Neuroprotective metabolites via fungal biotransformation of a novel sapogenin, cyclocephagenol

Cyclocephagenol (1), a novel cycloartane-type sapogenin with tetrahydropyran unit, is only encountered in Astragalus species. This rare sapogenin has never been a topic of biological activity or modification studies. The objectives of this study were; (i) to perform microbial transformation studies on cyclocephagenol (1) using Astragalus endophyte, Alternaria eureka 1E1BL1, followed by isolation and structural characterization of the metabolites; (ii) to investigate neuroprotective activities of the metabolites; (iii) to understand structure–activity relationships towards neuroprotection. The microbial transformation of cyclocephagenol (1) using Alternaria eureka resulted in the production of twenty-one (2–22) previously undescribed metabolites. Oxidation, monooxygenation, dehydration, methyl migration, epoxidation, and ring expansion reactions were observed on the triterpenoid skeleton. Structures of the compounds were established by 1D-, 2D-NMR, and HR-MS analyses. The neuroprotective activities of metabolites and parent compound (1) were evaluated against H2O2-induced cell injury. The structure–activity relationship (SAR) was established, and the results revealed that 1 and several other metabolites had potent neuroprotective activity. Further studies revealed that selected compounds reduced the amount of ROS and preserved the integrity of the mitochondrial membrane. This is the first report of microbial transformation of cyclocephagenol (1).

When 1 H-and 13 C-NMR spectra of 16 were inspected in detail, two additional low-field protons (δ H 3.35 and 3.93) were observed in the low-field region and two extra down-field carbon signals at δ C 81.0 and 83.4 were also noted. The 2D-NMR spectra were examined in detail to deduce hydroxylation locations. Key long-range correlations from H 3 -18 (δ H 1.35) and H-17 (δ H 2.32) to 83.4 ppm substantiated that one of the hydroxy groups was located at C-12 based on the HMBC spectrum. The second hydroxy group was located at C-11 (δ C 81.0) based on the COSY correlation of H-11 (δ H 3.35) with H-12 (δ H 3.93) together with the 3 J H-C correlations from H 2 -19 (δ H 0.57 and 0.83) and H-12 (δ H 3.93) to C-11. The relative configurations at C-11 and C-12 were determined based on the 2D-NOESY data. The orientation of C-12(OH) was found to be β based on NOESY cross-peak between H-12 (δ H 3.93) and α-oriented H 3 -30. The strong correlation of H-11 with one of the β-oriented C-19 protons (δ H-19a 0.83) revealed that the hydroxy group at C-11 was α-oriented. Based on this evidence, the structure of compound 16 was elucidated as 20,25-epoxy-3β,6α,11α,12β,16β,24α-hexahydroxycycloartane.
When the 13 C-NMR and HMBC spectra of 10 and 11 were examined, signals at δ C 210-220 range suggested two keto carbonyl groups in the structure of both compounds. The HMBC correlations from H 2 -11 (δ H 1.96 and 2.64) and H 3 -18 (δ H 1.73) to δ C 211.3 for 10 and H 2 -11 (δ H 2.09 and 2.83) and H 3 -18 (δ H 2.06) to δ C 210.2 for 11 confirmed the location of the first ketone group to be at C-12. Moreover, the low-field signal of H-3 in 10 was absent in the 1 H-NMR spectrum, whereas the H-16 signal disappeared in that of 11. The carbon signal δ C 216.1 had long-range correlations with H 3 -28 and H 3 -29, readily assigned to C-3 in the HMBC spectrum of 10, while the HMBC correlations of H-17 (δ H 3.08) and H 2 -15 (δ H 2.18 and 2.52) with the carbonyl carbon at δ C 214.4 confirmed the oxidation at C-16 to establish the structure of 11. Hence, the structure of 10 was deduced as 20,25-epoxy-6α,16β,24α-trihydroxycycloartan-3,12-dione, and the structure of 11 was established as 20,25-epoxy-3β,6α,24α-trihydroxycycloartan-12,16-dione.
The HR-ESI-MS spectrum of 17 showed a major ion peak at m/z 541.31528 [M + Na] + (C 30 H 46 NaO 7 ). The oxymethine proton at C-3 was lost in the 1 H-NMR spectrum. A detailed inspection of 13 C-NMR and HMBC spectra suggested that the C-3 (δ C 216.3) secondary alcohol had been oxidized and the signal at δ C 211.4 suggested an additional keto carbonyl group in 17, as in 10. Unlike metabolite 10, a broad singlet observed at δ H 3.71 displaying a long-distance correlation with the C-12 (δ C 211.4) suggested an additional oxymethine group. With these findings, a monooxygenation at C-11 was suggested. The configuration of C-11(OH) was deduced based on the 2D-NOESY data. The correlation of H-11 with α-oriented H 3 -30 (δ H 0.66) revealed that the configuration of the OH group at C-11 was β-oriented. Thus, the structure of 17 was determined to be 20,25-epoxy-6α,11β,16β,24α-tetrahydroxycycloartan-3,12-dione.
Ring cleavage and ring expansion products. In the 1 H-NMR spectra of metabolites 19, 20, 21 and 22, the characteristic 9,19-cyclopropane ring signals were absent, suggesting a ring cleavage reaction in the triterpenoid skeleton.  6 , 529.35051) indicating a 16 amu increase over 1, suggesting being a monohydroxy derivative. An additional oxymethylene group at δ C 68.5 and two olefinic carbon signals (δ C 134.5, 132.7) were observed in the low field of the 13 C-NMR spectrum. In the HSQC spectrum, double bond carbons showed no correlation with any proton, denoted a tetrasubstituted olefinic system. Based on these findings, the structure of 19 was proposed to have a C-9(10) double bond with a primary alcohol substitution at C-11 based on our previous biotransformation studies, and this assumption was confirmed by the HMBC experiment 20,23,27,28 . In conclusion, metabolite 19 was determined to be 20,25-epoxy-3β,6α,16β,19,24α-pentahydroxy-ranunculan-9(10)-ene.
The HMBC spectra of 20, 21 and 22 displayed a cross-peak between H-3 (δ H 3.82) and C-10 (δ C 88.7), indicative of an oxygen bridge between C-3, which correlated with H 3 -28 and H 3 -29, and C-10. Based on this evidence and previous biotransformation studies 20,29 , ring B was inferred as a seven-membered ring system. Also, olefinic carbon resonances were detected in the 13 C-NMR spectra. The nature of double bonds was determined by examining the correlation of olefinic carbons with protons in the HSQC spectra.
The molecular formula of 20 was established as C 30 H 48 O 6 by HR-ESI-MS analysis (m/z 527.33874 [M + Na] + , calcd for C 30 H 48 NaO 6 , 527.33486). In the 13 C-NMR spectrum, two additional oxymethine carbons at δ C 80.2 and 88.7 and two olefinic carbon resonances at δ C 126.7 and 139.5 were detected. From the HSQC spectrum, the nature of the double bond was determined as a tetra-substituted olefinic system. The δ C 126.7 and 139.5 resonances were assigned to C-9 and C-8, respectively, based on the long-distance correlations in the HMBC spectrum (C-9 to H 2 -12; C-8 to H 2 -12 and H 3 -30). The resonance observed at δ H 4.39 suggested an additional oxymethine group corresponding to carbon at δ C 80.2 in the HSQC spectrum. A detailed inspection of the 1 H-and 13 C-NMR spectra showed down-field shifts for H-6 and C-6 signals (ca. 0.64 and 8.6 ppm, respectively) when compared to that of 1; therefore, a hydroxylation at C-7 was suggested. The COSY and HSQC spectra revealed a spin system of H-5 (δ H 1.47) → H-6 (δ H 4.42) → H-7 (δ H 4.39), justifying this assignment. In the 2D-NOESY spectrum, the orientation of C-7(OH) was found to be β based on NOESY cross-peaks between H-7 (δ H 4.39) and the α-oriented H 3 -30 and H-5. Consequently, the structure of 20 was established as 3β,10β;20,25-diepoxy-6α,7β,16β,24α-tetrahydroxy-9,10-seco-cycloartan-8-ene.
The HR-ESI-MS data of metabolite 21 displayed a sodium adduct ion at m/z 509.32640 [M + Na] + (calcd. 509.32429 for C 30 H 46 NaO 5 ). The oxymethine proton at C-6 was absent in the 1 H-NMR spectrum. Also, an additional hydroxymethine signal at δ H 4.45 was observed corresponding to carbon at δ C 70.1 in the HSQC spectrum. The 13 C-NMR spectrum of 21 exhibited four olefinic carbon resonances (δ C 128.7, 131.8, 132.1 and 138.5). Locations of the olefinic double bonds were assigned from the correlations in the HMBC spectrum and with the combined use of COSY, HSQC and HSQC-TOCSY spectra. The long-distance correlation from H-5 (δ H 1.96) to the olefinic methine carbon at δ C 128.7 confirmed the location of the double bond between C-6 and C-7. The other tetrasubstituted double bond was positioned between C-8 and C-9 based on the HMBC correlations from H 2 -11a to the olefinic carbon at δ C 131.8 (C-9), and H 2 -11a and H 3 -30 to the second double bond carbon at 138.5 ppm (C-8). The 3 J-HMBC correlations of the oxymethine proton at δ H 4.45 with the C-18 signal revealed oxygenation at C-12. The hydroxy group at C-12 was deduced to be β-oriented based on the NOESY correlation of H-12 (δ H 4.45) with the α-oriented H 3 -30 (δ H 0.94). As a result, metabolite 21 was established as 3β,10β;20,25-diepoxy-12β,16β,24α-trihydroxy-9,10-seco-cycloartan-6,8-diene.
In addition to their protective effect against oxidative injury in SH-SY5Y, the selected compounds were also screened for their effect against 6-OHDA induced neurotoxicity in in vitro Parkinson's disease model. Despite the lower potency of compounds in this model compared to the H 2 O 2 -induced neurotoxicity model, the tested metabolites still showed a statistically significant protective effect, suggesting that they may act as general protective agents against neurotoxicity mediated by a different mechanism of action ( Fig. S 213).

Selected compounds decreased H 2 O 2 -mediated oxidative stress.
Excessive ROS was reported to cause severe cell damage and induce cell death 30 . Since H 2 O 2 is well known to increase the level of ROS, we aimed to determine the effect of compounds on H 2 O 2 -mediated increased ROS levels. Six molecules, including parent compound (1), were selected to detect their potential in rescuing H 2 O 2 -induced oxidative stress, considering their cell viability results, structure, and available quantity. Results showed that treatment with H 2 O 2 significantly increased ROS levels compared to control. In line with the cell viability assay, all selected compounds reduced H 2 O 2 -induced ROS levels in cells (Fig. 3). Parent 1 was the least potent, while 6 and 13 were the most effective compounds reducing ROS at all concentrations (Fig. 3). Interestingly, 2 enhanced ROS production via H 2 O 2 at 10 nM, but higher concentrations significantly decreased ROS level (Fig. 3). tion is one of the most emerging pathological processes in neurodegenerative diseases 31 . Since mitochondrial membrane potential was reported as an indicator to detect mitochondrial dysfunction, we next evaluated the effect of 1 and four selected compounds on mitochondrial membrane potential by using Mitotracker Red. As expected, the fluorescence intensity significantly decreased after treatment with H 2 O 2 treatment, which suggested that H 2 O 2 could induce mitochondrial dysfunction. All selected compounds efficiently protected cells from H 2 O 2 -mediated mitochondrial damage (Fig. 4a,b).

Discussion
In recent years, endophytic fungi have received great attention as a whole-cell catalyst because of their capability to produce enzymes necessary for their colonization. It is anticipated that the endophytic biocatalysts will match chemical reactions even as powerful as conventional chemical methods in near future 3,[13][14][15] . Additionally, studies on fungal biotransformation of plant secondary metabolites with the plant's own endophytes are very  [19][20][21][22][23] . In this study, the biocatalysis of cyclocephagenol (1) by A. eureka yielded twenty-one metabolites. A. eureka was found to be capable of modifications including monooxygenation, dehydration, methyl migration, epoxidation, and ring expansion resulted in the formation of metabolites that would be difficult or impossible to prepare by conventional synthetic methods. Although fungal biotransformation has been used in the modification of natural products for a long time, demonstration of endophytic fungi's utilization in biotransformation is essential for the field not only for utilizing endophytes as potent catalysis systems but also for proving the potential of the plant's own microbiota for transformation studies.
The regioselective hydroxylation at C-12 position was the most prevalent reaction in the cycloartane skeleton. We suggest that A. eureka first catalyzes α-or β-hydroxylation at C-12, then performs further modifications. The other monooxygenation locations were identified as C-7 and C-11. Although hydroxylation at C-7 has been encountered with steroidal sapogenins 19,[21][22][23] , this modification is reported for the first time on the triterpenoid framework by A. eureka.
One of the most interesting biotransformation was the dihydroxylation at C-11 and C-12. Previously, 11α-hydroxy,12β-acetoxy steroids were obtained from the gorgonian Isis hippuris 32 . This is the first report of C-11 and C-12 dihydroxylated products obtained via microbial biotransformation. Additionally, 3(10)-epoxy formation and ring expansion modifications have been reported in the previous studies 20,29,33 , whereas the 6,8diene system is being reported for the first time in triterpenoid chemistry. www.nature.com/scientificreports/ Natural products have the potential for the prevention and treatment of neurodegeneration. In recent years, cycloartane-type saponins, especially astragaloside IV and cycloastragenol, have been reported as a new class of neuroprotective agents 17,18 . Based on the promising bioactivity of cycloartane-types saponins, we first evaluated the neuroprotective activity of cyclocephagenol (1) against H 2 O 2 -induced SH-SY5Y cell death. Compared to cycloastragenol, the neuroprotective activity of 1 started at lower concentrations. Since the neuroprotective activity of cyclocephagenol was noteworthy, a biotransformation study on 1 was carried out to develop a molecule library and to investigate structure-activity relationships by A. eureka.
Among the oxygenated metabolites, 2 and 6 possessing hydroxy group at position 12 (12β and 12α, respectively) showed potent neuroprotective activity. While the most active concentration of 2 was 10 nM, the neuroprotection by 6 was observed at a higher concentration (100 nM). Also, the metabolite 13 possessing hydroxy group at position 11 demonstrated potent neuroprotective activity at higher concentrations (100 and 1000 nM). On the other hand, a dihydroxy group in ring C (16; 11α,12β-dihydroxy) led to a loss of neuroprotection suggesting that the hydroxylation pattern was critical for the activity.
As a result of SAR studies, we conclude that i) monooxygenation at positions 11 (13) and 12 (2 and 6) is significant bioactivity; ii) oxidation at C-12 (9, 10, 17) improves neuroprotective activity; iii) further increase of hydrophobicity (5, 8, 11, 12) and hydrophilicity (16, 18) diminishes bioactivity; iv) 3(10)β-epoxy-9,10-secocycloartane products formed by ring expansion and epoxidation reactions could be potential neuroprotective agents as long as they have conformational flexibility. Further studies revealed that selected compounds reduced the amount of ROS and preserved the integrity of the mitochondrial membrane.

Conclusion
Collectively, the biocatalyst potential of A. eureka, which plays an important role in expanding our cycloartane molecule library, has been demonstrated once again by producing 21 new metabolites. In addition to chemical diversity, biotransformation provided several novel compounds having potent protective activity against H 2 O 2and 6-OHDA-induced neurotoxicity. Further studies are warranted to establish a mechanism of action of the bioactive metabolites.

Methods
General experimental procedures. The spectroscopic (NMR and HR-ESI-MS) and chromatographic procedures were described previously 26 . Microorganism and starting compound. Cyclocephagenol (1) and cycloastragenol were provided by Bionorm Natural Products, Ltd. (İzmir, Turkey). The fungal endophyte used in this study was isolated from leaves of Astragalus angustifolius and the original strain (Deposit number: 20131E1BL1) was banked at the Bedir Laboratory 20 . Before biotransformation process, the stock culture of A. eureka was pre-cultivated on PDA in Petri dishes for 10 days at 25 °C.

Microbial biotransformation procedures. A one-stage microbial biotransformation process was car-
ried out at a preparative scale using a biotransformation medium 26 . A preparative scale biotransformation study was performed utilizing 1850 mg of 1 with A. eureka for 13 days (25 °C and 180 rpm).

Biological activities. Determination of cell viability. SH-SY5Y cell line was maintained in high-glucose
Dulbecco's modified Eagle medium (DMEM) containing 10% FBS at 37 °C, and 5% CO 2 . The cells were homogenously seeded in 96 well plate (20,000 cells/well) and incubated for 24 h. Following 2 h incubation with compounds or vehicle (DMSO), the cells were treated with 70 µM H 2 O 2 . For the 6-OHDA mediated toxicity experiments, cells were treated with 50 µM 6-OHDA after 8 h of treatment with the compounds. For both experiments, cell viability was determined after 24 h via the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Briefly, the cells were incubated with MTT (0.5 mg/ml final concentration) for 4 h. Then, all the media was pulled out, and DMSO was added to wells. Photometric absorbance was measured at a wavelength of 590/690 nm by using Varioscan flash spectrophotometer by Thermo Scientific. The statistical significance of Determination of ROS levels. SH-SY5Y cells were seeded onto the 6 well plates and were treated with either compounds or the vesicle for 2 h. Then, cells were treated with 70 µM H 2 O 2 . Following procedures were performed as described previously 34 .
Determination of mitochondrial membrane potential. SH-SY5Y cells were seeded onto the coverslip. After 2 h pretreatment with 100 nM of each compound, cells were exposed to 70 µM H 2 O 2. Following 24 h incubation, cells were treated with 100 nM MitoTracker ® Red FM (Thermo Fisher Scientific, US) for 30 min at 37 °C. Then cells were washed with PBS. After mounting, cells were immediately observed using a fluorescence microscope (Olympus IX70). All photographs were taken under the same conditions, and the fluorescence intensity of the mitochondria relative to the cell volume was calculated in 30 cells using the ImageJ software.

Data availability
The data that support the findings of this study are available from the corresponding authors (EB and PBK) upon reasonable request.  www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.