New Pregnane Glycosides from Mandevilla dardanoi and Their Anti-Inflammatory Activity

Mandevilla Lindl. is an important genus of the Apocynaceae family, not only as ornamental plants but also for its medicinal uses. In Brazil, Mandevilla species are indicated to treat asthma and skin infections, their anti-inflammatory potential and wound healing properties are also reported in the literature. Concerning their chemical composition, this group of plants is a conspicuous producer of pregnane glycosides. Mandevilla dardanoi is an endemic species from the Brazilian semiarid region not studied by any phytochemical methods. In view of the medicinal potential of Mandevilla species, this study aimed to isolate new pregnane glycosides from M. dardanoi. To achieve this main goal, modern chromatography techniques were employed. Five new pregnane glycosides, dardanols A-E, were isolated from the roots of M. dardanoi by HPLC. Their structures were determined using extensive 1D and 2D-NMR and mass spectrometry (MSn and HRESIMS) data. The cytotoxicity and the anti-inflammatory potential of these compounds were evaluated. The first was evaluated by measuring proinflammatory cytokines and nitric oxide production by stimulated macrophages. Dardanols were able to inhibit the production of nitric oxide and reduce IL-1β and TNF-α. The current work demonstrates the chemodiversity of Brazilian semiarid species and contributes to amplifying knowledge about the biological potential of the Mandevilla genus.


Introduction
Mandevilla Lindl. is one of the largest neotropical genera of the Apocynaceae family, this group comprises approximately 170 species [1][2][3]. First registered in 2017, M. dardanoi M.F. Sales, Kin.-Gouv. & A.O. Simões is an endemic species from the Brazilian semiarid region, occurring in the states of Pernambuco and Paraíba [4], there are no studies regarding their chemical composition or pharmacological activities [4]. Rich in flavonoids, steroids, and pregnane glycosides Mandevilla species have been used by folk medicine in therapy for snakebites, wound healing, and to treat skin infections and inflammation [5][6][7][8][9][10].
Composed of a steroidal scaffold, the pregnane (C 21 ) and seco-pregnane-type glycosides mainly occur in Apocynaceae, Malpighiaceae, Ranunculaceae, and Zygophyllaceae families [11]. This class of compounds has demonstrated remarkable biological activities, including anticancer, antinociceptive, anti-inflammatory, antiviral, and antibacterial properties [12][13][14][15][16]. Considering the medicinal potential of Mandevilla species, the current study aimed to isolate novel bioactive pregnane derivatives from the roots of M. dardanoi. Five new pregnane glycosides, dardanols A-E, were isolated and characterized by spectroscopic and spectrometric analysis. The cytotoxicity and anti-inflammatory activity of dardanols A, B, C, and E were also assessed. All of the compounds tested were active in the models applied.  3 -19), methyl hydrogens, associated with their respective carbons at δ C 104.2 (C-16), 122.1 (C-6), 78.9 (C-20), 77.5 (C-3), 16.9 (C-18) and 19.7 (C-19), are in agreement with the values described for the aglycone illustrol, a seco-norpregnane derivative, isolated from M. illustris and reported only once [17]. The five anomeric hydrogen signals at 4.76 (dd, J = 9.5, 2.0 Hz, H-1 ), 5.47 (dd, J = 4.4, 2.2 Hz, H-1 ), δ H 5.15 (dd, J = 9.6, 1.6 Hz, H-1 ), δ H 5.08 (dd, J = 9.6, 1.7 Hz, H-1 ) and 4.71 (dd, J = 9.7, 2.0 Hz, H ), as well as the presence of the methyl hydrogen signals at 1.49 (3H, d, J = 6.4 Hz, C-6 ), 1.33 (6H, d, J = 6.2 Hz, C-6 and C-6 ), 1.39 (3H, d, J = 6.2 Hz, C-6 ) and 1.29 (3H, d, J = 6.4 Hz, C-6 ), and the hydrogens of the methoxy groups at δ H 3.33 (s, OCH 3 ), δ H 3. 39 (s, OCH 3 ), δ H 3.60 (s, OCH 3 ), δ H 3.46 (s, OCH 3 ), and δ H 3.38 (s, OCH 3 ) indicated the existence of five osidic units in the structure of 1. This evidence was corroborated by the presence of the signals in the 13 C NMR spectrum corresponding to five anomeric carbons at δ C 102. 9, 100.9, 100.8, 99.0, and 97.3, five methyl carbons at δ C 19.0, δ C 18.9, δ C 18.4, δ C 17.5, and δ C 17.2, and five methoxy carbons at δ C 59.3, δ C 58.9, δ C 56.8, δ C 56.7, and δ C 56. 5. In the HSQC spectrum, the anomeric hydrogen at δ H 4.76 (dd, J = 9.5, 2.0 Hz) correlated with the carbon signal at δ C 99.0. In the HMBC spectrum, this same hydrogen showed a long-distance correlation with the carbon at δ C 77.6 (C-3), thus being assigned to H-1 . Conversely, the hydrogen signal at δ H 3.81 (H-3) correlated with the carbon at δ C 99.0, confirming the bonding of the first glycosyl unit to the aglycone portion. Furthermore, this assignment was corroborated through cross-correlations of δ H 3.81 (H-3) and δ H 5.41 (H-6) with δ C 39.6 (C-4). In the COSY spectrum, a correlation of the signal at δ H 4.76 (H-1) with δ H 2.22 (H-2 ) and of that with δ H 3.41(H-3 ) was observed. In the HSQC spectrum, H-2 and H-3 correlated with the carbons at δ C 28.7 (C-2 ) and δ C 81.3, respectively. These data compared with the literature allowed us to assign the osidic unit I as being diginopyransideo [18]. The H-1 coupling constants at 9.7 and 2.0 Hz are compatible with β-glycosidic bonding. According to the literature, a chemical shift of C-2 of the osidic units smaller than 34 ppm corresponds to an L configuration and larger than 35 ppm to a D configuration [19]. This effect is associated with the orientation of the ether group in the axial present in C-4, which causes a gamma shielding effect at C-2 . Thus, with the chemical shift of C-2 being equal to δ C 28.7, it was possible to assign the configuration of the osidic unit I as β-L-diginopyranosyl.  In the HSQC spectrum, the anomeric hydrogen at δ H 5.47 (dd, J = 4.4 and 2.2 Hz) correlates with the carbon at δ C 97.3. In the HMBC spectrum, the same signal correlated with the carbon at δ C 71.0 (C-4 ) was assigned to H-1", confirming the union of the sugars through 1-4 bonds. In the COSY spectrum, a correlation of the hydrogen H-1" was observed with the signal at δ H 2.18 and of this with the signal at δ H 3.96, which were assigned to H-2" and H-3". In the HSQC spectrum, H-2" and H-3" correlate with the carbons at δ C 33.8 and δ C 76.3, respectively. Thus, by comparing the other data that are presented in Table 1 with the data present in the literature, it is possible to assign the osidic unit II as being the sarmentopyranoside [11]. The coupling constants at 4.4 and 2.2 Hz are compatible with α-glycosidic bonding and the C-2" value at δ C 33.8 with the L configuration for acidic unit II, which was assigned as β-L-sarmentopyranosyl.

Structural Assignment
In the HSQC spectrum, the anomeric hydrogen at δ H 5.15 (dd, J = 9.6 and 1.6, H-1 ) correlated with the carbon at δ C 100.8. In the HMBC spectrum, a correlation of the signal at δ H 3.73 (H-4 ) with the carbon at δ C 100.8 was observed, confirming the binding of osidic unit II to III via 1-4 bonds. Similar to assignments performed previously, the COSY and HSQC spectra were analyzed together to perform the 1 H and 13 C assignments of osidic unit III (Tables 1 and 2). By comparing these data with the literature, it is possible to assign osidic unit III as a cymaropyranoside [11]. The coupling constants at 9.6 and 1.6 Hz are compatible with β-glycosidic bonding, and the chemical shift value of C-2 at 37.0 allowed us to assign the configuration of osidic unit III as β-D-cymaropyranosyl.
For osidic unit IV, the signal of the anomeric hydrogen at δ H 5.08 (dd, J = 9.6 and 1.7 Hz, H-1 ) was observed to correlate in the HSQC spectrum with the carbon at δ C 100.9. In the HMBC spectrum, a correlation of the signal at δ H 3.48 (H-4 ) with the carbon at δ C 100.9 was observed, which was assigned to C-1 , confirming the union of osidic units III and IV. In the COSY spectrum, a correlation of δ H 5.08 (H-1 ) with δ H 2.35 (H-2 ) and of this with δ H 4.01 (H-3 ) was observed. In the analysis of the HSQC spectrum, it was possible to assign the respective carbons of the IV osidic units ( Table 2). By comparing the data in Tables 1 and 2 with the literature, it was possible to mark osidic unit IV as oleandropyranose [11]. The coupling constant at 9.6 and 1.7 Hz was compatible with β-glycosidic bonding, and the value of C-2 at δ C 38.1 allowed the assignment of the configuration of osidic unit IV as β-D-oleandropyranosyl.
Finally, anomeric hydrogen was also observed at δ H 4.71 (dd, J = 9.7 and 2.0 Hz, H-1 ) correlating in the HSQC spectrum with the carbon at δ C 102.9. In HMBC, a correlation of the signal at δ H 3.48 (H-4 r) with the carbon at δ C 102 was observed. 9 was observed, confirming the bonding between osidic units IV and V ( Figure 1). A correlation of δ H 5.15 (H-1 ) with δ H 2.25 (H-2 ) was observed in COSY, whose corresponding carbon was signaled by the correlation observed in the HSQC spectrum with the carbon at δ C 34.2 (C-2 ). The other data (Tables 1 and 2) compared with the literature allowed us to assign the glycosidic unit V as simentopyranoside. The coupling constant at 9.7 and 2.0 Hz was compatible with β-glycosidic bonding, and the value of C-2 at δ C 34.2 allowed the assignment of the configuration of the osidic unit V as β-L-sarmentopyranosyl. The signal at δ H 5.38 (d, J = 2.9 Hz) was assigned to H-4 , whose corresponding carbon was marked at δ C 68.5 by HSQC. In HMBC, a correlation of the signal at δ H 1.29 (d, J = 6.4 Hz, 3H-6 ) with δ C 68.5 (C-4 ) and of δ H 5.38 (H-4 ) with the carbon at δ C 171.2 was observed, confirming the position of the acetyl group in osidic unit V at C-4 ( Figure 2). After extensive NMR analysis, the structure of 1 was determined to be illustrol-  Compound 2 was isolated as a white powder with a positive optical rotation of [α] 25 D +18 (c 0.1, pyridine). Its molecular formula was determined to be C50H78O17 by HRESIMS, with m/z 973.5086 [M + Na] + (calcd for C50H78NaO17, 973.5131, Δ = 4.7 ppm), indicating 12 hydrogen deficiency indices. The 1 H and 13 C NMR data were similar to compound 1 being assigned to illustrol-type aglycone (Tables 1 and 2). When compared to compound 1, a difference of 144 Da was observed in the HRMS spectrum of compound 2, attributed to  Compound 2 was isolated as a white powder with a positive optical rotation of [α] 25 D +18 (c 0.1, pyridine). Its molecular formula was determined to be C50H78O17 by HRESIMS, with m/z 973.5086 [M + Na] + (calcd for C50H78NaO17, 973.5131, Δ = 4.7 ppm), indicating 12 hydrogen deficiency indices. The 1 H and 13 C NMR data were similar to compound 1 being assigned to illustrol-type aglycone (Tables 1 and 2). When compared to compound 1, a difference of 144 Da was observed in the HRMS spectrum of compound 2, attributed to The 1 H and 13 C NMR data of the osidic units also resembled compound 1, allowing us to establish 2 as illus-trol-  20 , 1109.6254, ∆ = 1.0 ppm), indicating 12 hydrogen deficiency indices. The NMR data resembled that of the aglycone illustrol; however, some differences were observed for the carbons at δ C 38.9, 46 and 62.5, assigned to C-8, C-13 and C-17, respectively, with C-8 and C-17 being deprotected and C-13 protected when compared to the chemical shifts of these carbons in compound 1. These data were associated with one lower hydrogen deficiency index compared with compound 1, suggesting the opening of the C-14-O-C-16 epoxide. The signal at δ H 6.88 (s), uncorrelated in the HSQC spectrum, was assigned to the OH located at C-14. This signal showed correlations in the HMBC spectrum with the signals at δ C 46.0 and 110.1 that were assigned to C-13 and C-14, respectively. Additionally, in the HMBC spectrum, we observed correlations of the signal at δ H 1.40 (3H-18) with the carbons at δ C 110.1 (C-14) and in the HMBC spectrum at δ C 46.0 (C-13) and with δ C 62.5, which was assigned to C-17. The signal at δ H 5.17, whose corresponding carbon in the HSQC spectrum was δ C 105.8, was assigned to C-16. The signal at δ H 5.17 correlated with δ C 46.0 (C-13) and with δ C 62.5 (C-17). A correlation of this signal with δ C 71.4, assigned to C-21, was also observed. A correlation was also observed with the signal at δ C 54.5 that was assigned to methoxy bound at position 16. The coupling constant of H-17 (d, J = 8.0) and a singlet for H-16 demonstrated near 90-degree angulation between these hydrogens, and thus, the aglycone was defined as shown in Figure 1. As far as we have searched, no records were found for this type of aglycone. The NMR data (Tables 1 and 2), together with literature data, comparisons with compound 1 data and high-resolution mass spectrometry confirmed the presence of five 1-4 bonded osidic units identical to compound 1 and inserted in C-3 (Table 2).
To reject the possibility that the aglycone had been formed in the process of separating the compounds, a direct infusion on the

Biological Activity
To assess the anti-inflammatory potential of compounds, J774 macrophages were used as an in vitro model. Macrophages play a key role in inflammation and immune regulation processes, contributing to tissue homeostasis [21,22]. They are tissue-resident or infiltrated immune cells activated upon stimulation of a great number of pro-inflammatory mediators, such as chemokines, cytokines, and nitric oxide (NO) [21]. Here, the nitric oxide production by macrophages stimulated with Lipopolysaccharides (LPS) and Interferon gamma (IFN-γ) was assessed. Stimulated macrophage that received vehicle as treatment (control group) show an increase in NO levels in comparison to non-stimulated macrophages (basal group, p < 0.01; Figure 3A Figure 3A; p < 0.01). Plus, compounds 3 and 5 were able to lower the production of NO at the range of 200 µM to 50 µM ( Figures 1D and 3C, respectively; p < 0.01), in a dose-dependent manner. Dexamethasone (20 µM), the goldstandard drug, was also able to reduce the levels of NO when compared to untreated cells, as expected (Figure 3A-D; p < 0.001). Remarkably, at the concentration of 200 µM compounds 5 and 1 showed greater efficacy in comparison to dexamethasone (p < 0.05), while 2 and 3 exhibited a similar efficacy to that of dexamethasone (p < 0.05). Importantly, cell toxicity assays ( Figure S86; Supplementary Material) show that there was no reduction in cellular viability. These data corroborate the interpretation of the NO assay results, as the decrease of cellular viability could reduce the production of inflammatory mediators, and it could be wrongly acknowledged as anti-inflammatory activity. NO has a ubiquitous role in the maintenance of homeostasis, but upon inflammatory stimuli, it will have mainly a proinflammatory role. During the inflammatory response, it will act as an oxidant agent or a signaling mediator, by activating cascades that lead to the production of more inflammatory mediators [23]. Therefore, the ability to reduce the production of this mediator during inflammation is an interesting feature for anti-inflammatory compounds. The modulatory effect of 1, 2, 3 and 5 on the pro-inflammatory cytokine production by stimulated macrophages was further assessed. The untreated cells stimulated with LPS and IFN-γ (control group) show an increase in Tumor necrosis factor alpha (TNF-α) and Interleukin 1 beta (IL-1β) levels in comparison to non-stimulated macrophage (basal group, p < 0.01; Figure 4A-H).
Treatment with all tested concentrations (25-200 μM) of the tested compounds resulted in inhibition of TNF-α production ( Figure 4A-D, p < 0.01). Within the tested range, most of the molecules showed a dose-dependent effect, except for compound 2 which did not show any difference in the magnitude of its effect among the tested concentrations. Dexamethasone, the gold standard drug, also inhibited the production of TNF-α. Importantly, the effect of compounds 1, 2, 3 and 5 ( Figure 4A; p < 0.01) was comparable to that of dexamethasone. The modulatory effect of 1, 2, 3 and 5 on the pro-inflammatory cytokine production by stimulated macrophages was further assessed. The untreated cells stimulated with LPS and IFN-γ (control group) show an increase in Tumor necrosis factor alpha (TNF-α) and Interleukin 1 beta (IL-1β) levels in comparison to non-stimulated macrophage (basal group, p < 0.01; Figure 4A-H).
Treatment with all tested concentrations (25-200 µM) of the tested compounds resulted in inhibition of TNF-α production ( Figure 4A-D, p < 0.01). Within the tested range, most of the molecules showed a dose-dependent effect, except for compound 2 which did not show any difference in the magnitude of its effect among the tested concentrations. Dexamethasone, the gold standard drug, also inhibited the production of TNF-α. Importantly, the effect of compounds 1, 2, 3 and 5 ( Figure 4A; p < 0.01) was comparable to that of dexamethasone.
The production of IL-1β was also modulated by the tested compounds ( Figure 4E-H).   Together with NO, TNF-α and IL-1β are important mediators of the inflammatory response. TNF-α is a pro-inflammatory cytokine produced primarily by monocytes/macrophages and it plays a key role in the modulation of immune responses and induction of inflammation. Upon activation of its receptor, a cascade of events leading to the production of more pro-inflammatory mediators is initiated [24]. Moreover, TNF-α is a target to treat several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel diseases, ankylosing spondylitis, and psoriasis [25]. Similarly, IL-1β is also recognized as an important cytokine for inflammatory events. It is primarily a pro-inflammatory cytokine capable of inducing its own production, in a positive feedback loop that amplifies the inflammatory signaling [26]. Plus, the enhanced secretion of IL-1β has been associated with the pathogenesis of autoinflammatory diseases, metabolic syndromes, acute inflammation, chronic inflammation, and malignancy [27].
Therefore, the ability of 1, 2, 3 and 5 to reduce the production/release of IL-1β and TNFα reinforces the potential anti-inflammatory activity previously displayed by reducing the amount of NO released by stimulated macrophages. Interestingly, velutinol A, an aglycone steroid compound with a structure similar to the tested compounds, was reported to inhibit kinin B1 receptor-mediated inflammatory responses in vivo [28]. Kinin B1 receptor is an inducible receptor that has been implicated in the process of stimulation and release of IL-1β and TNF-α from macrophages [29].
Data presented here demonstrate that compounds 1, 2, 3 and 5 inhibit the production/release of pro-inflammatory mediators, which is an important attribute of antiinflammatory compounds. Moreover, the tested compounds show a similar or even greater efficacy in comparison to that of dexamethasone, considered the gold-standard drug in the tests. Therefore, these results evidence the anti-inflammatory potential of these compounds.
The analytical-scale high-performance liquid chromatographic (HPLC) analyses were performed on a Shimadzu Prominence chromatograph, (flow rate of 600 µL-min −1 ) and injections of 20 µL, using a reversed-phase analytical column (YMC, 250 × 4.6 mm and particle size of 5µC 18 ). The preparative HPLC analysis was used on Shimadzu Proeminence equipment and reverse phase column (YMC, 250 × 21.2mm and particle size of 5µC 18 ). The solvents used were acetonitrile (HPLC grade, Tedia ® , Cincinnati, OH, USA) and ultrapure water obtained with a Milli-Q (Millipore ® ).
1D and 2D NMR experiments were performed using Bruker Avance III HD (400 and 100 MHz for 1 H and 13 C, respectively) and Varian NMR (500 and 125 MHz for 1 H and 13 C, respectively) spectrometers. The residual peaks of the deuterated solvents were taken as reference points and chemical shifts were given in ppm. Mass spectrometry analyses were performed on an HRMS microTOFII ESI-TOF.
A HPLC Shimadzu (Kyoto, Japan) coupled with an Amazon X (Bruker Daltonics, Billerica, MA, USA) with an electrospray ion (ESI) source, was used to perform ESI-MSn. The analysis parameters were as follows: capillary 4.5 kV, ESI (positive mode for samples from the chloroform phase and negative mode for samples from the ethyl acetate phase), final plate offset 500 V, 40 psi nebulizer, dry gas (N 2 ) with a flow rate of 8 mL/min and a temperature of 200 • C. Collision-induced dissociation (CID) fragmentation was achieved in the Amazon X in auto-MS/MS mode using the enhanced resolution mode. The mass spectra (m/z 50-1300) were recorded every 2 s. Moreover, these samples were injected again into an HPLC system coupled to a micrOTOF II mass spectrometer (Bruker Daltonics, Billerica, MA, USA) for high resolution electrospray ionization mass spectrometry (HRESIMS) analyses using the same method as previously reported [30].

Plant Material
The roots of M. dardanoi were collected at Serra do Jatobá (07 •

Extraction and Isolation
The roots of M. dardanoi were dried in a circulating air oven at 45 • C for 92 h and then ground in a knife mill, obtaining 1.78 kg of powder. This material was macerated with 95% ethanol for 72 h in five repetitions and the extracted solution was concentrated in a rotary evaporator (40 • C), resulting in 303 g of the crude ethanolic extract (BSE-Md). Subsequently, 290 g of the BSE-Md was solubilized in MeOH/H 2 O (7:3, v/v). This solution was subjected to partitioning with solvents of an increasing degree of polarity (using 2 L of each solvent: hexane, chloroform, ethyl acetate, and n-butanol). The partitioning allowed obtaining the following phases: hexane (18.2 g), chloroform (12 g), ethyl acetate (4.2 g), and n-butanol (15 g).
For fractionation of the chloroform phase, 3 g were submitted to Sephadex LH-20 gel permeation chromatography, with isocratic elution (MeOH). This method allowed the isolation of 10 fractions (Md-S1 to Md-S10), which were analyzed by CCD. The fractions Md-S1-Md-S5 were subjected to reversed-phase chromatography (C18), with gradient elution using MeOH/H2O ( Nuclear magnetic resonance (NMR) spectra were obtained using spectrometer Bruker 400 MHz ( 1 H) and 100 MHz ( 13 C) and Varian NMR (500 and 125 MHz for 1 H and 13 C, respectively) at the Center of Characterization and Analysis of the Federal University of Paraíba. Deuterated solvent (pyridine-d 5 (C 5 D 5 N) was used in the solubilization of the samples for NMR e chemical shifts (δ) were recorded in ppm (parts per million) and coupling constants (J) in Hz.

Assessment of Cytokine and Nitric Oxide Production by Macrophages
For cytokine and nitric oxide evaluations, J774 cells were seeded in 96-well tissue culture plates at 2 × 10 5 cells/well in DMEM medium supplemented with 10% of FBS and 50 µg/mL of gentamycin for 2 h at 37 • C and 5% CO 2 , as described previously [32]. Cells were then stimulated with LPS (500 ng/mL) and IFN-γ (5 ng/mL) in the presence of 1, 2, 3 or 5 at different concentrations (25 to 200 µM), medium (control group), or dexamethasone (20 µM, gold-standard drug), and incubated at 37 • C. Cell-free supernatants were collected 4 h after the incubation for TNF-α quantification, or 24 h after the incubation for IL-1β and nitrite quantification. Cytokine concentrations in supernatants from J774 cultures were determined by enzyme-linked immunosorbent assay (ELISA), using the DuoSet kit from R&D Systems (Minneapolis, MN), according to the manufacturer's instructions. The results were expressed in picograms/mL of IL-1β. Quantification of nitrite as an indicator of nitric oxide production was performed using the Griess method [33].

Statistical Analysis
Data are presented as mean ± standard deviation (SD) of 3 replicates. Comparisons between groups were made using one-way ANOVA with Tukey post-hoc test. Analyses were performed using Prism 8 Computer Software (GraphPad, San Diego, CA, USA), with a statistical significance of p < 0.05.

Conclusions
Five new pregnane steroidal glycosides (dardanols A-E) were isolated from the ethanolic extract of Mandevilla dardanoi by modern chromatographic techniques and characterized by comprehensive spectroscopic data. Among them, compounds 3 and 4 contained a novel seco-pregnane-type aglycone. Dardanols A, B, C and E showed anti-inflammatory potential by inhibiting the production of nitric oxide and reducing the pro-inflammatory cytokines IL-1β an d TNF-α in stimulated macrophages. These findings enrich the knowledge of the chemodiversity and biological potential of Caatinga species.