Six New Compounds from the Herbaceous Stems of Ephedra intermedia Schrenket C. A. Meyer and Their Lung-Protective Activity

Six new compounds, (7R,8S,8′R)-balanophorone (1), (7′S,8′R,8R)-yunnanensin A (2), (3S)-thunberginol C (3), (8R,8′R)-maninsigin B (4), (7S,8R)-4,7,8-dihydroxy-9,9-dimethyl-chroman (5), and 4-hydroxy-1-(4-hydroxy-3-methoxyphenyl)butan-1-one (6), along with eight known compounds (7–14), were isolated from the herbaceous stems of Ephedra intermedia Schrenket C. A. Meyer. Their structures were elucidated based on their spectroscopic (MS, NMR, IR, and UV) data, and their absolute configurations were determined by comparing their calculated and experimental electronic circular dichroic (ECD) spectra. Moreover, compounds 1 and 3–6 were evaluated for their ability to protect human pulmonary epithelial cells (BEAS-2B) from injury induced by lipopolysaccharide (LPS) in vitro. The results showed that compound 6 exhibited a significant protective effect against LPS-induced injury in BEAS-2B, and compound 5 exhibited a slightly protective effect at the concentration of 10 μM.


Introduction
Ephedra has a long history of medicinal use in China, first recorded in the Shennong Materia Medica [1,2], with the efficacy of Fahan Sanhan and Xuanfei Pingchaun.According to the 2020 edition of the Chinese Pharmacopoeia [3,4], the herbaceous stems of Ephedra intermedia Schrenket C. A. Meyer, Ephedra sinica Stapf, and Ephedra equisetina Bunge are considered to be the original plants of Ephedrae Herba (Mahuang).These plants are extensively distributed in Northeast and Northwest China [5][6][7], growing in arid deserts, sandy beach areas, arid mountain slopes, or grassland at elevations ranging from several hundred meters to more than 2000 m above sea level.Ephedra intermedia Schrenket C. A. Meyer, also known as "the treasure of the desert," plays an important role in maintaining the balance of the ecological environment [8].
The NOESY experiment (Figure 1) showed that H-7 was correlated with H-8 ′ , which suggested that H-7 and H-8 ′ were co-facial.The absolute configuration of 1 was established as 7R,8S,8 ′ R by comparing its experimental and calculated ECD spectra (Figure 2).Finally, the structure of 1 was determined and it was termed (7R,8S,8 ′ R)-balanophorone.
Compound 3 was obtained as colorless needles, and its molecular formula was assigned as C 15 The interpretation of the NMR data of 3 suggested that compound 3 had the same planar structure as thunberginol C but different stereochemistry [23].SeonJu Park reported the CD data and showed that the characteristic curve for dihydroisocoumarins had a 3R configuration, i.e., a negative Cotton effect at 247 nm, a positive Cotton effect at 253 nm, and a negative Cotton effect at 305 nm, which is in contrast to the CD data of compound 3 (a positive Cotton effect at 247 nm, a negative Cotton effect at 253 nm, and a positive Cotton effect at 305 nm).Meanwhile, we determined its absolute configuration to be S by comparing its predicted and experimental ECD spectra (Figure 2).Therefore, compound 3 was determined and named as (3S)-thunberginol C.
Compound 4 was yielded as a yellow oil with a molecular formula of C The 1 H-1 H COSY correlations (Figure 3) between H-8 and H-9 and between H-7, H-8 ′ , and H-8 and H-7 ′ , as well as the HMBC correlations (Figure 3) from H-7 to C-1 and H-8 ′ /9 to C-9 ′ , indicated the determination of one spin system [CH 2 (9)-CH(8)-CH 2 (7), CH(8)-CH(8 ′ )-CH 2 (7 ′ )] and the presence of a five-membered oxygen ring, and the methylene (C-7) was attached to C-1.In the NOESY spectrum (Figure 1), cross-peaks between H-8 and H-8 ′ indicated that H-8 and H-8 ′ were co-facial.The absolute configuration of 4 was established as 8R,8 ′ R by comparing its experimental and calculated ECD spectra (Figure 2).Thus, compound 4 was elucidated and named (8R,8 ′ R)-maninsigin B.  SeonJu Park reported the CD data and showed that the characteristic curve for dihydroisocoumarins had a 3R configuration, i.e., a negative Cotton effect at 247 nm, a positive Cotton effect at 253 nm, and a negative Cotton effect at 305 nm, which is in contrast to the CD data of compound 3 (a positive Cotton effect at 247 nm, a negative Cotton effect at 253 nm, and a positive Cotton effect at 305 nm).Meanwhile, we determined its absolute configuration to be S by comparing its predicted and experimental ECD spectra (Figure 2).Therefore, compound 3 was determined and named as (3S)-thunberginol C.
Additionally, eight known compounds (Figure 4  We have previously evaluated the protective effects against LPS-induced BEAS-2B cell injury of compounds isolated from C. tinctorius [34].Meanwhile, some studies have reported that MaHuang possesses the efficacy of Xuanfei Pingchaun and may have potential lung-protective effects.Therefore, to further investigate the lung-protective activity of the new compounds, they were evaluated for their protective effects against LPS-induced BEAS-2B cell injury.As shown in Table 3, the results indicated that compound 6 exhibited a significant protective effect (* p < 0.05) against LPS-induced BEAS-2B cell damage at a concentration of 10 μM, and compound 5 exhibited slightly protective activity.We have previously evaluated the protective effects against LPS-induced BEAS-2B cell injury of compounds isolated from C. tinctorius [34].Meanwhile, some studies have reported that MaHuang possesses the efficacy of Xuanfei Pingchaun and may have potential lung-protective effects.Therefore, to further investigate the lung-protective activity of the new compounds, they were evaluated for their protective effects against LPS-induced BEAS-2B cell injury.As shown in Table 3, the results indicated that compound 6 exhibited a significant protective effect (* p < 0.05) against LPS-induced BEAS-2B cell damage at a concentration of 10 µM, and compound 5 exhibited slightly protective activity.

Plant Material
The herbaceous stems of Ephedra intermedia Schrenket C. A. Meyer were the focal plant material for this study.They were purchased from Xinjiang Xiyu Mocao Chinese Medicinal Materials Development Limited Company, Xinjiang, China, in October 2020, and authenticated by Prof. Cheng-Ming Dong of Henan University of Chinese Medicine.For archival purposes, a specimen (No. 20201111) was deposited at the Department of Natural Medicinal Chemistry, Henan University of Chinese Medicine, Zhengzhou, China.

Extraction and Isolation
The dried herbaceous stems of Ephedra intermedia Schrenket C. A. Meyer (45.0 kg) were cut into small segments and extracted with 50% aqueous acetone at room temperature.Then, the evaporation of the solvent under reduced pressure yielded a crude residue (11.1 kg), which was dissolved in water and partitioned by CH 2 Cl 2 , EtOAc, and n-BuOH fifteen times, respectively.

MTT Assay
The BEAS-2B cells cultured to the log phase were plated into 96-well flat-bottomed culture plates at a concentration of 4 × 10 3 cells per well.Then, the cells were divided into a normal group (NC), a model group (M, 0.5µg mL −1 ), and a group for each compound (compounds 1, 3-6, 10 µM, 10 µM + LPS 0.5 µg mL −1 ).After incubation for 24 h, 20 µL MTT (5 mg/mL) was added to each well and the plates were incubated for 4 h.Then, dimethyl sulfoxide (DMSO, 150 µL) was added, and the solution was removed.Finally, the optical density (OD) values were measured at 490 nm with a microplate reader (Thermo Scientific, Boston, MA, USA).

Statistical Analysis
All data were analyzed using the SPSS software version 26.0 and presented as the mean ± standard deviation (x ± sd).A one-way analysis of variance (one-way ANOVA) was used for comparisons between groups.The differences were considered significant when p < 0.05 and very significant when p < 0.01.

ECD Spectra Calculations
Conformational analyses of compounds 1-4 were performed using the GMMX software 6.0, which uses the MMFF94 force field.After they were optimized at the B3LYP/6-31G (d,p) level, the conformers with a Boltzmann distribution ≥ 1% were imported into the Gaussian 16 software.Then, calculations of the ECD curves were performed with the TDDFT method at the B3LYP/6-311G (d,p) level in CH 3 OH solution.Finally, based on the Boltzmann weighting of each conformer with a half-band width of 0.25 eV, the ECD spectra were simulated by the SpecDis 1.70.1 software.