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. 2024 May 31;14(1):12540.
doi: 10.1038/s41598-024-63110-y.

Bio-synthesis, purification and structural analysis of Cyclosporine-A produced by Tolypocladium inflatum with valorization of agro-industrial wastes

Fereshteh Falah  1 , Ali Samie  2   3 , Seyed Ali Mortazavi  4 , Abolghasem Danesh  5 , Farideh Tabatabaei Yazdi  1 , Mohammad Ramezani  6
Affiliations

Bio-synthesis, purification and structural analysis of Cyclosporine-A produced by Tolypocladium inflatum with valorization of agro-industrial wastes

Fereshteh Falah et al. Sci Rep. .

Abstract

Cyclosporine A (CyA) holds significant importance as a strategic immunosuppressive drug for organ transplant patients. In this study, we aimed to produce pure and cost-effective Cyclosporine A (CyA) by fermenting a culture medium containing dairy sludge, using Tolypocladium inflatum PTCC 5253. Following the fermentation stage, ethyl acetate extraction and fast protein liquid chromatography were employed for sample purification. The initial evaluation of the effectiveness of CyA obtained from these processes was performed through bioassay, wherein the antimicrobial clear zone diameter was found to be larger compared to the sample obtained from the fermentation culture. The concentration of CyA was determined using high-performance liquid chromatography, yielding values of 334 mg/L, 456 mg/L, and 578 mg/L for the fermented, extracted, and purified samples, respectively. Further analysis utilizing liquid chromatography tandem mass spectrometry (LC/MS/MS) confirmed a purity of 91.9% and proper agreement with the standard sample based on the ion intensity of Z/m 1205. To validate the structure of CyA, nuclear magnetic resonance spectroscopy, Fourier-transform infrared (FT-IR), and Raman spectroscopy were employed. X-ray diffraction and differential scanning calorimetry analyses demonstrated that the purified CyA exhibited a crystal structure similar to the standard sample, characterized by two broad peaks at 2θ = 9° and 20°, and comparable glass transition temperatures (57-68 °C for the purified sample; 53-64 °C for the standard sample). Dynamic light scattering analysis confirmed a uniform particle size distribution in both the purified and standard samples. The zeta potentials of the purified and standard samples were determined to be - 25.8 ± 0.16 and - 23.63 ± 0.12 mV, respectively. Our results demonstrate that dairy sludge can serve as a suitable culture medium for the production of (CyA).

Keywords: Tolypocladium inflatum; Chemical structure; Cyclosporine; Purification.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) TLC of purified (A), and standard (B) CyA samples at UV-254 nm. (b) The images of the plates obtained from the bioassay test related to the plate of the optimal sample (before extraction) (A), and the purified sample (B).
Figure 2
Figure 2
LC/MS/MS chromatograms of purified (up), and standard (down) CyA samples.
Figure 3
Figure 3
1H-NMR spectrum of purified (A), and standard (B) CyA samples.
Figure 4
Figure 4
The FT-IR spectra of purified, and standard CyA samples.
Figure 5
Figure 5
The complete Raman spectrum of purified sample.
Figure 6
Figure 6
XRD pattern of purified (A), and standard (B) CyA samples.
Figure 7
Figure 7
DSC curved of purified (A), and standard (B) CyA samples.
Figure 8
Figure 8
DLS curve related to purified (A), and standard (B) samples of CyA.
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