**2.1 The anticancer effect of** *G. biloba* **seeds**

*G. biloba* seeds have been used in traditional Chinese medicine for centuries. The seeds have orange flesh shell, which are toxic as raw forms. The annual global yield of seeds is over 14 kt, more than 90% of which is produced in China [27]. As a traditional Chinese medicinal material, the ginkgo seeds have been used for clinical diseases such as asthma, coughs, cancers and etc. *G. biloba* seeds formed by the development of a fertilized ovule, contains an embryo and nutrient reserves that enable a new plant to grow. Only a few researches on the anticancer effects of *G. biloba* seeds extract. *G. biloba* seeds extracts positively induce cytochrome P450 (CYP) 1B1 expression, inhibiting the proliferation of breast cancer cells [28]. In this vitro study, polysaccharide derived from *G. biloba* seeds was isolated by ethanol fractionation, which decrease the percentages of G2-M cells, inhibiting the hepatoma cells proliferation. In addition, *G. biloba* seeds polysaccharides also make microvilli thinner and form apoptosis bodies on and around the spherical cells to promote apoptosis in hepatoma cells. While the hepatoma cells without *G. biloba* seeds polysaccharide treatment were of shuttle shape and small proportion of cells was of spherical shape [29].

### **2.2 The anticancer effect of** *G. biloba* **exocarps extract**

*G. biloba* exocarp is the outermost layer of seeds, which was also called seed coat or sarcotesta in some studies. Exocarp was previously regarded as a waste material. It smells of rancid butter, causesing air pollution. Phenolic acid in exocarp can contaminate soil and poison fish and shrimp [30, 31]. Exocarp is rich in nutrients: the percent of polysaccharide is 10% and ginkgolic acids is more than 4%. Recently, constituents extracted from the exocarp shows antitumor effect [32].

The extracts prepare from the exocarps of *G. biloba* (GBEE) enhanced the ratio of Bax/Bcl-2. Meantime, the translocation of Bax/Bcl-2 to mitochondria was also increased accompanied by the release of cytochrome C. Consequently, the protein

expression of cleaved-caspase-3, Fas, FasL, p-p38, and the mRNA levels of Fas were all increased, finally inducing apoptosis in lewis lung carcinoma cells (LLC). In vivo study further demonstrated the anticancer effect of GBEE on LLC [33]. GBEE increased the activation of acidic vacuole, the content of Atg5 protein and the ratio of LC3-II/LC3-I protein by AMPK induced inactivation on mTOR/p70S6k, which promoted the formation of autophagosomes in LCC, finally it induced autophagic cell death in LCC [34].

Despite of the directly effect on inducing cancer cell death, GBEE can also suppress the processes of angiogenesis and metastasis. It was reported that GBEE inhibited tumor metastasis in LLC mice model, characterized by the suppression on CD34 and microvessel density (MVD). This anti-metastasis effect might be due to the inhibition on angiogenesis, which mediated by downregulation on Wnt/βcatenin- vascular endothelial growth factor (VEGF) signaling pathway, including the inhibition on Wnt3a, β-catenin, VEGF, VEGF2 and p-AKT/AKT [35]. The suppression of GBEE on CD34 and MVD was also found in B16 melanoma. Meanwhile, GBEE attenuated the mRNA and protein levels of VEGF, hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor receptor 2 (VEGFR2), p-PI3K and p-Akt. Finally, it exerted antiangiogenesis by inhibiting PI3K/Akt/HIF-1α/VEGF signaling pathways [36]. *G. biloba* exocarp extracts also have anti-metastasis effect in skin cancer via perturbing the expression of p-PI3K, p-Akt, NF-κB, and MMP-9 [37].

The polysaccharides isolated from *G. biloba* exocarp (GBEP) had therapeutic effect on cancer patients [33, 38, 39]. For instance, the area of tumors in patients with GBEP capsules were significantly reduced. Meanwhile, the ultrastructural of tumor cells in these patients observed by transmissional electron microscope revealed that abundant heterochromatins were observed in nuclei, swollen mitochondria and dilated rough endoplasmic reticulum were observed in cytosol, indicating apoptosis was triggered by GBEP [40]. The anticancer effect of GBEP was demonstrated in tumor bearing mice [39]. The mechanism involved in the anticancer effect of GBEP was illustrated in gastric cancer. GBEP downregulated the expression of c-myc and bcl-2, upregulated the level of c-fos genes, which inhibited proliferation and induced apoptosis on gastric cancer [40].

Botanical constituents extracted from the exocarp of *G. biloba* promoted ROS generation, which inducing G0/G1 phase arrest, apoptosis and autophagy in colon cancer cells. RT-qPCR analysis showed that Ginkgolic acid (GA) decreased Cyclin D1, CDK2, CDK4, and Cyclin E1 mRNA levels. The study also found that decreased p-mTOR, pp70s6k and p-pras40 protein levels induced by GA were reversed by NAC pretreatment [41]. GA extracted from the *G. biloba* exocarp promoted the activation of AMPK, decreased the level of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) involved in lipogenesis. Finally, it plays a positive role in inhibiting pancreatic cancer cells proliferation, migration and invasion. The study examined the effects of GA on the viability of pancreatic cancer cell by MTT assay, found that GA can inhibit the growth of pancreatic cancer cells. Wound-scratch assay and scratch assay showed that GA inhibited the migration and invasion capacities of pancreatic cancer cells in vitro [42]. GA activated caspase-3, decreased the expression of Bcl-2 protein and increased the expression of Bax protein, finally causing apoptosis in laryngeal cancer cells [43]. GA also had positive anticancer effects in gastric cancer cells and liver cancer [44, 45]. After treatment with GA, the morphology of liver cancer cell was shrinkage and formed nuclear fragmentation, activating caspases-3 and promoting Bax expression. Finally, it induced apoptosis in liver cancer cells [45]. GA perturbed the proliferation of human cervical cancer cells and enhanced immune function on immunocompromised S180. But the

correlation of GA promoting S180 immune function needs to be further studied [46]. A vitro experiment verified that with the increase of GA concentration on cancer cells, GA inhibited the growth of cancer cells but the toxic effect on normal cells also increases. The anti-tumor effect of GA needs to be further proved by animal experiments [47].
