Structure elucidation of constituents

A total of sixty-three constituents, including seven new compounds (including two flavonoid glycosides 1–2, one phenylpropionate 3, two fatty acids 4–5 and two alkaloids 6–7), were isolated and identified from T. chinense. The isolation and identification of new compounds have deepened our understanding of the chemical composition of T. chinense, enhancing the in-depth analysis of the drug-target network. This leads to a better discovery of active ingredients and a more comprehensive understanding of T. chinense's mechanism of action in treating lung inflammation.

Compound 1 (thesiuside A) was obtained as a yellowish solid, and the molecular formula was deduced as C42H38O21 based on HRESIMS and the protonated molecular ion [M + H] + at m/z 879.1991 (calcd. for 879.1978). The 1H NMR spectrum (Table 1) displayed two groups of AA′BB′ aromatic spin system signals [ring B, δH 7.35 (2H, d, J = 8.9 Hz), 6.78 (2H, d, J = 8.9 Hz); ring E, δH 8.12 (2H, d, J = 9.0 Hz), 6.90 (2 H, d, J = 9.0 Hz)]. In addition, ring A was a tetrasubstituted aromatic ring [δH 5.96 (1H, d, J = 2.1 Hz), 5.90 (1H, d, J = 2.1 Hz)] and ring D was an 1,2,3,4,5-pentasubstituted aromatic ring [δH 6.57 (1H, s)]. Besides, two anomeric protons were identified at δH 5.74 (1H, d, J = 7.7 Hz) and 5.23 (1H, d, J = 1.9 Hz), and a series of proton signals on sugars were identified at δH 3–4, which revealed a β-glucopyranosyl moiety and an α-rhamnopyranosyl moiety. The 13C NMR spectrum (Table 1) exhibited a carbonyl signal (δC 192.5), an α, β-unsaturated carbonyl group (δC 179.6), two olefinic protons (δC 158.8, 134.7), 24 aromatic carbon signals. It was worth noting that the carbon resonance at δC 119.5 and δC 81.7 were ascribed to C-2 and C-3 bearing oxygen atoms. According to the molecular formula, C-2 and C-3 should form a ternary ring through one oxygen atom. In addition, further analyses of HMBC associations of H-2′ to C-2 and H-2′′′ to C-2′′ revealed that ring B and ring C were connected as well as ring D and ring E were connected. Thus, the relative configuration of 1 was proposed as a biflavone glycoside, which was formed by a structure of kaempferol (ring A, B and C), an epoxy flavanone (ring D, E and F) and two sugar substituents. And the C-3 in ring C and C-8′′ in ring D were the only possible connection mode of these two flavonoid structures. The 2D NMR data (HSQC, 1H-1H COSY and HMBC) of 1 showed the signal attribution of the β-glucopyranosyl moiety [δH 5.74 (1H, d, J = 7.7 Hz), 3.73 (1H, dd, J = 12.0, 2.1 Hz), 3.63 (1H, dd, J = 9.2, 7.7 Hz), 3.55 (1H, dd, J = 9.2, 8.7 Hz), 3.49 (1H, dd, J = 12.0, 5.9 Hz), 3.27 (1H, dd, J = 9.8, 8.7 Hz) and 3.23 (1H, ddd, J = 9.8, 5.9, 2.1 Hz), and δC 100.1, 79.7, 79.0, 71.9, 78.5 and 62.9] and the α-rhamnopyranosyl moiety [δH 5.23 (1H, d, J = 1.9 Hz), 3.98 (1H, dd, J = 3.4, 1.9 Hz), 3.98 (1H, overlap), 3.73 (1H, dd, J = 9.5, 3.4 Hz), 3.33 (1H, overlap) and 0.91 (3H, d, J = 6.2 Hz), and δC 102.5, 72.4, 72.3, 74.0, 69.9 and 17.5]. The HMBC cross-peak from H-1′′′′ to C-3′′ suggested the position of the β-glucopyranosyl moiety (Fig. 1A). It was further confirmed that β-glucopyranosyl moiety was linked at C-2′′′′ to α-rhamnopyranosyl moiety at C-1′′′′′ based on the HMBC spectrum.

Fig. 1

Key HMBC and 1H-1H COSY correlations. A compounds 1–2. B compound 3. C compound 4. D compound 5

The determination of the absolute configuration of compound 1 was elucidated via the ECD spectrum. The ECD spectrum (Additional file 1: Fig. S8) showed positive cotton effects for the π → π* transition at 320 nm indicating the presence of para substituted phenolic motif and a negative cotton effect for the n → π* transition at 288 nm proving the presence of ketone carbonyl group. The relative configuration of 1 was analogous to the structure of [(2S,3S)-2,3-epoxy-5,7,4′-trihydroxyflavanone] -(3→8)-kaempferol 3″-O-β-D-glucopyranoside, which has reported the ECD spectrum of its hydrolysate, and the ECD spectrum of 1 has similar cotton effects with its ECD spectrum [24]. Consequently, the absolute configuration of C-2 and C-3 was determined as S. Based on these results, compound 1 was proposed to be [(2S,3S)-2,3-epoxy-5,7,4′-trihydroxyflavanone]-(3→8)-kaempferol 3″-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside, and named as thesiuside A.

Compound 2 (thesiuside B) was isolated as a yellowish solid and its molecular formula was determined to be C42H38O21 by the HRESIMS, which exhibited the molecular ion [M + H] + peak at m/z 879.1991 (calcd. for 879.1978). Intriguingly, the 1D (1H NMR and 13C NMR) and 2D NMR (HSQC, 1H-1H COSY and HMBC) data of 2 (Table 1 and Fig. 1A) resembled those of 1, which revealed that 2 was a diastereomer of 1. Their relative configurations were identical, and the only difference was the absolute configuration at positions C-2 and C-3. The ECD spectrum of 2 (Additional file 1: Fig. S15) and 1 had opposite cotton effect at 320 nm and 288 nm, which suggests that the absolute configuration of 2 at C-2 and C-3 was R. Accordingly, the structure 2 was established as [(2R,3R)-2,3-epoxy-5,7,4′-trihydroxyflavanone]-(3 → 8)-kaempferol 3″-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, and named as thesiuside B.

Compound 3 was purified as a colorless amorphous solid. The molecular formula was deduced as C34H40O18 by HRESIMS, which displayed the molecular ion [M − H] − at m/z 735.2159 (calcd. for 735.2142). The 1H NMR data (Table 2) showed characteristic signals for one oxygenated methyl group [δH 2.04 (3H, s)], three oxygenated methylenes [δH 4.48 (1H, d, J = 6.8 Hz) and 4.47 (1H, d, J = 3.7 Hz); 4.15 (1H, dd, J = 12.1, 5.5 Hz) and 4.13 (1H, dd, J = 12.1, 2.9 Hz); 3.72 (2H, s)], eight oxygenated methines [δH 5.47 (1H, d, J = 3.8 Hz), 4.93 (1H, dd, J = 10.2, 9.2 Hz), 4.40 (1H, ddd, J = 10.2, 5.5, 2.9 Hz), 4.19 (1H, d, J = 8.2 Hz), 4.14 (1H, overlap), 4.03 (1H, overlap), 4.02 (1H, dd, J = 9.7, 9.2 Hz), 3.58 (1H, dd, J = 9.7, 3.8 Hz)], four olefinic protons [δH 7.63 (1H, d, J = 16.0 Hz), 7.62(1H, d, J = 16.0 Hz), 6.39 (1H, d, J = 16.0 Hz), 6.33 (1H, d, J = 16.0 Hz)], which were two trans double bonds on the basis of coupling constants, two aromatic rings of ABX spin system [δH 7.13 (1H, d, J = 2.0 Hz), 7.03 (1H, dd, J = 8.2, 2.0 Hz) and 6.80 (1H, d, J = 8.2 Hz); 7.12 (1H, d, J = 2.0 Hz), 7.02 (1H, dd, J = 8.2, 2.0 Hz) and 6.78 (1H, d, J = 8.2 Hz)]. The 13C NMR and DEPT spectrum (Table 2) exhibited 34 carbon signals, corresponding to three ester carbonyl carbons (δC 172.7, 169.0, 168.3), sixteen sp2 carbons (δC 147.4, 147.1, 115.2, 115.2, 150.7, 150.7, 149.3, 149.3, 127.7, 127.6, 124.2, 124.1, 116.5, 116.5, 112.0, 111.8), three methyl carbons (δC 56.5, 56.5, 20.8) and twelve oxygenated carbons related to sugar groups (δC 105.9, 81.0, 78.9, 76.5, 66.4, 63.6; δC 93.5, 73.4, 72.6, 72.6, 69.9, 64.5). In addition, the 2D NMR data (HSQC, 1H-1H COSY and HMBC) suggested the existence of two identical trans ferulic acid structures and two sugars. Further analysis of the anomeric protons [δH 5.47 (1H, d, J = 3.8 Hz), 3.72 (1H, s)] and anomeric carbons (δC 93.5, 63.6) revealed that the structures of the two sugars were one β-D-fructose moiety and one α-D-glucose moiety, respectively. The HMBC cross-peak (Fig. 1B) from H-1′ to C-2 established the linkage of the two sugar moieties, indicating the presence of a sucrose moiety. After the glycohydrolysis experiments, it was determined that the sugar was sucrose analyzed by TLC and HPLC. The HMBC spectrum exhibited key correlations from H-6 to C-γ′′, from H-4′ to C-γ′′′ and from H-4 to carbonyl carbon (δC 172.7), which further revealed the connecting positions of two ferulic acids and one acetyl group. This compound was similar to compound helonioside B [25], with the main difference being the different connection positions of the trans ferulic acid groups and the acetyl group. Therefore, 3 was established as 4-acetyl-6, 4′-diferuloylsucrose.

Compound 4 was obtained as a yellowish oil. HRESIMS measurements of this compound showed a [M − H] − ion peak at m/z 155.0706 and the molecular formula was assigned as C8H12O3 (calcd. for C8H11O3, 155.0714), deducing three degrees of unsaturation. The 1H NMR spectrum (Table 3) indicated that 4 contained one methyl group [δH 0.94 (3H, t, J = 7.4 Hz)], one methylene [δH 1.56 (2H, m)], one oxygenated methine [δH 4.08 (1H, q, J = 6.1)] and four olefinic protons [δH 7.22 (1H, dd, J = 15.4, 11.0 Hz), 6.39 (1H, dd, J = 15.2, 11.0 Hz), 6.10 (1H, dd, J = 15.2, 11.0 Hz), and 5.89 (1H, d, J = 15.4 Hz)]. The 13C NMR data (Table 3) of 4 confirmed the presence of 22 carbon resonances, including one methyl carbon (δC 10.1), one methylene carbon (δC 30.9), one oxygenated methine carbon (δC 74.0), four olefinic carbons (δC 145.8, 144.8, 128.8 and 124.0) and one carboxylic carbon (δC 171.8). 1H-1H COSY correlations (Fig. 1C) of H-2/H-3/H-4/H-5/H-6/H-7/H-8 and the HMBC cross-peaks (Fig. 1C) of H-2/C-4; H-3/C-1, C-3; H-6/C-4; H-7/C-5 and H-8/C-6 proved that the compound bears a long-chain fatty acid structure. Furthermore, the E configurations of the C-2/C-3 and C-4/C-5 double bonds were determined by the coupling constants, which also explained the presence of an α, β, γ, δ-unsaturated carbonyl moiety. The absolute configuration of the hydroxyl group at C-6 was assigned to be R by measuring the specific rotation of 4 ([α] 20D -3.3), which is opposite to that of the similar compound previously reported [26]. Finally, compound 4 was elucidated as (2E,4E,6R)-6-hydroxy-2,4-oxtadienoic acid.

Compound 5 was isolated as a yellow oil. It had a molecular formula of C14H20O4 inferred from the [M − H] − ion peak at m/z 251.1279 (calcd. for 251.1289), which was deduced from the HRESIMS spectrum, and it required three degrees of unsaturation. The 1H NMR spectrum (Table 3) gave signals for one methyl group [δH 3.65 (3H, s)], seven methylenes [δH 2.40 (2H, td, J = 7.0, 2.0 Hz), 2.32 (2H, t, J = 7.4 Hz), 1.62 (2H, m), 1.56 (2H, m), 1.42 (2H, m), 1.35 (2H, m), 1.35 (2H, m)] and two olefinic protons [δH 6.69 (1H, dt, J = 15.9, 2.0 Hz), 6.10 (1H, d, J = 15.9 Hz)]. Its 13C NMR and DEPT spectrum (Table 3) suggested the presence of one methyl carbon (δC 52.0), seven methylene carbons (δC 34.7, 30.0, 29.8, 29.7, 29.4, 25.9, 20.2), two olefinic carbons (δC 131.5, 126.8), two carbonyl carbons (δC 176.0, 169.7) and two acetylenic carbons (δC 101.2, 78.8). A long methylene chain unit was established by the 1H-1H COSY spectrum (Fig. 1D), which showed the correlations at H-6/H-7/H-8/H-9/H-10/H-11/H-12. The HMBC cross-peaks (Fig. 1D) of H-3/C-1, C-5; H-2/C-1; H-6/C-2, C-3, C-4, and H-7/C-5 confirmed that one end of the acetylenic bond was connected to the fat chain and the other to the α, β-unsaturated carbonyl group, which could also be identified by the 1H-1H COSY cross-peaks from H-3 to H-6. The existence and position of the methyl ester group were supported by the HMBC data, which indicated the cross-peaks from methyl hydrogen to C-13 and from H-11 to C-13. Furthermore, the coupling constants between H-2 and H-3 (J = 15.9 Hz) suggested the C-2/C-3 double bonds were E geometry. The structure of compound 5 was similar to the known compound anacolosine, the main difference was the presence of methyl ester unit (δH 3.65, δC 52.0) in compound 5 [27]. Therefore, compound 5 was named as (E)-13-methoxy-13-oxotridec-2-en-4-ynoic acid.

Compound 6 was obtained as a colorless oil. The [M + H] + ion was observed at 247.1805 m/z (calcd. for 247.1805) by HRESIMS and the molecular formula of 6 was deduced as C15H22N2O with 6 degrees of unsaturation. The 1H NMR spectrum (Table 4) and HSQC spectrum revealed the presence of two olefinic protons [δH 6.64 (1H, ddd, J = 9.8, 4.7, 3.8 Hz) and 5.84 (1H, dt, J = 9.8, 2.0 Hz)], eight methylenes [δH 3.46 (1H, overlap) and 2.89 (1H, overlap), 1.97 (1H, overlap) and 1.72 (1H, overlap), 1.85 (1H, m) and 1.80 (1H, overlap), 2.01 (1H, overlap) and 1.72 (1H, overlap), 2.07 (1H, overlap) and 1.72 (1H, overlap), 3.46 (1H, overlap) and 2.89 (1H, overlap), 2.79 (1H, m) and 2.34 (1H, m) as well as 4.25 (1H, dd, J = 14.0, 5.0 Hz) and 3.23 (1H, dd, J = 14.0, 12.9 Hz)], and four methines [δH 4.13 (1H, ddd, J = 11.8, 8.4, 7.3 Hz), 3.39 (1H, t, J = 3.2 Hz), 2.11, (1H, overlap) and 2.07, (1H, overlap)]. The analysis of 13C NMR and DEPT spectrum (Table 4) showed 15 carbon resonances, including eight methylene carbons (δC 57.0, 56.9, 42.0, 28.0, 26.8, 25.2, 20.2, 19.8), four methines (δC 64.6, 52.0, 41.2, 34.8), two olefinic carbons (δC 140.7, 124.1) and one carbonyl carbon (δC 167.6). The 1H-1H COSY cross-peaks of H-14/H-13/H2-12/H-11, together with the HMBC correlations (Fig. 2A) from H-14 to C-15, from H-13 to C-15 and from H-11 to C-13/C-15 established the location of the α, β unsaturated ketone. The 1H-1H COSY cross-peaks of H-11/H-7/H-6/H-5/H2-17, H-7/H2-8/H2-9/H2-10 and H-5/H2-4/H2-3/H2-2 together with the HMBC correlations from H-11 to C-15, from H-17 to C-15, from H-17 to C-11, from H2-2 to C-6, from H2-10 to C-6 and from H2-2 to C-10 revealed that the skeleton of 6 was a matrine-type alkaloid. The relative configuration of 6 was deduced by the NOESY spectrum (Fig. 2A), where correlations of H-17b/H-7 and H-11, H-6/H-8b as well as H-11/H-8a exhibited the H-7 and H-11 were β-orientations. The cross-peaks of H-3b/H-5 and H-6 suggested the α-orientations of H-5 and H-6.

Fig. 2

A Key HMBC, 1H-1H COSY correlations and key NOESY correlations, and experimental and calculated ECD spectra of compound 6; B Key HMBC, 1H-1H COSY correlations and key NOESY correlations, and experimental and calculated ECD spectra of compound 7

The C-5, C-6, C-7, and C-11 of the compound were chiral carbon atoms, and there may be two absolute configurations of the compound, namely enantiomers 6A (5R, 6R, 7R, 11S) and 6B (5S, 6S, 7S, 11R), respectively. The ECD spectra of the two configurations were obtained by the method of ECD calculations. It was found that the calculated ECD curves of 6B (5S, 6S, 7S, 11R) were basically consistent with those of compound 6 measured by experiments (Fig. 2A). Thus, its absolute configuration was determined as 5S, 6S, 7S, 11R. Compound 6 was established as a new structure and named as thesiumine A.

Compound 7 was obtained as a colorless oil. The [M + H] + ion was observed at 249.1960 m/z (calcd. for 249.1961) by HRESIMS and the molecular formula of 7 was deduced as C15H24N2O with 5 degrees of unsaturation. The significant difference between the 1H NMR and 13C NMR data (Table 4) of 7 and those of 6 was the absence of a double bond. The NOESY correlations (Fig. 2B) of H-17b/H-6, H-7 and H-11 exhibited the H-11, H-7 and H-6 were β-orientations. The cross-peaks of H-2b/H-6 and H-2a/H-5 indicated the α-orientations of H-5. The absolute configuration was also determined by ECD calculations with the same method of compound 6. The experimental ECD spectrum of compound 7 was compared with the calculated ECD spectrum (Fig. 2B and Additional file 1: Fig. S51) of compound 7A (5S, 6R, 7S, 11R) and 7B (5R, 6S, 7R, 11S). The results showed that the ECD curves of 7A (5S, 6R, 7S, 11R) configuration were in good agreement with those of compound 7. Therefore, its absolute configuration was determined as 5S, 6R, 7S, 11R. Compound 7, which was an enantiomer of tetrahydroneosophoramine [28], was identified as a new compound named thesiumine B.

The other 56 known compounds (Fig. 3) were identified by comparing their spectroscopic data with those reported in the literature (Additional file 1: Table S2), including kaempferol (8), luteolin (9), quercetin (10), tricin (11), astragalin (12), isoquercitrin (13), rhamnetin-3-O-β-D-glucopyranoside (14), kaempferol-3-O-(6′′-O-acetyl)-β-D-glucopyranoside (15), quercetin-3-O-(6′′-O-acetyl)-β-D-glucopyranoside (16), kaempferol-3-O-(3′′-O-acetyl)-β-D-glucopyranoside (17), tiliroside (18), kaempferol-3-O-glucorhamnoside (19), kaempferol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[6-O-acetyl]-β-D-glucopyranoside (20), kaempferol-3-O-α-L-rhamnopyranosyl-(1 → 2)-[3-O-acetyl]-β-D-glucopyranoside (21), ajugasterone C (22), 20-hydroxyecdysone (23), calonysterone (24), esculetin (25), caffeic acid (26), (E)-ferulic acid (27), (E)-p-coumaric acid (28), syringin (29), 4,5-di-O-caffeoylquinic acid 1-methyl ether (30), geniposide (31), phaseic acid (32), uridine (33), 1-(β-D-ribofuranosyl)-1H-1,2,4-triazone (34), 3-hydroxypyridine (35), methyl-5-hydroxypyridine-2-carboxlate (36), isohematinic acid (37), uracil (38), protocatechuic acid (39), p-hydroxybenzoic acid (40), vanillic acid (41), p-hydroxyphenethyl alcohol (42), gallic acid (43), 3,4-dihydroxybenzyl alcohol (44), 1H-indole-3-carboxaldehyde (45), ( +)-syringaresinol (46), lirioresionol (47), ( +)-medioresinol (48), ( +)-pinoresinol (49), 5-methoxy-( +)-isolariciresinol (50), ( +)-isolariciresinol (51), ( +)-lyoniresinol (52), (7S, 8R)-dihydrodehydrodiconiferyl alcohol (53), 5-methoxydehydroconiferyl alcohol (54), isoscopoletin (55), scopoletin (56), isofraxidin (57), ( +)-dehydrovomifoliol (58), ( −)-loliolide (59), ( +)-isololiolide (60), p-hydroxyacetophenone (61), dihydroconiferylalcohol (62), acetovanillone (63). Notably, forty-seven previously unseparated compounds (10–11, 13–18, 20–24, 26–27, 29–39, 41–58, 60, and 62–63) were discovered from the plant in addition to the compounds 1–7.

Fig. 3

Chemical structures of compounds 1–63 isolated from the T. chinense

Inhibition of NO production by isolated compounds

The in vitro anti-inflammatory activity of isolated compounds was evaluated by detecting NO level in LPS-induced RAW 264.7 macrophages. Cytotoxicity of the compounds was determined by MTT assay to confirm that the reduction in NO production was not due to inhibition of cell proliferation.

New compounds 1, 2, 4 and 5 displayed NO production inhibition activity. Flavonoid aglycones 8–11 and fatty acid 5 displayed strong anti-inflammatory activity and their MIRs either exceeded or were equal to that of DIDOX (Fig. 4). Flavonoid glycosides 1 and 2, fatty acid 4, alkaloids 7 and 34, phenylpropionic acid 27, simple aromatics 39, lignans 44–54, coumarin 57 and terpenoid 58 demonstrated moderate anti-inflammatory activity. It was worth mentioning that although flavonoid glycosides were the main constituents of T. chinense, compounds 12–21 exhibited no inhibitory activity of NO production.

Fig. 4

Heat map analysis of inhibitory activity of compound on LPS-induced NO production in RAW 264.7 cells. NO level was measured after treatment with compounds at indicated doses along with LPS (1 μg/mL) for 24 h. The data were normalized using Z-score (https://www.omicshare.com/tools). The color of the spots represents the level of NO production in each group, with red indicating a higher level and blue indicating a lower level. C: control group

Targets of T. chinense against lung inflammation based on network pharmacology analysis

For further exploration of the potential targets of T. chinense against lung inflammation, we conducted a network pharmacology analysis of T. chinense against lung inflammation. A total of 88 constituents in T. chinense were obtained from literature reviews and our identified constituents in this article, and 10 active constituents were obtained after screening by Swiss target ADME. 236 drug targets and 1768 disease targets were collected from the HERB, TCMSP, and Genecards databases. We matched targets of the ingredients and the disease, 147 common targets were obtained and shown with a venn diagram (Fig. 5A; Additional file 1: Table S4). To better demonstrate the connection between drugs and disease targets, a T. chinense-constituents-targets-lung inflammation network diagram was constructed (Fig. 5B). The active ingredients were sorted by degree and displayed in Fig. 5C, and of which, five constituents (6–7 and 9–11) in the top ten active constituents were new compounds or firstly isolated from T. chinense. Protein–protein interaction (PPI) network was constructed to further explore the protein–protein interaction relationship between common targets. The key proteins for T. chinense treating lung inflammation included TP53, AKT1, STAT3, TNF, JUN, IL6, HSP90AA1, SRC, MAPK3, EGFR (Fig. 5D). Gene ontology (GO) enrichment analysis demonstrated that T. chinense may intervene lung inflammation through participating in inflammation-related biological processes containing response to molecule of bacterial origin, response to xenobiotic stimulus, regulation of defense response, response to oxygen levels, regulation of cellular response to stress, and cellular response to cytokine stimulus (Fig. 5E). KEGG enrichment analysis showed that T. chinense intervened lung inflammation via regulating TNF signaling pathway, FoXO signaling pathway, NF-κB signaling pathway, HIF-1 signaling pathway, TGF-β signaling pathway, and other inflammation-related pathways (Fig. 5F). All these data indicated the enormous potential of T. chinense therapy for lung inflammation and potential therapeutic targets and mechanisms.

Fig. 5

Results of network pharmacology prediction of T. chinense for lung inflammation. A Venn diagram of lung inflammation target and T. chinense target. B The “constituent-target-disease” network of T. chinense. The pink oval represents T. chinense, the green oval represents the active constituents in T. chinense, yellow oval represents predicted target, and blue diamond represents lung inflammation. C Active components sorted by degree. D Common target protein interaction network diagram. E GO biological process enrichment analysis of the potential targets of T. chinense against inflammation. F KEGG signaling pathway enrichment analysis on the potential targets of T. chinense against inflammation

The constituents, extract and preparation of T. chinense attenuate LPS-induced ALI in mice

Based on network pharmacology analysis, flavonoids and their glycosides play a crucial role in treating lung inflammation by T. chinense and Bairui Granules. The predominant and representative constituents were flavonoid glycosides KN (Bairuisu I, 19) and AG (Bairuisu II, 12) with contents of 15.072 mg/g and 8.014 mg/g in T. chinense, and 4.23 mg/g and 1.87 mg/g in Bairui Granules. Although they did not rank among the top ten most active constituents, their common glycoside, kaempferol, was predicted by network pharmacology analysis. In addition, 12 and 19 did not display an inhibitory effect against NO production. Thus, the two constituents (19 and 12) were further evaluated for their anti-inflammatory activity in vivo. Moreover, KF (Bairuisu III, 8) with potent inhibition on NO production, as well as BG and CE have also been determined.

As displayed in Fig. 6A, LPS exposure led to the persistent infiltration of inflammatory cells, accumulation of neutrophils in the alveolar and interstitial space, the thickened alveolar and airway walls. While, these symptoms of inflammation were obviously alleviated after KN (19), AG (12), KF (8), BG and CE. Furthermore, compared with the control group, the count of leukocytes and neutrophils in peripheral blood increased significantly in LPS-treated group (Fig. 6B-C). And the number of these inflammation-related cells was reduced in mice treated by KN (19), AG (12), KF (8), BG and CE. Abnormal activation of NOD-like receptor protein 3 (NLRP3) inflammasome could lead to the high expression of caspase-1 and the secretion of IL-1β, leading to the occurrence of inflammatory diseases [29]. Acute inflammation could lead to high expression of cyclooxygenase-2 (COX-2) and damage tissues [30]. Thus, we measured the levels of the inflammatory cytokine IL-1β in the BALF using ELISA assay and the mRNA levels of NLRP3, caspase-1, IL-1β and COX-2 using RT-PCR. As depicted in Fig. 6D, treatment with KN (19), AG (12), KF (8), BG and CE blocked the LPS-stimulated increase of IL-1β in BALF. Similarly, they significantly inhibited the LPS-stimulated upregulation of mRNA levels of NLRP3, caspase-1, IL-1β and COX-2 in the lung tissues (Fig. 6E-H). These data suggested that KN (19), AG (12), KF (8), BG and CE were capable of alleviating lung inflammation. KN (19), AG (12) and KF (8) are responsible for the traditional and clinical application of Thesium chinense Turcz. and Bairui Granules against inflammation-related diseases. Nevertheless, there is a deficiency of established quality control standards and markers for T. chinense. It is proposed that KN (19) and AG (12) could serve as foundational elements to enhance quality standards or act as markers for assessing the quality of T. chinense and its preparations.

Fig. 6

Effects of T. chinense on lung inflammation induced by LPS. A H&E staining of lung tissue sections. Scale bar, 50 µm. B, C Peripheral blood leukocyte and neutrophil count. D The level of IL-1β in BALF. E–H Relative mRNA level of NLRP3, caspase-1, IL-1β and COX-2. Data were expressed as mean ± SD (n = 3), * p < 0.05 as compared to LPS group

SafetyClinical observations and body weight including food intake.

In the subacute toxicity assessment, we conducted precise and systematic observations to detect potential signs of toxicity. These observations were conducted at specific time intervals, including 0, 30 min, 1, 3 and 6 h, followed by daily evaluations over a 28-day period. Notably, no fatalities or adverse indicators of toxicity, such as emesis, lethargy, paw jerking, and cutaneous lesions, were observed across all administered doses when compared to the normal control group (Table 5). Furthermore, despite the observed progressive increase in body weight throughout the experiment, we found no statistically significant differences in either body weight or food intake among the experimental groups (Additional file 1: Fig. S12A-B).

Table 5 Effects of Control, CE, BG-L and BG-H on physical and behavioral parameters in mice during a 28-day subacute toxicity studyOrgan coefficients and morphological examination

Hearts, livers, lungs, kidneys and spleens were dissected, rinsed in saline, and then placed on a blank background under the white light for the photographic documentation. The results demonstrated the structural integrity and healthy appearance of the organs across all four groups of mice, with no significant differences observed (Fig. 7C). The organ coefficient, calculated as the ratio of organ mass to body weight, is a commonly employed parameter in toxicology experiments. In this study, there were no statistically significant differences in organ coefficients among the control group, CE, BG-L and BG-H group (Fig. 7D-G).

Fig. 7

Effects of Control, CE, BG-L, BG-H on mice growth and organ characteristics in a 28-day subacute toxicity study. A Body weight. B Food intake. C The gross appearance of heart, liver, spleen, lung and kidney. D–H Organ coefficients of heart, liver, spleen, lung and kidney. I H&E staining of heart, liver, spleen, lung, stomach and colon tissue sections. Scale bar, 50 µm. Data are illustrated as mean ± SD (n = 6); * p < 0.05 as compared to the control group

Histopathological assessment

Significant histological changes were not observed in the control and treatment groups.

Heart: The microscopic analysis of the heart in all treatment groups displayed normal architecture of the cardiac muscle fibers with intact length and regular cell striation and nuclei and was bereft of significant cellular infiltration or degeneration.

Liver: The tissue slices displayed intact hepatic parenchyma including hepatocytes, central vein and portal triad. No significant mixed macro and micro vesicular steatosis, vacuolated hepatocytes or any kind of infiltration was observed in the liver tissue (hepatocytes). Hepatocytes were organized in cords with intact cellular and nucleus borders.

Lung: All treated groups showed normal lung architecture. No significant damage (such as granulomas, increased alveolar cell wall, inflammatory cells) was observed in the lung tissue of any treated group rats. The section showed an intact alveolar membrane.

Spleen: No significant damage was observed in the spleen tissue of any treated group. The graph showed the normal white and red pulp areas with no cell breakage in the splenic parenchyma.

Kidney: In all treatment groups, the kidneys exhibited tightly arranged and well-organized glomerular and tubular structures.

Stomach: No significant damage (such as focal necrosis, mucus wall disruption, extensive congestion in the mucosa and haemorrhagic bands) was observed in the inner lining of stomach of any treated group. The inner mucus membrane was completely remained intact.

Colon: No significant damage (villi atropy, inflammation, superficial erosion and crypt hyperplasia) was observed in the tissues of small intestine of any treated groups rats. The histology result displays no abnormalities in the microscopic structure of small intestine.

Blood biochemistry and routine blood indicators

Blood supernatants were analyzed using a blood biochemistry analyzer, which revealed no statistically significant differences in cardiac function parameters, including CK and LDH, between the treatment and control groups (Additional file 1: Fig. S52A-D). Similarly, no statistically significant differences were observed in TBA, ALP, ALT and AST, which serve as indicators of liver function (Additional file 1: Fig. S52C-F). There is no significant difference in creatinine and urea nitrogen levels, which are indicative of renal function (Additional file 1: Fig. S52G-H).

The results of the blood routine analysis are presented in Additional file 1: Fig. S52. In this analysis, there were no statistically significant differences observed between the experimental group and the control group in various indicators used for assessing infection and immune function, such as white blood cell count, red blood cell count, lymphs, eosinophils, and neut/gran. Furthermore, parameters such as hemoglobin, hematocrit, mean corpuscular volume, mean platelet volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and platelet count, which are indicative of anemia, blood viscosity, and platelet function, also showed no significant differences. In summary, the administration of CE, BG-L and BG-H did not have significant impacts on the blood and serum biochemical indices of the mice.