Anti-Inflammatory Triterpenoids from the Stems of Microtropis Fokienensis

Three new ursane- and four new oleanane- type triterpenoids 1–7 were isolated, along with six known compounds 8–13, from the methanolic extract of Microtropis fokienensis. All structures were elucidated by mass and NMR spectroscopic methods. The isolates 4–10 and known compounds 14–17 that were previously isolated from this material were evaluated for anti-inflammatory activity based on effects against superoxide anion generation and elastase release by neutrophils in response to fMLP/CB. 11α,30-Dihydroxy-2,3-seco-olean-12-en-2,3-dioic anhydride (7) was the first triterpene anhydride from the genus of Microtropis to have the ring A expanded to a seven-membered ring; it showed significant anti-inflammatory activity against superoxide anion generation and elastase release. Unexpectedly, 30-hydroxy-2,3-seco-lup-20(29)-ene-2,3-dioic acid (17) showed the best effect against superoxide anion generation and elastase release with IC50 values of 0.06 ± 0.01 and 1.03 ± 0.35 µg/mL, respectively. Compound 17 had a dioic acid function, and compound 7 had an anhydride function modification in ring A; both showed promising activity in the target assays.

Compound 1 was obtained as a white amorphous solid. Its IR spectrum showed absorption bands for hydroxyl groups (3425 cm −1 ) and an α,β-unsaturated ketone system (1655 cm −1 ). The molecular formula was determined to be C 30    Therefore, 1 was suggested to have an urs-12-ene frame and the α,β-unsaturated ketone system in ring C was similar to 3β-hydroxyurs-12-en-11-one, except for the presence of one more oxymethine [16][17][18]. The MS fragmentations at m/z 248, where each bond was cleaved at the α,β-unsaturated ketone system in ring C, indicated that the other hydroxyl group was seemed to be located in either ring D or E. (Figure 2) [19]. The hydroxyl group was assigned to C-16, because of the low field shifted signals of δ C 60.7 (C-18), and the HMBC correlation of H-18 (δ H 1.79) and H-15 (δ H 2.11)/C-16 (δ C 64.7) (Figure 3). The NOESY experiment showed the NOE effect of H-3 and H-16 with H-5 and H-27 ( Figure 4), respectively, indicating that the relative stereochemistry of the two hydroxyl groups at C-3 and C-16 were resolved as the β form. Therefore, the structure of 1 was determined and named 3β,16β-dihydroxyurs-12-en-11-one.  (1)  (1)  Table 1). The 13 C-NMR and the DEPT spectra of 2 suggested the presence of a cyclic ketone at δ C 216.0, two olefinic carbons at δ C 116.5 and 145.0, two oxygenated carbons at δ C 67.8 and 77.3, a methylene carbon at δ C 66.8, and a methoxy carbon at δ C 51.4 (Table 2). Based on the molecular formula of C 31 H 50 O 5 , the degrees of unsaturation of 2 were determined to be seven, including one ketone and double bond. This spectral evidence suggested that 2 was a derivative of the pentacyclic triterpenoid urs-12-en-3-one, with a tetrasubstituted double bond, two oxygenated methines, one hydroxymethylene, and one methoxy moiety [5]. Based on the evidence of the HMBC spectrum (Figure 3), a secondary hydroxyl, a methoxy, and a tertiary hydroxyl groups were deemed to be attached at C-6, C-11, and C-12. The chemical shifts of C-4 (δ C 54.7) and CH 3 -24 (δ C 20.5) led to the assignment of the CH 2 OH unit at the C-23α position [20]. The NOESY ( Figure 4) correlations illustrated the stereochemistry of 2. Therefore, the structure of the new compound 2 was determined to be 6β,12,23-trihydroxy-11α-methoxyurs-12-en-3-one.
Compound 3, isolated as white amorphous powder, showed IR absorption bands for hydroxyl (3418 cm −1 ) and carbonyl (1694 cm −1 ) groups. The molecular formula was determined as C 30 Table 2). This spectral evidence suggested that 3 was an urs-12-en-3-one derivative with a tetrasubstituted double bond, and two oxygen-bearing functional groups [5]. The partial structure of 3 was solved by the HMBC correlations of the eight methyl groups. Detailed analysis of other correlations in the HMBC spectrum determined the locations of the followings substituents: three hydroxyl groups were located at C-11, C-12, and C-16, and a double bond between C-12 and C-13 ( Figure 3). The stereochemistry of 3 was further established from the NOESY spectrum (Figure 4), and the structure 11α,12,16β-trihydroxyurs-12-en-3-one was assigned.
Compound 4 was assigned a molecular formula of C 30 H 48 O 3 and seven degrees of unsaturation, as deduced from the HRESIMS (m/z 479.3501 [M+Na] + , calcd. for 479.3503) and 13 C-NMR spectra. The IR spectrum showed a hydroxyl group (3421 cm −1 ) and an α,β-unsaturated ketone system (1652 cm −1 ). The 13 C-NMR and DEPT spectra displayed 30 carbon signals, with signals for two carbons bearing oxygen observed at δ C 72.3 (2 × C) and signals for an α,β-unsaturated ketone system observed at δ C 128.6, 169.7, and 200.9. A comparison of the 1 H and 13 C-NMR spectra data of 4 with those of 1 (Tables 1 and 2) revealed that 4 differed from 1 in the presence of eight tertiary methyls and had an oleanane skeleton [21]. The substituted functions and planar structure of 4 were established in extensive interpretation of its 2D NMR spectra (Figure 3). Based on the NOESY spectrum (Figure 4), the configurations of the H-1 and H-3 were assigned to be β and α orientation. Therefore, the structure of 4 was determined to be 1α,3β-dihydroxyolean-12-en-11-one.
The IR spectrum of compound 5 showed a hydroxyl group (3441 cm −1 ), a carbonyl group (1697 cm −1 ), and an α,β-unsaturated ketone system (1648 cm −1 ). The molecular formula C 30 1 and 2) indicated the presence of a carbonyl and a hydroxymethylene on the 12-oleanen-11-one chemical frame instead of two hydroxymethines in 4 [19]. The key HMBC were shown on Figure 3 in order to determine the locations of the substituents and the planar structure. Based on this evidence and the NOESY ( Figure 4), a primary alcohol group was deemed to be located at C-30. Therefore, the structure of compound 5 was determined to be 30-hydroxyolean-12-en-3,11-dione.
Compound 7 was isolated as a white amorphous powder. Its molecular formula, C 30 Table 1). The 13 C-NMR spectrum indicated the presence of 30 carbon atoms, including one trisubstituted double bond (δ C 121.4 and 149.8), seven methyls, nine methylenes (one oxygenated), four methines (one oxygenated), and eight quaternary carbons (two carbonyl carbons) ( Table 2). Previous studies [28,29] showed two carbonyls groups at δ C 170.6 and 182.0 together with the absence of absorption bands for carboxylic groups in the IR spectrum, which indicated the presence of an anhydride moiety in the molecule. Eight degrees of unsaturation were implied by the molecular formula. As well as two carbonyls and one olefinic group, compound 7 possesses a pentacyclic system. This data indicated that this compound belongs to the olean-12-ene family of triterpenoids, with an embedded anhydride function and two additional hydroxyl groups [28,29]. In the HMBC spectrum of 7, the correlations for H-11 (δ H 4.91)/C-12 (δ C 121.4), and for H-12 (δ H 5.49)/C-9 (δ C 45.9) and C-18 (δ C 47.2) indicated that a hydroxyl group was attached at C-11. The HMBC correlations of both H-29 and H-30 with C-19 and C-20 also indicated that the hydroxymethylene group was attached at C-20. On the basis of the previous study [30], the chemical shift of the C-29 (equatorial) hydroxymethylene group resonated at around 75 ppm and the value of the C-30 (axial) methyl group was found at around 20 ppm in the 13 C-NMR spectrum. In contrast, the chemical shift of the C-30 hydroxymethylene group appeared at around 67 ppm, while the C-29 methyl group appeared at around 28 ppm. Therefore, the remaining hydroxyl group in 7 was placed at C-30. Detailed analysis of the correlations of the HMBC spectrum gave the location of the anhydride at C-2 and C-3 ( Figure 3). The orientation of a hydroxyl group at C-11 was assumed to be α on the basis of the NOESY correlations between H-11 and H-25. In according with all these data, the structure of 7 was established as 11α,30-dihydroxy-2,3-seco-olean-12-en-2,3-dioic anhydride.
Although a triterpene anhydride has been reported from species of the Celastraceae family, 11α,30dihydroxy-2,3-seco-olean-12-en-2,3-dioic anhydride (7) represents the first example of a triterpene anhydride with the ring A expanding to a seven-membered ring from the genus of Microtropis.

Compounds
Superoxide anion Elastase IC 50 (g/mL) a or (Inh %) IC 50 (µg/mL) a or (Inh %) 4 125.9 ± 6.72 Percentage of inhibition (Inh %) at 10 µg/mL concentration. Results are presented as mean ± S.E.M. (n = 3-5).  p  0.01,  p  0.001 compared with the control value; a Concentration necessary for 50% inhibition (IC 50 ); b 4, 5, 9, and 10 induced superoxide anion generation by human neutrophils in absence of fMLP/CB; c 4 induced elastase release by human neutrophils in present of CB; d Diphenyleneiodonium (DPI, a NADPH oxidase inhibitor) and phenylmethylsulfonyl fluoride (PMSF, a serine protease inhibitior) were used as the positive controls in the generation of superoxide anion and release of elastase, respectively.

General
The IR spectra were obtained using a Mattson Genesis II spectrometer. 1 H and 13 C-NMR spectra were recorded using Varian VNMR-600, Varian INOVA 500, Varian Unity Plus 400, or Varian Gemini 200 NMR spectrometers. Chemical shifts (δ) are reported in parts per million, and coupling constants (J) are expressed in Hertz. The LREIMS and LRESIMS were measured using a VG Biotech Quattro 5022 mass spectrometer. The HRESIMS were measured using a Bruker Daltonics APEX Π mass spectrometer. Silica gel 60 (Merck, 230-400 mesh) and Sephadex LH-20 were used for column chromatography and TLC analysis was performed on silica gel GF 254 pre-coated plates and detection used 50% H 2 SO 4 followed by heating on a hot plate. HPLC was performed using a Hitachi L-7100 pump and a D-7000 interface equipped with a Bischoff RI detector using ODS (Hypersil ® , 250 × 4 mm; Hypersil ® , 250 × 10 mm) columns.

Plant Material
The dried stems of Microtropis fokienensis were collected from Taichung County, Taiwan, in June 2004, and identified by a botanist, Dr. Hsin-Fu Yen. A voucher specimen (Microtropis-01) was deposited at the Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.

Extraction and Isolation
The dried stems (2.85 kg) of M. fokienensis were extracted four times with MeOH (10 L) overnight at room temperature, followed by removal of the solvent under reduced pressure, to yield a dried MeOH extract (130 g). The MeOH extract was dissolved in H 2 O and extracted using EtOAc. Using nhexane and 80% MeOH, the EtOAc-soluble fraction was divided into n-hexane and aqueous MeOH layers. The aqueous MeOH layer (38.0 g) was chromatographed on silica gel using mixtures of nhexane-EtOAc of increasing polarity as eluants to afford eighteen fractions. Fr. 10 (2.5 g) was chromatographed on Sephadex LH-20, using

The Preparation of Human Neutrophils
Blood was taken from healthy human donors (20-35 years old) by venipuncture using a protocol approved by the Institutional Review Board at Chang Gung Memorial Hospital. Neutrophils were isolated using a standard method as previously described [31,32].

The Measurement of Superoxide Generation
SOD inhibition was measured by the reduction of ferricytochrome c [31,32]. Neutrophils in 0.5 mg/mL ferricytochrome c and 1 mM Ca 2+ were equilibrated at 37 °C for 2 min and then incubated with drugs for 5 min. The cells were activated using formyl-methionyl-leucyl-phenylalanine (fMLP, 100 nM)/cytochalasin B (CB, 1 µg/mL) for 10 min. The absorbance was continuously monitored at 550 nm using a double-beam, six-cell positioned spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations were based on the differences in absorbance with and without SOD (100 µ/mL) divided by the extinction coefficient for the reduction of ferricytochrome c (ε = 21.1/mM/10 mm).

The Measurement of Elastase Release
The degranulation of azurophilic granules was determined by elastase release as described previously [32]. Neutrophils (6 × 10 5 /mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-pnitroanilide (100 µM), an elastase substrate, at 37 °C for 2 min and then incubated with drugs for 5 min. The cells were activated using fMLP (100 nM) in the presence of CB (0.5 µg/mL), and changes in the absorbance at 405 nm were continuously measured to monitor elastase release. The results are expressed as a percentage of the initial rate of elastase release in the fMLP/CB-activated, drug-free control system.

Conclusions
In this study, a bioassay-guided separation of a MeOH extract of M. fokienensis stems resulted in the isolation of seven new triterpenoids 1-7 and six known compounds 8-13. Their isolation, purification, and structural determination are reported. Compounds 4-10 and 14-17 were tested in an anti-inflammatory assay for effects against superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB. Compounds 7 and 17 showed significant anti-inflammatory activity against superoxide anion generation and elastase release. Compounds 5 and 6 exhibited a selective inhibitory effect on elastase release. Compounds 8 and 15 exhibited good anti-inflammatory activity against superoxide anion generation. Interestingly, compound 17 had a dioic acid function and compound 7 had an anhydride function modification in ring A. Both perform well in the target assays. We propose that the modified dicarbonyl A-ring systems of triterpenoids may play a pivotal role in exhibiting anti-inflammatory biological functions on human neutrophils in response to fMLP/CB. This is also the first report of the anti-inflammatory effects of a plant from this genus, and these compounds may lead to pharmaceutical advances in the near future.