Ginkgo biloba: A Potential Anti-Cancer Agent

Abstract

Ginkgo biloba is generally considered as safe herbal extract in clinical application. Ginkgo Folium is a living fossil plant, which has been used in record by over few thousands of years. The extract of G. biloba, has been used extensively for the treatment of diseases related to the central nervous system and psychiatric disorders. Recently, different lines of evidence indicated that G. biloba exhibited anti-cancer effects. The potential therapeutic effect may due to antioxidant, anti-angiogenic and gene regulatory actions. In addition, Ginkgo Folium was studied in pharmacodynamic interactions induced by herb-drug interactions. These studies indicated that G. biloba usually exhibits synergistic effect. The extracts derived from G. biloba exhibits promising anticancer effect, including flavonoids, ginkgolide, and phenolic acids etc. This chapter will discuss the anticancer effect and mechanism of extracts derived from various parts of G. biloba, the possible usage as an adjuvant therapy in cancer treatment, and the development of G. biloba as potential novel anticancer drugs.

1. Introduction

Almost everyone talks about cancer discoloration, and no matter what kind of cancer it is, it can be life-threatening. But fortunately, cancer can also be treated, in addition to mastering the rhythm of life, you can also choose Chinese herbal medicine to strengthen the immunity and enhance the ability to anti-cancer. The active gradients from several plants have anticancer effect, such as Elemene, vincristine and ginsenosides [1, 2, 3, 4, 5, 6]. Elemene, isolated from the volatile oil of traditional Chinese medicine Curcuma wenyujin, has broad-spectrum anticancer activities and mild side effects. Vincristine is derived from periwinkle, which is used for the treatment of acute lymphoblastic leukemia, breast cancer. Ginsenosides is extracted from ginseng. Ginseng has been regarded as a famous traditional Chinese medicine since ancient times. After treatment with ginsenosides, ginsenosides inhibited cancer cell proliferation, invasion, and migration in several cancers, such as breast, brain, liver, gastric, and lung cancer [5].

In recent years, more and more studies have shown that G. biloba also has anti-tumor effects. G. biloba appeared on earth more than 200 million years ago and is the only living species in the order Ginkgoales [7, 8, 9]. Once G. biloba was growing everywhere on earth, but it became nearly extinct during the last ice age, and only survived in Asia. G. biloba has been used as a medicinal plant for a long time. It was firstly recorded in “Shennong Ben Cao Jing, but the medicinal value of ginkgo leaves recorded from the Song Dynasty. The medical use of G. biloba was first recorded in “Ben Cao Gang Mu” by Li Shizhen in Ming Dynasty [10]. G. biloba leaves are fan-shaped, flat, and have an indentation in the middle, giving birth to a species name “Biloba,” a Latin word meaning bi-lobed [11]. Male flower pollens are carried by the wind to the female tree, which produces ovules that fertilize and grow into seeds. G. biloba takes 15 to 20 years to produce fruits, which have a rancid, nasty odor. However, the seeds contain certain mild toxic chemicals [12, 13, 14]. G. biloba has been a beloved plant in Asian countries as an ornamental tree in the gardens and as a medicinal plant, particularly in China, Korea, and Japan [15, 16].

Scientists from Japan and Germany made pioneering and important contributions to research and development. The development history of G. biloba fully embodies the determination of innovation and has become a model for the research and development of traditional Chinese medicine and botanic medicine. In 1929, the Japanese first isolated a flavonoid from G. biloba leaves [17]. In the 1960s, Dr. Schwab firstly extracted the active ingredients (ginkgo flavone and terpenoid lactone) from Chinese G. biloba leaves, which were processed into tablets. These active ingredients quickly became the first world’s plant medicine, which were listed as the third generation of G. biloba leaves preparation. In 1972, Dr. Willmar Schwabe Company developed EGb761, a patent extract of G. biloba leaves. EGb761 contains 24% flavonoids and 6% terpenoids, which is widely used today [18]. From the 1920s to the 1930s, Chinese herbal medicine research upsurge, medical scholars began to analyze the chemical components of G. biloba, pharmacodynamics and toxicology research, with G. biloba tablet, known as the first generation of preparation. In the 1970s, Germany, France and other European countries carried out in-depth research on G. biloba leaves. Flavonoids and lactones extracted G. biloba have therapeutic effects on cardiovascular and cerebrovascular diseases [19]. In the 1990s, Professor Xie Delong, director of Shanghai Institute of Traditional Chinese Medicine, discovered a safer and more effective combination than the German ginkgo leaf invention patent, and innovated the process to raise the clear effective component of the extract to more than 50%, which was listed as the fifth generation of ginkgo leaf preparation.

At present, flavonoids (quercetin, kaempferol, isorhamnetin, lignin, etc.) and terpenoids (ginkgolide A, Ginkgolide B, ginkgolide C, ginkgolide J, etc.) have been found to be active pharmacological components in G. biloba [20]. It is recorded in Chinese Pharmacopoeia that G. biloba has the effects on promoting blood circulation and removing stasis, clearing collaterals and relieving pain, strengthening lungs and relieving asthma, removing turbidity and lowering lipids, and is used to treat blood stasis and blocking collaterals, chest paralysis and heartache, stroke hemiplegia, lung deficiency, cough and asthma, hyperlipidemia and other diseases [21, 22, 23]. In recent years, studies have also found that the pharmacological effect of G. biloba lies in the synergistic effect of various components, rather than a component to play a determined role. Studies have shown that G. biloba extract plays a significant role in the treatment of Alzheimer’s disease, neurodegenerative diseases, brain dysfunction, eye diseases, cardiovascular diseases and other diseases. Moreover, recent studies have shown that extract from different part of G. biloba may also be useful in treating cancer [24].

2. Anticancer effect of extracts derived from various parts of G. biloba

Plants have provided a rich source of therapeutic agents and bases for synthetic drugs. G. biloba is dioecious. Male ginkgo biloba release pollen in spring to fertilize the female ginkgo biloba, which produces a large amount of ginkgo drup-like seeds in fall [25]. The seeds of G. biloba have been used for the treatment of cancer thousands of years ago in China [26], which is first mentioned in herbals in the Yuan dynasty. Mature ginkgo seeds covered with a fleshy thick outer layer, which named as exocarps, or seed coat, sarcotesta in some research papers. Seed mainly consists of mesosperm, membranous endopleura and kernel. The mesosperm is hard and white shell. The innermost layer of the seed is membranous endopleura, which is red and consists of 1–2 layers of parenchymal cells. The kernal of G. biloba is fleshy and pale yellowish-green, consisting of endosperm and embryo, which is an edible part of the seed. The leaves of G. biloba (Ginkgo folium) are fan-shaped, which are unique among seed plants.

Studies reported that extract or active ingredient from different parts of G. biloba, including seeds, exocarps, kernel and flowers exert anticancer effect. In recent years, the anticancer effect of Ginkgo folium was extensively reported. We summarized the current reports on the anticancer effect and potential mechanism of the extracts from the different parts of G. biloba.

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/β-catenin- 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].

2.3 The anticancer effect of G. biloba kernel extract

G. biloba kernels called Baiguo in China are the main edible part of the seed, which consist of endosperm and embryo [48]. Ginkgo kernel is consumed as a delicious food in China, Japan and Korea after grilled or boiled [49]. Ginkgo kernels contain 60–70% starch, 10–20% protein, 2–4% lipids, 0.8–1.2% pectin, and about 6% sucrose [50, 51]. Raw or cooked the ginkgo kernel has been shown to cause allergic reactions or death [52]. The medicinal value of ginkgo kernel was overlooked. A few studies shed light to the anticancer effect of kernel. The medicinal value of ginkgo kernel was overlooked. A few studies shed light to the anticancer effect of kernel [53]. However, the role of total G. biloba kernel extract on anticancer was reported recently. A study firstly reported that G. biloba kernel extracts exhibited cytotoxic effects in colon cancer and melanoma, characterized by inhibiting cancer cells proliferation and vitality [54]. Melanoma cells are more sensitive to kernel extracts. Meantime, the cytotoxic effect was not found in normal cell line McCoy-Plovdive. However, the anticancer effect of kernel extract needs to be observed in other cancer types, and the molecular mechanism remains to be observed in detail in future.

2.4 The anticancer effect of G. biloba flowers extract

G. biloba is an ancient dioecious gymnosperm, which apply to worldwide for landscaping and medical usage. The male flowers with catkin blossom from late March to the middle of April for only three to seven days, varying in different areas of China [55]. The chemical constituents and bioactivities of the flowers contain high contents of nutritional and medicinally relevant components, such as amino acids, vitamins, unsaturated fatty acids, flavonoids, and lactones [56]. The extracts of ginkgo flowers can exhibit anticancer effects. Some phytochemical studies of G. biloba flowers enrich the diversity of Ginkgo chemical constituents and broaden its application in phytotherapy. The bioflavonoids from G. biloba male flowers promoted cell cycle arrest in the G2/M phase, inhibiting the proliferation of cervical cancer cells [57]. A study showed that biflavonoids bilobetin and isoginkgetin isolated from G. biloba flowers exhibited cytotoxic activities on cervical cancer, pancreatic cancer, lung cancer, Lymphoma, and ovarian cancer. The most sensitive cancer cell to these two compounds is cervical cancer. Furthermore, the morphological changes, apoptosis and cell cycle arrest were observed in cervical cancer cell. After treated with bilobetin and isoginkgetin, nuclear condensation together with the decrease on the ratio of Bcl2/BAX and the increase on cleaved-caspase 3, apoptosis rates were observed, indicating that apoptosis was triggered with these two compounds. In addition, cell cycle arrest via promoting G2/M phase arrest was also found in cervical cancer cell treated with bilobetin and isoginkgetin [55].

2.5 The anticancer effect of Ginkgo Folium extract

The anticancer effect of Ginkgo Folium was studied extensively in recent years. The tumor inhibition effect of Ginkgo Folium was demonstrated in mouse S180 mouse sarcoma, which might be due to the elevation on free radical scavenger enzymes [58]. The main reported death way induced by Ginkgo Folium is caspase dependent apoptosis that was observed in several cancer types, including cervical cancer [59], colon cancer [60], gastric cancer [61], melanoma [62]. The mechanism responsible for Ginkgo Folium induced apoptosis is the disruption on the balance of antiapoptotic protein Bcl-2 and proapoptotic protein BAX, characterized by the decrease on Bcl-2 and the increase on BAX.

Despite of apoptosis, Ginkgo Folium can also arrest cell cycle and inhibit cancer cell migration and invasion. The cell cycle arrest induced by Ginkgo Folium was due to the suppression on G0/G1 phase in gastric cancer [61, 63]. The suppression on cancer cell migration and invasion were observed in several cancer types through multiple ways. First, upregulation on E-cadherin via lincRNA-p21 mediated suppression on E-cadherin degradation in colon cancer [64]. Second, downregulation on ERK/NF-kB signaling in gastric cancer [65]. Third, inhibition on heat-shock protein 27 (HSP27) mediated by AKT and p38 MAPK pathways in NSCLC [66].

The extracts of Ginkgo Folium can combine with herbal formulation to exhibit synergistic effect on anticancer. For instance, it was reported that a new formulation consisted of Ginkgo Follium and an herbal mixture Yu Ping Feng San can sensitize cisplatin resistant lung cancer cells through WT1/MVP mediated stabilization on mTOR/AKT pathway [67]. There are few clinical studies on anticancer effect of Ginkgo. Considering Ginkgo Folium extract has the standard commercial product EGb761, thus, it will be alliable to conduct clinical research with EGb761 to observe whether Ginkgo could serve as an adjuvant therapy on anticancer.

3. Conclusions

G. biloba has been used to treat age-related disorders and improve blood circulation. Recently, the extract from ginkgo exocarp, seed, kernel, flowers and Folium showed anticancer effect in various cancer types. The reported researches are mainly focus on the in vitro and in in vivo studies. Clinical studies are need to be further conducted to verify their anticancer effect. Furthermore, the detailed mechanism and the active ingredient responsible for the anticancer effect are still need to be elucidated.

Acknowledgments

This work is supported by the Key Programme of National Natural Science Foundation of China [81730108, 81973635]; the National Natural Science Foundation of China [82174024]; the Natural Science Foundation of Zhejiang Province [LY20H280011]; and the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission [2020367195, 2022508369].

Conflict of interest

The authors declare no conflict of interest.