Boswellianols A–I, Structurally Diverse Diterpenoids from the Oleo-Gum Resin of Boswellia carterii and Their TGF-β Inhibition Activity

Olibanum, a golden oleo-gum resin from species in the Boswellia genus (Burseraceae family), is a famous traditional herbal medicine widely used around the world. Previous phytochemical studies mainly focused on the non-polar fractions of olibanum. In this study, nine novel diterpenoids, boswellianols A–I (1–9), and three known compounds were isolated from the polar methanolic fraction of the oleo-gum resin of Boswellia carterii. Their structures were determined through comprehensive spectroscopic analysis as well as experimental and calculated electronic circular dichroism (ECD) data comparison. Compound 1 is a novel diterpenoid possessing an undescribed prenylmaaliane-type skeleton with a 6/6/3 tricyclic system. Compounds 2–4 were unusual prenylaromadendrane-type diterpenoids, and compounds 5–9 were new highly oxidized cembrane-type diterpenoids. Compounds 1 and 5 showed significant transforming growth factor β (TGF-β) inhibitory activity via inhibiting the TGF-β-induced phosphorylation of Smad3 and the expression of fibronectin and N-cadherin (the biomarker of the epithelial–mesenchymal transition) in a dose-dependent manner in LX-2 human hepatic stellate cells, indicating that compounds 1 and 5 should be potential anti-fibrosis agents. These findings give a new insight into the chemical constituents of the polar fraction of olibanum and their inhibitory activities on the TGF-β/Smad signaling pathway.


Introduction
Olibanum is the shining and golden oleo-gum resin collected from the trunk incisions of species from the Boswellia genus (Burseraceae family), which comprises 24 accepted species and is distributed in the dry and tropical regions of Asia and Africa [1].Boswellia carterii and Boswellia hhaw-dajiana, native to Somalia and Ethiopia along the Red Sea [2], are officially regarded as the medicinal material olibanum according to Pharmacopoeia of the People's Republic of China.Olibanum has been used worldwide as a traditional herbal medicine to treat swelling and pain from injuries, rheumatoid arthritis, and dysmenorrhea for thousands of years [3][4][5][6].Olibanum extract has shown its hepatoprotective activity by inhibiting liver fibrosis and downregulating the expression of cytokines including transforming growth factor β (TGF-β), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) [7].Olibanum combined with other herbs has also been reported to treat pulmonary and interstitial fibrosis by inhibiting the TGF-β/Smad pathway [8,9].In addition, β-boswellic acid, a main triterpenoid from olibanum, showed anti-inflammation, anti-tumor, and anti-interstitial fibrosis properties, partly involving the Klotho/TGF-β/Smad signaling pathway [10][11][12][13].

Chemical Constituents from Olibanum of Boswellia carterii
Olibanum was sequentially extracted with petroleum ether and methanol to provide non-polar and polar fractions.The polar methanolic fraction was then separated by silica gel column chromatography and further purified by MPLC and semi-preparative HPLC to obtain 12 diterpenoids (Figure 1), including nine undescribed diterpenoids, named boswellianols A-I (1-9).Three known compounds were identified as boscartin D (10) [27], boscartol O (11) [22], and cneorubin A (12) [28] by comparing their NMR data with those of compounds reported in the literature.

Chemical Constituents from Olibanum of Boswellia carterii
Olibanum was sequentially extracted with petroleum ether and methanol to provide non-polar and polar fractions.The polar methanolic fraction was then separated by silica gel column chromatography and further purified by MPLC and semi-preparative HPLC to obtain 12 diterpenoids (Figure 1), including nine undescribed diterpenoids, named boswellianols A-I (1-9).Three known compounds were identified as boscartin D (10) [27], boscartol O (11) [22], and cneorubin A (12) [28] by comparing their NMR data with those of compounds reported in the literature.2) corresponding to four methyls, four methylenes, eight methines [including three alkenyl sp 2 carbons (δ C 156.5, 120.3, and 150.0), one aldehyde carbon (δ C 195.0), and one oxygenated sp3 carbon (δ C 78.3)], and four quaternary carbons [including an alkenyl sp 2 carbon (δ C 138.4) and an oxygenated sp 3 carbon (δ C 72.1)].The above spectroscopic data indicated that 1 is a diterpenoid with three rings (one aldehyde and two double bonds occupying three of the six degrees of unsaturation).The planar structure of 1 was elucidated by the analysis of HSQC, HMBC, and 1 H-1 H COSY spectra (Figure 2).Two spin-coupling systems were first established by 1   2).The NMR data on the prenylaldehyde moiety in 1 were consistent with those of boscartol F [16], further confirming the presence of the α, β, γ, δ-unsaturated aldehyde in 1.The 1,4-dihydroxy-maaliane unit and prenylaldehyde moiety were connected by C-15 (δ C 156.5) and C-16 (δ C 120.3), corresponding to the 1 H-1 H COSY correlation of olefinic protons H-15/H-16 (Figure 2).The aforementioned data analysis elucidated that compound 1 is a new diterpenoid with an undescribed prenylmaaliane skeleton.The relative configuration was further analyzed by the NOESY spectrum.The NOESY cross peaks of Me-11/Me-12, Me-11/H-6, and H-6/H-7 indicated that H-6, H-7, Me-11, and Me-12 are arranged in an α-orientation, based on the same H-6α and H-7α of the cyclopropane unit in prenylaromadendrane-type diterpenoids [22,30] isolated from the Boswellia genus.NOESY interactions between H-1/H-5 and H-5/Me-14 suggested the β-orientation for H-1, H-5, and Me-14.The 15E and 17E were assigned by the large coupling constant (J H-15, H-16 = 15.0Hz) and the correlation between H-17 and H-19 in the NOESY spectrum, respectively.The absolute configuration of 1 was deduced as 1S,4S,5S,6R,7R,10S,13S by comparing experimental electronic circular dichroism (ECD) recorded in MeOH with the calculated ECD spectra of the two possible stereoisomers by TD-DFT (Figure 3).Thus, the structure of 1 was established as shown.
Plants 2024, 13, 1074 5 of 15 by comparing experimental electronic circular dichroism (ECD) recorded in MeOH with the calculated ECD spectra of the two possible stereoisomers by TD-DFT (Figure 3).Thus, the structure of 1 was established as shown.2) including two double bonds at δC 106.6, 131.1, 138.6, and 153.5, a secondary alcohol at δC 62.1, and a quaternary oxygenated carbon at δC 80.7.The 1 H and 13 C NMR data of 2 (Tables 1 and 2) were similar to those of cneorubin A (12), a known prenylaromadendrane-type [28]  The NOE enhancement of Me-14/H-5 indicated that Me-14 and H-5 were β-oriented, while H-1, H-6, H-7, and Me-11 were α-oriented according the NOESY correlations of H-1/H-6, H-6/H-7, and H-6/Me-11 (Figure 5).It was observed that the NMR data of C-16 to C-19 in 2 and the large coupling constant (J = 8.0 Hz) between H-16 and H-17 were identical with those of boscartol E, a similar compound with the same prenyl moiety isolated from this plant [16].Considering the same skeleton and biogenetic pathway, the orientation of 16-OH was deduced as 16β-OH, which is the same as that of boscartol E [16].The 17-Z configuration was assigned by the NOESY correlations of H2-19/H-16 and H-17/Me-20 (Figure 5) as well as the similar NMR data of C-16 to C-19 between 2 and boscartol E [16].Thus, the structure of 2 was established as shown and named boswellianol B.  1 and 2) were similar to those of cneorubin A (12), a known prenylaromadendrane-type [28]     Compound 1 is the first example of a prenylmaaliane-type diterpenoid.The biosynthesis pathway of prenylaromadendrane-type diterpenoids is unclear.It was proposed (Figure 5) that compounds 1 and 2 could originate from the same intermediate cneorubin Y, a bicyclogermacrene-type diterpenoid, which should be formed from geranylgeranyl pyrophosphate (GGPP) by enzyme-catalyzed cyclization and deprotonation [31,32].The 1,3-deprotonation and cyclization of intermediate i could be essential steps to construct the primary scaffold with a cyclopropane ring in these diterpenoids.The key step to form the 6,6-bicyclic system of the prenylmaaliane-type intermediate iii from cneorubin Y by oxygenation and 1,6-cyclisation (shown as the blue arrow in Figure 6) would be similar to the reported enzyme-catalyzed cyclization to generate the maaliane-type sesquiterpene skeleton from bicyclogermacrene-type sesquiterpenoids [33,34].Subsequently, the intermediate iii may undergo further oxygenation and deprotonation to form compound 1.On the other hand, the oxygenation, 1,5-cyclisation(shown as the red arrow in Figure 6), and double bond rearrangement of cneorubin Y could produce the prenylaromadendrane-type diterpenoid, cneorubin X [33,34], which could be further oxidized to form compound 2 (Figure 6).

OR PEER REVIEW 7 of 15
Compound 1 is the first example of a prenylmaaliane-type diterpenoid.The biosynthesis pathway of prenylaromadendrane-type diterpenoids is unclear.It was proposed (Figure 5) that compounds 1 and 2 could originate from the same intermediate cneorubin Y, a bicyclogermacrene-type diterpenoid, which should be formed from geranylgeranyl pyrophosphate (GGPP) by enzyme-catalyzed cyclization and deprotonation [31,32].The 1,3-deprotonation and cyclization of intermediate i could be essential steps to construct the primary scaffold with a cyclopropane ring in these diterpenoids.The key step to form the 6,6-bicyclic system of the prenylmaaliane-type intermediate iii from cneorubin Y by oxygenation and 1,6-cyclisation (shown as the blue arrow in Figure 6) would be similar to the reported enzyme-catalyzed cyclization to generate the maaliane-type sesquiterpene skeleton from bicyclogermacrene-type sesquiterpenoids [33,34].Subsequently, the intermediate iii may undergo further oxygenation and deprotonation to form compound 1.On the other hand, the oxygenation, 1,5-cyclisation(shown as the red arrow in Figure 6), and double bond rearrangement of cneorubin Y could produce the prenylaromadendranetype diterpenoid, cneorubin X [33,34], which could be further oxidized to form compound 2 (Figure 6).Compound 3 shared the same formula as 2 deduced by its sodium adduct ion at m/z 343.2250 [M + Na] + (calculated for C20H32NaO3 as 343.2244) and protonated ion at m/z 321.2411 [M + H] + (calculated for C20H33O3 as 321.2424), indicating that 3 could be an isomer of 2 bearing the same prenylaromadendrane skeleton.The 1 H and 13 C NMR spectrum of 3 showed similar proton and carbon chemical shifts of aromadendrane moiety with 2 (Tables 1 and 2).The major difference between the two compounds was the prenyl group side chain.The 2,3-dihydroxy-2-methylpent-4-ene side chain in 3 was established by 1    1 and 2).The major difference between the two compounds was the prenyl group side chain.The 2,3-dihydroxy-2-methylpent-4-ene side chain in 3 was established by 1    4).The 15E double bond was confirmed by the big coupling constants (J 15,16 = 15.1 ).The 17E was suggested by the NOE correlation of H-17/H-19 and the NMR data comparison with those of 1 and boscartol F [16].The relative configuration of compound 4 was defined by NOESY interactions of H-1/H-11, H-12 and H-6/H-1, H-7 suggesting the same α-orientation as 2 and 3 (Figure 5).
Compound 7 was obtained as a white amorphous solid with a molecular formula of C 22 H 38 O 6 established by HR-ESI-MS sodium adduct ion at m/z 421.2540 (calculated as 421.2561 for C 22 H 38 NaO 6 ).The 1 H and 13 C NMR data of 7 (Tables 2 and 3) were similar to those of boscartin Z [19], except for the absence of the ketone (C-5) and the C3-C4 double bond of boscartin Z and the presence of two hydroxy groups at C-3 (δ C 71.0) and C-4 (δ C 75.1) in 7, which was supported by the HMBC correlations from Me-18 to C-3 and C-5 and from H-3 to C-1 and C-4 (Figure 4), as well as the molecular formula of 7. The double bond [δ H 5.56 (1H, td, J = 15.8, 7.0 Hz, H-6) and 5.77 (1H, td, J = 15.9, 1.4 Hz, H-7)] was assigned at the C-6 and C-7 positions based on 1 H-1 H COSY correlations of H-5/H-6/H-7 and the HMBC correlation from Me-19 to C-7, C-8, and C-9 (Figure 4).It has been reported that cembrane-type diterpenoids with 3-α-OH and quaternary C-4 have the smaller coupling between H-3 and H-2 (J H-2, H-3 = ~7.6-8.8Hz) than those with 3-β-OH (J H-2, H-3 = 10.0-10.8Hz) isolated from the Boswellia genus [15,19].Considering the similar coupling constant between H-3 and H-2 (J H-2, H-3 = 8.5 Hz in 7) and biogenesis, H-3β of 7 was deduced.The obvious NOE correlations of H-11/H-13a, Me-20/Me-16, and H-3/Me-18 (Figure 5), and the absence of the NOE correlation of H-11/Me-20 in the NOESY spectrum of 7, as well as the similar C-11-C-13 NMR data with those of boscartin Z [19], suggested that the Me-20, Me-18, and 11-OAc group were in β-orientation in 7. The orientation of Me-19 was not determined because of a lack of convincing evidence.Therefore, the structure of 7 was established as shown and named boswellianol G.

TGF-β Inhibition Assessments of the Isolated Compounds
Liver fibrosis is the result of a chronic wound-repairing response after injury [37].While injured by viruses, toxins, or cholestasis, the quiescent hepatic stellate cells (HSCs) are targeted by profibrogenic cytokines TGF-β and activated as myofibroblasts to produce fibrotic-related marker collagen type I, fibronectin (FN), N-cadherin (N-cad), and α-smooth muscle actin [38].The hepatoprotective activity against the hepatic fibrosis of the isolated compounds was evaluated in TGF-β-induced LX-2 HSCs by Western blot.The results showed that compounds 1, 5, 9, and 11 inhibited the TGF-β-induced Smad3 phosphorylation at 25 µM in LX-2 cells (Figure S89).Compounds 1 and 5 were then chosen for the further analysis of their effects on the TGF signaling pathway at different concentrations, using SB-431542 (a TGF-β inhibitor) as a positive control.Compound 5 showed significant inhibitory effects on the TGF-β-induced Smad3 phosphorylation at 12.5, 25, and 50 µM, while a weaker inhibitory effect of 1 on the TGF-β-induced Smad3 phosphorylation was observed in LX-2 cells (Figure 7).The TGF-β-induced upregulation of N-cad and FN was suppressed by 1 and 5 in a dose-dependent manner (Figure 6).These data indicated that 1 and 5 could inhibit the LX-2 cell epithelial-mesenchymal transition by inhibiting the TGF/Smad signaling pathway and should be potential anti-fibrosis agents.

TGF-β Inhibition Assessments of the Isolated Compounds
Liver fibrosis is the result of a chronic wound-repairing response after injury [37].While injured by viruses, toxins, or cholestasis, the quiescent hepatic stellate cells (HSCs) are targeted by profibrogenic cytokines TGF-β and activated as myofibroblasts to produce fibrotic-related marker collagen type I, fibronectin (FN), N-cadherin (N-cad), and αsmooth muscle actin [38].The hepatoprotective activity against the hepatic fibrosis of the isolated compounds was evaluated in TGF-β-induced LX-2 HSCs by Western blot.The results showed that compounds 1, 5, 9, and 11 inhibited the TGF-β-induced Smad3 phosphorylation at 25 µM in LX-2 cells (Figure S89).Compounds 1 and 5 were then chosen for the further analysis of their effects on the TGF signaling pathway at different concentrations, using SB-431542 (a TGF-β inhibitor) as a positive control.Compound 5 showed significant inhibitory effects on the TGF-β-induced Smad3 phosphorylation at 12.5, 25, and 50 µM, while a weaker inhibitory effect of 1 on the TGF-β-induced Smad3 phosphorylation was observed in LX-2 cells (Figure 7).The TGF-β-induced upregulation of N-cad and FN was suppressed by 1 and 5 in a dose-dependent manner (Figure 6).These data indicated that 1 and 5 could inhibit the LX-2 cell epithelial-mesenchymal transition by inhibiting the TGF/Smad signaling pathway and should be potential anti-fibrosis agents.

General Experimental Procedures
Optical rotations were measured on a Rudolph Research Analytical Autopol I (Rudolph, Hackettstown, USA) automatic polarimeter.UV spectra and ECD spectra were detected on a JASCO High Performance J-1500 CD spectrometer (JASCO, Tokyo, Japan).NMR spectra were obtained at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR,

Cell Lines and Cultures
The human hepatic stellate cell line LX-2 was provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).The cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) in a 5% CO 2 humidified atmosphere at 37.0 • C.

Western Blot Analysis
The LX-2 cells were seeded into a 6-well plate and incubated for 24 h.Cells were pretreated with or without selected compounds (50 µM), SB431542 (2 µM, positive control), or 12.5, 25, and 50 µM of compounds 1 and 5 for 2 h and then stimulated with 5 ng/mL TGF-β1 (Sigma-Aldrich, St. Louis, MI, USA) for 45 min (for Phospho-Smad3 and Smad2/3) or 48 h (for FN and N-cad).Cells were collected and lysed in radioimmunoprecipitation

Figure 6 .
Figure 6.The plausible biosynthesis pathway of compounds 1 and 2. Compound 3 shared the same formula as 2 deduced by its sodium adduct ion at m/z 343.2250 [M + Na] + (calculated for C 20 H 32 NaO 3 as 343.2244) and protonated ion at m/z 321.2411 [M + H] + (calculated for C 20 H 33 O 3 as 321.2424), indicating that 3 could be an isomer of 2 bearing the same prenylaromadendrane skeleton.The 1 H and 13 C NMR spectrum of 3 showed similar proton and carbon chemical shifts of aromadendrane moiety with 2 (Tables1 and 2).The major difference between the two compounds was the prenyl group side chain.The 2,3-dihydroxy-2-methylpent-4-ene side chain in 3 was established by1 H-1 H COSY correlation between H-15 (δ H 5.36, d, J = 15.5 Hz), H-16 (δ H , 5.41, dd, J = 15.5, 7.6 Hz), and H-17 [δ H 3.84 (1H, d, J = 7.6 Hz), as well as HMBC correlations from H-17 to C-18/C-19/C-20 (Figure 4).The NOESY correlations of Me-14/H-5, H-6/H-7, and Me-11/ H-1, H-6 (Figure 5) suggested that the relative configuration of the aromadendrane moiety in 3 was the same as that of 2. Because of the free rotation of the prenyl moiety, the configuration of C-17 was not determined in this study.Therefore, the structure of 3 was determined as shown and named boswellianol C. Boswellianol D (4) has the same molecular formula of C 20 H 30 O 3 as 1.The 1D and 2D NMR data demonstrated that 4 shared the same prenylaromadendrane skeleton as 2 and 3.The presence of the four methyl singlets, a quaternary oxygenated carbon at C-10 (δ C 74.8), and the absence of two terminal olefinic methylene protons in 4 showed that the exocyclic double bonds at C-10 of 2 and 3 were oxidized, which was proved by HMBC correlations from Me-12 (δ H 1.24, s) to C-1, C-9, and C-10.The remarkable α, β, H-1 H COSY correlation between H-15 (δ H 5.36, d, J = 15.5 Hz), H-16 (δ H , 5.41, dd, J = 15.5, 7.6 Hz), and H-17 [δ H 3.84 (1H, d, J = 7.6 Hz), as well as HMBC correlations from H-17 to C-18/C-19/C-20 (Figure 4).The NOESY correlations of Me-14/H-5, H-6/H-7, and Me-11/ H-1, H-6 (Figure 5) suggested that the relative configuration of the aromadendrane moiety in 3 was the same as that of 2. Because of the free rotation of the prenyl moiety, the configuration of C-17 was not determined in this study.Therefore, the structure of 3 was determined as shown and named boswellianol C. Boswellianol D (4) has the same molecular formula of C 20 H 30 O 3 as 1.The 1D and 2D NMR data demonstrated that 4 shared the same prenylaromadendrane skeleton as 2 and 3.The presence of the four methyl singlets, a quaternary oxygenated carbon at C-10 (δ C 74.8), and the absence of two terminal olefinic methylene protons in 4 showed that the exocyclic double bonds at C-10 of 2 and 3 were oxidized, which was proved by HMBC correlations from Me-12 (δ H 1.24, s) to C-1, C-9, and C-10.The remarkable α, β, Plants 2024, 13, 1074 8 of 15 O 5 ) in the HR-ESI-MS spectrum, implying four indices of hydrogen deficiency.The 1 H and 13 C NMR data of 6 (Tables

Figure 7 .
Figure 7.The inhibitory effects of compounds 1 and 5 on TGF-β/Smad signal pathway in LX-2 cells.The effects of compounds 1, 5, and SB-431542 (positive control) on Smad3 phosphorylation (a) and expression of EMT-related proteins, including FN and N-cad (c) in the TGF-β-induced LX-2 cells by Western blot.The rate of Smad3 phosphorylation (b), relative expression of FN (d), and N-cad (e) were calculated as the means ± SD (n = 3).Statistical significance was assessed by t-test for two groups comparison.* p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the TGF-β-induced group.

Figure 7 .
Figure 7.The inhibitory effects of compounds 1 and 5 on TGF-β/Smad signal pathway in LX-2 cells.The effects of compounds 1, 5, and SB-431542 (positive control) on Smad3 phosphorylation (a) and expression of EMT-related proteins, including FN and N-cad (c) in the TGF-β-induced LX-2 cells by Western blot.The rate of Smad3 phosphorylation (b), relative expression of FN (d), and N-cad (e) were calculated as the means ± SD (n = 3).Statistical significance was assessed by t-test for two groups comparison.* p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the TGF-β-induced group.