Endophytes from Ginkgo biloba and their secondary metabolites

Ginkgo biloba is a medicinal plant which contains abundant endophytes and various secondary metabolites. According to the literary about the information of endophytics from Ginkgo biloba, Chaetomium, Aspergillus, Alternaria, Penicillium and Charobacter were isolated from the root, stem, leaf, seed and bark of G. biloba. The endophytics could produce lots of phytochemicals like flavonoids, terpenoids, and other compounds. These compounds have antibacteria, antioxidation, anticardiovascular, anticancer, antimicrobial and some novel functions. This paper set forth the development of active extracts isolated from endophytes of Ginkgo biloba and will help to improve the resources of Ginkgo biloba to be used in a broader field.


Background
Ginkgo biloba (G. biloba) is a deciduous tree belonging to the ginkgo genus, which is also known as Gongsunshu, etc. G. biloba is one of the most ancient plants on earth dating back more than 200 million years. Commonly Ginkgo biloba has been used for a medicinal plant and its seeds, leaves and fruits can be used for medicines with biological activities involving antibacteria, antioxidation, anticardiovascular and others. However, Ginkgo trees grow slowly and under natural conditions they need more than 20 years from planting to fruiting, which is a restricting point for its development; while its endophytics provide physiological metabolic pathways to produce numerous novel medicinal compounds which have become a hotspot [1].
The endophytics play important roles in the process of host plant growth and systematic evolution [1,2]. During the whole life, endophytics protect their host from infectious diseases and also help to survive in adverse environment [3]. Since the unique relationships between the host plant and associated endophytes, endophytes in G. biloba have been recognized as important sources of a variety of novel secondary metabolites with anticancer, antimicrobial and other biological activities [4,5].
Secondary metabolites are the chemical bank which provides a huge quantity of diverse commercial products for human medicines. First report about endophytics is that Stierle et al. isolated Taxomyces andreanae from phloem of Taxus brevifolia, which can produce taxol and related chemicals at the concentration of 24-50 ng/L [6]. From then on, more and more endophytics from pharmaceutical plants, such as Camptotheca acuminata [7], pine [8] and Taxus plants [9][10][11] were isolated. As to G. biloba, various endophytics including Chaetomium, Aspergillus, Alternaria, Penicillium and Charobacter were isolated from the root, stem, leaf, seed and bark of G. biloba. They produce lots of phytochemicals like flavonoids, terpenoids, and other compounds [12,13]. 50% of these isolates showed antimicrobial activities against various pathogens. Some secondary metabolites such as 2-hexenal have been involved in the plant's defense against pests. These bioactive metabolites are attractive to developing the commercial prodrugs and agricultural/ industrial production. Most importantly, as a therapeutic drug, G. biloba has no side effects even after long periods of use and its phytopharmaceuticals are readily accessible throughout the world. For better using endophytic and secondary metabolites from ginkgo trees, we summarize the data previously reported.

Endophytes in Ginkgo biloba
The whole plant of G. biloba can be used as medicine. In its root, stem, leaf, seed and bark of Ginkgo biloba, various endophytes have been isolated and their biological function was investigated. The conventional procedure of endophytes isolation is to wash the roots, stems or leaves of ginkgo firstly with 75% alcohol for 3 min, rinse with sterile water 3-5 times, 0.1% mercury sterilized for 2 min, rinsed with sterile water 3-5 times, cut into 0.5 cm × 0.5 cm pieces. The cutting pieces were inoculated in PDA medium at 28 °C for 4 days. After purification, ginkgo endophytes were isolated.

Endophytic procaryotes in Ginkgo biloba
From the previous reports, around 50 species of endophytic procaryotes were found including Bacillus subtilis, Lactobacillus sp., Fusobacterium sp., Gemella sp., Neisseria sp., Pseudomonas sp., Rothia sp., Veillonella sp., etc. Basing on 16S RNA sequence of endophytic procaryotes from previous literatures, the phylogenetic tree was constructed in Fig. 1. Amongst these procaryotes, the community structure or compositional differences at different taxonomic levels was presented in Fig. 2.
Sphingomonadaceae are a family of the Alphaproteobacteria and most abundant in G. biloba. An important feature is the presence of sphingolipids in the outer membrane of the cell wall [14]. In this family, some species are phototrophic which may have high nutritional value. The phototrophic bacteria are rich in amino acids, folic acid and vitamins, especially vitamin B12, biotin and coenzyme Q. Some other species are known as the ability to degrade some aromatic compounds which has the interests for environmental remediation [11].
Other abundant species are family Hyphomicrobiaceae, Burkholderiaceae, Methylobacteriaceae, Enterobacteriaceae, Neisseriaceae and Micrococcaceae. The family Hyphomicrobiaceae is affiliated with Alphaproteobacteria and members of this family are distributed everywhere in soils, freshwater, and also under the marine. This family is highly diverse morphologically and physiologically.
Most are aerobic chemoheterotrophs and a few can grow anaerobically by denitrification or mixed-acid fermentation.
The Methylobacteriaceae comprises a large family of Alphaproteobacteria and contains three genera including Methylobacterium, Microvirga, and Meganema. Methylobacterium species are ubiquitous in the natural environment. Some species induce plant leaf and root nodule formation, and can promote plant growth by production of auxins [15]. Most of Methylobacterium are methylotrophs and they can use methanol or other one-carbon compounds as energy sources to produce proteins [16]. Otherwise, in Methylobacterium, common fatty acids were contained especially ubiquinone Q-10, a popular dietary supplement.
Family Enterobacteriaceae contains a large number of genera that are biochemically and genetically related to one another. Many of them are pathogens, such as Salmonella, Shigella or Yersinia, because they produce endotoxins. Endotoxins reside in the cell wall and when the cell dies and the cell wall disintegrates, endotoxins are released [9].
Family Burkholderiaceae belongs to the order Burkholderiales within the class Betaproteobacteria. This family is characterized by the presence of ecologically extremely diverse organisms and contains truly environmental saprophytic organisms, phytopathogens, opportunistic pathogens, as well as primary pathogens for humans and animals.
Family Neisseriaceae and Micrococcaceae are widespread in soil, subterranean cave silts, sea, glacier silts, sewage, water sludge, aerial surfaces of plants, vegetables, and various animal species and are even more distantly related to the human pathogens.

Endophytic eukarya in Ginkgo biloba
The phylogenetic tree of endophytic eukarya (Fig. 3) was constructed basing on ITS sequence of roots and leaves of Ginkgo biloba from previous literatures. Amongst these endophytic eukarya, the community structure at different taxonomic levels was presented in Fig. 4.
Amongst eukarya, family Pleosporaceae belongs to sac fungi. The taxonomic relationship of this family to associated genera is still not determined. The classification of Pleosporaceae has been a challenge because of the lack of the importance of morphological characters and reference strains. From the present knowledge, the family Pleosporaceae includes numerous saprobic, opportunistic human and plant pathogenic taxa [17].
Phaeosphaeriaceae is a large and important family of fungi in the order Pleosporales. Species in this family have a cosmopolitan distribution, and are generally nectrotrophic or saprobic on a wide range of plants [18]. This family includes economically important plant pathogens and previously accommodated 35 sexual and asexual genera and comprised more than 300 species with a range of morphological characters [19].
The Xylariaceae are a family of mostly small ascomycetous fungi. It is one of the most commonly encountered groups of ascomycetes and is found throughout the temperate and tropical regions of the world. They are typically found on wood, seeds, fruits, or plant leaves, some even associated with insect nests. Most decay wood and many are plant pathogens. Phylogenetic analyses suggest that there are two main lineages in this family, Hypoxyloideae and Xylarioideae [20,21].

Secondary metabolites of endophytics in Ginkgo biloba
A series of compounds were obtained by fermentation, extraction, and isolation from endophytics of G. biloba, amongst which 115 metabolites were found in the fermentation broth of Chaetomium fungi, 44 metabolites were found from Aspergillus, 43 metabolites found in the genus Xylaria. The amount from these three genera accounted for 72% of the secondary metabolites from endophytic procaryotes and 21% were isolated from Fusarium, Alternaria and Penicillium. The number of metabolites of each genus is shown in Fig. 5.
Many metabolic products from G. biloba have strong inhibitory effects on pathogenic bacteria Staphylococcus Fig. 1 The phylogenetic tree of endophytic procaryotes from soil, root and leaf of Ginkgo biloba. 50 most abundant OTUs are used for display. If a number appears before the species name, it represents the total number of sequences of this OTU. If it is a graph, the graph size represents the relative abundance (percentage), and the black dot on the branch represents the bootstrap confidence greater than 95% Fig. 2 The community structure at different taxonomic levels. (1) The community structure at different phylums; (2) the community structure at different classes; (3) the community structure at different families; (4) the community structure at different genus. The percentage in parentheses indicates that only the group with the average abundance greater than this ratio is listed. All other groups are classified in others
Chaetomugilin A and D, both are a kind of azaphilone isolated from Chaetomium globosum and has been shown to exhibit inhibitory activity against the brine shrimp (Artemia salina) and Mucor miehei [22]. Chaetomugilide A isolated from Chaetomium globosum Fig. 3 The phylogenetic tree of endophytic eukarya from soil, root and leaf of Ginkgo biloba. 50 most abundant OTUs are used for display. If a number appears before the species name, it represents the total number of sequences of this OTU. If it is a graph, the graph size represents the relative abundance (percentage), and the black dot on the branch represents the bootstrap confidence greater than 95% TY1 has strong activity against hepatoma cell HepG-2, and the IC 50 value is only 1.7 μmol/L [23]. Chaetoglobosin A is a Chaetomium secretion with the anticancer activity in vitro [24] and it derivates into other bilobalide compounds MBJ-0038, MBJ-0039, and MBJ-0040 [25]. Chaetoglobosin E is a cytochalasan alkaloid found in Chaetomium globosum and Chaetomium subaffine. It is a cytochalasan alkaloid, a member of indoles, a macrocycle The community structure at different taxonomic levels. (1) The community structure at different phylums; (2) The community structure at different classes; (3) The community structure at different families; (4) the community structure at different genus. The percentage in parentheses indicates that only the group with the average abundance greater than this ratio is listed. All other groups are classified in others and a secondary alpha-hydroxy ketone. It has a role as a Chaetomium metabolite and an antineoplastic agent.
One new cytochalasan alkaloid, chaetoglobosin V(b), together with two structurally related known compounds, chaetoglobosin V and chaetoglobosin G, were isolated from the ethyl acetate extract of a culture of the endophytic fungus Chaetomium globosum, associated with the leaves of G. biloba tree. The structures of the isolated compounds were elucidated by spectroscopic methods including 1D and 2D NMR and mass spectrometry. The absolute conStruration of chaetoglobosin V(b) was established by means of electronic circular dichroism (CD) spectroscopy. The correlation between compounds was demonstrated by a biomimetic transformation of chaetoglobosin G under mild conditions in chaetoglobosins V and V(b). The isolated metabolites were tested against some phytopathogens [22].
The compound flavipin isolated from Chaetomium globosum CDW 7 has strong antioxidant activity [23]. Chaetomium globosum ZY-22 could produce two polyhydroxylated steroids [24] and two other important compounds bilobalide, ginkgolides are to be beneficial to human health [26]. Bilobalide has neuroprotective effects [27] as well as inducing the liver enzymes CYP3A1 and 1A2 which may be partially responsible for interactions between gingko and other herbal medicines or pharmaceutical drugs; while ginkgolide has been investigated for its potential to reducing migraine frequency [28]. Ergosterol peroxide (5α,8α-epidioxy-22E-ergosta-6,22dien-3β-ol) is a steroid derivative. It has been reported to exhibit immune-suppressive, anti-inflammatory, antiviral, trypanocidal and antitumor activities in vitro [27].

Secondary metabolites of Alternaria
Alternaria is a very common fungus. It is an important pathogen for plants, human and animal diseases. It is a biological resource with great application potential as well. According to the existing literatures, 17 metabolites were isolated from the fermentation products of Alternaria (see Table 3). Alterperylenol inhibits human telomerase activity. Alterperylenol can inhibit telomerase activity (IC 50 = 30 μM), but altertoxin I (dihydroalterperylenol), a structurally related compound, did not affect activity at 1 mM. Moreover, alterperylenol and altertoxin I show phytotoxic and antifungal activity [31].
In these metabolites, botulinum toxin and botulinum toxin II have strong cytotoxic activity. When the concentration is 10 μg/mL, the mortality rate of brine shrimp is 68.9% and 73.6%, respectively [32]. Alternaria No. 28 could produce cytotoxic metabolites which have inhibitory potential against some different protein kinases [7].

Secondary metabolites of Penicillium
Penicillium is widely distributed in nature and generally has a strong biological activity. According to the existing literatures, 17 secondary metabolites were found from the fermentation products of Penicillium sp. in G. biloba (Table 4), and some metabolites were biologically active. The compound arcacic acid is isolated from the fermentation broth of Penicillium commune, which has antibacterial activity and has inhibition activities on 12 kinds of plant pathogens, especially has strong inhibitory activity against Bacillus licheniformis and Sclerotinia sclerotiorum, and the IC 50 values are only 39.28 mg/L and 60.62 mg/L [33].

ZY-22 An antioxidant and is believed
to reduce the risk of stomach cancer by reducing the formation of carcinogenic nitrosamines [33] 100 Octanoic acid, methyl ester 111-11-5

T16
Used to make paint, varnish and rust removers, as a solvent and as an ingredient in pesticides [45] 108 Succinic acid

Chaetomium globosum
No. 16 Used as a solvent and as an ingredient in gasoline and diesel and jet fuel [45] 112 Triethylene glycol monomethyl ether acetate

Secondary metabolites of Xylaria
43 kinds of compounds were isolated from the fermentation products of Xylaria in Ginkgo biloba (Table 5), in which the compound 7-amino-4-methylcoumarin was isolated from the fermentation product of Xylaria sp. YX-28 [36]. It has antibacterial activity and also has strong inhibitory activity against 13 kinds of human susceptible pathogens, which is significantly higher than the positive controls ampicillin, gentamicin and tetracycline.

Secondary metabolites of Fusarium
Fusarium is one of the dominant bacteria, which can be isolated from different parts of Ginkgo cultivated in various areas. According to the literatures, 25 kinds of compounds were isolated from the fermentation products of Fusarium (Table 6). Since Fusarium of G. biloba can produce ginkgolides B, it can be used as a new source of ginkgolides B [37]. Some studies have shown that Fusarium oxysporum GF521 can produce rutin and kaempferol, and the total flavonoids production of endophytic fungi is 21.10 ± 1.30 mg/L, which indicates that Fusarium genus also have a high ability of producing flavonoids [37].

Secondary metabolites of other genus
53 compounds were isolated from the fermentation products of other genus in G. biloba (Table 7), some of which can also produce other valuable compounds. From the endophytic Muscodor albus GBA, 19 kinds of volatile components can be separated [24], which normally have a strong ecological effect. Some volatile components can inhibit the pathogenic microorganisms and enhance the disease resistance of plants.

Application of secondary metabolites from Ginkgo biloba
Following the discovery by Schwabe of Germany that Ginkgo biloba contains active ingredients-ginkgo flavonoids and ginkgolides for the prevention and treatment of cardiovascular, cerebrovascular and neurological diseases, the researches about ginkgo has become more popular. Germany and France were the first countries in the world to develop ginkgo leaf products. In the mid-1970s, they first developed Ginkgo biloba leaves for the treatment of cardiovascular diseases. Since then, there are more than 50 kinds of ginkgo products on the market.
In the application, Ginkgo can be used with the extracts. Some examples, a substance EGb 761 extracted from Ginkgo biloba has shown to be effective against Noise-induced hearing loss (NIHL) in an animal model. This substance is assumed to protect the cochlea from hair cell loss after intensive noise exposure by reducing reactive oxygen species (ROS). Further effects of EGb 761 on the cellular and systemic levels of the nervous system make it a promising candidate not only for protection against NIHL but also for its secondary comorbidities like tinnitus [41]; One Ginkgo biloba extract (GbE) was used as a nontoxic natural reducing and stabilizing agent for preparing cytocompatible graphene. The as-prepared GbE-reduced graphene oxide (Gb-rGO) showed significant biocompatibility with cancer cells. Addition of GbE makes rGO producing procedure cost-effective and green. This method could be used for various biomedical applications, such as tissue engineering, drug delivery, biosensing, and molecular imaging [42]. Some application has been using a part of the plant. Another example, Ginkgo tea is a kind of health food produced from Ginkgo biloba leaves. Two kinds of glycosidase were used to improve the flavor of Ginkgo tea, and three kinds of bioactivities were selected to investigate the health care function of the tea infusion [43].
The Ginkgo preparation mainly includes capsules, tablets, granules, tea bags. Capsules and tablets are most popular in the formulation of the product. Recently, new preparation like shampoo, facial cleanser and hair moisturizer have been introduced in cosmetics applications. Most of the ginkgo products on the market are registered as health foods and a few are registered as over-the-counter drugs.
In many existing products, especially in the medicines, 24% of total flavonoids and 6% of ginkgolides are the basic quality requirements for Ginkgo biloba extracts. Some famous manufacturers proposed higher standards. They appended ginkgolides A, B, C, J and biloba lactone as the quality indicators and generally required the content of ginkgolides A, B, C, J greater than 2.5%, the content of biloba lactone greater than 2.6%.
On the basis of data about the endophytes and secondary metabolites in G. biloba, the catalogue is diverse in terms of structural complexity and lots of them have promising biological activities, which have the potential to be a source of new pharmaceutical agents which have a constant, critical need to combat cancers, viral infections, infectious diseases, and autoimmune disorders. There is also a growing need to fight insect-borne diseases of both animals and plants as climatological changes provide conditions conducive to more intensive outbreaks of these events. The fight against any disease is a dynamic equilibrium between advances in chemotherapy and natural selection in infectious or invasive agents. If the scientific community is to maintain parity in this never-ending struggle, then new sources of novel, bioactive chemotherapeutic agents must be found.
It appears that the mechanism by which endophytes produce secondary metabolites that mimic those produced by their host plants is far from clear. Even though efforts to unravel the pathway genes in the endophytes, it has failed to detect critical genes corresponding to those existing in plants, our understanding of the mechanisms associated with the development of different diseases increases, our ability to use this knowledge to select for ever more potent and selective compounds should increase commensurately. Endophytes of G. biloba will continue to provide a fertile arena for these quests.

Prospects
With human aging process is accelerating, it has been common pursuit for a healthy and high-quality living. Since Ginkgo biloba preparations have a worldwide reputation as natural medicines and healthy products, Ginkgo development and the prospects are attractive. In the United States, Ginkgo biloba extracts have been on the list of imported drugs. Ginkgo products on the market are almost all products of American companies, and few products have been seen in Europe. At present, the European market is basically occupied by French and German products. Most of the Ginkgo extracts on the US market are produced by Japan and South Korea, a small portion is purchased from China.
Although comparing with the developed countries, China market is not competitive and too weak to take the risks, the potential of China's Ginkgo development is still worth looking forward to. China is the birthplace and main producing area of the world's Ginkgo. Many excellent Ginkgo germplasm resources are valuable treasures for China. With the sharp increase in Ginkgo resources and products output in China, the market has become more concerned at present (Fig. 7). At present, the Ginkgo products in China have low added-value and quality. In the development of ginkgo industry in China, it is necessary to increase the quality standardization and to improve the scientific research efforts and the production technology of Ginkgo preparations. It deserves to initiate new and technological products on flavonoids, bilobalide, polyisoprene, etc. Especially some new application in other industries should be explored, such as supplying in cytocompatible graphene preparation.
Chinese people have a tradition to have Ginkgo preparation as healthy products. China's population accounts for about a quarter of the world's total population. Therefore, the Ginkgo products in China should have more concerns on the domestic market and at the same time expand the international market with high-quality and featured products.