09 54.4 ± 1.08 200 12.14 ± 1.02 31.04 ± 1.09 3 Axinastatin 1 100 82.68 ± 1.04 122.4 ± 1.02 200 74.40 ± 1.09 102.2 ± 1.10 Axinastatin 1 can be easily synthesized under normal laboratory conditions. The synthesis is cost effective and time saving. The structure of the cyclic peptide was confirmed by IR, 1 H MR, FABMASS and elemental analysis. The compound was screened for in vitro antiinflammatory activity and it showed potent antiinflammatory activity as compared to the standard, ibuprofen. Axinastatin 1 was screened for antimicrobial activity against four strains of bacteria and two strains of fungi and it showed potent antibacterial activity only against the Gram positive bacteria. The compound was also screened for anthelmintic activity and it showed moderate activity as compared to the standard drug, mebendazole. ACKWLEDGEMETS We are grateful to the VIT-SIF Lab, School of Advanced Sciences, VIT University, Vellore, for the support of this research. REFERECES 1. Das M. and Himaja M., Synthesis, antioxidant and anthelmintic activity of the linear tetrapeptide L-(Leu-Pro-Gly)-D-Ala (LPGA), Int. J. Pharm. Pharm. Sci., 2013, 5(Suppl 3), 713. 2. Das M, Agarwal DS, Sharma SK, Das P and Rout PK., Synthesis, docking studies and free radical scavenging activity of the linear tetrapeptide VFPF, Int. Res. J. Pharm., 2012; 3(6): 138. 3. Himaja M, Sreekanth K., Munirajsekhar D, Ramana MV. and Mukesh S, Computer- aided design, synthesis and antioxidant activity of linear tetrapeptide D-Phe-L-(Ala-Tyr-Val), J. Pharm. Res. 2011; 4(8): 2581. 4. Tadahiro T, Tayuka K, oriko S, Yukio and Machiko M, Synthesis of triglycosyl tetrapeptides and a hexaglycosyl tetrapeptide, Carbohydr. Res., 1996, 283, 93. 5. Daniele B, Andrea B, Matteo C, Gianni P and Sergio S, Synthesis and conformational preferences of unnatural tetrapeptides Universal Journal of Pharmacy, 04(05), September-ctober 2015 36
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