Anti-Pulmonary Fibrosis Activities of Triterpenoids from Oenothera biennis.

Five new triterpenoids, oenotheralanosterols C-G (1-5), with seven known triterpenoidcompounds, namely 2α,3α,19α-trihydroxy-24-norurs4,12-dien-28-oic acid (6), 3β,23-dihydroxy-1-oxo-olean-12-en-28-oic acid (7), remangilone C (8), knoxivalic acid A (9), termichebulolide (10), rosasecotriterpene A (11), androsanortriterpene C (12), were extracted and separated from the dichloromethane part of Oenothera biennis L. The anti-pulmonary fibrosis activities of all the compounds against TGF-β1-induced damage tonormal human lung epithelial (BEAS-2B) cells were investigated in vitro. The results showed that compounds 1-2, 6, 8, and 11 exhibited significant anti-pulmonary fibrosis activities, with EC50 values ranging from 4.7 μM to 9.9 μM.

1. Introduction

Pulmonary fibrosis (PF) is an end-stage change of a large group of lung diseases which can lead to death if it is not properly treated [1]. The main causes of PF are the inhalation of foreign particles (such as dust and asbestos fibers), infections (such as COVID-19), autoimmune diseases (such as systemic autoimmune diseases of the connective tissue), and exposure to radiation treatment (such as radiation therapy for lung or breast cancer) [2]. Currently, PF is mainly conventionally treated using anti-inflammatory and immunosuppressant drugs (such as pirfenidone and nintedanib). However, these drugs can only retard the progression and relieve the symptoms of PF [3]. At the same time, an increasing number of Traditional Chinese Medicine therapies have been proven effective in treating PF [4,5,6,7]. Particularly, triterpenoids were proven to be bioactive structures against PF [8].

Oenothera is a genus that includes more than 200 species that are distributed in various regions of the world. These species can be found in the southern parts (northeast) and mountainous areas of China [9]. These species of Oenothera biennis are rich in a variety of natural compounds, including flavonoids, tannins, fatty acids, and terpenoids [10,11,12,13,14]. Modern pharmacological research has shown that these species possess antibacterial, anti-inflammatory, antioxidant, blood lipid lowering, blood sugar lowering, and anti-tumor properties, as well as other pharmacological effects [15,16,17,18,19,20,21]. Among these species, Oenothera biennis is a perennial herbaceous plant which is commonly used in folk medicine [22]. In consulting local books, we found that O. biennis can be used to treat lung-related diseases. For example, “Quanzhou Materia Medica” recorded that O. biennis can be used to treat lung disease and fistulas. Meanwhile, according to the literature, it is found that the triterpenoids in evening primrose have antiproliferative, antimicrobial efficacy, with free radical scavenging and ferric reducing activities [12,23]. In this study, we investigated and identified five new (1–5) and seven known triterpenoids (6–12) from the dichloromethane fraction of O. biennis (Figure 1). Furthermore, some compounds have shown significant protective effects against TGF-β1-induced PF in healthy human lung epithelial (BEAS-2B) cells, which suggests the potential anti-pulmonary fibrosis activities of these compounds.

Figure 1

Structures of triterpenoids 1–12.

2. Results and Discussion

Compound 1 (Figure 1) was isolated as a white amorphous powder (20.6 mg), and the molecular formula was determined as C30H48O5, based on the quasi-molecular ion at m/z 511.3397 [M + Na]+ (calculated for C30H48O5Na, 511.3399) in the HR-ESI-MS. The IR absorption bands confirmed the presence of -OH (3369 cm−1) and -COOH (1636 cm−1) in compound 1. The UV spectrum exhibited an absorption band at λ 206 nm corresponding to the carbonyl group. The 1H NMR spectrum (Table 1) showed three oxygenated methines at [δH 4.45 (1H, m, H-6), 3.70 (1H, m, H-2), and 2.86 (1H, d, J = 9.5 Hz, H-3)] and seven methyl groups at [δH 0.77 (3H, s, H-30), 0.93 (3H, s, H-29), 1.07 (3H, s, H-23), 1.16 (3H, s, H-24), 1.17 (3H, s, H-27), 1.17 (3H, s, H-26),and 1.32 (3H, s, H-25)]. The 13C NMR (Table 2) and DEPT 135 spectra exhibited 29 carbon resonances, including a carboxylic carbon at (δC 180.6), two olefinic carbons at (δC 139.1 and 129.8), three oxygenated methine carbons at (δC 68.8, 69.8, and 84.7), and seven methyl carbons at (δC 32.7, 28.8, 24.7, 21.8, 19.8, 19.5, and 18.7). The HMBC and 1H–1H COSY correlations of compound 1 are shown in Figure 2. The 1H–1H COSY spectrum, including δH 1.61, 1.52 (H-11), δH 1.62 (H-9), δH 2.81, and 1.88 (H-12), was consistent with a double bond in ring D. Meanwhile, it can be observed that the attachment of a double bond of C-13 and C-18 was established by HMBC correlations from δH1.17 (H3-27) to δC139.1 (C-13) and from δH 2.27 (H-19) to δC 129.5 (C-18). The analysis of the13C NMR data of compound 1 established its close structural resemblance to 2α, 3β-dihydroxy-yolean-13(18)-en-28-oic acid [24]; however, the only difference was the presence ofan oxygenated methine δC 68.8 (C-6) in compound 1 and the absence of a methylenesignal compared to 2α, 3β-dihydroxy-yolean-13(18)-en-28-oic acid. The HMBC correlations from δH 4.45 (H-6) to δC 56.8 (C-5), δC 43.1 (C-7), and δC 39.3 (C-10) were observed. The relative configuration of compound 1 was determined based on a NOESY experiment. The NOESY correlation (Figure 3) was observed between H-2and H3-25, which indicated the α-orientation of the hydroxy group at C-2. The correlations of H-3 and H-5 demonstrated the α-orientation of H-3, which indicated that the hydroxy group at C-3 was β-oriented. Additionally, the NOESY correlation was observed between H-6and H3-23/H-5, which indicated that the hydroxy group at C-6 was β-oriented [25,26]. Thus, the structure of compound 1 was determined to be 2α,3β,6β-trihydroxy-yolean-13(18)-en-28-oic acid, and it was named oenotheralanosterol C.

Table 1

H NMR data (500 MHz) for compounds 1–5 (δ, ppm, J, Hz) in CD3OD.

Position	1	2	3	4	5
1	2.04dd (4.8, 12.4), 0.90 m	2.56d (16.4)	-	-	2.36 s, 2.22 s
2	3.70 m	-	3.10 t (4.6), 2.26dd, (4.6, 12.1)	3.11 m, 2.30 m	-
3	2.86d (9.5)	-	3.36dd (4.6, 12.1)	3.82dd (4.9, 12.1)	-
4	-	-	-	-	-
5	0.83d (2.0)	2.42 m	0.90 m	1.40 m	3.06dd (3.0, 12.4)
6	4.45 m	1.90 m, 1.48 m	1.68 m, 1.61 m	1.60 m, 1.53 m	1.76 m, 1.67 m
7	1.64 m, 1.63 m	1.73 m, 1.43 m	1.51 m, 1.39 m	1.59 m, 1.30 m	1.59 m, 1.36 m
8	-	-	-	-	-
9	1.62 m	2.08 m	2.34 m	2.39 m	2.33 m
10	-	-	-	-	-
11	1.61 m, 1.52 m	2.07 m, 2.02 m	1.88 m, 1.54 m	2.46 m, 1.90 m	2.19 m, 2.04 m
12	2.82 m, 1.88 m	5.28 t (3.8)	5.27 t (3.6)	5.27 t (3.0)	5.35 t (3.8)
13	-	-	-	-	-
14	-	-	-	-	-
15	1.14 m, 1.06 m	1.81 m, 1.06 m	1.82 m, 1.76 m	1.78 m, 1.02 m	1.70 m, 1.11 m
16	1.91 m, 1.55 m	2.71 m, 1.57 m	2.57 m, 2.44 m	2.57 m, 2.45 m	2.00 m, 1.97 m
17	-	-	-	-	-
18	-	2.76 s	2.50 s	2.50 s	2.76 brs
19	2.47dd, 1.76dd (2.2, 14.0)	-	-	-	3.22d (3.8)
20	-	1.69 m	1.34 m	1.32 m	-
21	1.28 m, 1.23 m	2.31 m, 1.19 m	1.75 m, 1.26 m	1.73 m, 1.25 m	1.80 m, 1.22 m
22 23	2.15 m, 1.34 m 1.07 s	1.84 m, 1.55 m 1.85d (2.0)	1.77 m, 1.63 m 1.01 s	1.72 m, 1.63 m 3.35d, 3.51d (11.2)	3.91dd (4.4, 11.6) 2.23 s
24	1.17 s	-	1.04 s	0.88 s	-
25	1.32 s	0.93 s	1.32 s	1.35 s	1.09 s
26	1.16 s	0.85 s	0.86 s	0.86 s	0.84 s
27	1.17 s	1.39 s	1.36 s	1.37 s	1.34 s
28	-	-	-	-	-
29	0.93 s	1.14 s	1.22 s	1.22 s	0.97 s
30	0.77 s	1.00d (6.6)	0.94d (6.7)	0.94d (6.6)	1.03 s

Table 2

C NMR data (125 MHz) for compounds 1–5 (δ, ppm, J, Hz) in CD3OD.

Position	1	2	3	4	5
1	50.5	53.2	215.3	215.3	44.4
2	69.8	195.7	45.1	44.8	175.4
3	84.7	145.3	79.7	73.4	-
4	41.1	133.1	40.4	44.1	215.6
5	56.8	49.9	55.8	47.7	57.5
6	68.8	22.0	19.0	18.6	23.1
7	43.1	33.2	34.1	33.6	32.2
8	41.6	40.6	40.9	40.8	43.3
9	52.6	44.9	40.1	40.0	40.8
10	39.3	42.3	53.8	53.3	40.6
11	23.0	24.8	26.6	26.6	24.6
12	26.5	128.1	130.0	130.0	125.1
13	139.1	140.0	139.4	139.4	143.8
14	45.9	42.9	42.8	42.9	40.6
15	28.2	29.5	29.6	29.6	29.0
16	34.2	27.2	26.5	26.5	20.8
17	49.4	49.0	49.3	49.3	53.0
18	129.8	48.0	55.3	55.3	46.4
19	42.0	74.2	73.5	73.5	82.0
20	33.6	43.3	43.1	43.1	36.8
21	38.0	25.2	27.3	27.3	38.0
22	36.9	32.8	38.9	38.9	72.4
23	28.8	13.4	16.6	66.0	31.6
24	18.7	-	29.0	13.3	-
25	19.5	14.4	15.4	15.9	18.4
26	19.8	17.7	18.1	18.1	17.5
27	21.8	24.5	24.8	24.8	25.2
28	180.6	182.2	182.2	182.2	180.7
29	32.7	29.5	27.0	27.0	28.8
30	24.7	16.2	16.6	16.6	26.2

Figure 2

Key 1H-1H COSY and HMBC correlations of compounds 1–5.

Figure 3

Key NOESY correlations of compounds 1–5.

Compound 2 (Figure 1) was isolated as a white amorphous powder (11.0 mg), and its molecular formula was established as C29H42O5 from the molecular ion peak at m/z 493.2934 [M + Na]+ (calculated for C29H42O5Na, 493.2930) in the HR-ESI-MS. The IR absorption bands confirmed the presence of -OH (3380 cm−1) and -COOH (1644 cm−1) groups. The UV spectrum exhibited an absorption band at λ 204 (278 nm), corresponding to the carbonyl group. The 1H NMR spectrum (Table 1) showed an olefinic proton at δH 5.28 (1H, t, J = 3.8 Hz) and six methyl groups [(δH 1.85, (3H, d, J = 2.0 Hz, H-23), 1.39 (3H, s, H-27), 1.14 (3H, s, H-29), 1.00 (3H, d, J = 6.6 Hz, H-30), 0.93 (3H, s, H-25), and 0.85 (3H, s, H-26)]. The 13C NMR (Table 2) and DEPT 135 spectra revealed 29 signals, with a ketocarbonyl carbon at (δC 195.7), a carboxylic carbon at (δC 182.8), two double bonds at (δC140.0, 128.1 and 145.3, 133.1), an oxygenated quaternary carbon at (δC 74.2), and six methyl carbons at (δC 29.5, 24.5, 17.7, 16.2, 14.4, and 13.4). The HMBC and 1H–1H COSY correlations of compound 2 are illustrated in Figure 2. Meanwhile, an analysis of the 1H and 13C NMR data of compound 2 established its close structural resemblance to 2-oxo-3J,19α-dihydroxy-24-nor-urs-12-en-28-oic acid [27]. The only difference between the compound and the resemblance structure is the presence of a double bond at C-3/C-4 in compound 2. It can be observed that the attachment of a double bond to C-3 and C-4 was established by the HMBC correlations from δH 2.56 and 2.16 (H-1) to δC 195.7 (C-2), δC 145.3 (C-3), δC49.9 (C-5), and δC39.3 (C-10) and from δH 1.00 (H3-23) to δC 145.3 (C-3), δC133.1(C-4), and δC49.9 (C-5). The relative configuration of compound 2 was determined based on a NOESY experiment. The NOESY correlations (Figure 3) of H3-29/H-12/H3-26 showed the J-orientation of H3-29, which indicated that the hydroxy group at C-29 was α-oriented [28]. Thus, compound 2’s structure was determined to be 2-oxo-3,19α-dihydroxy-24-nor-urs-12-en-28-oic acid, and it was named oenotheralanosterol D. The data are available in supplementary materials.

Compound 3 (Figure 1) was isolated as a white amorphous powder (12.8 mg), possessing the molecular formula of C30H46O5, based on HR-ESI-MS at m/z 509.3237 [M + Na]+ (calculated for C30H46O5Na, 509.3243). The IR absorption bands confirmed the presence of -OH (3368 cm−1) and -COOH (1686 cm−1) groups. The UV spectrum showed an absorption bandat λ 204 nm, corresponding to the carbonyl group. The 1H NMR spectrum (Table 1) showed an olefinic proton at δH 5.27 (1H, t, J = 3.6 Hz, H-12), an oxygenated methine at δH 3.36 (1H, dd, J = 4.6, 12.2 Hz, H-3), and seven methyl groups [δH 0.86 (3H, s, H-26), 0.94 (3H, d, J = 6.7 Hz, H-30), 1.01 (3H, s, H-23), 1.04 (3H, s, H-24), 1.22 (3H, s, H-29), 1.32 (3H, s, H-25), and 1.36 (3H, s, H-27)]. The 13C NMR (Table 2) and DEPT 135 spectra revealed 30 signals, with a ketocarbonyl carbon at (δC 215.3), a carboxylic carbon at (δC 182.2), a double bond at (δC139.4 and 130.0), an oxygenated quaternary carbon at (δC 73.5), an oxygenated methine carbon at (δC 79.7), and seven methyl carbons at (δC 29.0, 27.0, 24.8, 18.1, 16.6, 16.6, and 15.9). Furthermore, an analysis of the13C NMR data of compound 3 established its close structural resemblance to 2-oxo-pomolic acid [29], which only differed in the position of the ketocarbonyl (δC 215.3, C-6) and replaced (δC 215.3, C-1) in the compound. The HMBC correlations from δH 3.10, 2.26 (H-2) to δC 215.3 (C-1), δC 79.7 (C-3), and δC40.4 (C-4) were observed. The relative configuration of compound 3 was determined based on a NOESY experiment. The NOESY correlations (Figure 3) of H-3/H3-23/H-5 demonstrated that the H-3 and H3-23 were α-oriented, indicating the J-orientation of the hydroxy group at C-3. The correlations of H3-29/H-12/H3-26 demonstrated that H3-29 was J-oriented, indicating the α-orientation of the hydroxy group at C-29 [30,31]. Thus, the structure of compound 3 was determined to be 3J,19α-dihydroxy-1-oxo-olean-12-en-28-oic acid, and it was named oenotheralanosterol E.

Compound 4 (Figure 1) was isolated as a white amorphous powder (11.7 mg), and itsmolecular formula was determined to be C30H46O6, based on the quasi-molecular ion at m/z 525.3180 [M + Na]+ (calculated for C30H46O6Na, 525.3192) in the HR-ESI-MS analysis. The IR absorption bands confirmed the presence of -OH (3366 cm−1) and -COOH (1688 cm−1) groups. The UV spectrum showed an absorption band at λ 203 nm, corresponding to the carbonyl group. The 1H NMR (Table 1) and 13C NMR (Table 2) data of compound 4 were similar to those of compound 3, differing only in the absence of a methyl group, which was replaced by an oxygenated methylene carbon (δC 66.0, C-23) in compound 4. The HMBC and 1H–1H COSY correlations of compound 4 are illustrated in Figure 2. The HMBC correlations from δH 3.51, 3.35 (H3-23) to δC 79.7 (C-3), δC 40.4(C-4), δC 55.8 (C-5), and δC 13.3 (C-24) were observed. The relative configuration of compound 4 was determined based on a NOESY experiment. The NOESY correlations (Figure 3) of H-3/H3-23/H-5 demonstrated that H-3 and H3-23 were α-oriented, implying the J-orientation of the hydroxy group at C-3. The correlations of H3-29/H-12/H3-26 demonstrated the J-orientation of H3-29, indicating the α-orientation of the hydroxy group at C-29 [32]. Thus, the structure of compound 4 was determined to be 3J,19α-dihydroxy-1-oxo-olean-12-en-23-ol-28-oic acid, and it was named oenotheralanosterol F.

Compound 5 (Figure 1) was isolated as a white amorphous powder (3.4 mg), and its molecular formula was established as C28H42O7 from the molecular ion peak at m/z 513.2832 [M + Na]+ (calculated for C28H42O7Na, 513.2828) in the HR-ESI-MS. The IR absorption bands confirmed the presence of -OH (3371, 2951 cm−1) and -COOH (1650, 1388 cm−1) groups. The UV spectrum exhibited an absorption band at λ 204 nm, corresponding to the carbonyl group. The 1H NMR spectrum (Table 1) showed an olefinic bond at δH 5.35 (1H, t, J = 3.8 Hz, H-12), two oxygenated methines at [δH 3.22 (1H, d, J = 3.8 Hz, H-22) and 3.91 (1H, dd, J = 4.4, 11.6 Hz, H-19)], and six methyl groups at [δH 0.84 (3H, s, H-26), 0.97 (3H, s, H-29), 1.03 (3H, s, H-30), 1.09 (3H, s, H-25), 1.34 (3H, s, H-27), and 2.23 (3H, s, H-23)]. The 13C NMR (Table 2) and DEPT 135 spectra revealed 28 signals, with a ketocarbonyl carbon at (δC 215.3), two carboxylic carbons at (δC 180.7 and 175.2), a double bond at (δC143.8 and 125.1), two oxygenated methine carbons at (δC 72.4 and 82.0), and six methyl carbons at (δC 31.6, 28.8, 26.2, 25.2, 18.4, and 17.5). The HMBC and 1H–1H COSY correlations of compound 1 are shown in Figure 2. Furthermore, an analysis of the1H and 13C NMR data of compound 5 established its close structural resemblance toivorengenin B [33], and the only difference was that the oxygenated methine (δC 72.4, C-22) replaced the methylene signal (δC 32.5, C-22). The HMBC correlations from δH 3.91 (H-22) to δC 53.0 (C-17), δC 182.2 (C-28), and δC38.0 (C-21) were observed. The relative configuration of compound 5 was determined based on a NOESY experiment. The NOESY correlations (see Figure 3) of H-22/H-18/H3-27 showed that H-18 and H-22 were a-oriented, indicating the J-orientation of the hydroxy group at C-22. The correlations of H-19/H-12/H3-26 showed that H-19 was J-oriented, indicating the a-orientation of the hydroxy group at C-19 [34]. Thus, the structure of compound 5 was determined to be 19a,22J-dihydroxy-4-oxo-3,24-dinor-2,4-secoolean-12-ene-2,28-dioic acid, and it was named oenotheralanosterol G.

In addition, the known compounds 6–12 were also obtained from the dichloromethane part of O. biennis. Comparing the NMR spectroscopic data of compounds 6–12 with the reported literature, the known compounds were identified as 2α,3α,19α-trihydroxy-24-norurs4(23),12-dien-28-oic acid (6) [35], 3J,23-dihydroxy-1-oxo-olean-12-en-28-oic acid (7) [36], remangilone C (8) [37], knoxivalic acid A (9) [38], termichebulolide(10) [39], rosasecotriterpene A (11) [40], and rosanortriterpene C (12) [41], respectively.

The TGF-J1-induced lung slice fibrosis model provided an experimental basis for the study of the pathological mechanism of PF and therapeutic drugs. In this paper, we used this model to explore the anti-pulmonary fibrosis activities of the isolated compounds through real-time cell analysis. The results indicated that compounds 1–2, 6, 8, and 11 significantly decreased the damage of BEAS-2B cells induced by TGF-J1, with EC50 values ranging from 4.7 μM to 9.9 μM (Table 3). It is speculated that these compounds may have potential activities against PF.

Table 3

Anti-pulmonary fibrosis activities of compounds 1–12 against BEAS-2B cell damage induced by TGF-J1.

Group	EC50 (μM)	Group	EC50 (μM)
CON	NA	6	4.7
M	NA	7	53.3
Pirfenidone	12.7	8	7.9
1	8.4	9	NA
2	9.9	10	NA
3	48.3	11	9.6
4	NA	12	NA
5	NA

Note: CON: Normal group; M: Model group; Pirfenidone: Positive control compound.

According to our experimental results, the lung-protective activities are influenced by multiple structural factors. For example, compound 6 exhibited the most effective anti-pulmonary fibrosis activities compared to the other compounds; therefore, the double bond at C-4/C-23 might be an active group that could increase the structures’ activities. Similarly, compound 10 showed weaker anti-pulmonary fibrosis activities compared to the other compounds, which means the esterification of the carboxyl group at C-28 could decrease the compounds’ activities.

3. Materials and Methods

3.1. General Procedures

The NMR spectra were recorded using a Bruker AVANCE III 500 nuclear magnetic resonance instrument and mass spectra was completed with a Bruker maxis HD time-of-flight mass spectrometer (Bruker, Germany). The UV and IR spectra were recorded on a Thermo EVO 300 spectrometer (Thermo, Waltham, MA, USA) and a Thermo Nicolet IS 10 spectrometer (Thermo, Waltham, MA, USA). The separation of compounds was achieved on an LC-52 HPLC (separation Beijing Technology, SP-5030 semi-preparative high-pressure infusion pump, UV200 detector, easy Chromchromatographic workstation, with a COSMOSIL C18-MS-II chromatographic column of 250 mm × 20 mm, 5 μm). A Multiskan MK3 microplate reader (Thermo Fisher, Waltham, MA, USA) was used in the bioassay, along with a carbon dioxide type 3111 incubator (Thermo, Waltham, MA, USA) and a Centrifuge-5804R high speed centrifuge (Eppendorf, Germany). Column chromatography (CC) was performed using an MCI gel CHP-20 (TOSOH Corp, Tokyo, Japan), a Sephadex LH-20 (40–70 mm, anAmersham Pharmacia Biotech AB, Uppsala, Sweden) and silica gel (200–300 mesh, Marine Chemical Industry, Qingdao, China). The chemical reagents were supplied by the Beijing Chemical Plant (Beijing, China), and the RTCA from Agilent, Santa Clara, CA, USA and TGF-J1 from PeproTech, Cranbury, NJ, USA.

3.2. Plant Material

The Oenothera biennis L. specimens were collected in August 2019 from the Funiu Mountains in Henan Province, China. The plants were identified and authenticated by prof. Cheng-ming Dong of the Henan University of Chinese Medicine. A voucher specimen (YJC-201908) was deposited in the Engineering and Technology Center for Chinese Medicine Development of Henan Province, Zhengzhou, China.

3.3. Extraction and Isolation

The air-dried Oenothera biennis L. (40.0 kg) was subjected to extraction with an aqueous solution containing 50% acetone of tissue fragmentation (two times each 85 L, overnight). The extract (3.3 kg) was dispersed in H2O (15 L) and sequentially extracted with PE (10 × 15 L), CH2Cl2(10 × 15 L), EtOAc (10 × 15 L), and n-BuOH (10 × 15 L). The CH2Cl2 part (49.6 g) was separated using silica gel column chromatography (CC, 12 × 130 cm) involving a mobile phase of PE/EtOAc (100:0 to 0:100, v/v), yielding 10 fractions (Fr.1–Fr.10).

Subfraction Fr.8 (2.47 g) was fractionally eluted via silica gel column containing PE/EtOAc (20:1 to 0:1, v/v), which yielded nine subfractions (Fr.8-1–Fr.8-9). SubfractionFr.8-6 (536.0 mg) was repeatedly separated via silica gel column containing CH2Cl2/MeOH (50:1 to 10:1, v/v), yielding seven subfractions (Fr.8-6-1–Fr.8-6-7). Subfraction Fr.8-6-5 (356.5 mg) was applied to a Sephadex LH-20 column containing MeOH/H2O (70:30, v/v), and the product was purified by semipreparative HPLC (MeOH/H2O 66:34), yielding compound 5 (3.4 mg, t = 33.2 min) and compound 12 (16.2 mg, tR = 36.0 min). Then, the subfraction Fr.8-9 (950.0 mg) was fractionally separated via silica gel column containing CH2Cl2/MeOH (100:1 to 10:1, v/v), yielding four subfractions (Fr.8-9-1–Fr.8-9-4).Then, subfraction Fr.8-9-3 (251.6 mg) was fractionally separated via silica gel column containing CH2Cl2/MeOH (50:1 to 10:1, v/v) and purified using semipreparative HPLC (MeOH/H2O = 66:34), yielding compound 1 (20.6 mg, t = 35.7 min) and compound 2 (11.0 mg, tR = 50.4 min). The subfraction Fr.8-9-4 (341.5 mg) was chromatographed with Sephadex LH-20 MeOH/H2O (70:30, v/v) and purified using semipreparative HPLC (MeOH/H2O = 62:38), yielding compound 6 (14.7 mg, tR = 29.1 min) and compound 10 (4.6 mg, t = 34.0 min). Sequentially, the subfraction Fr.8-9-5 (208.5 mg) was separated via silica gel column containing CH2Cl2/MeOH (40:1 to 5:1, v/v) and purified using semipreparative HPLC (MeOH/H2O = 53:47), which yielded compound 7 (33.8 mg, t = 33.0 min).

In addition, subfraction Fr.9 (1.58 g) was fractionally separated via silica gel column containing CH2Cl2/MeOH (100:1 to 0:100, v/v), which yielded five subfractions (Fr.9-1–Fr.9-5). Then, the subfraction Fr.9-4 (0.78 g) was separated via silica gel column containing CH2Cl2/MeOH (50:1 to 10:1, v/v), yielding four subfractions (Fr.9-4-1–Fr.9-4-4). Further separation of the subfraction Fr.9-4-3 (304.2 mg) was chromatographed with Sephadex LH-20 MeOH/H2O (70:30, v/v) and purified using semipreparative HPLC (MeOH/H2O 65:35), producing compound 4 (11.7 mg, t = 30.6 min) and compound 3 (12.8 mg, t = 37.5 min). Meanwhile, subfraction Fr.9-4-4 (315.2 mg) was eluted via an MCI gel CHP-20 CC containing MeOH/H2O (0:100 to 100:0, v/v), yielding nine fractions (Fr.9-4-4-1–Fr.9-4-4-4). Then, subfraction Fr.9-4-4-2 (194.2 mg) was separated via silica gel column containing CH2Cl2/MeOH (40:1 to 10:1, v/v) and purified with semipreparative HPLC (MeOH/H2O 70:30), yielding compound 8 (26.4 mg, t = 31.3 min) and compound 9 (23.9 mg, t = 40.2 min). Subsequently, the subfraction Fr.9-5 (0.45 g) was separated via silica gel column containing CH2Cl2/MeOH (50:1 to 1:1, v/v), which yielded four subfractions (Fr.9-5-1–Fr.9-5-4). A Sephadex LH-20 column with MeOH/H2O (70:30, v/v) was further used to separate the subfraction Fr.9-5-2 (235.1 mg), and the obtained product was purified with semipreparative HPLC (MeOH/H2O = 48:52), which yielded compound 11 (12.8 mg, t = 28.1 min).

Oenotheralanosterol C (1): The white amorphous powder of compound 1 was characterized with [α − 71.397 (c 0.4120, CH3OH), mp 217 °C, HR-ESI-MS m/z: 511.3397 [M + Na]+ (calculated for C30H48O5Na, 511.3399) UV λmax(CH3OH)/nm (logε): 206 nm; IR (CH3OH)vmax: 3369, 1636, 1457, 1019, and 682 cm−1. 1H NMR (500 MHz, CD3OD) data can be found in Table 1 and 13C NMR (125 MHz, CD3OD) data can be found in Table 2.

OenotheralanosterolD (2): The white amorphous powder of compound 2 was characterized with [α + 1 00.290 (c 0.2192, CH3OH), mp 238 °C, HR-ESI-MS m/z: 493.2934 [M + Na]+ (calculated for C29H42O5Na, 493.2930) UV λmax (CH3OH)/nm (logε): 278, 204 nm; IR (CH3OH) vmax: 3380, 1644, 1457, 1397, 1018, and 699 cm−1. 1H NMR (500 MHz, CD3OD) datacan be found in Table 1 and 13C NMR (125 MHz, CD3OD) data can be found in Table 2.

OenotheralanosterolE (3): The white amorphous powder of compound 3 was characterized with [α + 69.888 (c 0.2336, CH3OH), mp 247 °C, HR-ESI-MS m/z: 509.3237 [M+Na]+ (calculated for C30H46O5Na, 509.3243) UV λmax(CH3OH)/nm (logε): 204 nm; IR (CH3OH)vmax: 3368, 2941, 1686, 1459, 1383, 1238, 1018, 656, 603, and 557 cm−1.1H NMR (500 MHz, CD3OD) data can be found in Table 1 and 13C NMR (125 MHz, CD3OD) data can be found in Table 2.

OenotheralanosterolF (4): The white amorphous powder of compound 4 was characterized with [α + 62.971 (c 0.2552, CH3OH), mp 246 °C, HR-ESI-MS m/z: 525.3180 [M+Na]+ (calculated for C30H46O6Na, 525.3192) UV λmax(CH3OH)/nm(logε): 203 nm; IR (CH3OH)vmax:3366, 2939, 1688, 1455,1385, 1239, 1021,and 655 cm−1.1H NMR (500 MHz, CD3OD) data can be found in Table 1 and 13C NMR (125 MHz, CD3OD) data can be found in Table 2.

OenotheralanosterolG (5): The white amorphous powder of compound 4 was characterized with [α + 62.971 (c 0.2552, CH3OH), mp 219 °C, HR-ESI-MS m/z: 513.2832 [M+Na]+ (calculated for C28H42O7Na, 513.2828) UV λmax(CH3OH) /nm(logε): 204 nm; IR (CH3OH)vmax: 3371, 2951, 1650, 1388, 1202,1018, 669,and 554 cm−1.1H NMR (500 MHz, CD3OD) data can be found in Table 1 and 13C NMR (125 MHz, CD3OD) data can be found in Table 2.

3.4. In Vitro Cell Experiment

In the previous stage, the research group conducted relevant studies on the anti-pulmonary fibrosis activities of the total extracts and some monomers of Oenothera biennis L. Based on this, the RTCA method was used to detect the effect of monomer compounds on TGF-J1-induced BEAS-2B cell damage to explore its anti-pulmonary fibrosis active ingredients.

An xCELLigence instrument (Acea Biosciences, Inc., San Diego, CA, USA) was used for the real-time cell analysis (RTCA) assay. BEAS-2B cells were plated in 16-well plates (2.5 × 104 cells/well) for 24 h at 37 °C in a humidified atmosphere of 5% CO2. Then, these compounds, or pirfenidone at various concentrations (0.1, 1, 10, 50, and 100 μM), were added to the standard medium of TGF-J1 (1 ng/mL) and incubated for 24 h. Each experiment was repeated four times to obtain the mean values. Finally, the EC50 values of these compounds were calculated by GraphPadSigmoidal dose-response.

4. Conclusions

In conclusion, we successfully isolated five new triterpenoids and seven known compounds from Oenothera biennis L. The bioassay indicated that compounds 1–2, 6, 8, and 11 exhibited significant anti-pulmonary fibrosis activities to TGF-J1-induced BEAS-2B cells, with EC50 values ranging from 4.7 μM to 9.9 μM. These results provide a certain theoretical basis for the further development and utilization of Oenothera biennis L.