Sea buckthorn belongs to the Elaeagnaceae family and Hippophae genus, and is a small tree or shrub. Sea buckthorn flavonoids are one of the most important active ingredients in sea buckthorn, with quercetin, isorhamnetin, and kaempferol being the main single flavonoids. It has the effects of anti-tumor, anti-inflammatory, protecting cardiovascular and cerebrovascular systems, and improving the body’s immunity. Lung cancer is one of the most common cancers in the world at present, with high incidence rate and high mortality. The main reason for its high mortality is that lung cancer patients often metastasize and develop resistance to radiotherapy and chemotherapy after their first diagnosis. In recent years, research on the anti-tumor properties of seabuckthorn has gradually gained recognition, and the intestinal absorption characteristics of total flavonoids in seabuckthorn are significantly better than those of the single flavonoid compound isorhamnetin. It has been proved that flavonoids in seabuckthorn have inhibitory effects on colon cancer HT29 cells, prostate cancer PC-3 cells, breast cancer Bcap-37 cells and other tumor cells. In non-small cell lung cancer, flavonoids have a sensitizing effect on some chemotherapy drugs such as cisplatin. In this study, three species of seabuckthorn plants of the genus Hippophae were used as materials to detect the inhibitory effect of their total flavonoids on the proliferation of A549, further analyze the impact of the total flavonoids of Xizang seabuckthorn on the invasion and migration of lung cancer cells, and preliminarily explore the relevant molecular mechanisms, in order to provide a theoretical basis for the efficient development and utilization of seabuckthorn resources.

Different concentrations of total flavonoids of Xizang seabuckthorn (Hippophae tibetana Schlecht), Chinese seabuckthorn (H. rhamnoides L. subsp. sinensis Rousi) and costal seabuckthorn (H. neurocarpa) were selected to act on A549 cells. Cell relative viability was detected by MTT assay, cell colony formation ability was assessed by plate colony formation assay and soft agar colony formation assay, and cell apoptosis rate was detected by AnnexinV/PI double staining flow cytometry. The best effect of Xizang Hippophae rhamnoides total flavonoids was selected to apply the cell scratch test and Transwell test to analyze the effect of this compound on the migration and invasion of lung cancer cells. Western blot was used to detect the expression of invasion and migration related proteins such as MMP9 and E-cadherin. Knocking down the E-cadherin gene to detect the effect of total flavonoids in seabuckthorn on cell migration ability.

Lung cancer is one of the malignant tumors with high incidence rate and mortality in China in recent years. The main reason for its high mortality is that it is easy to relapse and metastasis. As is well known, the invasion and migration ability of tumor cells is the key to measuring tumor recurrence and metastasis. The metastasis of tumors is also one of the difficulties in cancer treatment. Therefore, there is an urgent need to find a suitable adjuvant chemotherapy drug to reduce the risk of metastasis and significantly improve the survival rate of lung cancer patients. In recent years, plant derived compounds have become a hot topic in current drug research. Therefore, it is of great significance to search for a compound with low toxicity and high anti-cancer effect from medicinal and edible plants or herbs for cancer adjuvant therapy.

This study used the leaves of three species of seabuckthorn plants in the seabuckthorn genus as materials and found that all three types of seabuckthorn total flavonoids have inhibitory effects on proliferation and pro apoptotic effects on lung cancer A549 cells. The inhibitory effect of the three kinds of total flavonoids of seabuckthorn is as follows: Xizang seabuckthorn>China seabuckthorn>costal seabuckthorn. There are literature reports that the flavonoid glycoside components of total flavonoids in seabuckthorn leaves are mainly isorhamnetin, quercetin, and kaempferol. Previous studies have confirmed that all three types of glycosides can exert inhibitory and pro apoptotic effects on cell proliferation in lung cancer A549, and can induce the mitochondrial dependent pathway mediated apoptosis process by Bcl-2/Bax. Further research has found that during the process of cell apoptosis, the mechanism of action of isorhamnetin is to promote the release of CytoC, leading to the cessation of ATP synthesis and triggering membrane potential abnormalities. And it was found that isorhamnetin and quercetin have a synergistic effect, and the proliferation inhibition effect of mixing the two in proportion is significantly better than that of a single compound.

HPLC analysis detected that all three types of sea buckthorn flavonoids contained flavonoids such as quercetin, isorhamnetin, and kaempferol, but there were significant differences in the proportion of each flavonoid glycoside. Among them, the ratios of kaempferol to quercetin and isorhamnetin to quercetin of Xizang seabuckthorn are much higher than those of Chinese seabuckthorn and costal seabuckthorn. The anti-cancer effects of the three glycosides are not the same. For lung cancer A549, treatment with 10 μ mol/L isorhamnetin can reduce the cell proliferation rate to around 50%. The median lethal concentration of kaempferol is 47 μ mol/L, while quercetin is 74 μ mol/L. In this study, it was found that the ratios of kaempferol to quercetin and isorhamnetin to quercetin of Xizang seabuckthorn were higher than those of Chinese seabuckthorn and costal seabuckthorn, which better explained that the total flavonoids of Xizang seabuckthorn had obvious advantages in inhibiting proliferation and invasion migration. This research result can provide experimental basis for the efficient development and utilization of seabuckthorn resources.

In further research, we selected the total flavonoids of Xizang seabuckthorn to study the invasion and migration of A549 cells. This study shows that the total flavonoids of Xizang Hippophae rhamnoides can significantly inhibit the migration and invasion of lung cancer A549 cells. The results of this study suggest that total flavonoids of seabuckthorn have the potential to inhibit the metastasis of lung cancer cells and provide assistance for the treatment of advanced lung cancer.

The invasion and migration of tumor cells are the result of multiple interactions between tumor cells, host cells, and extracellular matrix. The absence of E-cadherin is a major characteristic of epithelial mesenchymal transition. Its expression is reduced, resulting in weakened adhesion ability between cells and changes in cell polarity, leading to the migration of tumor cells. In this study, total flavonoids from seabuckthorn can upregulate the expression of E-cadherin and reverse the increased migration caused by E-cadherin deficiency. This proves that total flavonoids in seabuckthorn inhibit lung cancer EMT, thereby suppressing invasion and migration. In addition, the study found that A549 cells treated with total flavonoids from seabuckthorn showed a decrease in the expression levels of TGF – β and MMP9 in the cells. MMP9 is a key component in the degradation of extracellular matrix, promoting tumor infiltration and invasion of surrounding tissues by degrading extracellular matrix such as type IV collagen and laminin in the basement membrane. And previous studies have shown that metalloproteinases MMPs can cleave E-cadherin located on the cell surface and release soluble E-cadherin into the culture medium, thereby affecting cell invasion and migration. The expression of MMP9 is influenced by different cytokines, including TGF – β. As one of the important components in the tumor microenvironment, TGF – β can affect cell invasion and migration by mediating MMP9/MMP2. In summary, this study suggests that total flavonoids from seabuckthorn inhibit A549 invasion and migration by reducing TGF – β expression in cells, suppressing MMP9 expression, and preventing EMT in lung cancer.