Abietane Diterpenes from Medusantha martiusii and Their Anti-Neuroinflammatory Activity

Seven new abietane diterpenoids, comprising medusanthol A–G (1–3, 5, 7–9) and two previously identified analogs (4 and 6), were isolated from the hexane extract of the aerial parts of Medusantha martiusii. The structures of the compounds were elucidated by HRESIMS, 1D/2D NMR spectroscopic data, IR spectroscopy, NMR calculations with DP4+ probability analysis, and ECD calculations. The anti-neuroinflammatory potential of compounds 1–7 was evaluated by determining their ability to inhibit the production of nitric oxide (NO) and the proinflammatory cytokine TNF-α in BV2 microglia stimulated with LPS and IFN-γ. Compounds 1–4 and 7 exhibited decreased NO levels at a concentration of 12.5 µM. Compound 1 demonstrated strong activity with an IC50 of 3.12 µM, and compound 2 had an IC50 of 15.53 µM; both compounds effectively reduced NO levels compared to the positive control quercetin (IC50 11.8 µM). Additionally, both compounds significantly decreased TNF-α levels, indicating their potential as promising anti-neuroinflammatory agents.


Introduction
The Lamiaceae family, which consists of plants and shrubs, is composed of approximately 258 genera and 7193 species.In Brazil, over 500 species from the Lamiaceae family are distributed across 46 genera, with nearly half of these species belonging to the subfamily Nepetoideae [1,2], which is a well-known source of abietane-type diterpenoids [3,4].The broad spectrum of biological activities associated with these compounds has garnered special attention, with many demonstrating anti-inflammatory [5], anticancer [6], antimicrobial [7], and antiprotozoal [8] properties.
Medusantha martiusii (Benth.)Harley and J. F. B. Pastore (syn: Hyptis martiusii), commonly known as "cidreira brava" or "cidreira-do-campo," is a shrub native and endemic to Brazil and belongs to the subfamily Nepetoideae.This species is predominantly found in the Northeast region, specifically in the Caatinga, a unique semiarid biome exclusive to Brazil.In traditional Brazilian medicine, the infusion or decoction of M. martiusii leaves is used to combat intestinal and stomach disorders, while the decoction of roots is commonly used to combat inflammation of the ovaries [9,10].Previous phytochemical investigations of M. martiusii have documented the isolation of abietane diterpenes from its roots and aerial parts, as well as the phytochemical profiling of its essential oil and its pharmacological activities [11][12][13][14].However, to the best of our knowledge, there is no evidence in the literature with respect to the anti-inflammatory activity of the isolated compounds.As part of our investigation into species from the Brazilian semiarid region, we conducted a chemical reinvestigation of M. martiusii, which led to the isolation of nine abietane diterpenes, including seven previously undescribed compounds named medusanthol A-G (1-3, 5, 7-9) and two known analogs (4 and 6).Here, we describe the isolation, structural elucidation, and anti-neuroinflammatory activity of these isolates.

Structure Elucidation of the Compounds
The hexanic extract of the aerial parts was fractionated into six fractions by vacuum liquid chromatography.The EtOAc fraction was purified using HPLC, yielding seven previously unknown (1-3, 5, 7-9) and two known (4 and 6) abietanes (Figure 1).
Molecules 2024, 29, x FOR PEER REVIEW 2 of 16 knowledge, there is no evidence in the literature with respect to the anti-inflammatory activity of the isolated compounds.As part of our investigation into species from the Brazilian semiarid region, we conducted a chemical reinvestigation of M. martiusii, which led to the isolation of nine abietane diterpenes, including seven previously undescribed compounds named medusanthol A-G (1-3, 5, 7-9) and two known analogs (4 and 6).
Here, we describe the isolation, structural elucidation, and anti-neuroinflammatory activity of these isolates.
No. ) of compounds 1-3, 5, and 7-9.Furthermore, the correlations of the signals at δ H 1.07 and 0.91 (6H, s, H 3 -18/H 3 -19) with the carbon signals at δ C 38.9, 82.8, and 48.3 identified the two geminal methyl groups linked to C-4 and defined the chemical shifts of carbons C-4, C-3, and C-5, respectively.The presence of vicinal hydroxyl groups at C-2 and C-3 in the A ring of compound 1 was supported by the HSQC correlations of the oxymethine proton at δ H 3.01 (1H, d, J = 9.6 Hz, H-3) with carbon at δ C 82.8 (C-3), as well as the spin system H 2 -1(δ H 3.01)/H-2/H-3 observed in the COSY spectrum.The correlation between the signals at δ H 4.79 (1H, dd, J = 10.2, 7.5 Hz, H-7) and δ C 67.9 (C-7) in the HSQC spectrum suggested the presence of a third hydroxyl group at C-7.The correlation of H-5/H 2 -6/H-7 in the COSY spectrum established the location of the hydroxyl group at this position.The correlations of the methine proton H-12 (δ H 6.36, d, J = 1.2 Hz) with C-9, C-14, and C-15 and H-15 (δ H 2.97, m) with C-12, C-14, C-16, and C-17 in the HMBC spectrum substantiated the attachment of the isopropyl group to para-benzoquinone and suggested that the quaternary carbon at δC 188.0 was linked to C-11.
Analysis of the NMR data of compound 2 indicated a close resemblance to 6, identified as 2α-hydroxysugiol, a recognized aromatic abietane [19].The only distinction between the two compounds was the replacement of a methylene carbon signal at δ C 50.4 in C-3 with an oxymethine carbon at δ C 83.5, suggesting the presence of an additional hydroxyl group at this position in compound 2. In the HMBC spectrum, the correlation between δ H 1.06 and 0.98 (6H, s, H 3 -18/H 3 -19) and C-3 (δ C 83.5) confirmed the presence of the proposed connectivity (Figure 2).Similar to medusanthol A (1), compound 2 also contained vicinal hydroxyl groups attached to C-2 (δ C 69.4) and C-3 (δ C 83.5).Additionally, the correlation between the signal at δH 7.80 (1H, s, H-14) and the carbon at δ C 200.6 in the HMBC spectrum confirmed the insertion of the carbonyl group at C-7.The NOESY correlations were consistent with the same relative configuration as medusanthol A (1).The NOESY correlations of H-2/H 3 -19/H 3 -20 and H-3/H-5, as well as the coupling constant 3 J H-2/H-3 = 9.2 Hz, suggested that H-2/H 3 -19/H 3 -20 were β-oriented, while H-3/H-5 adopted the α orientation (Figure 3).NMR shift calculations and DP4+ probability analysis supported the relative configuration assigned, with 100% probability ascribed to the 2R*,3R*,5R*,10S*-2b isomer (Figure S153).The absolute configuration was determined by comparing the experimental and calcu-lated ECD data, and the products were assigned as 2R,3R,5R,10S (Figure 4).Accordingly, compound 2 was designated medusanthol B.  Analysis of the NMR data of compound 2 indicated a close resemblance to 6, identified as 2α-hydroxysugiol, a recognized aromatic abietane [19].The only distinction between the two compounds was the replacement of a methylene carbon signal at δC 50.4 in C-3 with an oxymethine carbon at δC 83.5, suggesting the presence of an additional hydroxyl group at this position in compound 2. In the HMBC spectrum, the correlation between δH 1.06 and 0.98 (6H, s, H3-18/H3-19) and C-3 (δC 83.5) confirmed the presence of the proposed connectivity (Figure 2).Similar to medusanthol A (1), compound 2 also contained vicinal hydroxyl groups a ached to C-2 (δC 69.4) and C-3 (δC 83.5).Additionally, the correlation between the signal at δH 7.80 (1H, s, H-14) and the carbon at δC 200.6 in the HMBC spectrum confirmed the insertion of the carbonyl group at C-7.The NOESY correlations were consistent with the same relative configuration as medusanthol A (1).The NOESY correlations of H-2/H3-19/H3-20 and H-3/H-5, as well as the coupling constant 3 JH-2/H-3 = 9.2 Hz, suggested that H-2/H3-19/H3-20 were β-oriented, while H-3/H-5 adopted the α orientation (Figure 3).NMR shift calculations and DP4+ probability analysis supported the relative configuration assigned, with 100% probability ascribed to the Compound 3, a needle crystal, was shown to have a molecular formula of C 20 H 30 O 2 according to its HRESIMS peak at m/z 659.4276 ([2M + Na] + , calcd for C 40 H 60 NaO 6 , 659.4282, ∆ = 0.9 ppm), indicating six degrees of hydrogen deficiency.In the IR spectrum, characteristic absorption bands were observed for a hydroxyl group (3361 cm −1 ) and an aromatic ring (1618 and 1425 cm −1 ).The 1D and 2D NMR data revealed that compound 3 is also an aromatic abietane, displaying significant structural similarity to medusanthol B (2), except for the substitution of the carbonyl group at δ C 200.6 for the methylene carbon at δ C 31.1 in C-7 (Table 1).In the 1 H NMR spectrum, the shielding of the aromatic proton H-14 (δ H 6.75, 1H, s) compared to that of medusanthol B (2) (Table 2), along with the cross-peak of H-14 with C-7 (δ C 31.1) in the HMBC spectrum, supported the absence of a carbonyl group at C-7 (Figure 2).
Further comprehensive analysis of the NMR data revealed that compound 3 shares an identical planar structure with 2,3-dihydroxyferruginol, which was isolated from the leaves of Podocarpus nagi [20].The only distinction between these compounds is observed in the configuration at the C-2 center, suggesting a potential stereoisomer.In the NOESY spectrum, correlations between H-2 and H 3 -19/H 3 -20, along with those between H-3 and H-5/H 3 -18, allowed the determination of the α orientation of the 2-OH in compound 3, which contrasts with the β orientation reported for this group in 2,3-dihydroxyferruginol.Furthermore, the 9.6 Hz coupling constant between H-2 and H-3 in compound 3 is distinct from the 2.9 Hz observed for these protons in 2,3-dihydroxyferruginol, further substantiating the aforementioned proposition.NMR calculations and DP4+ analyses supported the relative configuration of 3 as 2R*,3R*,5R*,10S*-3b with a probability of 100% (Figure S156).Ultimately, the absolute configuration was determined to be 2R,3R,5R,10S by comparing the experimental and calculated ECD data (Figure 4), suggesting that 3 is an epimer of 2,3-dihydroxyferruginol. Biogenetically, the configuration of 3 is proposed to be the same as that of 1 and 2. Therefore, compound 3 was identified as a new abietane named medusanthol C.
Compound 5, a white amorphous powder, exhibited a molecular formula of C 22 H 30 NaO 5 (m/z 397.1974 [M + Na] + , calcd for C 22 H 30 NaO 5 , 397.1985, ∆ = 2.8 ppm), suggesting the presence of an aromatic abietane with eight degrees of hydrogen deficiency.The infrared spectrum displayed characteristic absorptions at 3431 cm −1 (hydroxyl), 1735 and 1269 cm −1 (ester), 1710 cm −1 (carboxylic acid), and 1658, 1510, and 1421 cm −1 (aromatic ring).The 13 C NMR data of the compound indicated a significant resemblance to medusanthol C (3) (Table 1).However, the presence of a single oxygenation on ring A for compound 5 was suggested by the replacement of the oxymethine carbon at δ C 84.3 (C-3) in medusanthol C with a methylene carbon at δ C 47.7.Furthermore, the 1D NMR spectrum revealed the deshielding of the oxymethine proton H-2 (δ H 5.42, 1H, tt, J = 11.7,4.5 Hz), as well as the presence of characteristic signals for an acetoxy group at δ H 2.02 (3H, s), δ C 21.4, and δ C 172.6 (Tables 1 and 2).This finding was consistent with the presence of a 2-OCOCH 3 group in 5, similar to miltiorin A, which is isolated from the roots of Salvia miltiorrhiza [21].In the HMBC spectrum, the correlation between the signals at δ H 1.02 and 0.93 (6H, s, H 3 -18/H 3 -19) and the signal at δ C 47.7 assigned to C-3 confirmed the absence of a hydroxyl group at this position (Figure 2).According to the 13 C NMR spectrum, compound 5 also differed from compound 3 in that it displayed signals for only four methyl groups, indicating the absence of a signal corresponding to CH 3 -20, as observed for compound 3 (δ C 26.3).Therefore, the presence of a signal at δ C 179.0 was attributed to C-20, indicating oxidation to a carboxylic acid at this position.The HMBC correlations of H-1 (δ H 3.09, 1H, ddd, J = 12.1, 4.5, 2.8 Hz) with C-2 (δ C 70.8) and C-20 (δ C 179.0) confirmed the localization of the acetoxy and carboxylic acid functionalities, respectively (Figure 2).
Compound 7 was obtained as a white amorphous powder with a molecular formula of C 22 H 28 O 6 , as determined by its HRESIMS peak at m/z 799.3652 [2M + Na] + (calculated for C 44 H 56 NaO 12 , 799.3664, ∆ = 1.5 ppm), implying nine degrees of hydrogen deficiency.The IR spectrum displayed characteristic bands for hydroxyl (3446 cm −1 ) and lactone (1741 cm −1 ) groups.The 1D and 2D NMR data of 7 showed similarities to those of medusanthol D (5), with an acetoxy group at C-2, a tetra-substituted aromatic ring, and four methyl groups in its structure, as shown in Table 1.However, differences between the two compounds were also detected.The 1 H NMR and HSQC spectra of compound 7 revealed an extra oxymethine signal at δ H 5.94 (1H, d, J = 6.5 Hz) and correlations for only three methylene hydrogen groups.Similarly, the HMBC correlation of the signal at δ H 1.85 (1H, t, J = 11.7 Hz, H-1) with the carbon at δ C 170.9 suggested oxidation at the C-20 position, consistent with a lactone carbonyl [22] (Figure 2).In particular, the data of 7 notably differed from those of 5 due to the presence of a carbon at δ C 95.4 and the deshielding of C-8 (∆δ C + 17.1 ppm) (Table 1).
Moreover, the correlation between δ H 5.94 and δ C 95.4 in the HSQC spectrum, along with the spin system H-5/H 2 -6/H-7 as determined by the COSY spectrum, provided substantial evidence for the presence of an acetal group at C-7.The aforementioned data, along with the HMBC correlations of the acetalic hydrogen H-7 (δ H 5.94, d, J = 6.The relative stereochemistry of compound 7 was deduced from NOESY correlations, similar to those observed for medusanthol E (5).The cross-peaks between H-2/H 3 -19 and H-5/H 3 -18 in the NOESY spectrum suggested that H-2 and H 3 -19 adopted a β orientation, while CH 3 -18 and H-5 assumed an α orientation (Figure 3).To further determine the relative configuration of compound 7, the NMR data of two candidates (7a and 7b) were calculated.DP4+ analyses indicated that (2S*,5S*,7S*,10R*)-7b was highly likely at 99.81% (Figure S168).Furthermore, the absolute configuration of 7 was determined to be 2S,5S,7S,10R through comparison of the experimental and calculated ECD spectra (Figure 4).Ultimately, 7 was denominated medusanthol E.
Compound 8 was isolated as a white amorphous powder with the molecular formula C 22 H 26 O 6 , as deduced from its HRESIMS signal at m/z 409.1612 [M + Na] + (calcd for C 22 H 26 NaO 6 , 409.1622, ∆ = 2.3 ppm), corresponding to ten degrees of hydrogen deficiency.In the IR spectrum, characteristic absorption bands for hydroxyl (3446 cm −1 ), lactonic (1786 cm −1 ), ester (1720 cm −1 ), and conjugated ketone (1695 cm −1 ) groups were observed.The 13 C NMR spectrum of 8 showed signals between δ C 189.1 and 111.0, similar to those observed for compound 2, which were attributed to the aromatic carbons of the C ring and the carbonyl of the ketone at C-7 (Table 1).On the other hand, compound 8 also exhibited structural similarities to compound 7, as evidenced by the signals detected at δ C 176.0 and δ C 66.4, suggesting the presence of a lactone group at C-20 and an acetoxy group at C-2, respectively.In addition, the signals at δ C 22.3, 22.4, 22.7, and 31.4,corresponding to the four methyl groups, also align with those found in compound 7.However, the absence of signals at δ C 95.4 and 145.8 and the presence of a signal at δ C 81.4 in the 13  The relative stereochemistry of C-6 was determined by analyzing the dihedral angle between singlets H-5 (δ H 2.41, 1H) and H-6 (δ H 4.77, 1H).These protons displayed an approximately 90 • dihedral angle, indicating a pseudoequatorial arrangement for H-6, while H-5 exhibited an α-axial disposition.However, the relative configuration of the C-2 chiral center could not be conclusively determined by NOESY.In this way, the quantum GIAO method was utilized to calculate the 13 C and 1 H NMR chemical shifts of two potential isomers, (2R*,5S*,6S*,10R*)-8a and (2S*,5S*,6S*,10R*)-8b.Subsequently, comparison of these computed values with experimental data through DP4+ probability analysis indicated that the most likely relative configuration was (2S*,5S*,6S*,10R*)-8b, with a 76.92% probability (Figure S171).To determine the absolute configuration, the calculated and experimental ECD data were compared (Figure 4).The calculated ECD spectrum of 8b aligned closely with the experimental curve for 8, suggesting the absolute configuration of 2S,5S,6S,10R.Ultimately, its structure was denoted as medusanthol F.
Compound 9 was obtained as a yellow amorphous powder, with an HRESIMS peak at m/z 799.3637 [2M + Na] + (calcd for C 44 H 56 NaO 12 , 799.3664, ∆ = 3.4 ppm), indicating a molecular formula of C 22 H 28 O 6 and suggesting nine degrees of hydrogen deficiency.The infrared spectrum exhibited absorption bands for hydroxyl (3427 cm −1 ), lactonic (1791 cm −1 ), and aldehydic (1724 cm −1 ) groups. 13C NMR analysis of compound 9 indicated similar chemical shifts in the A ring to those of compounds 5-8.However, differences in the chemical shifts of the B and C rings were observed compared to those of compounds 1-8 identified in this study (Table 1).According to the 13 C NMR and DEPT spectra of compound 9, six aromatic carbons were identified, including two oxygenated carbons at δ C 150.2 and 146.6, two methine carbons at δ C 110.8 and 108.6, and two nonhydrogenated carbons at δ C 137.1 and 128.Furthermore, the resonance observed at δ C 177.9 in the 13 C NMR spectrum was assigned to C-20, indicating the presence of a lactone carbonyl in 9.In the HMBC spectrum, the correlation of the signal at δ H 3.16 (1H, sept., J = 6.9 Hz, H-15) with the signals at δ C 108.6 and 150.2 confirmed the chemical shifts of C-12 and C-14, respectively (Figure 2).Consequently, the HSQC correlation between the proton at δ H 6.57 (1H, s) and the carbon at δ C 110.2, as well as the signal at δ H 6.86 (1H, s) with the carbon at δ C 108.6, confirmed the chemical shifts of the two hydrogenated aromatic carbons at C-8 and C-12, respectively.According to the information provided, it is suggested that the lactone ring in compound 9 formed via the C ring.HMBC correlations from H-8 (δ H 6.57, 1H, s) to C-14 (δ C 150.2), C-11 (δ C 146.6), C-13 (δ C 137.1), C-9 (δ C 128.3), and C-10 (δ C 53.1) confirmed that C-9, C-10, C-11, and C-20, along with an oxygen atom, formed a γ-lactone ring (Figure 2).
In addition, the presence of a signal at δ H 9.19 (1H, t, J = 1.5 Hz) in the 1 H NMR spectrum, along with the correlation of this proton with the carbon at δ C 199.5 in the HSQC spectrum, suggested the presence of an aldehydic group in 9.The correlation of the aldehydic proton (δ H 9.19, 1H, t, J = 1.5 Hz) and H-5 (δ H 2.34, 1H, dd, J = 6.1, 4.6 Hz) with the C-6 carbon (δ C 41.8) in the HMBC spectrum (Figure 2), along with the COSY spin system H-5/H-6/H-7, determined the position of the aldehyde group at C-7.The conjunction of these correlations established that 9 is a 7-8-seco-abietane.
Furthermore, the structures of the identified known diterpenoids, salviol (4) [23] and 2α-hydroxysugiol (6) [19], were confirmed by comparing their spectroscopic data with reported values in the literature.Here, we present the 1D NMR, HRESIMS, ECD, and IR data, along with 13 C and 1 H NMR calculations and DP4+ probability analysis for compounds 4 and 6 (see Supplementary Materials).

Biological Activity Anti-Neuroinflammatory Activity
Neuroinflammation is characterized by the prolonged activation of glial cells and the influx of immune cells into the nervous system and plays a significant role in the progression of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and traumatic brain injury [24].Research has shown that abietanes in the Lamiaceae family have the potential to reduce neuroinflammation and act as antioxidants [25,26].
The noncytotoxic concentrations of compounds 1-7 in BV2 cells were determined using the MTT assay.Our results showed that at 50 µM, most compounds reduced cell viability by more than 20%.On the other hand, at 12.5 and 25 µM, cell viability greater than 80% was observed for all the compounds (Table 3).Therefore, 12.5 µM was considered a safe concentration for assessing the anti-neuroinflammatory effects of compounds 1-7.Results are expressed as the mean ± SEM (n = 5) of two independent experiments.
The anti-neuroinflammatory effects of compounds 1-7 were initially evaluated by determining the levels of nitrite, a stable metabolite of NO.As shown in Figure 5, the LPS/IFN-γ-induced inflammatory response was greater in the control group than in the basal group (unstimulated).At 12.5 µM, compounds 1-4 and 7 significantly reduced nitrite levels compared to those in the control group.No significant effect was recorded for compounds 5 and 6.As expected, the positive control quercetin (20 µM) also significantly reduced nitrite levels in stimulated BV2 cells.Nitric oxide plays a crucial role in inflammation, including its involvement in neurodegenerative diseases [27].Thus, our results suggest that compounds 1, 2, 3, 4, and 7 exert anti-neuroinflammatory effects.Table 4.The IC50 values of compounds 1 and 2 on nitric oxide production inhibition in LPS and IFNγ-stimulated BV2 cells.
Compounds 1 and 2 significantly reduced TNF-α levels in stimulated BV2 cells compared to those in the control group (Figure 6).Data from the literature have shown that inflammation induced in BV2 cells increases the activation of signaling pathways such as the NF-κB and MAPK pathways, leading to the production of cytokines, including TNF-α [29].TNF-α is a proinflammatory cytokine that modulates the immune system and plays a role in  4).The IC 50 value for the positive control quercetin was 11.8 µM.These results support the potent anti-neuroinflammatory effect, especially for compound 1.Moreover, considering that TNFα acts as an important inflammatory mediator [28], the inhibitory effects of compounds 1 and 2 on LPS-and IFN-γ-induced TNF-α release from BV2 cells were assessed.
Compounds 1 and 2 significantly reduced TNF-α levels in stimulated BV2 cells compared to those in the control group (Figure 6).Data from the literature have shown that inflammation induced in BV2 cells increases the activation of signaling pathways such as the NF-κB and MAPK pathways, leading to the production of cytokines, including TNF-α [29].TNF-α is a proinflammatory cytokine that modulates the immune system and plays a role in all types of inflammatory disorders, such as central nervous system disorders [30].Therefore, the anti-neuroinflammatory effects of compounds 1 and 2 are linked to the inhibition of NO and TNF-α release from BV2 cells.11.8 ± 1.5 1 IC50 means half maximal (50%) inhibitory concentration.Results are presented as the mean ± SEM (95% confidence interval). 2Quercetin was used as positive control.
Compounds 1 and 2 significantly reduced TNF-α levels in stimulated BV2 cells compared to those in the control group (Figure 6).Data from the literature have shown that inflammation induced in BV2 cells increases the activation of signaling pathways such as the NF-κB and MAPK pathways, leading to the production of cytokines, including TNF-α [29].TNF-α is a proinflammatory cytokine that modulates the immune system and plays a role in all types of inflammatory disorders, such as central nervous system disorders [30].Therefore, the anti-neuroinflammatory effects of compounds 1 and 2 are linked to the inhibition of NO and TNF-α release from BV2 cells.

General Experimental Procedures
Optical rotations were measured on a JASCO P-2000 polarimeter (JASCO, Tokyo Japan).Infrared (IR) spectra were recorded on a Shimadzu IRPrestige-21 spectrometer (Shimadzu, Kyoto, Japan) using the KBr disk method.NMR data were acquired on Bruker Ascend 400 MHz and Bruker AvanceNeo 500 MHz spectrometers (Bruker, Billerica, MA, USA) using the residual nondeuterated solvent peaks as an internal standard.The experimental ECD spectra were obtained on a JASCO J-1100 CD Spectrometer (JASCO, Tokyo Japan).The vacuum-liquid chromatography (VLC) system was constructed in a Büchner funnel, and an Erlenmeyer flask was connected to a vacuum system using silica gel (60-200 µm, 70-230 mesh, SiliaCycle, Quebec, QC, Canada) as the packing material.High-resolution electrospray ionization mass spectrometry (HRESIMS) analyses were carried out using a Bruker micrOTOF II spectrometer (Bruker, Billerica, MA, USA) operating in positive mode.Analytical high-performance liquid chromatography (HPLC) was performed on a Prominence Shimadzu instrument (Shimadzu, Kyoto, Japan) equipped with an SPD-M20A diode array detector and a YMC C-18 (250 mm × 4.6 mm × 5 µm) column.Semipreparative HPLC separations were conducted on a Shimadzu 10AVP instrument (Shimadzu, Kyoto, Japan) with an SPD-M10AVP detector on a Venusil XBP C-18 (259 mm × 10 mm × 10 µm) column.For preparative HPLC isolations, a Shimadzu apparatus with an SPD-M10A diode array detector and a YMC-Triart ® C-18 (250 mm × 20 mm × 5 µm) column was used.

Plant Material
The aerial parts of Medusantha martiusii (Benth.)Harley and J. F. B. Pastore were collected in July 2019 at Maturéia, a Caatinga region of Paraíba, Brazil (07 • 16 ′ 01 ′′ S, 37 • 21 ′ 05 ′′ W).The sample was authenticated by Maria de Fátima Agra.A specimen is housed under the code JPB 37884 at the Herbarium Prof. Lauro Pires Xavier (JPB) at the Federal University of Paraíba (UFPB), Brazil.This species was registered under the code AB7F3C9 in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen-Brasil).

NMR and ECD Calculations
The three-dimensional molecular structures of the compounds were obtained using ChemSketch software version C25E41 [30].Stochastic conformational searches were performed for all possible stereoisomers of each compound using the Monte Carlo method and the molecular mechanic force field (MMFF) in SPARTAN'10 software version 1.1.0[31].All conformers within a relative free energy window of 10 kcal mol −1 were reoptimized using the B3LYP/6-31G(d) level of theory.The conformations within the energy range of 2.5 kcal mol −1 above the minimum energy conformer, corresponding to more than 90% of the total Boltzmann population, were selected for the GIAO NMR calculations and the simulations of the ECD spectra.To simulate nuclear magnetic shielding, the GIAO-mPW1PW91/6-31+G(d,p) level of theory was used, employing a polarizable continuum model with integral equation formalism (IEF-PCM) to implicitly simulate chloroform as a solvent.The 1 H and 13 C NMR chemical shifts (δ) were obtained using δ i = σ 0 − σ i after the calculation of the shielding constant of the tetramethylsilane (σ 0 ) using the same of theory.For the application of the DP4+ method, as recommended by the author, the nuclear magnetic shields for all candidates of each compound were added to the DP4+ Excel spreadsheet [32].For the ECD simulations, TD-DFT was performed in acetonitrile at the CAM-B3LYP/TZVP level.The IEF-PCM model for acetonitrile was used.The final ECD spectra were obtained based on the weighted average Boltzmann statistics of the selected conformers and plotted using Origin 8 software [33].All quantum-mechanical calculations were performed using the Gaussian 09 software package [34].

Nitric Oxide (NO) and TNF-α Measurement
To determine the NO and TNF-α levels, BV2 cells were seeded in 96-well plates (1 × 10 6 cells/mL) in RPMI medium supplemented with 10% FBS and 1% penicillinstreptomycin in a 5% CO 2 incubator at 37 • C.After four hours, the cells were exposed to LPS (500 ng/mL, Sigma Aldrich) and IFN-γ (5 ng/mL, Thermo Fisher) in the absence or presence of compounds 1 to 7 at a final concentration of 12.5 µM (for NO and TNF-α measurement) or 3.125-25 µM (to calculate the IC 50 values on NO production inhibition), in five replicates.Quercetin (20 µM) was used as a positive control.After 24 h, cell-free supernatants were collected for NO quantification using the Griess method [36] or stored at −80 • C for cytokine concentration determination.The TNF-α concentrations in the BV2 cell culture supernatants were assessed via enzyme-linked immunosorbent assay (ELISA) with an Invitrogen kit (Thermo Fisher, Viena, Austria).

Figure 5 .
Figure 5. Effects of compounds 1-7 (12.5 µM) on the nitric oxide measurement in LPS and IFNγ-stimulated BV2 cells.Results are expressed as the mean ± SEM (n = 5) of two independent experiments.B: basal.C: control.Q: quercetin (positive control, 20 µM).# p < 0.05 versus basal group; * p < 0.05 versus control group.Considering the promising results for compounds 1 and 2, a new set of experiments was performed to calculate the IC 50 values at concentrations of 3.125, 6.250, 12.5, and 25 µM.Compounds 1 and 2 exhibited IC 50 values of 3.12 and 15.53 µM, respectively (Table4).The IC 50 value for the positive control quercetin was 11.8 µM.These results support the potent anti-neuroinflammatory effect, especially for compound 1.Moreover, considering that TNFα acts as an important inflammatory mediator[28], the inhibitory effects of compounds 1 and 2 on LPS-and IFN-γ-induced TNF-α release from BV2 cells were assessed.

Table 4 .
The IC 50 values of compounds 1 and 2 on nitric oxide production inhibition in LPS and IFN-γ-stimulated BV2 cells.

Table 4 .
The IC50 values of compounds 1 and 2 on nitric oxide production inhibition in LPS and IFNγ-stimulated BV2 cells.