Anti-Pulmonary Fibrosis Activities of Triterpenoids from Oenothera biennis

Abstract

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.

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. 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

) showed three oxygenated methines at [δ_H 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 [δ_H 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. ¹³C 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

) and DEPT 135 spectra exhibited 29 carbon resonances, including a carboxylic carbon at (δ_C 180.6), two olefinic carbons at (δ_C 139.1 and 129.8), three oxygenated methine carbons at (δ_C 68.8, 69.8, and 84.7), and seven methyl carbons at (δ_C 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 δ_H 1.61, 1.52 (H-11), δ_H 1.62 (H-9), δ_H 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 δ_H1.17 (H₃-27) to δ_C139.1 (C-13) and from δ_H 2.27 (H-19) to δ_C 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 δ_C 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 δ_H 4.45 (H-6) to δ_C 56.8 (C-5), δ_C 43.1 (C-7), and δ_C 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.

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 δ_H 5.28 (1H, t, J = 3.8 Hz) and six methyl groups [(δ_H 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 (δ_C 195.7), a carboxylic carbon at (δ_C 182.8), two double bonds at (δ_C140.0, 128.1 and 145.3, 133.1), an oxygenated quaternary carbon at (δ_C 74.2), and six methyl carbons at (δ_C 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 δ_H 2.56 and 2.16 (H-1) to δ_C 195.7 (C-2), δ_C 145.3 (C-3), δ_C49.9 (C-5), and δ_C39.3 (C-10) and from δ_H 1.00 (H₃-23) to δ_C 145.3 (C-3), δ_C133.1(C-4), and δ_C49.9 (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 δ_H 5.27 (1H, t, J = 3.6 Hz, H-12), an oxygenated methine at δ_H 3.36 (1H, dd, J = 4.6, 12.2 Hz, H-3), and seven methyl groups [δ_H 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 (δ_C 215.3), a carboxylic carbon at (δ_C 182.2), a double bond at (δ_C139.4 and 130.0), an oxygenated quaternary carbon at (δ_C 73.5), an oxygenated methine carbon at (δ_C 79.7), and seven methyl carbons at (δ_C 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 (δ_C 215.3, C-6) and replaced (δ_C 215.3, C-1) in the compound. The HMBC correlations from δ_H 3.10, 2.26 (H-2) to δ_C 215.3 (C-1), δ_C 79.7 (C-3), and δ_C40.4 (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 (δ_C 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 δ_H 3.51, 3.35 (H₃-23) to δ_C 79.7 (C-3), δ_C 40.4(C-4), δ_C 55.8 (C-5), and δ_C 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 δ_H 5.35 (1H, t, J = 3.8 Hz, H-12), two oxygenated methines at [δ_H 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 [δ_H 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 (δ_C 215.3), two carboxylic carbons at (δ_C 180.7 and 175.2), a double bond at (δ_C143.8 and 125.1), two oxygenated methine carbons at (δ_C 72.4 and 82.0), and six methyl carbons at (δ_C 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 (δ_C 72.4, C-22) replaced the methylene signal (δ_C 32.5, C-22). The HMBC correlations from δ_H 3.91 (H-22) to δ_C 53.0 (C-17), δ_C 182.2 (C-28), and δ_C38.0 (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. 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.

). It is speculated that these compounds may have potential activities against PF.

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_R = 33.2 min) and compound 12 (16.2 mg, t_R = 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_R = 35.7 min) and compound 2 (11.0 mg, t_R = 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, t_R = 29.1 min) and compound 10 (4.6 mg, t_R = 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_R = 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_R = 30.6 min) and compound 3 (12.8 mg, t_R = 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_R = 31.3 min) and compound 9 (23.9 mg, t_R = 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_R = 28.1 min).

Oenotheralanosterol C (1): The white amorphous powder of compound 1 was characterized with [α${]}_{\mathrm{D}}^{20}$ − 71.397 (c 0.4120, CH₃OH), mp 217 °C, HR-ESI-MS m/z: 511.3397 [M + Na]⁺ (calculated for C₃₀H₄₈O₅Na, 511.3399) UV λ_max(CH₃OH)/nm (logε): 206 nm; IR (CH₃OH)v_max: 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${]}_{\mathrm{D}}^{20}$ + 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) v_max: 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${]}_{\mathrm{D}}^{20}$ + 69.888 (c 0.2336, CH₃OH), mp 247 °C, HR-ESI-MS m/z: 509.3237 [M+Na]⁺ (calculated for C₃₀H₄₆O₅Na, 509.3243) UV λ_max(CH₃OH)/nm (logε): 204 nm; IR (CH₃OH)v_max: 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${]}_{\mathrm{D}}^{20}$ + 62.971 (c 0.2552, CH₃OH), mp 246 °C, HR-ESI-MS m/z: 525.3180 [M+Na]⁺ (calculated for C₃₀H₄₆O₆Na, 525.3192) UV λ_max(CH₃OH)/nm(logε): 203 nm; IR (CH₃OH)v_max: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${]}_{\mathrm{D}}^{20}$ + 62.971 (c 0.2552, CH₃OH), mp 219 °C, HR-ESI-MS m/z: 513.2832 [M+Na]⁺ (calculated for C₂₈H₄₂O₇Na, 513.2828) UV λ_max(CH₃OH) /nm(logε): 204 nm; IR (CH₃OH)v_max: 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.