The interaction of anti-inflammatory and anti-tumor components in the traditional Chinese medicine Solanum lyratum Thunb

Abstract Solanum lyratum Thunb is a traditional Chinese medicinal with a significant clinical outcome for tumor treatment; however, chemicals or fractions separated from the herb did not exhibit strong and comparable efficacy. To investigate the potential synergy or antagonism among chemicals in the extract, we obtained the compounds solavetivone (SO), tigogenin (TI) and friedelin (FR) from the herb. The anti-tumor effects of these three monomer compounds alone or in combination with the anti-inflammatory compound DRG were also tested in this study. SO, FR and TI used alone did not inhibit the proliferation of A549 and HepG2 cells, but the combination of the three achieved 40% inhibition. In vitro anti-inflammatory analysis showed that DRG had a stronger anti-inflammatory effect than TS at the same concentration, and the combination of DRG with SO, FR or TI inhibited the anti-tumor effect of DRG. This is the first study that documented the synergistic and antagonistic interactions between different compounds in a single herb. Graphical Abstract


Introduction
Solanum lyratum Thunb is a traditional Chinese medicine that has been used for >1000 years.S. lyratum has significant pharmacological activity, which includes antitumor, anti-inflammatory, antibacterial and anti-allergic effects due to the saponins, alkaloids, terpenes and other chemical substances that this plant contains (Murakami et al. 1981;Yahara et al. 1986;Xu et al. 2018;Li et al. 2020;Liang et al. 2021).Studies showed that the extract of S. lyratum, which can improve immune function and survival rate of tumour-bearing mice, exhibiting certain in vivo anti-tumour effect (Guan et al. 2013;Xiao et al. 2013).In recent years, researchers have tried to separate more chemicals with anticancer, anticholinesterase and antibacterial activity from S. lyratum (Yao et al. 2013;Li et al. 2014;Nie et al. 2014;Xu et al. 2018;Chen et al. 2020;Zhang et al. 2022).For example, aspidistrin, one of the steroidal glycosides, showed significant cytotoxic activity against gastric cancer SGC7901 and liver cancer BEL-7402 cells (Xu et al. 2018).Two new sesquiterpenoids were isolated and exhibited significant cytotoxicity against three kinds of cancer cell lines from S. lyratum (Nie et al. 2014).In this study, we tried to trace the main active parts and separated three compounds with different structures from the total extract of S. lyratum.We also studied the antitumor effect of single components or interaction among these components.

Results and discussion
In this study, we isolated and identified three compounds from S. lyratum.Solavetivone (SO, 20 mg) and friedelin (FI, 15 mg) were the first time isolated and identified from S. lyratum, but tigogenin (TI, 30 mg) is a compound previously known to occur in S. lyratum (Figure 1).Then, anticancer activity was conducted on A549 and HepG2 cells for 24 h.Surprisingly, the three compounds only inhibited A549 and HepG2 cells a little (Supplementary material, Figure S2).But the combination of any two chemicals was more effective than any single compound (Supplementary material, Figure S3).This clearly indicated that there was synergy among the three compounds.On the other hand, our study also proved that there was an antagonism between components of S. lyratum.An alkaloid DRG, which was previously isolated from S. lyratum by anti-inflammatory drug screening platform based on the ICAM-1 signaling pathway (to be published), was adopted to test the cancer cellular toxicity between DRG and total saponins.The anti-inflammatory results showed that both groups inhibited the expression of the ICAM-1 protein and reduced the inflammation index after treatment (Supplementary material, Figure S4A).Nevertheless, the inflammation inhibition rate of the monomer compound group was significantly lower than that of the total saponins group at the same content of DRG.That is, the anti-inflammatory effect of total saponins was not as good as DRG.In addition, DRG also inhibited the proliferation of HepG2 cells with a concentration-effect gradient relationship (Supplementary material, Figure S4B).
Finally, the inhibitory effects of DRG in combination with the compounds SO, TI and FR on HepG2 cells were also tested.Based on the ratio of the four compounds in the plant, two concentrations were selected for comparison.Compared with DRG alone, adding TI or SO to DRG significantly reduced the efficacy for HepG2 cell toxicity (Supplementary material, Figure S4C).FR did not exhibit any combinatory effect with DRG in this regard.

Experimental
The experimental part is described in the supplementary materials.

Conclusions
In conclusion, we have separated pure compounds and important fractions from S. lyratum to explore the potential interaction between different molecules.The synergism was found in purified FR, SO and TI compounds against cancer cells.In the meantime, except for FR, the combination of SO, TI and DRG reduced the cellular toxicity to tumor cells.To our best knowledge, this is the first time that both synergism and antagonism between different compounds in one herbal medicine has been documented.The mystery of traditional medicine awaits further disclosure not only with respect to direct chemical interactions, but also the in vivo pharmacological details in treating diseases.