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

Investigating the effect of solvent extraction on CyA production efficiency during fermentation

During a 10-day microbial fermentation process, a culture medium was utilized. The initial separation of the target compound, CyA, was performed using a centrifuge. Subsequently, the quantification of CyA in the supernatant solution was carried out using HPLC. The measured concentration of CyA in the supernatant was determined to be 334 mg/L. Dairy sludge, which is a byproduct of dairy factories, shares similarities in composition with milk and contains various nutrients such as proteins, fats, carbohydrates, and minerals. This composition makes it a cost-effective fermentation environment for the production of fermented products37.

In the subsequent step of the process, ethyl acetate solvent was utilized for extraction, resulting in a measured CyA concentration of 456 mg/L in the sample. A comparison of the CyA levels before and after extraction revealed a 36% increase in the amount of the metabolite. The non-polar and hydrophobic nature of CyA can be attributed to the abundance of methyl groups present in its amino acid residues. This characteristic makes CyA more soluble in non-polar solvents, such as alcohols, compared to water. The solubility of CyA in water, ethanol, and methylene chloride is reported as 0.04, 10, and 100 mg/mL, respectively. This information demonstrates the higher solubility of CyA in non-polar solvents like methylene chloride, which explains the increased concentration observed after the extraction step using ethyl acetate38. The choice of extracting solvent affects the structural properties of CyA, leading to variations in its solubility. When a non-polar solvent is used, the intramolecular hydrogen bonds within the β-sheet structure of CyA remain intact. However, the use of a polar solvent disrupts the three-dimensional conformation of CyA39. CyA, produced by fungi, exists in two forms: freely available in the fermentation medium and sequestered within the fungal vacuole. To obtain a higher yield of CyA, it is necessary to release it from the fungal vacuole. This requires a solvent that can create pores in the cell wall, traverse the cell membrane, and enter the fungal vacuole. The cell wall’s rigid structure poses the primary barrier to material transport. Therefore, overcoming this resistance is crucial for successful extraction. Additionally, the solvent must possess the capability to dissolve the solid phase of CyA present in the vacuole. CyA is linked to lipoproteins and lipolytic molecules through various chemical and physical bonds within the mycelium. Thus, the solvent must be capable of breaking these bonds. Finally, the combination of the solvent and CyA needs to traverse the hyphal cellular compartments, the hyphal surface, and the solid–liquid interface through mass transfer. Ultimately, the solvent-CyA mixture should enter the liquid phase to complete the extraction process38,40. The use of ethyl acetate as a solvent for extracting CyA has shown promising results in increasing the yield of CyA. This solvent has demonstrated its effectiveness in traversing the cell wall, reaching the site where CyA accumulates, and overcoming barriers to mass transfer. Ethyl acetate has been successfully utilized by Balaraman and Mathew41 as well as Sharmila et al.42 for the isolation of CyA. Indeed, Tanseer and Anjum25 employed n-butyl solvent for the extraction of CyA from the culture medium of Aspergillus terreus strain. Likewise, Shetty et al.43 utilized ethyl acetate solvent for the extraction of antibiotics produced by Streptomyces parvulus. These studies highlight the versatility of different solvents in extracting bioactive compounds from various microbial sources43.


The purification process of CyA involved using FPLC packed with silica gel, and elution was carried out using a mixture of n-hexane and ethyl acetate in a 20:80 (v/v) ratio. This process resulted in obtaining a pure compound. To validate the samples obtained from the purification column, a TLC test was conducted. The sample that exhibited a line similar to that of the pure sample (Fig. 1a–A,B) was selected as the sample containing CyA. The CyA content of the pure sample was further measured using HPLC, and a value of 578 mg/L was determined. This represented a significant increase in the amount of CyA compared to the extracted and primary sample. The pure sample exhibited a 42% increase in CyA content compared to the initial sample before extraction, indicating the efficiency of the purification methods used. Consistent with previous research, TLC, column chromatography using silica gel, and HPLC were employed for the purification and measurement of CyA42. In addition to the purification process, the antimicrobial properties of the obtained sample were evaluated using a bioassay. The diameter of the clear zone formed around the fungal colony was examined before and after purification. It was observed that the diameter of the clear zone after purification was greater than that in the sample before purification (Fig. 1b–A,B). The size of the clear zone can be considered as an indicator of the concentration of CyA, as there is a direct relationship between the concentration and the size of the clear zone. This suggests that the purification process not only increased the concentration of CyA but also enhanced its antimicrobial activity, as evidenced by the larger clear zone observed in the bioassay. This indicates the potential of purified CyA as an effective antimicrobial agent22.

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).

Analysis of the structure of CyA

LC/MS/MS profiling

LC–MS/MS has indeed gained significant popularity and utility in clinical routine environments in recent years. This technique combines the separation power of liquid chromatography with the sensitivity and specificity of mass spectrometry. As a result, LC–MS/MS enables the precise identification and quantification of a wide range of analytes in complex biological samples. Its ability to handle diverse compounds and provide accurate measurements has made it an invaluable tool in various fields, including pharmacokinetic studies, drug monitoring, toxicology, and biomarker analysis. LC–MS/MS has revolutionized analytical chemistry and has become an indispensable technique in modern clinical research and diagnostics44. The LC–MS/MS method was utilized to identify metabolites from the purified fungal extract. The validation process involved analyzing accuracy and precision in three replicates. The standard analytical range of 9–1000 ng/mL was calculated for both the purified and standard CyA. Quantitative data was obtained using selected ion monitoring (SIM) of the hydrogen adduct of cyclosporine, with an m/z value of 1203, representing [M + H] + CyA. By comparing the LC–MS chromatogram of the standard CyA with the purified sample, the results confirmed the presence of CyA with an m/z value of 1203 [M + H]+, indicating successful identification. Based on the analysis of the chromatogram in terms of retention time for both the standard and purified samples, the retention time for CyA was found to be between 10 and 13 min (Fig. 2). This similarity in retention time between the two samples provides evidence for the presence of CyA. However, the standard CyA chromatogram appeared sharper than the purified sample chromatogram, indicating the presence of impurities in the sample. The purified sample was reported to have a purity value of 91.9%, as determined by LC/MS/MS. Furthermore, an investigation was conducted comparing the purified and standard CyA. It was observed that the peak area of the LC/MS chromatogram with an m/z value of 1203, corresponding to [M + H]+, was 2.1 times higher in the standard sample than in the purified one (Fig. 2). This difference in peak area suggests that the concentration of CyA in the purified sample is lower compared to the standard, indicating the need for further optimization of the purification process. Regarding the adducts ([M + H]+, [M + Na]+, [M + K]+), they refer to ions resulting from ion/molecule reactions, where the ion attaches itself to the molecule. These adducts can provide additional information about the molecular structure and behavior of the analyte.

Figure 2
figure 2

LC/MS/MS chromatograms of purified (up), and standard (down) CyA samples.

The use of LC–MS/MS for assessing purity and detecting other metabolites has been widely reported in various research articles across different fields45,46,47,48. Abrol et al.49 conducted a study using LC–MS/MS to investigate the production of CyA by 11 mutant strains of T. inflatum. The LC–MS/MS profile of the crude fungal extract confirmed that both the wild type and mutant strains (MT1-3538, MT2-3538) were indeed more suitable for CyA production. Through analysis of the ion intensity profile, the researchers discovered that the mutant strain MT2-3538, when cultivated in an optimal media composition, exhibited a significant 16-fold increase in CyA efficiency compared to the other strains. This finding suggests that the specific genetic mutation in the MT2-3538 strain, along with the optimized growth conditions, had a substantial impact on enhancing the production of CyA49. Lam et al.50 conducted a study where they employed tandem mass spectrometry (MS/MS) to differentiate between various forms of CyA analogues. Specifically, their focus was on distinguishing between CyA and CycH (CyH), which are enantiomers, as well as isoCyA, a structural isomer of CyA and CycH. By utilizing the MS/MS technique, Lam et al. were able to fragment and analyze the molecular ions of these CyA analogues, enabling their differentiation based on their distinct fragmentation patterns. The MS/MS analysis provided valuable information about the mass-to-charge ratio (m/z) values of the fragmented ions, which proved instrumental in distinguishing between the different forms of the analogues. This study highlights the practicality of tandem mass spectrometry in accurately characterizing and differentiating closely related compounds such as CyA, CycH, and isoCyA50.


H-NMR spectroscopy was utilized for the structural analysis and purity assessment of purified CyA. Figure 3 depicts the H-NMR spectra of both standard and purified CyA samples. CyA, with its intricate structure of cyclo-[-MeBmt1-Abu2-Sar3-MeLeu4-Val5-MeLeu6-Ala7-D-Ala8-MeLe9-MeLeu10-MeVal11], exhibits a highly complex proton spectrum in the high-field region. In both samples, the spectrum revealed the presence of four doublets in the 6–8 ppm range, corresponding to the amide protons. Additionally, approximately 23 protons, representing the protons of the 11 amino acids and olefinic protons, were observed in the 4–6 ppm range. Furthermore, the N-methyl protons were clearly distinguishable as seven distinct peaks within the 2.6–3.5 ppm range. Notably, the peaks’ intensity was higher in the 0.5–3 and 7–8 ppm range, which aligns with the findings reported in previous H-NMR studies on CyA. The utilization of H-NMR spectroscopy provided valuable insights into the structural characteristics of purified CyA and facilitated the evaluation of its purity. The observed spectral features are consistent with the findings reported in previous studies, thereby validating the reliability and conformity of the obtained results32,51,52.

Figure 3
figure 3

1H-NMR spectrum of purified (A), and standard (B) CyA samples.

Figure 3 illustrates the chemical shifts corresponding to the protons of each amino acid segment in CyA. The spectra characteristics of the purified sample are displayed as follows: DAL8 HβHα (0.76` ppm), MLE9 Hβ2Hα (0.78 ppm), MLE10 Hβ2Hα (0.83 ppm), MLE6 Hβ2Hα (0.85 ppm), MLE4 Hβ2Hα (0.88 ppm), ALA7 HβHα (1.1 ppm), BMT1 HƞHζ (1.14 ppm), ABU2 HβHα (1.19 ppm), BMT1 Hδα2Hε (2.5 ppm), MLE9 Hβ1Hα (2.59 ppm), VAL11 HβHα (2.7 ppm), MLE4 Hβ1Hα (2.77 ppm), MLE6 Hβ1Hα (2.84 ppm), MLE10 Hβ1Hα (2.95 ppm), VAL5 HβHα (2.99 ppm), BMT1 HδβHε (3.05 ppm), BMT1 HβHα (3.35 ppm), VAL5 HNHα (6.5 ppm), DAL8 HNHα (6.67 ppm), ALA5 HNHα (8.4 ppm), ABU2 HNHα (8.5 ppm). The Schiff chemical of standard sample was DAL8 HβHα (0.76 ppm), MLE9 Hβ2Hα (0.78 ppm), MLE10 Hβ2Hα (0.83 ppm), MLE6 Hβ2Hα (0.85 ppm), MLE4 Hβ2Hα (0.88 ppm), ALA7 HβHα (1.1 ppm), BMT1 HƞHζ (1.14 ppm), ABU2 HβHα (1.20 ppm), BMT1 Hδα2Hε (2.52 ppm), MLE9 Hβ1Hα (2.62 ppm), VAL11 HβHα (2.7 ppm), MLE4 Hβ1Hα (2.8 ppm), MLE6 Hβ1Hα (2.83 ppm), MLE10 Hβ1Hα (2.92 ppm), VAL5 HβHα (2.98 ppm), BMT1 HδβHε (3.04 ppm), BMT1 HβHα (3.35 ppm), VAL5 HNHα (6.58 ppm), DAL8 HNHα (6.67 ppm), ALA5 HNHα (8.38 ppm), ABU2 HNHα (8.5 ppm)52. These values are in accordance with those found by Sinnaeve et al.53.

The comparison of H-NMR spectra between the standard and purified samples indicates that the peaks in the standard sample exhibit sharper characteristics compared to those in the purified sample. This discrepancy can be attributed to the presence of impurities within the structure of the purified CyA extract. Similar observations have been reported by Price et al.19 and Ohta et al.54, and Gendron55 in their respective investigations of CyA structure using H-NMR. These studies corroborate the findings obtained in our own study, providing further validation of the consistency and reliability of the observed peaks.

FT-IR and Raman spectroscopy

To identify and detect the functional groups present in the structure of the purified CyA and compare them with the standard sample, we utilized FT-IR analysis and Raman spectroscopy. The results obtained from the FT-IR analysis, as depicted in Fig. 4, offer valuable insights into the functional groups present in the purified CyA sample.

Figure 4
figure 4

The FT-IR spectra of purified, and standard CyA samples.

In the structure of CyA, which consists of 11 different amino acids, there are numerous carboxyl and amine groups. During the FT-IR analysis, distinctive peaks corresponding to specific vibrational modes were observed in both the standard and purified samples. The N–H stretching and N–H bending vibrations, associated with the amine groups, display characteristic peaks in the broad absorption region of 3100–3500 cm−1 and 1410–1500 cm−1, respectively, in both samples. In the purified sample, specific peaks at 1206, 1266, and 1636 cm−1 indicate the C–O stretching vibrations of carboxyl groups, C–N stretching vibrations of amine groups, and C=O stretching vibrations of amide groups, respectively. Furthermore, the range of 2870–2960 cm−1 exhibits peaks that are characteristic of C–H stretching vibrations. Notably, in the purified sample chromatogram, distinct peaks at 791 and 1470 cm−1 can be attributed to the stretching and bending vibrations, respectively, of CH2 groups. These findings from the FT-IR analysis provide valuable insights into the presence and characteristics of various functional groups in the structure of purified CyA, allowing for a comparison with the standard sample56.

The Raman spectrum of the purified sample can be observed in Fig. 5. In this spectrum, two bands are observed at 2944 and 2939 cm−1, which correspond to the stretching vibration of CH2 and CH3 groups, respectively. Additionally, a band at 1453 cm−1 is assigned as the bending vibration of N–H. A strong band is observed at 1665 cm−1, which is assigned as an in-plane vibration involving the C=O bond. Furthermore, rocking of NH3+ is observed at 1117 cm−1, while the antisymmetric bending vibration of CNH2 appears at 11,246 cm−1.

Figure 5
figure 5

The complete Raman spectrum of purified sample.

The bending vibrations of C–H were observed at 1325 and 1333 cm−1. The signal at 1315 cm−1 is related to stretching vibrations of C–O groups. Jenkins et al.57 employed Raman spectroscopy to examine the bonds and functional groups within the structure of CyA. Their analysis revealed the presence of amine, carbonyl, carboxyl, hydroxyl, and methyl groups in the structure of CyA57.

Crystallinity and thermal analysis of the CyA

The XRD test was conducted using Cu-Kα radiation at a voltage of 40 kV and a current of 40 mA. The scanning range of 2θ was set from 5° to 50°. In XRD analysis, the presence or absence of sharp diffraction peaks in the pattern is used to characterize a pharmaceutical powder as either crystalline or amorphous. As depicted in Fig. 6, both samples of CyA exhibited distinct diffraction peaks within the range of 5° to 45° (2θ), indicating a crystalline nature.

Figure 6
figure 6

XRD pattern of purified (A), and standard (B) CyA samples.

The XRD patterns obtained from both the lyophilized CyA (standard) and the purified CyA exhibited two broad peaks at 2θ = 9° and 19°, accompanied by characteristic narrow diffraction peaks. These findings indicate the presence of a semi-crystalline state in CyA, suggesting the absence of long-range three-dimensional order. The presence of broad peaks in the XRD patterns signifies a degree of disorder or amorphous nature in the samples. However, the presence of narrow diffraction peaks suggests the existence of localized ordering or crystalline domains within the overall semi-crystalline structure of CyA. The semi-crystalline nature of CyA, with the lack of long-range order, may be attributed to various factors such as molecular packing, intermolecular interactions, or the presence of impurities. Further investigations are required to fully comprehend the implications of this semi-crystalline state on the properties and behavior of CyA58,59. The intensity of the peak observed at 2θ = 9° in the purified sample is higher than the intensity of the peak at 2θ = 19°, which is in contrast to the standard sample. This difference in peak intensities suggests the presence of impurities in the structure of the purified sample, as well as the presence of water within its structure. These impurities and water content could contribute to the altered intensities of the peaks. This finding aligns with the results reported by Jain et al.60, where changes in peak intensities were observed after drying a sample using a freeze dryer. The presence of impurities and water in the purified sample may affect its crystalline structure, leading to variations in peak intensities in the XRD pattern. Further analysis and characterization are necessary to better understand the specific impact of these factors on the structural properties of the purified sample60. Two broad peaks at 2θ = 9° and 20°, and characteristic narrow diffraction peaks, suggesting the existence of the semi-crystalline state of CyA, which is in accordance with the findings of this study61. In the tetragonal crystal structure, CyA has been described as having a twisted antiparallel β-sheet conformation. Within the orthorhombic crystal structure, the β-OH group of the MeBmt 1 residue is capable of forming a bond with a structural water molecule present in the crystalline monohydrate. This water molecule, in turn, participates in intramolecular hydrogen bonding with the carboxyl group of the MeLeu10 residue (as depicted in Fig. 6). However, in the dried thermotropic liquid crystal conformation, it is suggested that the β-sheet, g-loop, type II β-turn, and MeBmt1 turn structures remain intact. This conformation differs from the three-dimensional crystal structures due to the loss of a g-turn structure resulting from the absence of a hydrogen bond between the amide of D-Ala 8 and the carbonyl oxygen of MeLeu6 58. In line with the findings of this study, the crystal structure of CyA has been confirmed by Sun et al.35 and Guada et al.59 through XRD analysis.

DSC is a powerful method used to identify the physical and chemical structure of materials and perform thermal analysis. It quantitatively measures energy changes and is employed to determine melting temperature, latent heat of melting, glass transition temperature (Tg), and crystallization temperature. Notably, the solid-to-liquid transition exhibited glass transition characteristics. The glass transition temperature can serve as a measure of macromolecular mobility, providing insights into the molecular mobility of CyA and changes in its semi-crystalline state62. Figure 7 represented the DSC curves of standard and purified CyA.

Figure 7
figure 7

DSC curved of purified (A), and standard (B) CyA samples.

In this study, the glass transition temperatures of the purified and standard CyA samples were identified as 57–68 °C and 53–64 °C, respectively. The DSC thermogram revealed melting endothermic peaks ranging from 55 to 180 °C for the pure CyA sample and 70–200 °C for the standard CyA sample. Additionally, typical endothermic peaks indicating CyA crystallization temperature were observed at approximately 186–230 °C for the purified sample and 200–250 °C for the standard sample. The DSC curve analysis of CyA yielded consistent results, indicating that the glass transition temperature of pure CyA was found to be in the range of 51–55 °C61. The absence of variation in the lyophilized polymer and polymer of the device indicated an unchanged mobility from polymeric chains, and suggested the absence of detectable interactions between polymer and drug. In a study of Li et al.63, a temperature of 130 °C was reported for the crystallization of CyA. Sun et al.35 and Dubey et al.64 used DSC to investigate the thermal characteristics of CyA used in the nanofiber structure, which obtained similar results to this study. The study on CyA loading in lipid nanoparticles reported melting and crystallization temperatures of 140 °C and 200 °C, respectively, for pure CyA. These findings align with the results of the current study, indicating consistency in the temperature ranges for both melting and crystallization of CyA59. The DSC curve analysis of the purified CyA sample revealed a close resemblance to the standard sample. This similarity indicates that the thermal characteristics of these two samples are not significantly different from each other.

Investigation of particle distribution and surface characteristics

Particle size plays a crucial role in determining the solubility, spreadability, bioavailability, and effectiveness of materials. When particles are smaller, the ratio of surface area to volume increases. Consequently, this enhanced surface area provides more opportunities for substances to come into contact, react, and exert their effects, ultimately improving solubility and efficacy65. The analysis of the standard sample revealed an average particle diameter of 149.54 nm (intensity), 128.91 nm (volume), and 51.26 nm (number). In comparison, the refined sample exhibited an average particle size of 136.15 nm (intensity), 111.72 nm (volume), and 53.13 nm (number) (Fig. 8). These findings indicate a slight difference in particle size between the standard and purified samples. However, both samples displayed a uniform and similar particle size distribution. It is important to note that maintaining a narrow size distribution is crucial for preserving the functional characteristics and maximizing the effectiveness of particles. The extraction and purification processes employed in this research successfully generated particles with a uniform distribution, validating the accuracy of the downstream fermentation processes. The PDI (polydispersity index) values for both samples were low, indicating relatively homogeneous populations (less than 0.5). Furthermore, in a separate study, CyA-loaded micelles were reported to have a size of 137 nm and a PDI of less than 0.2, as per the results of this work64. Also, the particle size of 163 nm for encapsulated CyA was obtained60.

Figure 8
figure 8

DLS curve related to purified (A), and standard (B) samples of CyA.

Zeta potential measurements were carried out to assess the stability of the electrostatic colloidal dispersion of the purified CyA particles and the standard sample. Both the pure and standard CyA particles were characterized for surface charge using a zeta meter. The zeta potential values obtained were − 25.8 ± 0.16 mV for the pure sample and − 23.63 ± 0.12 mV for the standard sample. A higher negative zeta potential indicates increased electrostatic repulsion between particles, which contributes to enhanced stability and favorable colloidal properties. In this case, the zeta potential of the purified CyA was not significantly different from that of the standard sample. However, there was a slight difference, which can be attributed to the lower purity of the purified sample. It is worth noting that the purified CyA sample did not exhibit any noticeable changes in appearance after being stored in an ambient atmosphere. This lack of change may be attributed to the high zeta potential, which could potentially prevent significant aggregation in the suspension66.