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.


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, antiinflammatory, 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)(2)(3)(4)(5) and seven known triterpenoids (6)(7)(8)(9)(10)(11)(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.
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 EC 50 values ranging from 4.7 µM to 9.9 µM (Table 3). 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.

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-offlight 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 highpressure 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.

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.

In Vitro Cell Experiment
In the previous stage, the research group conducted relevant studies on the antipulmonary 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 × 10 4 cells/well) for 24 h at 37 • C in a humidified atmosphere of 5% CO 2 . 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 EC 50 values of these compounds were calculated by GraphPadSigmoidal dose-response.

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 EC 50 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.
Author Contributions: J.L., separation of compounds, structural identification, and writing-original draft. J.Z., separation of compounds. M.Z., study of the anti-pulmonary fibrosis activity of the compounds. M.L., S.X., X.Z. and W.F., writing-review and editing, structural identification, methodology. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.