Authentic plant material was purchased from Shaanxi Xing Sheng De Pharmaceutical Co., Ltd. (Shaanxi, China, Lot:20181201). Identification of the H. diffusa Willd. herb was confirmed by Dr Liu Shijun (Department of Processing, Shaanxi University of Traditional Chinese Medicine, Shaanxi, China).

H. diffusa Willd. (2.5 Kg) were extracted two times (each for 2 h) with 80% EtOH (100 L) under reflux. After evaporation of the combined ethanol extracts in vacuo, the resultant aqueous residues were dissolved in water and then extracted successively with petroleum ether and ethyl acetate to obtain the petroleum ether extract and the EtOAc extract. Once the solvent was removed, the residue was dissolved in distilled water and placed in the fridge overnight. The next day, the supernatant was collected and then subjected to column chromatography on macroporous resin (HPD-826, 2 BV/h), eluted with 80% aqueous EtOH. 80% ethanol extract was purified again with HPD-826 macroporous resin, eluted with 70% aqueous EtOH. After evaporation of the ethanol extracts in vacuo, ethanol extract was dissolved in anhydrous ethanol, and then activated carbon (10 mg/ml) was added in water for 30°C for decolorization and impurity removal until colorless (each for 30 min). After filtering the activated carbon, the filtrate was added with distilled water to precipitate and centrifuge. Total Triterpenoids were obtained.

Total Triterpenoids content was determined following the procedure described by Wei et al. with slight modifications (Wei et al., 2015). Briefly, after a 600 μl oleanolic acid reference substance and 2 ml sample solution in 10 ml volumetric flask respectively was heated to evaporate in a water-bath at 80°C, 0.2 ml of 5% vanillin-acetic solution and 0.8 ml of perchloric acid were added accurately, and incubated at 70°C water-bath for 15 min. Then the mixed solution was cooled in ice water for 5 min. Then, 5 ml glacial acetic acid was added, shaken well, and stood for 5 min. The absorbance was measured at 548 nm against blank using a spectrophotometer. The content was determined using the oleanolic acid standard calibration curve.

H1975, A549, and SW620 cell lines were purchased from Fenghui Biotechnology Co., Ltd. (Hunan, China). Cells were cultured in 90% RPMI-1640 (Solarbio, Beijing, China) media supplemented with 10% fetal calf serum (Corning), 100 U/ml penicillin and 100 U/ml streptomycin, and with 5%(v/v) CO₂ in 37°C incubator for 24 h.

Cells were inoculated with a density of 1.5×10⁵ cells/well into 96-well plates. After 24 h, H1975 (A549, SW620) cells were treated with different concentrations of TTH (1 µg/ml, 2 µg/ml, 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, 30 µg/ml, 50 µg/ml and 75 µg/ml) for 24 and 48 h, respectively. Then the cells were cultured with 20 μl MTT solution for 4 h (5 mg/ml). The MTT solution was removed and 150 µl of DMSO. After thorough mixing, absorbance of each well with a microplate reader (A490) (Molecular Device, Sunnyvale, CA, United States). Half-maximal inhibitory concentration (IC₅₀) values were calculated by SPSS.

When at 100% confluence, the cells followed by starvation in serum-free RPMI 1640 medium for 12 h to completely inhibit cell proliferation. A 200-μl sterile pipette tip was used to make a line in cell cultures. Cells were then incubated with different concentrations of TTH for 0, 24, and 48 h in incubators. The scratch gap width in each group was measured at different positions and compared to the gap width at 0 h which was arbitrarily set at 1.

H1975 cells culture on glass cover slips placed in 6-well dishes. After treatment cells were washed with PBS twice, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 1% Triton X-100 for 10 min. After being sealed with 5% BCA for 40 min, cells were incubated with anti-Ki67 (1:100) primary antibody at 4°C overnight. The next day, cells were incubated with a secondary antibody conjugated with FITC-conjugated secondary antibody (Invitrogen) for 1 h, and then mounted by using Prolong Gold Anti-fade reagent with DAPI (Boster) for 5 min. Immunofluorescence micrographs were produced by using an Olympus FSX100 Fluorescence Microscope (Olympus, Tokyo, Japan).

H1975 cells were washed with ice-cold PBS and lysed using RIPA buffer supplemented with protease and phosphatase inhibitor mixtures (Boster Biological Technology Co., Ltd., Wuhan, China) on ice. The Lysates were homogenized twice in 1.5 ml EP tube, 5–10 s each time, and then lysed on ice for 20 min. Lysates were separated by centrifugation at 4°C and 10,000 g for 10 min. Protein concentration was determined by BCA assay (Beyotime Biological Technology Co., Ltd., Shanghai, China). Finally, add loading buffer in proportion to the protein and store at −20°C. 40 mg total proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Bedford, MA, United States). The membranes were blocked for 2 h with 5% non-fat dry milk at room temperature, then incubated with rabbit mAbs specific for TIMP2 (1:500) (Abcam, ab180630), MMP9 (1:1,000) (Proteintech, 00083130), MMP2 (1:1,000) (Proteintech, 00082362), NF-ΚB/p65 (1:1,000) (CST, #8242), p-STAT3 (1:1,000) (CST, #9145), STAT3 (1:1,000) (CST, #30835), Bax (1:1,000) (Abcam, ab69643), Bcl-2 (1:1,000) (Abcam, ab32124), anti-caspase3 (1:1,000) (Abcam, ab2171) and anti-cleaved caspase-3 (1:1,000) (Abcam, ab32024) overnight at 4°C. Following washing and subsequent incubation with horseradish peroxidase conjugated secondary antibodies (1:3,000) 37°C for 1 h. Then immunoreactive proteins were visualized using ECL Western blotting detection reagent (Millipore, Billerica, MA, United States) and detected using MultiImage Light Cabinet Filter Positions (Alpha Innotech, San Leandro, CA, United States). ImageJ software version 1.0 was used for gray-scale value analysis and quantification of Western blots.

Cell cycle distribution was analyzed by flow cytometry (Becton, Dickinson and company, Franklin Lakes, NJ, United States). H1975 cells were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well, and divided into control, 2.5 µg/ml, 5 µg/ml and 10 µg/ml groups. After 48 h of treatment, cells were harvested, rinsed with PBS, fixed with 75% (v/v) ice-cold ethanol and re-suspended in staining buffercontaining FITC-Annexin V and propidium iodide (PI). The mixture was then incubated in the dark at 37°C for 30 min. DNA contents of stained nuclei were analyzed, and the cell numbers in each cycle phase were calculated.

Cell apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, United States) according to the manufacturer’s instruction. H1975 cells were treated with different concentrations of TTH for 48 h according to the cell cycle distribution experiment. Cells were washed twice with pre-cooled PBS, then digested with 0.25% trypsin free of EDTA, re-suspended in binding buffer and then incubated with Annexin V-FITC and PI for 10 min at 25°C in dark. Samples were then analyzed by FACS Calibur (BD Bioscience). The percentage of stained cells was determined using BD FACSDiva software (Becton, Dickinson and company, Franklin Lakes, NJ, United States).

Each experiment was repeated at least three times, and the data are expressed as the mean ± SEM. Statistical differences between groups were analyzed by Student’s t-test or the Mann-Whitney U test as appropriate using a SPSS 26.0 program. *p < 0.05 was considered statistically significant.

The standard curve equation was Y = 12.27X–0.0649 and expressed good linearity (r = 1) within the test ranges. According to the results, the content of TTH in 70% EtOH eluates was 83.12 ± 0.85%.

Cellular-based screening of TTH were performed on A549, SW620, and H1975 cell lines to determine the activity of the derivatives by the MTT assay in vitro. The IC50 values obtained are tabulated in Figure 1A. As shown in Figure 1A, after treatment with various concentrations (1, 2, 5, 10, 15, 20, 30, 50, 75 µg/ml) of TTH for 24 and 48 h, the proliferation of H1975 cells were inhibited moderately. Compounds TTH showed significant anticancer activity against A549, SW620, and H1975 with 24 h IC₅₀ values of 24.38, 28.29, and 19.73 µg/ml, respectively. The results showed that the IC₅₀ value of 48 h was 11.79, 23.35, and 9.64 µg/ml, respectively. In view of the good activity of TTH on H1975 cell lines, the antitumor activity of H1975 was further evaluated.

Cell proliferation plays an important role in tumors, and flow cytometry was used to analyze cell cycle distribution. As shown in Figures 1B,C, TTH significantly increased the number of cells in the G0/G1phase. Contrarily, the number of cells in the S phase were decreased along with the increase of the treatment concentration. The results showed that TTH could significantly arrest cell cycle at G0/G1 phase.

As showed in Figure 1D, the expression of Ki-67 decreased in H1975 cells in vitro after administration compared with control cells. The results showed that total Triterpenoids inhibited the proliferation of H1975 cells.

Flow cytometry was used to detect the apoptosis of H1975 cells treated with different doses of TTH for 48 h. As shown in Figure 2A, Annexin V-negative/PI negative cells (Q4 quadrant) represented viable cells; Annexin V-positive/PI positive cells (Q2 quadrant) indicated cells undergoing late-stage apoptosis; and Annexin V-positive/PI negative cells (Q3 quadrant) were considered to represent early apoptosis cells. Q2 and Q3 represented the apoptosis cells. After treatment with TTH for 48 h, cells in Q2 and Q3 were greatly increased, indicating that TTH could induce the apoptosis of H1975 cells.

Apoptosis is a complex and multi-stage process involving multiple genes. Due to the different initial stages of apoptosis, there are three pathways. The mitochondrial pathway is the first stronghold of apoptosis. This pathway includes the interaction between the Bcl-2 family containing the BH3 domain and the Bcl-2 family bound to the outer mitochondrial membrane, which changes the permeability of mitochondrial membrane, activates caspase pathway and induces apoptosis. In the present study, the protein expression levels of caspase-3, cleaved caspase-3, Bax and Bcl-2 were determined by Western blotting. As shown in Figures 2C,D, the results revealed the expression of Bcl-2 were decreased, while that of proapoptotic protein Bax were increased after TTH treatment. These results indicated that TTH could regulate the expression of Bcl-2/Bax and induce apoptosis. However, it had little effect on the activation of Caspase3.

Cell migration plays a central role in many physiological and pathological processes such as embryonic development, immune defense, injury repair, angiogenesis, and tumor metastasis. It exists in normal physiological processes such as tissue development, wound healing and rebirth. And it is also a key link in pathological development such as inflammation and tumor.

Abnormal cell migration can lead to normal physiological changes and diseases, such as the migration of vascular endothelial cells to the tumor to form new blood vessels, promoting tumor cell attachment and proliferation. Therefore, we investigated whether TTH could inhibit tumor cell migration. As shown in Figures 3A,B, after treatment 24 and 48 h, the scratch gap was significantly larger than that of the control group.

In this study, the protein expression levels of MMP-2, MMP-9 and TIMP-2 were detected. As shown in the Figures 3C,D, Group 1 was the control group, Group 2, Group 3 and Group 4 were treated with different concentrations of TTH. The results showed that the levels of MMP-2 and MMP-9 were decreased after TTH treatment, while the level of TIMP-2 was increased. These results suggested that the inhibition effects of TTH on cell migration were related to the downregulation of MMP-2 and MMP-9 and the upregulation of TIMP-2.

Recently, roles of NF-κB and STAT3 in many cancers have been extensively studied. They are two main factors for tumor monitoring and regulation of tumor angiogenesis and invasiveness in pre-tumor and malignant cells against apoptosis. The activation and interaction of these two pathways play an important role in communication between cancer cells and inflammatory cells. We investigated the effects of TTH on NF-κB and STAT3 signaling pathways, and detected the expression of NF-κB, p-STAT3, and STAT3.

As presented in Figure 4, TTH treatment significantly reduced phosphorylation level of STAT3 in H1975 cells, but the total STAT3 protein level had no significant change. However, the expression of NF-κB (nucleus) was decreased and the expression of NF-κB (cytoplasm) protein was increased, when compared with untreated control cells (p < 0.05). These results suggested that the inhibition effects of TTH on H1975 may be related to NF- κB and STAT3 signal pathway.