Eudesmane and Eremophilane Sesquiterpenes from the Fruits of Alpinia oxyphylla with Protective Effects against Oxidative Stress in Adipose-Derived Mesenchymal Stem Cells

Alpinia oxyphylla Miquel (Zingiberaceae) has been reported to show antioxidant, anti-inflammatory, and neuroprotective effects. In this study, two new eudesmane sesquiterpenes, 7α-hydroperoxy eudesma-3,11-diene-2-one (1) and 7β-hydroperoxy eudesma-3,11-diene-2-one (2), and a new eremophilane sesquiterpene, 3α-hydroxynootkatone (3), were isolated from the MeOH extract of dried fruits of A. oxyphylla along with eleven known sesquiterpenes (4–14). The structures were elucidated by the analysis of 1D/2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and optical rotation data. Compounds (1–3, 5–14) were evaluated for their protective effects against tert-butyl hydroperoxide (tBHP)-induced oxidative stress in adipose-derived mesenchymal stem cells (ADMSCs). As a result, treatment with isolated compounds, especially compounds 11 and 12, effectively reverted the damage of tBHP on ADMSCs in a dose-dependent manner. In particular, 11 and 12 at 50 µM improved the viability of tBHP-toxified ADMSCs by 1.69 ± 0.05-fold and 1.61 ± 0.03-fold, respectively.


Structure Elucidation
Compound 1 was isolated as a yellow oil. Its molecular formula, C15H22O3, with five degrees of unsaturation, was determined based on the high-resolution electrospray ionization mass spectrometry (HRESIMS) ion peak at m/z 251. 1643 3H)). The 13 C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra displayed fifteen carbon signals, including one carbonyl group at δ C 198.9; four quaternary carbons at δ C 162.0, 141.5, 86.7, and 37.9; four methylenes at δ C 54.1, 37.2, 28.7, and 26.7; an olefinic methylene at δ C 118.5; three methyls at δ C 22.1, 18.9 and 17.0; one methine at δ C 44.5; and one olefinic methine at δ C 127.2. The 1 H and 13 C-NMR spectra of 1 (Tables 1 and 2) were found to be similar to those of the known eudesmane sesquiterpene, 7-epi-teucrenone (6) [16], except for a substitution of a tertiary hydroperoxy group (δ C 85.9) [25,26] instead of a tertiary hydroxy group (δ C 74.4) [16] at C-7, which was supported by the downfield shifted signal of C-7 (δ C 86.7). Five degrees of unsaturation indicated a dicyclic ring system possesses one carbonyl group and two double bonds. The planar structure of 1 was established based on a comprehensive analysis of COSY, HSQC, and HMBC correlations, as shown in Figure 2. Key HMBC correlations from H-1 to C-2/C-5, H-3 to C-1/C-5/C-15, H-15 to C-3/C-4/C-5, and H-5 to C-15 were used to elucidate ring A. Ring B was confirmed by HMBC correlations from H-6 to C-5/C-7/C-8/C-10 and H-8 to C-10. The HMBC correlations from H-12 to C-7 and H-13 to C-7/C-11/C-12 suggested an isopropylidene group positioned at C-7. All figures of HRESIMS and 1D, 2D NMR of compound 1 were provided in supplementary material (Figures S1-S8).    Compound 2 was isolated as a white powder. Its molecular formula, C15H22O3, with five degrees of unsaturation, was confirmed based on the HRESIMS ion peak at m/z 251.1639 [M + H] + (calcd for C15H23O3 + , 251.1642) (S16). The fragmentation pattern showed the same pattern as mentioned in 1 to confirm the presence of a hydroperoxy group. The   The relative configuration of 1 was determined through the NOESY experiment (Figure 3). The NOE correlations between H-6β and H-12b as well as H-6α and H3-14 indicated that both the C-7 hydroperoxide and C-14 methyl groups were α-positioned. In addition, the NOE correlation between H-5 and H-12b suggested a β-oriented proton at C-5. The optical rotation was obtained to determine the absolute configuration of 1; a weak nega-  Compound 2 was isolated as a white powder. Its molecular formula, C15H22O3, with five degrees of unsaturation, was confirmed based on the HRESIMS ion peak at m/z 251.1639 [M + H] + (calcd for C15H23O3 + , 251.1642) (S16). The fragmentation pattern showed the same pattern as mentioned in 1 to confirm the presence of a hydroperoxy group. The ) correlations of 1-3.

Results and Discussion
The relative configuration of 1 was determined through the NOESY experiment ( Figure 3). The NOE correlations between H-6β and H-12b as well as H-6α and H 3 -14 indicated that both the C-7 hydroperoxide and C-14 methyl groups were α-positioned. In addition, the NOE correlation between H-5 and H-12b suggested a β-oriented proton at C-5. The optical rotation was obtained to determine the absolute configuration of 1; a weak negative specific rotation, [α] 25 D : −1.2 (c 0.25, CH 3 OH), was observed, indicating the presence of a racemic mixture. Based on these observations, the structure of 1 was elucidated as 7α-hydroperoxy eudesma-3,11-diene-2-one.  The relative configuration of 1 was determined through the NOESY experiment (Figure 3). The NOE correlations between H-6β and H-12b as well as H-6α and H3-14 indicated that both the C-7 hydroperoxide and C-14 methyl groups were α-positioned. In addition, the NOE correlation between H-5 and H-12b suggested a β-oriented proton at C-5. The optical rotation was obtained to determine the absolute configuration of 1; a weak nega-  Compound 2 was isolated as a white powder. Its molecular formula, C15H22O3, with five degrees of unsaturation, was confirmed based on the HRESIMS ion peak at m/z 251.1639 [M + H] + (calcd for C15H23O3 + , 251.1642) (S16). The fragmentation pattern showed the same pattern as mentioned in 1 to confirm the presence of a hydroperoxy group. The   Figure S16). The fragmentation pattern showed the same pattern as mentioned in 1 to confirm the presence of a hydroperoxy group. The comparison of the 1 H and 13 C-NMR spectroscopic data revealed that the planar structure of 2 was identical to that of 1, which was further supported by the analysis of the 2D NMR spectra, as shown in Figure 2 (Figures S9-S15). The relative configurations of a hydroperoxy group at C-7 in the eudesmane sesquiterpene were determined using 13 C-NMR chemical shift differences at C-11 and C-12 [16,17] as well as NOESY spectrum. The resonances for C-11 and C-12 in 2 were downfield and upfield shifted, respectively, both by 6 ppm, compared to those of 1, suggesting that the relative configurations at C-7 in 2 were different from those in 1. The H-6α was observed as dd with relatively large coupling constants of 14.0 Hz ( 2 J H-6α, H-6β ) and 13.2 Hz ( 3 J H-5, H-6α ), which indicated H-6α and H-6β are axial and equatorial orientations, respectively. The NOE correlations between H-6α and H-14, as well as between H-6α and H-12a revealed that an isopropylidene group at C-7 of 2 was positioned on the same side with a methyl group (H 3 -14). Again, to determine the absolute configuration of 2, the optical rotation was obtained, and a weak positive specific rotation, [α] 25 D : +3.16 (c 0.19, CH 3 OH), was observed, concluding the presence of a racemic mixture. Based on these observations, the structure of 2 was elucidated as 7β-hydroperoxy eudesma-3,11-diene-2-one, a 7-epimer of 1.

Biological Activities of Isolated Compounds on ADMSCs
In this study, we tested the protective effects of compounds 1-3 and 5-14 on oxidative stress-induced ADMSCs. The activity of compound 4 could not be evaluated due to amount limitations. Tert-butyl hydroperoxide (tBHP) was used as an inducer of ADMSC death by generating excessive intracellular reactive oxygen species and reducing the activity of endogenous defensive molecules, such as superoxide dismutase-SOD and glutathione [28]. It was found that tBHP triggered ADMSC death dose-dependently ( Figure 4). Particularly, the relative viability of ADMSCs after exposure to 0, 50, 75, 100, and 150 µM tBHP was 100.0 ± 3.3%, 103.3 ± 6.6%, 88.7 ± 6.4%, 53.8 ± 8.6% (p < 0.0001), and 25.1 ± 1.3% (p < 0.0001), respectively. To investigate the effects of the isolated compounds, 100 µM tBHP was chosen to induce oxidative stress in ADMSCs. Figure 5 shows the relative cell viability of the control (only tBHP treatment) and treated groups (co-treatment with tBHP and compounds). Interestingly, compounds 8 to 13 effectively prevented oxidative stress-induced ADMSC death, as indicated by a minimum 1.4-fold improvement in cell viability at 50 µM. Among these, compounds 11 and 12 exhibited the highest protective effects, which were improved by 1.69 ± 0.05-fold (p < 0.0001) and 1.61 ± 0.03fold (p < 0.0001), respectively, at 50 µM. Meanwhile, compounds 3 and 6 only slightly inhibited the toxic effects of tBHP on ADMSCs, with 1.21 ± 0.14-fold and 1.20 ± 0.12-fold higher cell viability at 50 µM, respectively. We found that all the remaining compounds were ineffective in recovering ADMSCs from chemically induced oxidative stress at all tested concentrations. Further analysis would be necessary to investigate the relationship between structure and its effect. on the viability of ADMSCs exposed to tBHP-induced oxidative stress. In neurodegenerative diseases, neuronal stem cells experience a high level of oxidative stress, which impairs their regenerative capacity [33]. Although we could not isolate neuronal stem cells for the test, our results provide clear evidence for the prevention of oxidative stress in ADMSCs using several potent isolated sesquiterpenes. Future studies would be required to investigate the impact of these compounds in in vivo neurodegenerative disease models.  The data represent two independent experiments. *p < 0.05, **p < 0.01, $p < 0.001, #p < 0.0001.
A. oxyphylla is commonly used for its antioxidant, anti-inflammatory, and neuroprotective effects, but the effects of its active compounds remain to be explored [29][30][31][32]. In the present study, we successfully proved the protective effects of 13 isolated sesquiterpenes on the viability of ADMSCs exposed to tBHP-induced oxidative stress. In neurodegenerative diseases, neuronal stem cells experience a high level of oxidative stress, which impairs their regenerative capacity [33]. Although we could not isolate neuronal stem cells for the test, our results provide clear evidence for the prevention of oxidative stress in ADMSCs using several potent isolated sesquiterpenes. Future studies would be required to investigate the impact of these compounds in in vivo neurodegenerative disease models.

General Procedures
Optical rotation was measured using a JASCO P-2000 polarimeter (Tokyo, Japan). The NMR spectra were acquired on a 400 MHz Agilent NMR spectrometer (DD2, Santa Clara, CA, USA) using CDCl 3

Plant Materials
The dried fruits of A. oxyphylla were purchased from the Insan Oriental Herbal Market in Seoul, Korea. A voucher specimen (No. EA323) was deposited at the Natural Product Chemistry Laboratory at Ewha Womans University, Seoul, Korea.

Culture of Mouse ADMSCs
Experiments on animals were performed according to the national guidelines and with the approval of the Institutional Ethical Committee, Yeungnam University, Republic of Korea. ADMSCs were obtained from C57BL/6 mice (male, 8-week age; Samtako, Republic of Korea). The cells were carefully characterized using specific surface markers and differentiation capacities, according to previous studies [34]. ADMSCs were cultured in MEM-α supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin 100X (Hyclone Laboratories, South Logan, UT, USA). The cells were used within passages 3-5.

Effect of Isolated Compounds on the Viability of tBHP-Damaged ADMSCs
The stock solution of each compound was prepared in advance by dissolving it in cell culture-grade DMSO (Sigma-Aldrich). To investigate the effect of the compounds, ADMSCs were chemically induced with tBHP (Tokyo Chemical Industry, Tokyo, Japan), and cell viability was measured using a cell counting kit 8 (CCK-8; Dojindo, Molecular Technologies Inc., Rockville, MD, USA). Briefly, ADMSCs were trypsinized and cultured in 96-well plates at a density of 5000 cells/well. The following day, the cells were incubated in a medium containing 100 µM tBHP and the compounds at various concentrations (3-50 µM) for 24 h in a CO 2 incubator at 37 • C. The cells were then washed twice using phosphate-buffered saline (pH = 7.4; Hyclone Laboratories) and incubated in a medium (120 µl/well) containing 5% CCK-8 reagent for 2 h at 37 • C. The absorbance of the supernatant in a 96-well plate was measured at 450 nm using a plate spectrophotometer (Spark 10M; Tecan, Männedorf, Switzerland). Relative cell viability was calculated by comparing the absorbance of the control and treated cells. The data were analyzed using the GraphPad Prism software version 8.4.2.

Conclusions
In this study, three new and eleven known sesquiterpenes were isolated from the dried fruits of Alpinia oxyphylla. Their structures were elucidated and identified by NMR spectroscopic methods. In the screening for biological activities on adipose-derived mesenchymal stem cells (ADMSCs) of the isolates 1-3, 5-14, compounds 8 to 13 effectively prevented oxidative stress-induced ADMSC death. Among these, compounds 11 and 12 showed the highest protective effect. These results supported that A. oxyphylla fruit-derived sesquiterpenoids and active compounds enriched extract can be applied to regenerative medicine therapies by prevention of oxidative stress in ADMSCs, which can be differentiated into neurons, osteoblasts, pancreatic cells, etc. Further examination using in vivo degenerative disease models is required in future studies.