AU2021105736A4 - Synthesis, characterization and biological evaluation of Cyclo-octapeptide, Cyclogossine B (77) - Google Patents
Synthesis, characterization and biological evaluation of Cyclo-octapeptide, Cyclogossine B (77) Download PDFInfo
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- AU2021105736A4 AU2021105736A4 AU2021105736A AU2021105736A AU2021105736A4 AU 2021105736 A4 AU2021105736 A4 AU 2021105736A4 AU 2021105736 A AU2021105736 A AU 2021105736A AU 2021105736 A AU2021105736 A AU 2021105736A AU 2021105736 A4 AU2021105736 A4 AU 2021105736A4
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- 238000003776 cleavage reaction Methods 0.000 description 1
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- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 108010084607 cordyheptapeptide A Proteins 0.000 description 1
- ZXPWAGZHBVVLJM-PPLXTMPISA-N cordyheptapeptide A Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CC(C)C)C(=O)N(C)[C@H](CC=2C=CC=CC=2)C(=O)N2CCC[C@H]2C(=O)N(C)CC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N1C)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZXPWAGZHBVVLJM-PPLXTMPISA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000002828 disc diffusion antibiotic sensitivity testing Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 239000003966 growth inhibitor Substances 0.000 description 1
- 239000012456 homogeneous solution Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- -1 methyl ester hydrochloride Chemical class 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 108010067594 psammosilenin A Proteins 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
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- A61K38/00—Medicinal preparations containing peptides
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- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF CYCLO
OCTAPEPTIDE, CYCLOGOSSINE B (77)
5 The present work reports the synthesis of cyclic octapeptide, Cyclogossine-B which was
previously isolated from latex of Jatropha gossypifolia, accomplished through coupling of tetra
peptide fragment (Boc-L-Gly-L-Gly-L-Trp-L-Leu-OMe) (72) with another tetra-peptide fragment
(Boc-L-Ala-L-Ala-L-Ile-L-Leu-OMe) (73), followed by cyclization of the linear octa-peptide unit
under alkaline condition. The formation of newly synthesized cyclic compound was confirmed by
.0 means of spectral techniques including FT-IR, 1H-NMR, Mass spectroscopy along with elemental
analyses. Cyclogossine-B was subjected for biological screening to evaluate antimicrobial and
anticancer activities. The anti-bacterial activity was carried out by using Gram +ve bacteria (B.
subtilis, S. epidermidis) and Gram -ve bacteria (E. coli, P. aeruginosa, S. aureus and K.
pneumonia). The anti-fungal activity was performed by using fungal strains like C. albicans, A.
.5 niger, T. mentagrophytes and M. audouinii. Similarly, the cytotoxic activity of synthesized cyclic
peptide was carried out through MTT assay using Doxorubicin as standard drug on HCT116 and
B16F10 cell lines. The cytotoxic effect was evaluated by determining the percentage inhibition of
growth of HCT116 and B16F10 cell-lines. Then CTC50 (Concentration of test drug needed to
inhibit cell growth by 50%) values were calculated by graphical extrapolation method. Different
20 concentration of test, control and standard drug (120-7.5 g/mL) were used for the cytotoxicity
study. It was observed that this cyclic peptide exhibited significant antimicrobial and cytotoxic
activity against cancer cell lines.
19
Description
Synthesis, characterization and biological evaluation of Cyclo-octapeptide, Cyclogossine B (77)
This Invention relates to Synthesis, characterization and biological evaluation of Cyclo
octapeptide, Cyclogossine B (77)
Therapeutic compounds are obtained from the natural resources. In drug discovery, the growth of
microbes is inhibited by micro-organisms by producing a large number of drug candidates [I]. A
variety of natural products produced by fungi, bacteria and plants with different biological
activities.
Recently, in molecular biology, peptides get predominance due to several reasons such as in
animals they promote the formation of anti-bodies. Another reason that these peptides can be
easily detected by mass spectroscopy, based on peptide mass. Recently, the protein structure and
their functions are studied by using peptides. In clinical research, inhibitory peptides are in trial
for becoming future drugs to treat cancer and other diseases [2].
Cyclic peptides are isolated from natural resources having numerous biological functions. Cyclic
peptides are isolated from invertebrate animals, micro-organisms of marine habitats and higher
plants, possessing unique structures and diverse pharmacological activities. Now a days, cyclic
peptides are becoming superior therapeutic agents due to several reasons such greater resistance
to enzymatic degradation (in vivo) and higher bio-availability. Some cyclic peptides are leucine
rich, other are proline while some are having other amino acids as their major constituents [3].
Peptidase enzymes degrade the linear peptides which limit their clinical applications and lead to
metabolic instability that restricts their oral delivery. The poor ability to cross the blood brain barrier is also a major problem for peptides. So, in order to overcome these problems several modifications have been considered in the peptide structures, one of the valuable modifications of linear peptides is 'cyclization', which reduces their flexibility, stabilizes secondary structure and increases the activity [4].
Higher plants provide a large number of natural congeners having wide spectrum of
biological activities [5, 6]. From these natural congeners, cyclic peptides and related congeners
have received special attention due to unique structure and wide biological spectrum of activity
[7, 8]. These cyclic peptides can overcome the problem of resistance associated with existing
drugs. A novel cyclic peptide, Cyclogossine-B, previously isolated from latex of Jatropha
gossypifolia which is used as HSV-1, anti-fungal and anti-malarial agent (Fig. 1).
The structure of this cyclopeptide was elucidated by IR, 1H-NMR and Mass
Spectrometery [9]. In continuation of our synthetic efforts on biologically active cyclopeptides
[10, 11], the present research work was aimed at the synthesis of a novel cyclic octa-peptide of
plant origin. By keeping in mind the biological potential of cyclic peptides i.e. anti-tumour [12]
, anti-dysentric activity [13], anti-mycobacterial [14], anti-microbial [15], Anthelmintic activity
[16] the above synthesized cyclic octa-peptide was subjected to cytotoxic and anti-microbial
activity studies.
US9133260B2 relates generally to novel GIP analogs and GIP hybrid polypeptides with
selectable properties, useful as agents for the treatment and prevention of metabolic diseases and
disorders, for example those which can be alleviated by control plasma glucose levels, insulin
levels, and/or insulin secretion, positive inotropic effects, reduction of catabolic effects, slowing
of gastric emptying. Such conditions and disorders include, but are not limited to, hypertension, dyslipidemia, cardiovascular disease, eating disorders, critical care, insulin-resistance, obesity, and diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes.
US8895498B2 relates generally to novel GIP analogs and GIP hybrid polypeptides with
selectable properties, useful as agents for the treatment and prevention of metabolic diseases and
disorders, for example those which can be alleviated by control plasma glucose levels, insulin
levels, and/or insulin secretion, positive inotropic effects, reduction of catabolic effects, slowing
of gastric emptying. Such conditions and disorders include, but are not limited to, hypertension,
dyslipidemia, cardiovascular disease, eating disorders, critical care, insulin-resistance, obesity,
and diabetes mellitus of any kind, including type 1, type 2, and gestational diabetes.
The present work reports the synthesis of cyclic octapeptide, Cyclogossine-B which was
previously isolated from latex of Jatropha gossypifolia, accomplished through coupling of tetra
peptide fragment (Boc-L-Gly-L-Gly-L-Trp-L-Leu-OMe) (72) with another tetra-peptide
fragment (Boc-L-Ala-L-Ala-L-Ile-L-Leu-OMe) (73), followed by cyclization of the linear octa
peptide unit under alkaline condition. The formation of newly synthesized cyclic compound was
confirmed by means of spectral techniques including FT-IR, 1H-NMR, Mass spectroscopy along
with elemental analyses. Cyclogossine-B was subjected for biological screening to evaluate
antimicrobial and anticancer activities. The anti-bacterial activity was carried out by using Gram
+ve bacteria (B. subtilis, S. epidermidis) and Gram -ve bacteria (E. coli, P. aeruginosa, S. aureus
and K. pneumonia). The anti-fungal activity was performed by using fungal strains like C.
albicans, A. niger, T. mentagrophytes and M. audouinii. Similarly, the cytotoxic activity of
synthesized cyclic peptide was carried out through MTT assay using Doxorubicin as standard
drug on HCT116 and B16F10 cell lines. The cytotoxic effect was evaluated by determining the percentage inhibition of growth of HCT116 and B6F10 cell-lines. Then CTC50 (Concentration of test drug needed to inhibit cell growth by 50%) values were calculated by graphical extrapolation method. Different concentration of test, control and standard drug (120-7.5 pg/mL) were used for the cytotoxicity study. It was observed that this cyclic peptide exhibited significant antimicrobial and cytotoxic activity against cancer cell lines.
Fig. (1). Structure of Cyclogossine-B.
Scheme 1. Synthetic route to Cyclogossine-B
Fig. 2 Cyto-toxic effects of Cyclogossine B (77) on HCT116 Cell line in comparison of standard
Fig. 3 Cyto-toxic effects of Cyclogossine B (77) on B16F10 Cell line in comparison of standard
The chemicals used for experimental work were commercially procured from diverse chemical organizations-E. Merck India Ltd., CDH, S.D. Fine Chem. Ltd. and have been of L.R. Grade and purified by using widespread manner earlier than their use. The amino acids used in this research work- L-Proline, L-Alanine, L-Tyrosine, L-Isoleucine, L-Glycine, L-Tryptophan, L-Threonine, L-Phenylalanine and L-Leucine. Di-tert-butyl dicarbonate (Boc2 0) was used to protect amino group and Dicyclohexylcarbodiimide (DCC) was used as coupling agent. N methylmorpholine (NMM) and p-nitrophenol are used as base. Trifluoroacetic acid (TFA) was used to deprotect amino group while Lithium hydroxide (LiOH) was used to deprotect carboxyl
group.
MR-VIS Visual melting point apparatus (LAB India) was used to record the melting points of synthesized compounds and were uncorrected. The IR spectra had been recorded on
Bruker Tensor-27, USA, spectrophotometer using KBr. 'H-NMR spectra was recorded by using Bruker NMR spectrophotometer at 400 MHz by dissolving the compounds in CDCl 3 in reference
to Tetramethylsilane as internal standard and shift (6) values are reported in parts per million (ppm). Water UPLC-TQD Mass spectrophotometer operated at 70.21 eV was used to record the mass data of compounds. The results are observed by molecular ion peaks. The spectral analysis was carried out at Punjab University, Chandigarh.
Elemental analyser (Thermo Scientific) was used to perform elemental analyses (C, H, N). It was also performed at Punjab University, Chandigarh. Progress of the chemical reactions
and the purity level of the compounds was evaluated using TLC plates by solvent systems: CHCl3:CH 30H - (9.5:0.5); C 4H 90H:CH 3COOH:H 20 - (4:1:1) and CHC 3 :CH30H - (9:1). The visualization of spots on TLC plates was carried out in iodine chamber and UV cabinet at long
wavelength under UV lamp.
General methods for synthesis of linear Tetrapeptide units (72 & 73)
In a beaker, methyl ester hydrochloride of amino acid/dipeptide methyl ester (10 mmol) was mixed in 20 ml of CHC 3.Then we added, 2.3 ml of N-methylmorpholine at 00 C and stirring was done of above mixture for 15 min (solution A). In another beaker, Boc-dipeptide (10 mmol) was mixed in 20 ml of CHC13 and 2.1 gm of DCC (10 mmol) (solution B). After that, solution (B) was added to solution (A) and stirred for 36 h. After that, filtration of reaction mixture was done and washed with 25 ml of NaHCO3 (5%) and 25 ml of NaCl solution to give title compounds which were further purified from the mixture of Chloroform-petroleum ether (b.p. 59-84 °C) followed by cooling at 0 °C.
Synthesis of Linear octapeptide methyl ester (Boc-Gly-Gly-L-Trp-L-Leu-L-Ala-L-Ala-L Ile-L-Leu-OMe) (76): This linear octapeptide (76) was synthesized by coupling of deprotected tetrapeptide at carboxyl end i.e. Boc-L-Gly-L-Gly-L-Trp-L-Leu-OMe (72) with Boc-L-Ala-L Ala-L-Ile-L-Leu-OMe (73), another tetrapeptide deprotected at amino end.
Percentage Yield - 79%; M.P.: 163-164° C
TLC (CHCl 3 :CH30H - 9.5:0.5); Rf - 0.56
IR (KBr) Vmax (cm-1 ): v 3410 (N-H stretch, indole ring of tryptophan), 3063, 3042 (w,-CH str, heteroarom. ring), 2953, 2823 (m,-CH str, asym and sym, -CH2), 2537 (s/br, NH str), 1767 (s,C=O str, ester), 1594, 1469 (m, skeletal bands, heteroarom. ring), 1522 (in, -NH bend), 1376, 1356 (m, -CH bend, tert-Butyl group), 1388, 1367 (s, C-H bend, isopropyl group), 1253 (s, C-O str, ester), 811 (m,C-H str,OCH 3). 1 H-NMR (CDC3) (ppm): 8.23-8.21 (s, 1H, -NH, indole ring), 7.74-7.76 (1H, d, J= 7.15 Hz,p
H, indole ring), 7.41-7.43 (2H, m, E &i-H's, indole ring), 7.19-7.21 (1H, t, (-H, indole ring), 5.86 (7H, br. S, -NH), 4.34 (3H, s,-OCH 3) 3.96-3.97 (1H, m, a-H, Trp), 3.71-3.73 (4H, d, J = 4.8 Hz, -CH 2, Gly), 3.63-3.65 (2H, m, a-H, Ala), 3.58-3.60 (1 H, d, J = 5.8 Hz, a-H, Ile) 3.54-3.56 (4H, t, -H's, Leu), 2.89-2.91 (2H, d, J=6.75 Hz, -H's, Trp), 2.24-2.26 (2H, m, y-H's, Ile), 1.79 1.81 (2H, m, y-H, Leu), 1.64 (9H, s, tert-Butyl group), 1.32-1.34 (6H, d, J= 4.2 Hz, P-H's, Ala), 1.26-1.28 (4H, m, - & y'H's, Ile), 0.99-1.01 (12H, d, J= 6.0 Hz, H's, Leu).
Synthesis of Cyclogossine B - [Cyclo (Gly-L-Trp-L-Leu-L-Ala- L-Ala L-Ile-L-Leu-L-Gly)] (77): This cyclic octapeptide was synthesized by cyclization of linear octapeptide unit Boc-Gly Gly-L-Trp-L-Leu-L-Ala-L-Ala-L-Ile-L-Leu-OMe (76) by deprotecting at carboxyl end.
SCHEME - 1I [Synthesis of Cyclogossine B (77)]
. Molecular Weight: 781.94; Yield: 72.8 %; M.P.: 148-150° C. The purity of compound was checked by TLC using CHCl 3:CH30H (9.5:0.5) as solvent system and iodine vapours as visualizing agent and only one spot was obtained at Rf value 0.62.
Elemental analysis:
Calculated for C 39 H 9N 90 1 6: C, 51.48; H, 6.54; N, 13.85; 0, 28.13 % Found: C, 51.52; H, 6.51; N, 13.88; 0, 28.09 %
Spectral Analysis:
IR (KBr) Vmax (cm-1 ): v 3479 (N-H stretch, indole ring of tryptophan), 3179-3158 (N-H
stretch, amide), 3071 (C-H stretch, aromatic ring), 2967-2929 (Asym. C-H stretch, CH 3 & CH2), 2892 (C-H stretch, CH), 2873-2858 (Sym. C-H stretch, CH 3 & CH2), 1679, 1647-1638 (C=O stretch, amide), 1588 & 1449 (skeletal bands), 1546, 1534-1527 (N-H def, amide), 1468 (C-H bending, CH2), 1457 (Asym bending, CH3 ), 1379 & 1370 (Sym C-H bending, iso-Propyl in leucine), 929, 685 (C-H def. oop, aromatic ring). 1 H-NMR (CDC 3) (ppm): 10.16 (s, 1H, NH-indole), 7.62 (d, 1H, J= 6.8Hz, CH-indole), 7.34 (d, 1H, J= 8.8 Hz, CH-indole), 7.18 (s, 1H, CH-indole), 7.12 (t, 1H, J= 8.4 & 9.7 Hz, CH indole), 3.86 (t, 1H, J= 11 & 7.8 Hz, CH-Trp), 3.64 (q, 2H, CH-Ala), 3.59 (s, 4H, CH-Gly), 3.45 (t, 3H, J= 8 & 4 Hz, CH-N-Ile & Leu), 3.33 (d, 1H, J= 8.9 Hz, CH-Trp), 3.05 (d, 1H, J= 8 Hz, CH-Trp), 2.17-2.11 (m, 1H, CH-Ile), 2.01 (s, 8H, NH), 1.77 (t, 4H, J= 0.4 & 6 Hz, CH2-Leu), 1.57-1.45 (m, 4H, CH & CH2 Leu & Ile), 1.23 (d, 6H, J= 7.2 Hz, CH 3- Ala), 1.13 (d, 3H, J= 7.2
Hz, CH 3- Ile), 0.90 (t, 15H, J= 2.2 & 7.4 Hz, CH3-Leu & Ile).
FABMS m/z: 782.9 [(M+H)*, 100], 754.9 [(782.9-CO)*, 12], 725.9 [(H- Leu-Ileu-Ala-Ala-Leu Trp-Gly)*, 59], 711.8 [(H- Leu-Trp-Gly-Gly-Leu-Ileu-Ala)*, 68], 697.9 [(725.9-CO)*, 12], 687.8
[(711.8-CO)', 16], 669.8 [(H-Ileu-Ala-Ala-Leu-Trp-G1y-Gly)*, 52], 668.1 [(H-Leu-Ileu-Ala Ala-Leu-Trp)*, 58], 641.8 [(669.8-CO)*, 11], 640.9 [(H- Leu-Trp-Gly-Gly-Leu-Ileu)*, 72], 640.1 [(668.1-CO)*, 12], 612.9 [(640.9-CO)*, 19], 612.2 [(H-Gly-Trp-Leu-Ala-Ala-Ileu)*, 69], 598.7 [(H- Ala-Ileu-Leu-Gly-Gly-Trp)*, 55], 596.7 [(H- Leu-Ala-Ala-Ileu-Leu-Gly-Gly)*, 62], 584.2 [(612.2-CO)*, 16], 570.7 [(598.7-CO)*, 11], 568.7 [(596.7-CO)*, 15], 556.6 [(H- Ala Ala-Leu-Trp-Gly-Gly)*, 63], 528.6 [(556.6-CO)*, 14], 482.6 [(H- Leu-Ileu-Ala-Ala-Leu)*, 74], 454.6 [(482.6-CO)*, 18], 412.5 [(H- Ala-Ieu-Leu-Gly-Gly)*, 68], 426.5 [(H-Ala-Ala-Ileu-Leu Gly), 65], 398.5 [(426.5-CO)*, 13], 384.5 [(412.5-CO)*, 15], 369.1 [(H- Leu-Ala-Ala-Ileu)*, 65], 341.9 [(H- Gly-Gly-Leu-Ileu)+, 62], 341.1[(369.1-CO)*, 12], 313.9[(341.9-CO)*, 12], 298.4 [(H- Leu-Ileu-Ala)*, 75], 270.4 [(298.4-CO)*, 16], 256.9 [(H- Leu- Ala-Ala)+, 72], 228.9
[(256.9-CO)*, 13], 228.2 [(H- Leu- Gly-Gly)*, 66], 227.3 [(H- Leu-Ileu)*, 30], 200.2 [(228.2 CO)+, 11], 199.3 [(227.3-CO)*, 20], 187.2 [(H-Trp)*, 12], 185.2 [(H-Leu-Ala)*, 28], 171.8 [(H Leu- Gly)*, 25], 159.2 [Trp immonium ion (CioHilN 2)*, 15], 157.2 [(185.2-CO)*, 18], 143.8
[(171.8-CO)*, 15], 143.1 [(H- Ala-Ala)*, 24], 115.1 [(H-Gly-Gly)*, 21], 115.8 [(143.8-CO)*, 14], 114.1 [(H-Leu/Ileu)*, 15], 87.1 [(115.1-CO)*, 12], 86.1 [Leu/Ileu immonium ion (C 5H 2N)*, 25], 72.0 [(H-Ala)*, 11], 71.1 [(C 6H5 )*, 12], 58.0 [(H-Gly)*, 12], 57.1 [(C 4H 9)*, 11], 45.1
[(C 2 H5 O)*, 12], 44.0 [(Ala immonium ion C 2H 6N)*, 12], 43.1 [(C 3H 7 )*, 11], 30.0 [(Gly immonium ion CH4N)*, 14], 29.1 [(C 2 H5 )*, 11], 15.0 [(CH 3 )*, 10]%.
Cyclic peptides have emerged as an important class of compounds which have broad biological profile. After the synthesis and characterization of cyclic peptides, is more rational to evaluate their pharmacological profile in details. Cyclic peptides have unique structures and a wide pharmacological profile [17]. So, cyclic peptides are established as drug of choice to where there is resistance to existing medicines.
Thus, keeping in view the advantage of bio-degradability and bio-compatibility of amino acids and peptides, these novel cyclic peptides have been synthesize to provide maxima therapeutic efficacy and no undesired effects [18]. As earlier reported that peptides of marine origin are natural growth inhibitors for cancer cells [19]. Hence, in this research work by keeping in mind the biological profile existing in theses natural cyclic peptides obtained from marine sources, plants and insect pathogenic fungus were synthesized with improved yields. The cyclopeptide was evaluated for their biological activities as follows:
Anti-microbial Activity
The anti-bacterial activity was carried out by using Gram +ve bacteria- B. subtilis (MUMC 408) and Gram -ve bacteria- E. coli (MUMC 106), P. aeruginosa(MUMC 266) and S. aureus (MUMC 377). The anti-fungal activity was carried out by using i.e. C. albicans (MUMC 29), A. niger (MUMC 96), T. mentagrophytes (MUMC 665) and M. audouinii (MUMC 545). To carry out the anti-microbial activity of newly formed cyclic peptides following nutrient media were used to culture the bacterial and fungal strains:
Preparation of Broth Media
The following broths were used in the present work having composition as follows [20, 21].
Composition of Nutrient broth:
Sr. No. Components Amount (gm/liter)
1 Peptone (bacteriological) 5 gm 2 Yeast extract 1.5 gm 3 Sodium chloride 5 gm 4 Beef extract 1.5 gm 5 pH 7.2 0.2
Preparation of nutrient broth
13 gm of the above medium was dissolved in distilled water and final volume was made up to 1000 ml with distilled water. It was sterilized by autoclaving at 15 psi of pressure (1210 C) for 15 minutes.
Composition of Sabouraud's broth
Sr. No. Components Amount (gm/liter)
1 Dextrose 40 gm 2 Peptone (mycological) 10 gm 3 Agar 15 gm 4 pH 5.6 0.2
Preparation of Sabouraud's broth
65 gm of the above medium was dissolved in distilled water and boiled to dissolve the medium completely and final volume was made up to 1000 ml with distilled water. It was sterilized by autoclaving at 15 psi of pressure (121 C) for 15 minutes.
Culturing of Microorganisms
For culturing the bacteria, the different bacterial strains were transferred aseptically in nutrient broth media for 24 h by maintaining aerobic conditions at 37 C and for culturing the fungi, the different fungal strains were transferred aseptically in Sabouraud's broth medium for 48 h by maintaining aerobic conditions at 25° C.
Procedure of Anti-microbial Activity: (Modified Kirby-Bauer method)
It is a disc diffusion method [22] which was used for the determination of anti-microbial efficacy of the cyclopeptides.
1. The test as well as standard drugs solutions were prepared in concentration of 10 mg/ml.
2. The previously grown bacterial and fungal strains were innoculated in perti plates and four wells, two for test compounds, one for standard drug and one for control. The standard drugs, ciprofloxacin and griseofulvin for anti-bacterial and anti-fungal activity, respectively and DMF as control was used.
3. Then filter disks previously sterilized in autoclave at 121° C temperature were impregnated with test solution, standard drug solution and with DMF (control), were placed in corresponding wells of perti plates.
4. Then petri plates were incubated for a period of 24 h for bacterial cultures at temperature 37° C and 48 h at a temperature of 25 C for cultures of fungi [23, 24].
5. The, zones of inhibition (in mm) were measured and the average was calculated for three sets of zones of inhibition (Fig. 2, 3). The zones of inhibition of test were compared with those of standard drugs i.e, ciprofloxacin for anti-bacterial and griseofulvin for anti-fungal activity. The data of average zones of inhibition is listed in Table 1.
Table 1. Results of Anti-microbial activity of Synthesized Cyclopeptide.
Compounds Average zone of inhibition (mm) Bacterial strains Fungal strains Bacillus Staphylococcus Pseudomonas Escherichia Candida Microsporum Aspergillus Trichophyyton subtilis aureus aeruginosa coli albicans audouinii niger mentagrophytes CYB (77) 12.42 18.92 14.55 16.53 16.43 15.44 15.08 18.59 Control - - - - - - -
(DMF) Ciprofloxacin 20.04 19.88 25.40 20.69 - - - (Standard) Griseofulvin - - - - 20.57 19.55 18.62 21.30 (Standard)
5 Anti-cancer activity
Cyto-toxic study of synthesized cyclic peptide - IG A (32) was carried out at Deshpandey Laboratories Pvt. Ltd., Bhopal, through MTT [(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)] assay by using Doxorubicin as standard drug on HCT116 and B16F1O, cell lines. HCT116 cell lines are taken from colorectal carcinoma of human where as B16F10 10 cell lines are taken from melanoma of skin of mouse.The cyto-toxic activity was evaluated by determination of % inhibition of growth of HCT116 & B16F10 cell-lines. Then, CTC5o (cyto toxic concentration at 50% of the cells are dead after drug exposure) values were calculated by graphical extrapolation method. The different conc. of 120-7.5 pg/mL used for test, control and standard drug [25].
15 Principle of method
It is based on colorimetric measurement of MTT conversion [(3-[4,5-dimethylthiazol-2 yl]-2,5-diphenyl-tetrazolium bromide)] in to crystals of formazan by the cells in living state, which determines the activity of mitochondria. After that, MTT is reduced to dark purple formazan complex which is insoluble, inside the cells. After that those cells are get mixed with 20 isopropanol and they release formazan reagent which is measured by spectrophotometer.
Requirements
• Incubator with CO 2 (5%) • Phosphate-buffered saline (PBS) • Reagent - MTT, Isopropanol with acidic pH • Plate with 96-well having flat bottom
Procedure
1. In 96-well flat bottomed tissue culture plate, HCT116 and B16F10 cells-lines containing 5000-10,000 cells were transferred along with 100 pl of RPMI-1640 as culture media.
2. Then, the above mixture was allowed to stand for a night for attachment of cells and then test cyclic peptides were added to respective wells. After that, 50 p1 of MTT reagent (5 mg/ml) was mixed to each well and allowed to incubate at 37 for 4 h for cleavage of MTT to impart colour.
3. A multichannel pipettor was used to add 0.1 ml of acidic isopropanol to the above mixture in each well and mixed thoroughly. A yellow colour was produced by HCl reaction with phenol red culture media that make the measurement of MTT-formazan easy. A homogeneous solution of blue colour was produced due to mixing of formazan in isopropanol.
4. After that, by using a plate reader at 570 nm for test and 630 nm for the standard drug, the optical density was measured (Table 2, Fig 4 and 5). Then, % growth inhibition was determined by using following formula:
Cell total-Cell dead X 100 % growth inhibition= Celltotai Table - 2: Results of Cyto-toxic effects of Synthesized Cyclopeptide Cyclogossine B (77)
HCT116 B16F1O Compou Conc.(pg/ Live % Live No. %
nd ml) cells No. of growth bCT cells of growth bCT count dead inhibiti C50 count dead inhibiti C50 ed cells on' (IM) ed cells on' (IM) 23+2. 17+2.7 42.5±2. 22+2. 18+3. 120 32 4 54 36 22 45±2.85 26+2. 14+2.4 25+2. 15+2. 37.5+2. 60 58 2 35+2.41 58 98 59
31+3. 09+2.4 22.5+2. 27+2. 13+3. 32.5+2. 30 02 8 98 51 10 36 34+2. 06+0.2. 36+2. 04+2. 15 14 69 15+2.21 73 93 10+2.44 37+3. 37+1. 7.5 02 3+0.48 7.5+1.2 2 03±.8 7.5+0.4 120 40 0 0 40 0 0 60 40 0 0 40 0 0 30 40 0 40 0 0 15 40 0 0 40 0 0 7.5 40 0 0 40 0 0 0 40+1.2 100+1.4 0 401. 100+1.4 120 3 7 36 2 0 40+1.7 100+1.2 0 40+1. 100+1.6 60 4 5 16 1 6+1.0 28+1.4 6+1.6 28+1. 30 4 5 70±1.44 3 43 70±1.65 15 12+1. 18+1.5 45±1.62 12+1. 18+1. 45+1.47 25 7 41 35 21+1. 12+1.3 21+1. 12+1. 7.5 11 2 30+1.22 13 45 30+1.54 a % growth inhibition = 100 - [{(Celltotai - Celldead) x100}/Cellotai; bCTC 5o = conc. inhibiting 50% of percentage growth RESULTS AND DISCUSSION
The synthesized cyclic peptide was synthesized by solution phase technique because it is very simple and cheaper as compared to solid phase synthesis. Cyclic peptide was synthesized by using NMM as base for cyclization and DCC as coupling agent and provided very good yield.
Chemical Analysis
The synthesis of cyclooctapeptide, Cyclogossine B (77) was accomplished with 72.8% yield and characterized by the prominent peaks at 3479 (N-H stretch, indole ring of tryptophan), 3179 3158 (-NH stretch for amide), 3071 (-CH stretch, Ar); singlet of peptide bond 2.01(s, 7H, NH) and by existence of ionic pseudo molecular peak at 782.9 (m/z).
Biological Evaluation
The biological studies of synthesized cyclic peptide - CYB (77) was carried out for anti bacterial, anti-fungal and cyto-toxic activities and the results are described as follows:
The anti-bacterial activity was carried out by using Gram +ve strains viz. B. subtilis, and Gram ve strains viz. E. coli, P. aeruginosa, S. aureus. The anti-fungal activity was carried out by using anti-fungal strains viz. C. albicans, A. niger, T. mentagrophytes and M. audouinii. The anti microbial activity was evaluated by Modified Kirby-Bauer method by using Ciprofloxacin as standard drug for anti-bacterial activity and Griseofulvin for anti-fungal activity at 10 mg mL-1 conc. respectively.
The observation of anti-microbial activity showed that the cyclic peptide (77) exhibited moderate anti-bacterial activity against gram -ve bacterial strains E. coli and S. aureus with inhibition values 19.22 and 18.92, respectively. Cyto-toxic study of synthesized cyclic peptides - CYB (77) was carried out at Deshpandey Laboratories Pvt. Ltd., Bhopal, through MTT assay by using Doxorubicin as standard drug on HCT116 and B16F10 cell lines. The cyto-toxic activity was evaluated by determination of % inhibition of growth of HCT116 and B16F10 cell lines. The conc. of 120-7.5 pg/mL used for test, control and standard drug.
• HCT116 cell lines are taken from colorectal carcinoma of human where as B16F10 cell lines are taken from melanoma of skin of mouse. The cyclopeptide CY B (77) showed maximum percent growth inhibitions 42.5 and 45 against HCT116 and B16F1O cell lines respectively at dose of 120 pg/mL. The CTC5 o values showed by cyclopeptide CY B (77) against HCT116 and B16F1O are 68.55 pM and 97.54 pM respectively. At lower doses percent growth inhibitions are 35 and 37.5 at 60,ug/mL respectively.
e The standard drug (doxorubicin) showed maximum percent growth inhibition 100 with CTC 5 o values 45.49 pM and 42.28 pM against HCT116 and B16F1O respectively.
• Control sample does not show any inhibition.
• Cyclogossine-B is a cyclic octa-peptide, which was previously isolated from latex of Jatropha gossypifolia. Its synthetic route involves the coupling of tetra-peptide fragment (Boc-L-Gly-L-Gly-L-Trp-L-Leu-OMe) (72) with another tetra-peptide fragment (Boc-L-Ala L-Ala-L-Ile-L-Leu-OMe) (73), followed by cyclization of the linear octa-peptide unit under alkaline condition. The structure of newly synthesized compound was characterized by means of spectral data including FT-IR,1 H-NMR, Mass spectroscopy and elemental analysis.
Cyclogossine-B was evaluated for antimicrobial and anticancer activities. The anti-bacterial activity was performed by using Gram+ve bacteria and Gram -ve bacteria whereas the anti fungal activity was determined by using fungal strains like C. albicans, A. niger, T. mentagrophytes and M. audouinii. Similarly, the cytotoxic activity study was carried out through MTT assay using Doxorubicin as standard drug on HCTl16 and B16F10 cell lines. It was observed that this cyclic peptide exhibited significant antimicrobial and cytotoxic activity against cancer cell lines.
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Claims (3)
1. A composition of Cyclo-octapeptide, Cyclogossine B (77) isolated from latex of Jatropha
gossypifolia.
2. The Composition as claimed in claim 1, wherein synthetic route involves the coupling of
tetra-peptide fragment (Boc-L-Gly-L-Gly-L-Trp-L-Leu-OMe) (72) with another tetra-peptide
fragment (Boc-L-Ala-L-Ala-L-Ile-L-Leu-OMe) (73), followed by cyclization of the linear
octa-peptide unit under alkaline condition.
3. The Composition as claimed in claim 1, wherein the structure of newly synthesized
compound was characterized by means of spectral data including FT-IR, IH-NMR, Mass
spectroscopy and elemental analysis. Cyclogossine-B was evaluated for antimicrobial and
anticancer activities.
4. The Composition as claimed in claim 1, wherein the anti-bacterial activity was performed
by using Gram +ve bacteria and Gram -ve bacteria whereas the anti-fungal activity was
determined by using fungal strains like C. albicans, A. niger, T. mentagrophytes and M.
audouinii.
5. The Composition as claimed in claim 1, wherein the cytotoxic activity study was carried
out through MTT assay using Doxorubicin as standard drug on HCT116 and B16F10 cell
lines.
6. The Composition as claimed in claim 1, wherein it was observed that this cyclic peptide
exhibited significant antimicrobial and cytotoxic activity against cancer cell lines.
NH H3C CH3 O O NH NH O NH O NH H3C 2021105736
NH O NH O H3C NH NH O OH3C CH3 CH3
CH3
Fig. (1). Structure of Cyclogossine-B.
NH O O CH3 H3C H3C O NH O NH O + NH O CH3 O O H3C O O CH3 O CH3 H3C i,ii,iii O H3C O NH NH (64) O O NH O H3C CH3 O (65) CH3 (72) H3C CH3 NH H3C CH3 CH3 O CH3 CH3 O CH3 O CH3 H3C O O NH O CH3 + CH3 NH NH CH3 CH3 O CH3 CH3 H3C NH H3C O NH NH CH3 H3C O O NH O O NH H3C O CH3 O CH3 (66) (67) O O O CH3
(73) CH3 O CH3 H3C O NH NH NH NH i,ii,iii O NH HN H3C CH3 O (72) + (73) O O H3C O NH H3C NH O NH i,ii i = LiOH, THF: H2O (1:1), r.t., 1h NH NH CH3 O iv,v O NH O O O CH3 NH O ii = CF3COOH, CHCl3, r.t., 1h O H3C H3C CH3 NH (76) O iii =DMF/ THF, r.t., 24 h CH3 NH O H3C NH iv = DCC, CHCl3, pnp, r.t., 12 h O NH OH3C CH3 CH3 v = TEA/NMM/pyridine, 0°C, 7d CH3 (77)
Scheme 1. Synthetic route to Cyclogossine-B
Effect of CY B (77) on HCT116 Cell line 120
% of inhibition (Cell Viability) 100 100 100
80 70
60 CY B (77) 45 42.5 40 35 Doxorubicin 30 22.5 2021105736
20 15 7.5 0 7.5 15 30 60 120 Concentration of Samples (µg/mL)
Fig.
2 Cyto-toxic effects of Cyclogossine B (77) on HCT116 Cell line in comparison of standard
Effect of CY B (77) on B16F10 Cell line 120 100 100 % of inhibition ( Cell viability)
100
80 70
60 CY B (77) 45 45 37.5 Doxorubicin 40 30 32.5
20 10 7.5 0 7.5 15 30 60 120 Concentration of Samples (µg/mL)
Fig.
3 Cyto-toxic effects of Cyclogossine B (77) on B16F10 Cell line in comparison of standard
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