Study on the harvesting timing and anticancer activity of different varieties of fig based on flavonoid components
Ficus carica L. is a temperate deciduous fruit tree belonging to the Ficus genus in the Moraceae family. It is native to the Euphrates and Tigris River basins and is one of the earliest cultivated fruit trees domesticated by humans, with a planting history of over 10000 years. Flavonoids have been proven to be a potential natural antioxidant with antibacterial and anti-tumor effects. The roots, stems, leaves, and fruits of figs can all be used as medicine. In the 1950s, Ullman proposed that treating animal intestines with external fig milk can cause significant capillary damage, which can lead to animal death in severe cases; But if taken orally, there is no toxicity. Four active components were isolated from fig milk, of which two components were injected subcutaneously or intravenously to inhibit the growth of transplanted sarcoma in mice. It was subsequently confirmed that fig emulsion could significantly inhibit the growth of primary breast cancer cells in mice, cause necrosis of cancer cells, delay the occurrence of transplanted adenocarcinoma, myeloid leukemia, lymphosarcoma and sarcoma, and cause sarcoma degeneration. In China, Wang et al. first reported that extracts from fig roots, stems, leaves, and fruits (including young and mature fruits) exhibited significant inhibitory activity against S180 sarcoma, lung cancer, liver cancer, and Ehrlich ascites carcinoma. They supplied fig water extract to tumor bearing mice or cancer patients, which can improve the immune function of the experimental subjects. Therefore, it is suggested that fig preparations can be used as a natural and non-toxic adjuvant drug to alleviate the pain of chemotherapy and radiotherapy patients and improve their quality of life. However, the effective anti-cancer ingredients of figs have not been elucidated yet. Takeuchi et al. first proposed that benzaldehyde is the main anti-cancer component of figs. Yin et al. believe that the anti-cancer effect of figs is the result of the combined action of coumarin compounds, benzaldehyde, and furan ring small molecule aromatic compounds, with benzaldehyde being only one of the enhancers or organic active molecules. Later, it was discovered that fig polysaccharides have anti-cancer activity. However, the structure of polysaccharides is complex, and their relationship with anti-tumor needs further exploration. On the other hand, the relationship between flavonoids and anti-cancer has attracted attention. Sharada et al. proposed that morin, a flavonoid substance found in figs, can inhibit the development of colon cancer induced by 1,2-dimethylhydrazine (DMH) in rats. There is a lack of follow-up reports on this research. Due to the numerous varieties of figs, the leaves of fruits that mature at different stages may have different functions. Therefore, this study collected leaves of three fig varieties from July to November 2019, extracted and refined flavonoids, and studied the inhibitory activity of fig flavonoids on human cancer cell proliferation in different months. The anti-cancer mechanism of fig flavonoids was explored, and the relationship between flavonoid substances and anti-cancer activity was analyzed, in order to provide theoretical basis for the application and development of fig flavonoids in anti-cancer medicine.

Flavonoids are a series of compounds composed of two benzene rings connected by three carbon atoms. They have a C6-C3-C6 structure, are insoluble or insoluble in water, and are easily soluble in organic solvents such as methanol, ethanol, and ethyl acetate. Therefore, fig alcohol extract and even fig wine contain a certain amount of flavonoids and have anti-cancer activity. In terms of anti-tumor effects, Bai et al. used fig ethanol extract to intraperitoneally inject S180 solid tumor bearing mice and found that it had a significant inhibitory effect on sarcoma growth, increased organ index, and enhanced T lymphocyte percentage, suggesting that fig ethanol extract may have anti-tumor effects by enhancing the immune function of tumor bearing mice. This is the earliest report of fig flavonoids inhibiting animal tumors. In this experiment, we observed that flavonoids from fig leaves had significant inhibitory activity on the proliferation of human gastric cancer cells SGC-7901, but had relatively low effects on the proliferation of gastric cancer BGC-823 and liver cancer Hepg-2 cells (see Figure 1). From the anti-cancer effects extracted from leaves collected from different varieties in different months, it can be seen that the inhibition rate of flavonoids on SGC-7901 cells is highest in the leaves of ‘Braunschweig’ in August, while the inhibition rates of flavonoids in leaves of other varieties and months are relatively low (see Table 2). It seems that only flavonoids from fig leaves of specific varieties and months have a significant effect on the proliferation of specific tumor cells. Some people have suggested that the ethanol extract of cicada cordyceps has the highest inhibition rate on cancer cell proliferation in SGC-7901. The results of this experiment are similar to this. As the concentration of flavonoids in fig leaves increased, the number of gastric cancer SGC-7901 cells decreased, became shorter and wrinkled, gradually detached from the cell plate, and its inhibitory effect showed a dose effect (see Figure 2). This provides a theoretical basis for determining the harvesting timing of fig leaves and developing the anti-cancer effects of flavonoids.
We used flow cytometry for the first time to observe the induction of cell apoptosis by flavonoids in fig leaves (see Figure 3). At a flavonoid concentration of 200 μ g/mL, the total apoptosis rate of cells was 39.08%; When the concentration reached 1600 μ g/mL, the total apoptosis rate reached 83.60%. Based on this, the semi lethal concentration of fig flavonoids on SGC-7901 cells was calculated to be 577 25 μ g/mL. This is similar to the semi inhibitory concentration of 653 μ g/mL proposed by Purnamasari et al. for Huh7it liver cancer cells in fig methanol extract. They found that the methanol extract from fig leaves can inhibit the proliferation of liver cancer cells, promote apoptosis, and lead to cell necrosis. Apoptosis involves the activation, expression, and regulation of a series of genes, mainly including endogenous mitochondrial pathways and exogenous death receptor regulatory pathways. The decrease in mitochondrial membrane potential is a hallmark event of early mitochondrial dependent apoptosis. Garc í a-Zepeda et al. demonstrated that resveratrol induces a decrease in mitochondrial membrane potential in cervical cancer cell lines and promotes cancer cell apoptosis. This experiment observed that fig flavonoids reduced the mitochondrial membrane potential level of human gastric cancer SGC-7901 cells (see Figure 4). This indicates that flavonoids in fig leaves promote apoptosis by inducing a decrease in mitochondrial membrane potential levels in SGC-7901 cells.
We also observed the inhibitory effect of flavonoids in fig leaves on the cell cycle of cancer cells and found that they mainly block the mitosis of SGC-7901 cells in the S phase, with some in the G2 phase (see Figure 5). It has been reported that capsaicin can block endometrial cancer cells in the S phase and cause an increase in apoptosis rate. According to research, the anticancer mechanism of capsaicin is to inhibit the expression of cell cycle dependent protein kinases CDK2, CDK4, and CDK6 proteins. Artificially synthesized flavonoid amino acid derivatives can also inhibit the G2/M phase of MGC-803 gastric cancer cell cycle, while downregulating the expression of anti apoptotic protein Bcl-2 and upregulating the expression of pro apoptotic protein Bax, thereby triggering cell apoptosis. This experiment analyzed the mechanism of fig flavonoids inhibiting apoptosis in SGC-7901 cells at the mRNA level. We found that the expression of CDK1, CDK2, CDK6, and CyclinD1 genes in SGC-7901 cells treated with fig leaf flavonoids significantly decreased, while the expression of E2F1 increased (see Figure 6B). In various tumor tissues, CDKs are in an abnormally activated state, and inhibiting CDKs expression can suppress uncontrolled tumor proliferation. In addition, treatment with flavonoids in fig leaves also affected the expression of tumor suppressor genes P53 and Bax, as well as the oncogene Bcl-2 (see Figure 6A). Among these three genes, Bax and Bcl-2 belong to the same gene family. Among them, Bcl-2 promotes the cell cycle, while Bax and Bcl-2 form a protein dimer that antagonizes the activity of the latter, inhibits the cell cycle, and causes cell apoptosis. According to reports, flavonoids from Tianshan Snow Lotus can upregulate the expression of CaEs-17Bax and downregulate the expression of Bcl-2 in human esophageal cancer cells, thereby inducing cell apoptosis. The flavonoids observed in fig leaves promote the expression of tumor suppressor genes P53 and Bax, while inhibiting the expression of tumor suppressor gene Bcl-2. This suggests that the apoptosis inducing effect of fig leaf flavonoids on gastric cancer cell line SGC-7901 may be achieved by regulating the expression of apoptosis related genes Bax, P53, and Bcl-2.
In order to explore the specific active ingredients of fig leaf flavonoids in inhibiting the proliferation of gastric cancer SGC-7901 cells, we selected three samples (‘Branrick’ in August, ‘Masyutaofen’ in September, and ‘Bojihong’ in September) for LCMS/MS qualitative and quantitative analysis based on the different inhibitory effects of fig leaf flavonoids on human gastric cancer SGC-7901 cell proliferation in different varieties and months. After screening, 12 differential metabolites highly correlated with cancer inhibition rate were obtained (see Table 3). Among them, there is a highly significant positive correlation between the anti-cancer rate of narcissin and fig flavonoids. It has been reported that narcissoside has antiviral activity and is also of great significance in the prevention and treatment of protein conformation diseases such as Alzheimer’s disease, type II diabetes, Parkinson’s disease, etc. Recently, it was proposed that narcissoside could be used to prevent and treat COVID-19. However, whether it has anti-cancer effects remains to be studied. The results in Table 3 also indicate a significant correlation between isoquercetin and the anti-cancer rate of fig flavonoids. It has been reported that isoquercetin can significantly inhibit the expression of miR-23a in glioma tissue, thereby suppressing cancer cell proliferation. Therefore, the relationship between isoquercetin in fig leaves and anti-tumor activity deserves further investigation. Thirdly, melanin can inhibit the growth of tumor cells and occurs in the S phase of the cell cycle. But some people also believe that blockade occurs in the G2/M phase. This study also detected the presence of melanin, and the correlation coefficient between its content and anti-cancer rate was r= 9923, P=0.08. Although it did not reach the 0.05 level, it is very close. Moreover, we observed that flavonoids from fig leaves inhibited the cell cycle effect of SGC-7901 cells in the S phase. This is similar to previous results. Due to the fact that figs belong to the Ficus genus in the Moraceae family, it is worth further studying whether this flavonoid substance is related to the anticancer activity of figs. In addition, we also observed 2 The contents of 6-dihydroxybenzoic acid, vanillophenone, cyanidin-3-O-rutinoside chloride, anthocyanins, epicatechin, sinapine, etc. were significantly correlated with the anticancer rate of fig flavonoids (see Table 3). It has been reported that catechins have inhibitory effects on human liver cancer. However, in this experiment, we observed that the inhibitory effect of fig leaf flavonoids on liver cancer HepG-2 was relatively low (see Figure 1), suggesting that its inhibitory effect on SGC-7901 may not be related to epicatechin. The relationship between other metabolites and anticancer effects is currently rarely reported.
In summary, this article first analyzed the inhibitory efficiency of flavonoids on the proliferation of different cancer cell lines in the leaves of three varieties, including ‘Branrick’, ‘Masyutaofen’, and ‘Bojihong’, from July to November. It was found that the ‘Branrick’ fig variety had the highest inhibitory rate of flavonoids on SGC-7901 gastric cancer in August. As the concentration of flavonoids increases, the density and adhesion of cancer cells decrease, the number of live cells decreases, and the proportion of dead cells and late apoptotic cells increases. Research on the mechanism of cancer inhibition has shown that flavonoids from fig leaves block the cell cycle in the S phase and a small amount in the G2 phase. The mitochondrial membrane potential of cells significantly decreases, and the expression levels of pro apoptotic genes Bax and p53, as well as cell cycle regulatory gene E1F2, significantly increase. The expression levels of anti apoptotic gene Bcl-2, as well as cell cycle related genes Cy-clinD1 and CDKs, significantly decrease. These are important mechanisms by which fig leaf flavonoids promote cancer cell apoptosis. In addition, with the help of LC-MS/MS technology and related analysis, we screened several differential metabolites highly correlated with cancer inhibition rates, such as narcissin, isorhamnoside, and mulberry pigment. This opens up new horizons for the development of anti-cancer drugs for fig leaves in the future.