Optical rotations were determined by a JASCO P-2000 polarimeter at 25°C. Infrared (IR) spectra were recorded on a Thermo Scientific Nicolet iS5 FTIR spectrometer. The ECD spectra were measured by a JASCO J-715 spectropolarimeter. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were established by a Thermo Scientific Ultimate 3000 UHPLC System with a Thermo Scientific Q Exactive™ Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer. NMR (nuclear magnetic resonance) spectra, including ¹H, ¹³C, DEPT, ¹H–¹H COSY, HMBC, HSQC, and NOESY, were recorded on Varian Unity Inova 500 MHz (5 mm SWPFG/TRPFG probe) or Varian VNMRS 600 MHz spectrometers (cold probe) and the chemical shifts were referenced by deuterated solvent methanol-d ₄. Silica gel 60 (Merck, 70–230 and 230–400 meshes), C₁₈ gel (Chromatorex, 40–75 mesh), Diaion HP-20 (Mitsubishi Chemical Co.), and Sephadex LH-20 (GE) were used for column chromatography. Preparative HPLC (high-performance liquid chromatography) was performed using a Shimadzu LC-8A pump and an SPD-10A VP UV detector (210 and 254 nm wavelengths) with a Cosmosil 5C₁₈ AR-II column (250 × 20 mm, Nacalai Tesque). TLC (thin-layer chromatography) analyses were conducted on pre-coated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 1 mm) and sprayed with anisaldehyde–sulfuric acid reagent and then heated at 100°C.

The whole plants of the air-dried M. procumbens Hemsley (8.0 kg) were purchased from Starsci Biotech company in September 2019. A voucher specimen (No. NRICM-20190901) was deposited in the Herbarium of Division of Chinese Materia Medica Development, NRICM, Taipei, Taiwan.

The air-dried plant of M. procumbens Hemsley (8.0 kg) was shredded into 5 mm and extracted with methanol (80 L) at 50°C thrice, and the combined extract was concentrated under reduced pressure removing methanol to obtain the methanolic crude extract. The crude extract (ca. 796.5 g) was dissolved in ddH₂O and then the aqueous solution was further sequentially partitioned with n-hexane and dichloromethane (CH₂Cl₂) to obtain the hexane, CH₂Cl₂, and aqueous extracts. The CH₂Cl₂ extract (ca. 47.6 g) was fractionated by a C₁₈ gel flash column (15 × 25 cm) eluting with a 20%, 40%, 60%, 80%, 90%, and 100% MeOH/H₂O, successively, to yield six fractions (Fr. I‒VI). Fr. IV was separated by chromatography on a flash column (silica gel, 60–230 mesh, 15 × 20 cm) eluting with CH₂Cl₂ and acetone (from 5% to 100% acetone) to obtain eight subfractions (Fr. IVA ∼ H). Fr. IVF was separated by preparative HPLC on a Cosmosil 5C₁₈ AR-II column (250 × 20 mm, flow rate: 10.0 ml/min) with 60% acetonitrile (ACN) in H₂O to afford seven subfractions (Fr. IVF-1∼7). Fr. IVF-4 was further purified by HPLC with Cosmosil 5C₁₈ AR-II column (flow rate: 10.0 ml/min), eluting with 45% ACN to afford 2 (2.1 mg, R _t: 33.7 min). Using the same HPLC and RP column, eluting with 50% ACN, compound 5 (2.6 mg, R _t: 21.3 min) was purified. Fr. IVE was fractionated by HPLC (Cosmosil 5C₁₈ AR-II column, 250 × 20 mm, flow rate: 10.0 ml/min) with 55% ACN to afford eight subfractions (Fr. IVE-1∼8). Fr. IVE-3 was further purified again with 45% ACN (flow rate: 10.0 ml/min) to yield 3 (1.9 mg, R _t: 18.3 min). Fr. IVD was subjected to preparative HPLC with 45% ACN (flow rate: 10.0 ml/min) to yield six subfractions (Fr. IVD-1∼6). Fr. IVD-4 was further repeatedly purified with 40% ACN (flow rate: 10.0 ml/min) to afford compounds 4 (2.3 mg, R _t: 17.9 min) and 7 (4.8 mg, R _t: 26.2 min). Fr. IVC was subjected to HPLC with 45% ACN (flow rate: 10.0 ml/min) to divide into eight subfractions (Fr. IVC-1∼5)_. Fr. IVC-2 was separated by a Cosmosil 5C₁₈ AR-II column (flow rate: 10.0 ml/min) to afford 1 (1.6 mg, R _t: 31.2 min). Fr. IVC-4 was also purified by HPLC using the same RP column with 40% ACN (flow rate: 10.0 ml/min) to afford 6 (2.5 mg, R _t: 49.9 min).

The determination of the absolute configuration of cyclic and acyclic vic-diols was achieved by employing a transition metal chelate reagent, dimolybdenum tetraacetate [Mo₂(OAc)₄]. Compound 7 was directly dissolved in a solution of Mo₂(OAc)₄ complex in DMSO in a molar ratio of Mo₂(OAc)₄/compound of about 1:0.3–1:0.7, and the mixture was subsequently measured for the induced CD spectra without the preparation and isolation of the complexes.

The RAW264.7 mouse macrophages were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in DMEM supplemented with 10% heat-inactivated FBS in a 5% CO₂ humidified incubator at 37°C. For viability assay, RAW264.7 cells were pretreated with various concentrations of the isolated compounds (0, 5, 10, and 20 μM) 1 h prior to LPS (1 μg/ml) stimulation. After 24 h treatment, cell viability was determined by Cell Counting Kit-8 (Dojindo, Rockville, MD, United States) according to the manufacturer’s instructions.

Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) was used to determine NO production. RAW264.7 cells were pretreated in the same manner described in cell viability assay. 100 µL of supernatant of each pretreated cell solution was transferred to a new microtiter plate, and each supernatant was mixed with 100 µL of Griess reagent. The microtiter plate was left at room temperature for 10 min for color development, and each solution was measured by a microplate reader at UV 540 nm. All experiments were performed in triplicate.

RAW264.7 cells were pretreated with compound 7 (0, 5, 10, or 20 μM) or quercetin (25 μM, Sigma-Aldrich) for 1 h and then stimulated with LPS for an additional 24 h. The cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer and protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Munich, Germany). The protein lysates were separated by SDS-PAGE and then transferred to a PVDF membrane. The blot was blocked with TBS containing 5% nonfat milk for 1 h at room temperature and then incubated with primary antibodies to iNOS, COX-2 or β-actin at 4°C overnight. The blots were washed three times with 0.1% TBST (0.1% Tween 20 in TBS) and then incubated with a peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing, the protein bands were detected using an ECL reagent and X-ray film.

The measurement of the IL-6 and TNF-α levels was performed using commercial ELISA kits (RAB0308-1KT and RAB0477-1KT (Sigma Chemical Co., St. Louis, MO, United States), respectively) according to the manufacturer’s instructions. Briefly, samples and standards were added to antibody-coated 96 wells and were incubated for 2.5 h at room temperature. After incubation, wells were washed with wash buffer four times, and then detection antibody was added and incubated for another 1 h at room temperature. After washing, 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added and incubated in the dark for 30 min at room temperature followed by adding stop solution and reading absorbance at 450 nm immediately.

Statistical analyses were performed using SPSS (SPSS, Chicago, IL, United States). Data are expressed as the mean ± standard deviations. Statistical significance was determined using one-way ANOVA analysis followed by Tukey’s test. p-values < 0.05 were considered statistically significant.

Compound 1 ([α] +12.9, c = 0.5, MeOH), a colorless amorphous powder, has a molecular formula of C₂₉H₄₄O₅ with 8 degrees of unsaturation (DOU) deduced from the sodiated HRESIMS pseudo-ion at m/z 495.3097 [M + Na]⁺ (calcd. for C₂₉H₄₄O₅Na, 495.3081). In ¹H-NMR spectrum (Table 1), four tertiary methyl signals (δ _H 0.76, 0.86, 0.92, and 1.21 s), one olefinic methylene signal (δ _H 4.67 brs and 5.00 brs), and one olefinic methine signal (δ _H 5.28 t, J = 3.6 Hz) were observed. Based on ¹³C-NMR and DEPTs spectra (Table 2), the 29 carbons can be categorized into 4 methyls, 11 methylenes (one oxymethylene at δ _C 73.0 and one olefinic methylene at δ _C 109.4), 6 methines (two oxymethines at δ _C 68.7 and 75.6 as well as one olefinic methine at δ _C 122.4), and 8 quaternary carbons (two sp² quaternary carbons at δ _C 144.1 and 151.0 and one carboxylic acid group at δ _C 180.3). The above two olefins and one carboxyl group accounted for three DOU, and the remaining five constrained 1 to a pentacyclic structure. The assignments of ¹H- and ¹³C-NMR spectroscopic data were completed by a combination of HSQC, ¹H–¹H COSY, and HMBC experiments. By comparison of carbon signals of 1 with that of oleanolic acid along with the analysis of the COSY and HMBC correlations of 1 altogether determined the carbon skeleton of 1, an oleanane-type nortriterpenoid (Kashiwada et al., 1998). In Figure 2, the COSY correlations of H-2 (δ _H 3.65 ddd, J = 3.6, 4.8, 12.0 Hz)/H-3 (δ _H 4.12 d, J = 3.6 Hz) confirmed that the attachments of these two hydroxyls are at C-2 and C-3, whereas the HMBC correlations from H₂-30 (δ _H 3.18 brs) to C-19 (δ _C 39.9), C-20 (δ _C 35.4), and C-21 (δ _C 27.9) revealed a primary alcohol at C-30. Furthermore, an exo-4 (23)-double bound was verified according to the HMBC correlations of olefinic methylene H₂-23 to C-3 (δ _C 75.6), C-4 (δ _C 151.0), and C-5 (δ _C 44.4); the tri-substituted Δ¹² double bond was confirmed by the COSY correlations of H-9 (δ _H 1.82 m)/H₂-11 (δ _H 1.94 and 2.02 m)/H-12 (δ _H 5.28 t, J = 3.6 Hz) and by the HMBC correlations from H-18 (δ _H 2.88 dd, J = 4.8 and 14.4 Hz) to C-12 (δ _C 122.4), C-13 (δ _C 144.1), and C-14 (δ _C 41.8). The spectroscopic evidence altogether indicated that 1 is a new 24-noroleanane-type triterpene acid.

The pentacyclic moiety of oleanane-type triterpenoids is rigid, showing the configurations with those of regular oleanane-type triterpenoids based on NOESY experiment, which provides clear assignments. The NOESY correlations of H-5/H-9, H-9/CH₃-27, CH₃-27/H_α-19, and H_α-19/H₂-30 indicated α-orientations, whereas those of CH₃-25, CH₃-26, H-18, and CH₃-29 presented β-directions with regard to H-18/CH₃-25, CH₃-25/CH₃-26, and H-18/CH₃-29 (Figure 3). Meanwhile, the NOESY correlations of H-2/H-3 and H-2/CH₃-25 and the small proton constant of H-3 (³ J _H-2,H-3 = 3.6 Hz) agreed with the α-oriented OH-2 and OH-3 within a cis-relationship. As a result, the chemical structure of mesonaic acid D (1) was established as 2α,3α,30-trihydroxy-24-norolean-4 (23),12-dien-28-oic acid.

Mesonaic acid E (2) was white amorphous powders with [α] +19.7 (c 0.5, MeOH). The molecular formula C₂₉H₄₂O₅ on par with 9 DOU was deduced based on a quasimolecular ion at m/z 469.2965 [M ‒ H]^‒ (calcd. for C₂₉H₄₁O₅, 469.2949) in the HRESIMS experiment. The characteristic ¹H- and ¹³C-NMR signals (Tables 1, 2), four tertiary methyls (δ _H 0.66 s/δ _C 13.5; δ _H 0.94 s/δ _C 32.0; δ _H 0.98 s/δ _C 22.7; δ _H 1.17 s/δ _C 20.0), two oxymethines C-2 (δ _H 3.68 ddd, J = 3.6, 4.8, 11.4 Hz/δ _C 68.8) and C-3 (δ _H 4.11 d, J = 3.6 Hz/δ _C 75.4), one exocyclic methylene involving C-4 (δ _C 150.5) and C-23 (δ _H 4.85 brs and 4.99 brs/δ _C 109.0), and one COOH-28 (δ _C 180.3) suggested that compound 2 is a derivative of 24-nor-oleanane triterpene acid possessing the same 2,3-diol ring A as 1. Deduction of DOU from the 4 (23)-double bond, COOH-28, and the pentacyclic ring moiety, there are two DOU unassigned. After a detailed comparison of ¹H-, ¹³C-NMR, and DEPTs spectra, we found that a tri-substituted Δ¹² double bond and the primary alcohol of C-30 disappeared, while two oxymethines (δ _H 4.00 dd, J = 2.4 and 5.4 Hz/δ _C 73.0 and δ _H 5.02 d, J = 5.4 Hz/δ _C 78.0), one aliphatic methylene (δ _H 0.74 d, J = 7.2 Hz/δ _C 15.7), and one aliphatic quaternary carbon (δ _C 33.8) emerged. By a combination of the COSY correlations of H-9/H₂-11/H-12 (indicating that olefinic methine C-12 converts to an oxymethine) and the HMBC correlations from another oxymethine H-15 to C-12, one DOU was contributed to an oxygen-bridge between C-12 and C-15. The remaining DOU is resulted from a cyclopropane unit assembled by one aliphatic methylene C-27 and two aliphatic quaternary carbons C-13 and C-14, which were confirmed by the HMBC correlations (Figure 2) of H-18 (δ _H 1.88 dd, J = 4.2 and 12.6 Hz)/C-12, C-13, C-17 (δ _C 45.2), C-27, and COOH-28 and H₃-26 (δ _H 1.17 s)/C-7 (δ _C 37.0), C-8 (δ _C 39.5), C-9 (δ _C 43.2), and C-14 (δ _C 36.1).

In terms of stereochemistry, the chiral centers at ring junctions of 2 are carried on as those at compound 1; The α-side of the cyclopropane unit (C-13, C-14, and C-27) which formed via a si-attack by α-oriented CH₃-27 were supported by the NOESY correlations (H-9/H₂-27, H₂-27/H_α-19, and H_α-19/CH₃-30) (Figure 3). The 2α,3α-diol is also confirmed by the NOESY correlations of H-2/H-3 and H-2/CH₃-25 as well as the small proton constant of H-3 (J = 3.6 Hz). The configurations of the oxygen bridge heads, C-12 and C-15, were determined to be at the β-side based on the NOESY correlations of H-12/H-18 and H-15/CH₃-26. Thereby, compound 2 was clearly elucidated as 2α,3α-dihydroxy-12 (15)-epoxy-13α,27-cycloolean-4 (23)-en-28-oic acid.

Mesonaic acid F (3) was obtained as a white powder with specific rotation [α] +25.1 (c 0.5, MeOH). Its IR spectrum showed the presence of a hydroxyl group (3369 cm⁻¹), a carboxyl group (1712 cm⁻¹), a conjugated carbonyl group (1630 cm⁻¹), and an olefinic group (1385 cm⁻¹). The molecular formula of 3 was determined to be C₂₉H₄₂O₆ (9 DOU) by the given HRESIMS ion at m/z 485.2919 [M ‒ H]^‒ (calcd. 485.2898 for C₂₉H₄₁O₆). The 29 carbons (Table 2) can be separated into 6 methyls (δ _C 11.9, 12.5, 13.0, 16.3, 22.5, and 25.1), 7 methylenes (δ _C 20.5, 23.4, 27.6, 28.4, 32.0, 43.4, and 51.8), 6 methines (δ _C 41.8, 43.4, 48.5, 54.1, 73.1, and 128.0), and 10 quaternary carbons (δ _C 39.1, 40.9, 41.6, 47.2, 75.2, 131.6, 138.2, 143.9, 180.1, and 194.2) by ¹³C-NMR and DEPTs spectra of 3. The protons of five methyls [δ _H 0.88, 0.93, 1.36 s, 1.16 d, (J = 6.6 Hz), and 1.84 d (J = 1.8 Hz)], one oxymethine (δ _H 3.90 dd, J = 3.0 and 6.0 Hz), and one olefinic methine (δ _H 5.33 t, J = 3.6 Hz) can be clearly observed in ¹H-NMR spectrum (Table 1). Combining the ¹H-, ¹³C-NMR, and HSQC experiments, compound 3 was figured out to be a pentacyclic triterpene acid containing one conjugated carbonyl group, one tri-substituted double bond, and one carboxylic acid, reconciling these functional groups and pentacyclic fused ring system with the 9 DOU. The COSY correlations of H-9/H₂-11/H-12 and the HMBC correlations from a junction proton H-18 (δ _H 2.69 brs) to C-12, C-13, C-14, C-17 and C-28 together made a tri-substituted double bond at the Δ¹² position clear; the ¹H–¹H COSY correlations of CH₃-30/H-20/H-21/H₂-22 alongside the HMBC correlations of CH₃-29/C-18, C-19, C-20 illustrated that 3 is a derivative of ursane-type triterpenoids. The most difficult part is that the α-hydroxy-β-methyl substitution in the α,β-unsaturated conjugated carbonyl part in ring A is deduced by the HMBC correlations of CH₃-23 to C-3, C-4, C-5 and CH₃-25 to C-1, C-9, C-10, revealing the ursane triterpenoid without the C-24 carbon in 3 (Figure 2). The chiral configurations of 3 were built up by the NOESY correlations (Figure 3) and comparison with a pomolic acid (Kashiwada et al., 1998). As a result, 3 is identified as 3,19α,21α-trihydroxy-2-oxo-24-norurs-3,12-dien-28-oic acid.

Compound 4, [α] +23.4 (c 0.5, MeOH), was purified as white amorphous powders. The molecular formula C₃₀H₄₆O₇ (8 DOU) was determined by the HRESIMS molecular ion at m/z 517.3176 [M-H]^‒ (calcd. for C₃₀H₄₅O₇, 517.3160). The ¹H-, ¹³C-NMR (Tables 1, 2), and HSQC spectra of 4 as a whole revealed the presence of six methyls (δ _H 0.79 s/δ _C 13.6; δ _H 0.92 d, J = 6.6 Hz/δ _C 15.2; δ _H 1.03 s/δ _C 15.9; δ _H 1.18 s/δ _C 25.6; δ _H 1.19 s/δ _C 16.2; and δ _H 1.38 s/δ _C 23.6), one tri-substituted olefinic double bond (δ _H 5.28 t, J = 3.6 Hz/δ _C 127.8 and δ _C 138.8), and two carboxyl groups (δ _C 178.5 and 180.8), the characteristics of urs-12-ene or olean-12-ene derivatives. In addition, two secondary hydroxyl groups at the C-2 and C-3 positions were amended for 4 because of the (C)−CH₂−CHOH−CHOH−(C) structural moiety (δ _H 1.34 and 1.53 m; 3.89 ddd, J = 3.0, 4.8, and 12.0 Hz; δ _H 3.73 brd, J = 3.0 Hz) established by COSY spectrum. The HMBC spectrum of 4 confirmed the above deductions. Furthermore, the two carboxyl groups were assigned at C-24 (δ _C 180.8) and C-28 (δ _C 178.5) according to the HMBC correlations from H-18 (δ _H 2.49 brs) toC-17 (δ _C 47.6) and carboxyl carbon C-28 (δ _C 180.8), from CH₃-23 to two oxymethine carbons [C-3 (δ _C 75.4) and C-4 (δ _C 51.8)], carboxyl carbon C-24 (δ _C 178.5), respectively. The HMBC correlation from CH₃-29 to oxygen-bearing quaternary carbon C-19 (δ _C 72.1) put forward that the tertiary hydroxyl group is at C-19 (Figure 2). The configuration of 4 was established by the NOESY correlations (Figure 3) and the coupling constant interpretations for the relevant protons. The 2α,3α-configurations of the two secondary hydroxyl groups of 4 were ascertained by the NOESY correlations of H-2/H-3, H-2/CH₃-25, H-3/CH₃-23, CH₃-23/CH₃-25, and the small proton coupling constant of H-3 (J = 3.6 Hz) agreed with their directions. These elucidations leaded compound 4 to 2α,3α,19α-trihydroxyurs-12-ene-24,28-dioic acid, a triterpene acid monomer named mesonaic acid G, the first of its kind isolated from M. procumbens (it was reported to be a unit in the dimeric triterpene glucoside sanguidioside A) (Liu et al., 2004).

Mesonaic acid H (5) was obtained as white powders ([α] +27.3 (c 0.5, MeOH) with a molecular formula C₃₀H₄₆O₈ (8 DOU) determined by the HRESIMS ion at m/z 533.3124 [M ‒ H]^‒ (calcd. for C₃₀H₄₅O₈, 533.3109). Compound 5 was thought to be a urs-12-ene derivative where one methyl doublet (δ _H 0.98 d, J = 6.5 Hz), seven methyls, one tri-substituted olefinic double bond (δ _H 5.31 t, J = 4.0 Hz/δ _C 128.4 and δ _C 138.0), and three carboxyl groups (δ _C 174.2, 179.0 and 182.4) were deduced by ¹H- and ¹³C-NMR spectra (Tables 1, 2). Except for one secondary hydroxyl group and two carboxyl groups, the overall similarity concerning the chemical shifts of remaining carbons made the chemical skeleton of ursane-type triterpene acid with seven unmodified methyls. Based on HMBC correlations (Figure 2), the secondary OH group was assigned at C-22 by the resonances of H-22 (δ _H 3.71 dd, J = 4.5 and 12.0 Hz)/C-17 (δ _C 53.5) and carboxyl C-28 (δ _C 179.0). The HMBC correlations of both CH₃-23 (δ _H 1.29 s) and CH₃-24 (δ _H 1.28 s) to carboxyl C-3 (δ _C 182.4), C-4 (δ _C 46.1), and C-5 (δ _C 48.4) pointed out that there are two carboxyl groups one at C-3 and the other at C-2. It means that the C‒C bond between C-2 and C-3 is broken likely by an oxidative cleavage, and compound 5 has a seco-ring A. The stereochemistry of 5 is identical to regular ursane triterpene acids in whichα-orientation of OH-22 is supported by the NOESY correlations as shown in Figure 3. Added together, compound 5 was determined to be 19α,22α-dihydroxy-2,3-secours-12-ene-28-oic acid.

Compound 6, [α] +21.8 (c 0.5, MeOH), was isolated as white amorphous powders and had a molecular formula of C₂₉H₄₄O₅ determined by sodiated quasimolecular ion at m/z 495.3072 [M + Na]⁺ (calcd. for C₂₉H₄₄O₅Na, 495.3081) in the HRESIMS experiment. The IR, ¹H, and ¹³C-NMR spectroscopic data are very similar to compound 4. Detailed analysis of their data, compound 6 possesses an exocyclic double bond (δ _H 4.68 brs, 5.01 brs; δ _C 151.1, 109.3), which replaced a carboxyl group and a singlet methyl in 4. The complete chemical structure and NMR assignment (Tables 1, 2) were further established and confirmed by 1D and 2D NMR to be identified as 2α,3α,19α-trihydroxy-24-norursa-4 (23),12-dien-28-oic acid (Jang et al., 2005).

Compound 7 was collected as an oil sample ([α] +9.1, c 0.5 in MeOH) with a molecular formula C₂₂H₃₂O₅ (on par with 7 DOU) deduced from the HRESIMS ion at m/z 399.2148 [M + Na]⁺ (calcd. for C₂₂H₃₂O₅Na, 399.2142). According to ¹³C-NMR and DEPT spectra (Table 3), the 22 carbons can be divided into three methyls, six methylenes (one oxymethylene at δ _C 67.7), eight methines [including one oxymethine at δ _C 69.3 and five sp² methines at δ _C 128.1 (2), 129.0 (2), and 132.7], and five quaternary carbons (two oxygenated ones at δ _C 72.0 and 74.0, one sp2 quaternary carbon at δ _C 130.4, and one ester carbonyl at δ _C 166.0). In the ¹H-NMR spectrum (Table 3), three methyl singlets (δ _H 1.02 s, 1.12 s, and 1.19 brs), one oxygenated methylene [δ _H 3.46 s (2H)] and one oxygenated methine [δ _H 3.46 s (2H) and 5.18 ddd, J = 4.2, 7.8, and 12.0 Hz] together with the multiple peaks of mono-substituted benzene protons (δ _H 7.45–7.98) distributed spread from upfield to downfield. Combining the information, compound 7 was reasoned to have a double-ring moiety in addition to a benzene and an ester carbonyl portion. A comprehensive analysis of ¹H–¹H COSY and HMBC spectra, an eudesmane-type sesquiterpene (Evans et al., 1982) was put forward by incorporating two COSY fragments (H₂-1/H-2/H₂-3 and H-5/H₂-6/H-7/H₂-8/H₂-9) and the correlations of HMBC at CH₃-14/C-1, C-5, C-9, and C-10 and CH₃-15/C-3, C-4, and C-5. Moreover, the attachments of an O-benzoyl (OBz) group and a propane-1,2-diol group were confirmed to be at C-2 and C-7 by the HMBC correlations: H-2, H-2′, and H-3′ to ester carbonyl C-1′; H₂-12 and H₃-13 to C-7 and C-11 (Figure 4A). Concerning the stereochemistry of 7, it was concluded to be a regular eudesmane-type sesquiterpene with anti-relationships of α-oriented H-5 and β-oriented CH₃-14. Furthermore, both the cross peaks of NOESY correlations for H-2/H_β-3, H-2/CH₃-14, and H-2/CH₃-15 suggested the β-orientation of H-2 and CH₃-14 but α-orientation of H-7 given H_α-3/H-5, and H-5/H-7 (Figure 4B). However, the conformation of C-11 was not unambiguous because it cannot be differentiated directly by the NOESY correlations of H-7/CH₃-13, H_α-8/CH₃-13, H_β-8/CH₃-14, and H_β-8/H₂-12. For this reason, the dimolybdenum tetra-acetate [Mo₂(OAc)₄]-modified CD analysis was applied to resolve the steric assignment of the acyclic propane-1,2-diol group, a prim, sec-glycol (Frelek et al., 1997). As in many vic-glycols with a rigid conformation, one can follow the “helicity rule” to interpret the CD curves, whereby a positive (negative) torsional angle in the (HO)‒C‒C‒(OH) moiety should lead to a positive (negative) Cotton effect of 300 nm. In Figure 4C, a negative CD band at 300 nm is obvious and accompanied by a second Cotton effect of the same sign at around 400 nm corresponding to a negative torsional angle in the (HO)‒C‒C‒(OH) moiety. A Newman projection for the propane-1,2-diol moiety in chelation with Mo₂(OAc)₄ demonstrated a counterclockwise (a negative torsional angle) relationship from OH-12 to OH-11. Finally, compound 7 was clearly identified as 2-O-benzoyl-proximadiol, named mesoeudesmol B.

M. procumbens extracts have been shown with pharmacological potentialities for many inflammation-associated disorders (Huang et al., 2012; Huang et al., 2021). Given the isolated compounds 1‒7 from M. procumbens, we examined these isolates to see whether they similarly exhibit anti-inflammatory activities by using the LPS-induced RAW 264.7 macrophage cell model. As shown in Table 4, mesonaic acids D (1) and E (2), 2α,3α,19α-trihydroxy-24-norursa-4 (23),12-dien-28-oic acid (6), and mesoeudesmol B (7) show lower EC₅₀ values than the positive control, quercetin (Li et al., 2016), suggesting that these four compounds possess better anti-inflammatory activity than those previously reported. Among them, mesoeudesmol B (7), a proximadiol derivative, displayed the highest inhibition activity on NO production with an EC₅₀ value of 12.88 ± 0.23 μM than that of quercetin (an EC₅₀ value of 24.12 ± 0.21 μM). Furthermore, all these seven compounds (at a concentration of 30 μΜ) showed an approximate 100% survival rate in cell viability assay, suggesting that they are, in general, safe with no major cytotoxicity (to RAW264.7 cells).

To better understand the anti-inflammatory mechanism of compound 7, we examined two key inflammation-mediated proteins in LPS-induced RAW264.7 cells, iNOS and COX-2 at the protein level (Murakami and Ohigashi, 2007). In terms of the expression level of iNOS and COX-2 shown in Figure 5A, the LPS-treated cells display a higher level as opposed to the non-treated cells, which show a low expression level. In addition, the protein level of the LPS-induced iNOS and COX-2 is negatively proportional to the addition of mesoeudesmol B (7) (5–20 μM) into the LPS-treated cells in a dose-dependent manner. Notably, 10 μM of 7 exhibits the lowest iNOS/COX-2 than 25 μM of quercetin does; moreover, the protein level of iNOS and COX-2 in the LPS-treated cells with the addition of 7 (20 μM) is nothing more than that in the non-treated cells, confirming that sesquiterpene 7 possesses stronger inhibitory activity than quercetin.

Given that TNF-α and IL-6 are major pro-inflammatory cytokines, they were further measured by ELISA in the LPS-stimulated murine macrophages. As shown in Figure 5B, it shows a dose-dependent suppression of the LPS-induced secretion of cytokines TNF-α and IL-6 with the addition of compound 7, which shows a similar trend to that of the LPS-induced expression of iNOS and COX-2. Likewise, 10 μM of 7 exhibits a lower level of TNF-α and IL-6 than 25 μM of quercetin does; the level of TNF-α and IL-6 in the LPS-treated cells with the addition of 7 (20 μM) is close to the basal one, once again confirming that 7 is a stronger inflammatory inhibitor than quercetin.