Analysis of metabolites of apigenin in rats and study of the anti-proliferative effect on gastric cancer cells

 Apigenin is a natural flavonoid widely found in plants and abundant in some fruits and vegetables. Metabolite Identification (MetID) plays a vital role in new drug development. In vivo drug metabolite analysis can provide a scientific basis for studying drug mechanisms of action and pharmacokinetics. 

Biological samples were collected after administering apigenin to rats. Then Apigenin metabolites were analyzed by high-resolution mass spectrometry, which can clarify the possible metabolites and metabolic pathways of apigenin and provide a theoretical basis for the pharmacology of apigenin. 

Many studies have shown that apigenin has significant antitumor activity, and apigenin has been found to modulate various molecular mechanisms related to cancer development in vitro and animal models.

Some researchers used a nude mouse tumor model to evaluate the effect of apigenin against SGC-7901 human gastric cancer cells in vivo and found that apigenin inhibited gastric cancer cell proliferation and induced apoptosis in human gastric cancer cells through upregulation of p53 and downregulation of bcl-2 expression.

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For the in vivo metabolite analysis of apigenin in rats, rats were divided into a blank group and a drug administration group (gavage of apigenin, 200 mg/kg), with six rats in each group. The urine and feces of the two groups were collected within 24 h of drug administration, treated accordingly, and analyzed by high-performance liquid-phase tandem ion trap time-of-flight multi-stage mass spectrometry in both positive and negative ion modes.

The results showed that nine metabolites were identified in the urine of rats from the dosing group, namely, apigenin underwent 2,3-position double bond reduction (U1, U7, U8, U9), binding to glucuronide (U2, U3, U4), attaching to sulfate (U5, U6, U7, U8, U9), and binding to glucose (U2); four metabolites were identified in the fecal samples of rats from the dosing group, namely, apigenin Four metabolites were identified from the fecal samples of rats in the administration group, which were apigenin undergoing double bond reduction at the 2,3 position (metabolite F3), binding to glucuronide (F2), and product bound to glucose (F1).

Therefore, apigenin still exists mainly as a prototype drug in rats, and it is presumed that the reduction of the 2,3-position double bond can occur under the action of intestinal bacteria, and glucuronide or glucose-binding products can be formed in both intestinal and in vivo excretion. In contrast, the formation of sulfate-binding products occurs only during in vivo excretion.

Research on the antiproliferative effect of apigenin on human gastric cancer cells

The effect of apigenin on the proliferation of 12 human malignant tumor cells in vitro was evaluated by the MTT method. A nude mouse tumor model was used to assess the effect of apigenin against SGC-7901 human gastric cancer cells in vivo. Transmission electron microscopy was used to observe the apoptosis of transplanted tumor tissues. Hoechst33342/PI fluorescence staining was used to monitor the effect of apigenin on the apoptosis of human gastric cancer cells. The development of apigenin on the expression of oncogene p53 and oncogene bcl-2 in SGC-7901 cells was determined by RT-PCR.

The results of the MTT method showed that apigenin inhibited the growth of 12 human malignant tumor cell lines in a dose-dependent relationship, among which SGC-7901 human gastric cancer cells were the most sensitive to apigenin with an IC50 of 27.14 μg/ml. 

The results of transplantation tumor experiments in nude mice showed that apigenin 600 mg/kg, 300 mg/kg, 150 mg/kg administered by gavage once/d for 10 d, the tumor weights were 1.282±0.159 g (P＜0.01), 1.397±0.401 g (P＜0.05), 1.477±0.362 g (P＞0.05), respectively, and the tumor inhibition rates were 31.78%, 25.66%, and 21.43%, respectively. After different concentrations of apigenin (10μg/ml, 20μg/ml, 40μg/ml) were applied to SGC-7901 cells, the mRNA expression of p53 was enhanced, and the p53 mRNA expression was significantly higher than that of the blank group (P<0.01); while the bcl-2 mRNA expression was reduced, with significant difference compared with the blank group P<0.01 and the effect had There is a certain dose-dependence of this effect.

Therefore, apigenin had a significant inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells in vitro, and also had a significant antitumor effect in vivo, and could lead to apoptosis of transplanted tumors. Apigenin can also induce apoptosis in human gastric cancer cells in vitro. 

The mechanism of the antitumor effect of apigenin is related to the upregulation of p53 and downregulation of bcl-2 expression.

In addition to its antiproliferative effect on gastric cancer cells, apigenin also has other antitumor and chemotherapy-sensitizing effects, such as:Ovary carcinoma,Pancreatic Cancer.

Apigenin inhibited the proliferation of pancreatic cancer cells in vitro, and the concentration of 100 μmol-L-1 significantly inhibited cell growth and down-regulated GLUT-1 mRNA expression. In pancreatic cancer cells under hypoxic conditions, apigenin could down-regulate GLUT-1 expression through HIF-1α and may inhibit cancer cell proliferation through this mechanism.

Chemotherapy sensitization:

Low concentrations of apigenin had no cytotoxic effect and could not effectively induce apoptosis in human acute myeloid leukemia (HL-60) cells. Still, its combination with different concentrations of cisplatin (DDP) could enhance the inhibitory effect of DDP on HL-60 cell proliferation, suggesting that apigenin may have a chemotherapy-sensitizing effect on HL-60; low concentrations of apigenin could also reduce the resistance of HL-60 cells to chemotherapy drug-induced apoptosis, and the mechanism may be related to the down-regulation of NF-κB and Bcl-2 expression.

Animal experiments or in vitro studies have shown that apigenin has antitumor effects and therefore has a broad development prospect, however, most of the current studies on the pharmacological effects of apigenin are still at the stage of in vitro tests and animal models, and there is still a certain distance from clinical applications, so further studies are needed.