Chemical Constituents from the Flowers of Carthamus tinctorius L. and Their Lung Protective Activity

A new flavonoid, saffloflavanside (1), a new sesquiterpene, safflomegastigside (2), and a new amide, saffloamide (3), together with twenty-two known compounds (4–25), were isolated from the flowers of Carthamus tinctorius L. Their structures were determined based on interpretation of their spectroscopic data and comparison with those reported in the literature. The protective effects against lipopolysaccharide (LPS)-stimulated damage on human normal lung epithelial (BEAS-2B) cells of the compounds were evaluated using MTT assay and cellular immunofluorescence assay. The results showed that compounds 2–3, 8–11, and 15–19 exhibited protective effects against LPS-induced damage to BEAS-2B cells. Moreover, compounds 2–3, 8–11, and 15–19 can significantly downregulate the level of nuclear translocation of NF-κB p-p65. In summary, this study revealed chemical constituents with lung protective activity from C. tinctorius, which may be developed as a drug for the treatment of lung injury.


Introduction
Carthamus tinctorius L., widely accepted as Safflower, belongs to the family of Asteraceae, mainly distributed in China, India, Iran, Egypt, and other countries [1]. It is an annual or biennial herbal plant mainly cultivated for its seeds, meals, and flowers, which primarily are rich in the orange-red dye (carthamin) and quality oil of polyunsaturated fatty acids [2]. Therefore, this plant is used for natural dyestuff, culinary, and textile purposes. More to the point, the dried flower of C. tinctorius is also clinically used to alleviate pain, increase circulation, and reduce blood-stasis syndrome with dysmenorrhoeal, amenorrhoea, trauma, and joint pain [3]. Pharmacological investigations have demonstrated that this plant possessed certain biological properties such as anti-inflammatory [4], cardioprotective [5], antitumor [6], anti-osteoporosis [7], and hepatoprotective effects [8]. Moreover, C. tinctorius shows effective outcomes in myocardial ischemia, coagulation, and thrombosis [9]. Concerning the phytochemistry of this plant, certain bioactive constituents have been isolated, such as flavonoids, phenylethanoid glycosides, coumarins, and polysaccharides [2].
Acute lung injury (ALI) is a continuum of pulmonary changes caused by various lung insults. The main pathological features of ALI are increased pulmonary vascular permeability, exudation of protein-rich fluid in the alveolar cavity, pulmonary edema, and hyaline membrane formation [10]. Lipopolysaccharide (LPS) can induce the apoptosis of lung epithelial cells and rapid influx of polymorphonuclear leukocytes (PMNs), causing the releases of proinflammatory cytokines, reactive oxygen species, and chemotactic factors [11]. The previous study demonstrated that flavonoids isolated from C. tinctorius could alleviate causing the releases of proinflammatory cytokines, reactive oxygen species, and chemotactic factors [11]. The previous study demonstrated that flavonoids isolated from C. tinctorius could alleviate acute lung injury induced by LPS [12], which attracted our interest to search for more natural products with lung-protective activity from this plant.

Biological Activity
Dexamethasone (DEX) is regarded as an effective drug to relieve the level of pneumonia, while it can trigger side effects, such as neuromuscular, cardiovascular, and gastric motility disorders [38]. In preliminary in vitro bioassays, all the isolated compounds were evaluated for their protective effects against LPS-induced BEAS-2B cell injury. The results indicated that compounds 2-3, 8-11, and 15-19 exhibited a significant protective effect against LPS-induced BEAS-2B cell damage at a concentration of 10 μM with the DEX as the positive control drug (Table 2). Hence, compounds 2-3, 8-11, and 15-19 isolated from C. tinctorius may be used as potential drugs to treat lung injury. Furthermore, NF-κB is a ubiquitous nuclear transcriptional activator in the body, which is involved in the occurrence of inflammation and cellular immunity. NF-κB p-p65 is a key functional isoform, whose nuclear translocation level is proportional to the degree of NF-κB activation [39]. The nuclear translocation level of NF-κB p-p65 in cells was detected by the cell immunofluorescence technique. As shown in Figure 3, compounds 2-3, 8-11, 15-19, and DEX can significantly downregulate the level of LPS-induced nuclear translocation compared with vehicle control, which indicated these compounds may reduce LPS-induced BEAS-2B cell damage by downregulating the nuclear translocation of NF-κB p-p65.

Biological Activity
Dexamethasone (DEX) is regarded as an effective drug to relieve the level of pneumonia, while it can trigger side effects, such as neuromuscular, cardiovascular, and gastric motility disorders [38]. In preliminary in vitro bioassays, all the isolated compounds were evaluated for their protective effects against LPS-induced BEAS-2B cell injury. The results indicated that compounds 2-3, 8-11, and 15-19 exhibited a significant protective effect against LPS-induced BEAS-2B cell damage at a concentration of 10 µM with the DEX as the positive control drug (Table 2). Hence, compounds 2-3, 8-11, and 15-19 isolated from C. tinctorius may be used as potential drugs to treat lung injury. Furthermore, NF-κB is a ubiquitous nuclear transcriptional activator in the body, which is involved in the occurrence of inflammation and cellular immunity. NF-κB p-p65 is a key functional isoform, whose nuclear translocation level is proportional to the degree of NF-κB activation [39]. The nuclear translocation level of NF-κB p-p65 in cells was detected by the cell immunofluorescence technique. As shown in Figure 3, compounds 2-3, 8-11, 15-19, and DEX can significantly downregulate the level of LPS-induced nuclear translocation compared with vehicle control, which indicated these compounds may reduce LPS-induced BEAS-2B cell damage by downregulating the nuclear translocation of NF-κB p-p65.     The previous study demonstrated that the total phenolics and flavonoids of the methanolic extracts of Pulicaria petiolaris (Asteraceae) decreased LPS-induced pulmonary inflammation, suggesting that P. petiolaris may be an important preventive strategy for the treatment of nonspecific pulmonary inflammation [40]. In addition, it reported that the sesquiterpenes of Eupatorium lindleyanum DC (Asteracea) significantly attenuated LPS- induced ALI [41]. This study indicates that flavonoids, sesquiterpenes, and alkaloids isolated from C. tinctorius (Asteracea) have a protective effect on ALI induced by LPS, indicating that plants in the Asteraceae family may have lung-protective potential, and that further research should focus on their use in pulmonary protection.

General Experimental Procedures
Optical rotations were recorded by using a Rudolph AP-IV polarimeter (Rudolph, Hackettstown, NJ, USA). UV spectra were recorded on a ThermoEVO 300 spectrometer (Thermo, Waltham, MA, USA). IR spectra were recorded on a Thermo Nicolet IS 10 spectrometer (Thermo, Waltham, MA, USA). NMR spectra were acquired using a Bruker Avance III 500spectrometer (Bruker, Berlin, Germany). MS spectra were obtained using a Bruker maXis HD mass spectrometer (Bruker, Germany). Semipreparative HPLC separations were performed on a Saipuruisi LC

Computational Analysis
The systematic random conformational analysis of each conformer of 2 was performed in the GMMX software by using an MMFF94 force field, which afforded a few conformers each, with an energy cutoff of 5 kcal/mol to the global minima. The obtained conformers were further optimized using density functional theory (DFT) at the mPW1PW91/6-31G(d) level in CH 3 OH in the Gaussian 16W. The 1 H and 13 C NMR chemical shifts of the optimized stable conformers were calculated with the GIAO method at the mPW1PW91/6-31G(d) level in CH 3 OH. The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory [42].

Cellular Immunofluorescence Assay
The BEAS-2B cells were distributed into 96-well plates. The final density per well was 2.5 × 10 4 cells in 200 µL of medium. Then the cells were added to the DMEM medium, the medium with LPS (10 µg/mL), the medium with DEX (1 µM), and the medium with test compounds (1-25) (10 µM) and LPS (10 µg/mL) group, respectively. After 24 h, the supernatant was aspirated, and 4% paraformaldehyde diluted with PBS was added, and it was fixed at room temperature for 15 min. Then, 0.25% TritonX-100 was added to each well and incubated for 10 min for permeabilization. Next, cell culture plates were incubated with 1% (BSA+PBST) for 30 min at room temperature. They were then incubated with primary antibodies NF-κB p-p65 overnight. After washing, the plates were incubated with the anti-rabbit IgG for 1h. Then, they were washed with 0.2% PBST twice, and DAPI (50 µL 2 µg/mL) was added in incubation for 5 min. Anti-fluorescence quenching mounting tablets were used for mounting, and performed detection and analysis in a high-content instrument [44].

Statistical Analysis
All data were analyzed by SPSS software version 26.0 (IBM, New York, NY, USA) and presented as the mean ± standard deviation. A one-way analysis of variance (One-Way ANOVA) was used for comparisons between groups. The differences were statistically significant when p < 0.05.

Conclusions
Three new compounds (1-3), together with twenty-two known compounds  were isolated from the flowers of C. tinctorius. Among the known compounds, 5, 9-17, 19, 21, and 24-25 were isolated from the plant for the first time. Most of the research on C. tinctorius. focuses on cardioprotective, antitumor, antithrombotic, anti-inflammatory, and hepatoprotective effects. In preliminary in vitro bioassays, the protective activities results showed that 2-3, 8-11, and 15-19 exhibited protective effects on BEAS-2B cell injury induced by LPS. Then, we will discover more bioactive compounds and carry out further research on the mechanism with potential compounds for the treatment of lung injury.