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 [9-11] 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.

For the endophytic procaryotes, on the total DNA as the template, 27F(AGAGTTTGATC-CTGGGTCAG)/1492R(GGTTACCTTGTTACGACTT) as a primer, 16S rDNA was amplified. For the endophytic eukarya, ITS5 (GAAG TAAAAG TCGTAACAAGG)/ITS4 (TCCTCCGC TTA TTGA TATGC) as a primer, ITS rDNA was amplified. According to the culturing and molecular analysis between different species, the endophytics residing in G. biloba belong to Chaetomium, Aspergillus, Alternaria, Penicillium, Charobacter, etc.

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.

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

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.

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%

Fig. 4

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

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.

Fig. 5

a The metabolite quantity of some major endophytics in Ginkgo biloba; b the metabolite quantity of some minor endophytics in Ginkgo biloba

Many metabolic products from G. biloba have strong inhibitory effects on pathogenic bacteria Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa. The secondary metabolites of Ginkgo, such as flavonoids and ginkgolides, are drugs or prodrugs used in the treatment of peripheral arterial diseases, neurological disorders, sclerosis of cerebral arteries, and cerebral ageing.

Secondary metabolites of Chaetomium

Chaetomium is the largest type of endophytic fungus from G. biloba and its secondary metabolites are biologically diverse. Chaetomium globosum is one of main endophytics. A total of 115 metabolites were isolated from the fermentation broth of Chaetomium globosum (see Fig. 6 and Table 1). Among them, chaetoglobosin A, chaetoglobosin C, chaetoglobosin E, chaetoglobosin G, chaetoglobosin Vb, chaetomugilin A, chaetomugilin D and ergosterol peroxide (peroxyergosterol; 5α, 8α-peroxy-(22E, 24R)-ergot-6,22-diene-3β-ol), which has been reported in many literatures, may be a research hotspot. Among these compounds, chaetomugilin A, chaetomugilin D, chaetoglobosin A and chaetoglobosin C have strong cytotoxic activity [22].

Fig. 6

The quantity of different kinds of metabolites from Chaetomium

Table 1

Secondary metabolites of Chaetomium in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	(22E, 24R)-ergosta-7,22-diene-3β,5α,6β-triol/cerevisterol	516-37-0		Chaetomium globosum		[33]
2	(22E, 24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol	88191-06-4		Chaetomium globosum		[44]
3	(7Z,11E)-7,11-Hexadecadien-1-yl acetate	53042-79-8		Chaetomium globosum No. 16	Pesticide	[45]
4	(E,E)-2,4-Decadienal	25152-84-5		Chaetomium globosum No. 16	Food_additive; fragrance	[45]
5	(Z)-9-Hexadecenoic acid, methyl ester	1120-25-8		Chaetomium globosum No. 16		[45]
6	(Z,Z)-9,12-Octadecadienoic acid	60-33-3		Chaetomium globosum No. 16	Biosynthesis of prostaglandins and cell membranes	[45]
7	1-(3-Acetyl-2,2-dimethylcyclopropyl)-2-methyl-1-propanone	77142-84-8		Chaetomium globosum T16		[49]
8	1-(3-Methoxy-2-pyrazinyl)-2-methyl-1-propanone	98618-81-6		Chaetomium globosum T16		[46]
9	1,3-Dioxolane, 2-methoxy	19693-75-5		Chaetomium globosum T16		[46]
10	1-Eicosene	3452-07-1		Chaetomium globosum No. 16		[45]
11	1-Trimethylsilyl methanol	3219-63-4		Chaetomium globosum T16		[46]
12	2,3,4-Trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran	1824584-79-3		Chaetomium globosum		[47]
13	2,4,5-Trimethyl-1,3-dioxolane	3299-32-9		Chaetomium globosum T16	Flavors	[46]
14	2,4-Decadienal	2363-88-4		Chaetomium globosum No.16	Food additive	[20, 21]
15	2′-O-Methyladenosine	2140-79-6		Chaetomium globosum	Inhibition of vaccinia virus growth	[47]
16	2′-Deoxyadenosine	958-09-8		Chaetomium globosum	Anti-tumor and antiviral nucleoside drugs (cladribine)	[44]
17	20-Dihydrochaetoglobosin A	149560-98-5		Chaetomium globosum		[47]
18	21 Methoxy-Chaetoglobosin F			Chaetomium globosum		[47]
18	2-Cyclohexyl-hex-5-en-2-ol	959261-17-7		Chaetomium globosum T16		[46]
19	2-Ethyl-5-propylphenol	72386-20-0		Chaetomium globosum T16		[46]
20	2-Methyl-5-propyl-2,4-dihydro-3H-pyrazol-3-one	31272-04-5		Chaetomium globosum T16		[46]
21	2-Octyl-cyclopropaneoctanal	56196-06-6		Chaetomium globosum No.16		[45]
22	3,4-Dihydroxyphenyl acetic acid	102-32-9		Chaetomium globosum	A metabolite of dopamine, Cytoplasm, Encephalitis, Hypothyroidism, Alzheimer’s disease, Colorectal cancer	[47]
23	3-Methylorsellinic acid	4707-46-4		Chaetomium globosum ZY-22	Neuroprotective Activity	[46]
24	4-Aminophenylacetic acid/p-aminophenylacetic acid/4-aminophenylacetic acid	1197-55-3		Chaetomium globosum	Anti-inflammatory Inhibition colitis	[47]
25	4-Methyl-1-hepten-5-one	26118-97-8		Chaetomium globosum		[46]
26	5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde	67350-50-9		Chaetomium globosum	Hapten, produces advanced glycation end-products (AGEs)	[47]
27	5′-Epichaetovirdin A	1308671-17-1		Chaetomium globosum No. 12		[45]
28	5′-Deoxy-5′-methylamino-adenosine	No cas no.		Chaetomium globosum		[47]
29	9(11)-dehyoergosterol peroxide	86363-50-0		Chaetomium globosum ZY-22		[44]
30	9,12-Octadecadien-1-ol	1577-52-2		Chaetomium globosum No. 16		[45]
31	Acetaldehyde, diethyl acetal	105-57-7		Chaetomium globosum T16	Used in fruit, rum and whisky flavour	[46]
32	Adenosine	58-61-7		Chaetomium globosum ZY-22	Vasodilatory, anti-arrhythmic and analgesic activities adenosine is an adenosine receptor agonist	[46]
33	Allantoin	97-59-6		Chaetomium globosum	Healing, soothing, and anti-irritating properties anti-acne products, sun care products, and clarifying lotions	[48]
34	alpha-Methylstyrene	98-83-9		Chaetomium globosum	Membrane adhesives and sealant chemicals	[48]
35	Anthranilic acid	118-92-3		Chaetomium globosum MX-0510	A water-soluble vitamin	[33]
36	Benzeneacetic acid	103-82-2		Chaetomium globosum No. 16	Used in the manufacture of penicillin and bendazol	[45]
37	Benzeneacetic acid, methyl ester	101-41-7		Chaetomium globosum No. 16	Used in the manufacture of atropine	[45]
38	Benzeneethanol/phenylethyl alcohol	60-12-8		Chaetomium globosum	Essence	[45]
39	Butyraldehyde, 4-phenyl	18328-11-5		Chaetomium globosum T16		[46]
40	Cerebroside B	88642-46-0		Chaetomium globosum ZY-22		[46]
41	Cerebroside C	98677-33-9		Chaetomium globosum ZY-22		[46]
42	Chaetoglobosin A	50335-03-0		Chaetomium globosum		[44, 49]
43	Chaetoglobosin B	50335-04-1		Chaetomium globosum CDW7		[48]
44	Chaetoglobosin C	50645-76-6		Chaetomium globosum		[26, 28]
45	Chaetoglobosin D	55945-73-8		Chaetomium globosum		[49]
46	Chaetoglobosin E	55945-74-9		Chaetomium globosum (CDW7)		[49]
47	Chaetoglobosin F	55945-75-0		Chaetomium globosum (CDW7)		[47]
48	Chaetoglobosin Fa	1599426-06-8		Chaetomium globosum		[47]
49	Chaetoglobosin Fex	149457-95-4		Chaetomium globosum		[47]
50	Chaetoglobosin G	65773-98-0		Chaetomium globosum (NM0066)		[47]
51	Chaetoglobosin R	777939-30-7		Chaetomium globosum		[49]
52	Chaetoglobosin V	1399682-37-1		Chaetomium globosum		[47]
53	Chaetoglobosin Vb	1399690-75-5		Chaetomium globosum (CDW7)		[48]
54	Chaetoglobosin Y	1608108-89-9		Chaetomium globosum		[48]
55	Chaetomugilide A	1418138-71-2		Chaetomium globosum		[45, 47]
56	Chaetomugilide B	1433976-48-7		Chaetomium globosum		[45]
57	Chaetomugilide C	1418138-70-1		Chaetomium globosum		[45, 47]
58	Chaetomugilin A	1041640-66-7		Chaetomium globosum		[45]
59	Chaetomugilin D	1098081-38-9		Chaetomium globosum		[25]
60	Chaetomugilin I	1187848-00-5		Chaetomium globosum		[25]
61	Chaetomugilin J	1187848-01-6		Chaetomium globosum		[25]
62	Chaetomugilin O	1187848-06-1		Chaetomium globosum		[25]
63	Chaetomugilin Q	1319729-85-5		Chaetomium globosum		[25]
64	Chaetomugilin S	1399093-77-6		Chaetomium globosum		[25]
65	Chaetoviridin C	128230-02-4		Chaetomium globosum		[15]
66	Chaetoviridin D	128230-04-6		Chaetomium globosum		[33]
67	Chaetoviridin E	1178875-15-4		Chaetomium globosum		[33]
68	Cyclo-(Phe-Gly)	5037-75-2		Chaetomium globosum		[33]
69	Cyclopentadecane	295-48-7		Chaetomium globosum No.16		[45]
70	Dimethyl phthalate	131-11-3		Chaetomium globosum No.16	Used in plastics, insect repellents, safety glass, and lacquer coatings	[45]
71	Epimwsokorwnone A	1073-96-7		Chaetomium globosum		[33]
72	Ergosta-4 6,8,22-tetraen-3-one/ergosta-4,6,8,22-tetraen-3-one	194721-75-0		Chaetomium globosum (ZY-22)		[33]
73	Ergosterol	57-87-4		Chaetomium globosum	Formation of vitamin D2	[49]
74	Ergosterol peroxide (5α,8α-epi-dioxy-(22E,24R) -ergosta-6,22-dien-3β-ol)	2061-64-5		Chaetomium globosum	An antineoplastic agent, an antimycobacterial drug and a trypanocidal drug	[33]
75	Ethanoic acid	64-19-7		Chaetomium globosum T16	Food additive, and in petroleum production	[46]
76	Ethyl 13-methyl-tetradecanoate	64317-63-1		Chaetomium globosum No. 16		[45]
77	Ethyl 2-heptenoate	2351-88-4		Chaetomium globosum T16		[45]
78	Ethylidene acetate	542-10-9		Chaetomium globosum T16		[45]
79	flavipin (1,2-benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl)	483-53-4		Chaetomium globosum CDW7	Antioxidant fungicides	[22]
80	Fumigaclavine B	6879-93-2		Chaetomium globosum		[47]
81	Fumitremorgin C	118974-02-0		Chaetomium globosum (NM0066)	A mycotoxin and a breast cancer resistance protein inhibitor	[33]
82	Gliotoxin	67-99-2		Chaetomium globosum (NM0066)	A mycotoxin, an immunosuppressive agent, an protein farnesyltransferase inhibitor, a proteasome inhibitor and an antifungal agent	[33]
83	Globosterol	1193319-70-8		Chaetomium globosum ZY-22		[44]
84	Glycerol formal	5464-28-8		Chaetomium globosum T16		[46]
85	Hexadecane	544-76-3		Chaetomium globosum	Used as a solvent and an ingredient in gasoline and diesel and jet fuels	[45]
86	Hexadecanoic acid, ethyl ester	628-97-7		Chaetomium globosum No. 16	Used as softener, lubricant, food additive	[45]
87	Hexadecanoic acid, methyl ester	112-39-0		Chaetomium globosum No. 16	Used as intermediate of emulsifier, wetting agent, stabilizer and plasticizer	[45]
88	Indole-3- carboxylic acid	771-50-6		Chaetomium globosum ZY-22	Used for synthesis of to rise tron and antiviral drugs	[33]
89	Indole-3-acetic acid	87-51-4		Chaetomium globosum	Plant growth stimulating hormone	[33]
90	Isopentyl alcohol, acetate	123-92-2		Chaetomium globosum T16	Used as a solvent and preparation of a variety of flavor food flavor	[22]
91	Lactic acid	50-21-5		Chaetomium globosum T16	Used to make some plasticizers, adhesives, pharmaceuticals and salts, used in the leather tanning industry and as a solvent	[46]
92	Lactic acid, 2-methyl-,ethyl ester	80-55-7		Chaetomium globosum T16		[46]
93	Maltol	118-71-8		Chaetomium globosum MX-0510	Food additive	[33]
94	Mannitol	87-78-5		Chaetomium globosum	Used as an osmotic diuretic	[33]
95	Methyl 13-methyltetradecanoate	5129-59-9		Chaetomium globosum No. 16		[45]
96	Methyl 9,12-heptadecadienoate	15620-59-4		Chaetomium globosum No. 16		[45]
97	Methyl vinylcarbinol	598-32-3		Chaetomium globosum	Food additive	[46]
98	Methylthiogliotoxin	74149-38-5		Chaetomium globosum (NM0066)		[33]
99	o-Coumaric acid	583-17-5		Chaetomium globosum 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		Chaetomium globosum No. 16	Food additive	[45]
101	Pentadecane	629-62-9		Chaetomium globosum No. 16	Used as a solvent and in some household pesticides	[45]
102	Pentadecanoic acid, methyl ester	7132-64-1		Chaetomium globosum No. 16	Fuels and fuel additives Intermediates, pesticide	[45]
103	p-Hydroxybenzoic acid	99-96-7		Chaetomium globosum	Used as preservatives, fungicides	[33]
104	Pseurotin A	58523-30-1		Chaetomium globosum (NM0066)	An azaspiro compound, an oxaspiro compound and a lactam	[33]
105	Quercetin	117-39-5		Chaetomium globosum GCZX015	Combined with chemotherapeutic drugs, produces anti-inflammatory and anti-allergy effects	[33]
106	Squalene	111-02-4		Chaetomium globosum (NM0066)	Investigated as an adjunctive cancer therapy, also used as cosmetics and dietary supplement	[33]
107	S-Tetrachloroethane	79-34-5		Chaetomium globosum T16	Used to make paint, varnish and rust removers, as a solvent and as an ingredient in pesticides	[45]
108	Succinic acid	110-15-6		Chaetomium globosum	A radiation protective agent, an anti-ulcer drug	[33]
109	Tetradecane	629-59-4		Chaetomium globosum No.16	Used as a solvent and some pesticide sprays	[45]
110	Thymine	65-71-4		Chaetomium globosum ZY-22	A pyrimidine nucleobase and a pyrimidone	[33]
111	Tridecane	629-50-5		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	3610-27-3		Chaetomium globosum T16		[46]
113	Uracil	66-22-8		Chaetomium globosum ZY-22	Use in the body to help synthesis of many enzymes, and the biosynthesis of polysaccharides and the transportation of sugars containing aldehydes	[49]
114	α-Guajene	3691-12-1		Chaetomium globosum No. 16		[45]

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 TY1 has strong activity against hepatoma cell HepG-2, and the IC50 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 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-6,22-dien-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 Aspergillus

Aspergillus is the dominant flora of endophytic fungi of G. biloba and was isolated from different parts of G. biloba which cultivated in various areas. A total of 44 metabolites were found in the fermentation broth of Aspergillus (see Table 2), among which 3-hydroxy-terphenyl, 4,5-dimethoxycandidusin A, prenylcandidusin C, and prenylterphenyllin were studied most popularly. For 4″-Deoxycandidusin A, 4″-deoxytripentin, 4′-deoxy-3-hydroxyrisperidone, aspergiloid A, coumarin A, and tribenzine, three articles reported about each compound, respectively. Among these metabolites, 3-hydroxy-terphenyl and 4″-deoxycandidusin A, 4″-deoxytripentin have strong inhibitory activity against neuraminidase [29]; 4′-deoxy-3-hydroxytripentin, 3-hydroxy-terphenyl, 4″-deoxycandidusin has moderate activity against human nasopharyngeal carcinoma cell KB, human gastric cancer cell SGC-7901, human colon cancer cell SW1116 and human lung cancer cell A549 [30].

Table 2

Secondary metabolites of Aspergilus in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	3-Hydroxyterphenyllin	66163-76-6		Aspergillus sp.	Induces apoptosis and S phase arrest in human ovarian carcinoma cells	[28, 50]
2	4″-Deoxycandidusin A	1354549-88-4		Aspergillus sp.		[51, 52]
3	4″-Deoxyterphenyllin	59904-04-0		Aspergillus sp.		[50]
4	4,5-Dimethoxycandidusin A/3,4-dimethoxycandidusin A	1354549-89-5		Aspergillus sp.		[50, 52]
5	4′-Deoxy- 3-hydroxyterphenyllin	1296205-84-9		Aspergillus sp.		[50, 52]
6	4′’-Deoxy-5′-desmethyl-terphenyllin	1354549-87-3		Aspergillus sp.		[50]
7	4′’-Deoxyprenylterphenyllin	959124-87-9		Aspergillus sp. IFB-YXS	Potential anticancer lead molecules	[50]
8	4-Hydroxy-3-(3′-methyl-2′-butenyl) benzoic acid	1138-41-6		Aspergillus sp. YXf3	Show potent inhibition of HLE	[50]
9	5′-Desmethylterphenyllin	1299485-87-2		Aspergillus sp.	An alpha-glucosidase inhibitor	[50]
10	Alternariol	641-38-3		Aspergillus sp. YXf3	An cholinesterase inhibitor and a mycotoxin	[52]
11	Alternariol monomethyl ether/alternariol-4-methyl ether	23452-05-3		Aspergillus sp. YXf3	An antifungal agent	[52]
12	Aspergiloid A	1354549-91-9		Aspergillus sp.		[50]
13	Aspergiloid B	1354549-92-0		Aspergillus sp.		[50]
14	Aspergiloid C	1354549-93-1		Aspergillus sp.		[50]
15	Aspergiloid D	1354549-94-2		Aspergillus sp.		[50]
16	Aspergiloid E	1579256-33-9		Aspergillus sp. YXf3		[52]
17	Aspergiloid F	1579256-35-1		Aspergillus sp. YXf3		[52]
18	Aspergiloid G	1579256-37-3		Aspergillus sp. YXf3		[52]
19	Aspergiloid H	1579256-39-5		Aspergillus sp. YXf3		[52]
20	Aspergiloid I	1887750-59-5		Aspergillus sp. YXf3	Anti-cancer and inhibition of plant pathogens	[50]
21	Candidusin A	81474-59-1		Aspergillus sp.		[50]
22	Candidusin C/4″-methoxycandidusin A	267007-58-9		Aspergillus sp.		[50]
23	Chlorflavonin	23363-64-6		Aspergillus sp. (strain no. YXf3)	An antifungal agent	[50]
24	Chlorflavonin A	1443055-96-6		Aspergillus sp. (strain no. YXf3)	An antifungal agent	[50]
25	Cyclo-(L-Leu-L-Trp)	15136-34-2		Aspergillus sp. YXf3		[50]
26	Ginkgolide B	15291-77-7		Aspergillus.fumigatus var. fumigatus FG 05	Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation	[52]
27	Ginkgolide C	15291-76-6		Aspergillus		[32]
28	Prenylcandidusin B	1297472-19-5		Aspergillus sp. IFB-YXS	An antineoplastic agent	[53]
29	Prenylcandidusin C	1297472-20-8		Aspergillus sp.	An antineoplastic agent	[53]
30	Prenylterphenyllin	959124-85-7		Aspergillus sp.	Exhibits cytotoxic activity, an antineoplastic agent	[53]
31	Prenylterphenyllin B	1297472-16-2		Aspergillus sp. IFB-YXS	Exhibits cytotoxic activity, an antineoplastic agent	[53]
32	Sphaeropsidin A	38991-80-9		Aspergillus sp. YXf3	larvicidal and biting deterrents against Aedes aegypti	[50]
33	Sphaeropsidin B	39022-38-3		Aspergillus sp. YXf3		[50]
34	Terphenolide	1354549-90-8		Aspergillus sp.		[50]
35	Terphenyllin	52452-60-5		Aspergillus sp.	A mycotoxin	[50]
36	Terreinol	669073-67-0		Aspergillus sp. YXf3		[31]
37	Xanthoascin	61391-08-0		Aspergillus sp. IFB-YXS		[53]
38	Prenylterphenyllin D	2079979-59-0		Aspergillus sp. IFB-YXS	Antibacterial activities, anti-phytopathogenic activities	[31]
39	Prenylterphenyllin E	2079979-60-3		Aspergillus sp. IFB-YXS	Antibacterial activities, anti-phytopathogenic activities	[31]
40	2′-O-Methylprenylterphenyllin	2079979-61-4		Aspergillus sp. IFB-YXS	Antibacterial activities, anti-phytopathogenic activities	[31]
41	4-O-Methylprenylterphenyllin	2079979-62-5		Aspergillus sp. IFB-YXS		[31]
42	[1,1′:4′,1′’-Terphenyl]-4,4′’-diol, 2′,3′,5′-trimethoxy-(9CI)	59914-89-5		Aspergillus sp. IFB-YXS		[31]
43	[1,1′:4′,1′’-Terphenyl]-2′,4′’-diol,3′,4,6′-trimethoxy-(9CI)	59903-93-4		Aspergillus sp. IFB-YXS		[31]
44	[1,1′:4′,1′’-Terphenyl]-2′,4-diol,3′,4′’,6′-trimethoxy-(9CI)	59903-92-3		Aspergillus sp. IFB-YXS		[31]

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 (IC50 = 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].

Table 3

Secondary metabolites of Alternaria in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	(22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol/cerevisterol	516-37-0		Alternaria tenuissima SY-P-07		[29]
2	(2R,3R)-3,5,7,3′,5′-pentahydroxyflavane	87592-94-7		Alternaria tenuissima SY-P-07		[29]
3	3β,5α,9α-Trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one	88191-14-4		Alternaria tenuissima SY-P-07		[29]
4	6-Epi-stemphytriol	1262797-65-8		Alternaria tenuissima SY-P-07		[29]
5	7-Epi-8-hydroxyaltertoxin I	1262797-64-7		Alternaria tenuissima SY-P-07		[29]
6	Alternariol	641-38-3		Alternaria No. 28	An cholinesterase inhibitor	[29]
7	Alternariol monomethyl ether/alternariol-4-methyl ether	23452-05-3		Alternaria No. 28	An antifungal agent	[29]
8	Alterperylenol	88899-62-1		Alternaria tenuissima		[45]
9	Altertoxin I (dihydroalterperylenol)	56258-32-3		Alternaria sp.		[29]
10	Ergosta-4,6,8,22-tetraen-3-one/ergosta-4,6,8,22-tetraen-3-one	194721-75-0		Alternaria No. 28		[29]
11	Ergosterol	57-87-4		Alternaria sp.	Formation of vitamin D2	[29]
12	Flazin	100041-05-2		Alternaria tenuissima SY-P-07		[47]
13	Solanapyrone G	220924-51-6		Alternaria tenuissima SY-P-07		[47]
14	Stemphyperylenol	102694-33-7		Alternaria tenuissima SY-P-07	An antifungal agent	[47]
15	Tenuazonic acid	610-88-8		Alternaria No. 28	An antibiotic with antiviral and antineoplastic, also as a mycotoxin	[29]
16	Vivotoxin II	1261267-71-3		Alternaria No. 28		[29]

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 IC50 values are only 39.28 mg/L and 60.62 mg/L [33].

Table 4

Secondary metabolite of Penicillium in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	2′-Deoxyuridine/uracil deoxyriboside	951-78-0		Penicillium sp. YY-25	Antimetabolite	[29]
2	3-Methylorsellinic acid	4707-46-4		Penicillium No. 97	Antibacterial activity	[29]
3	3-Methylpiperazine-2,5-dione	6062-46-0		Penicillium sp. YY-24		[29]
4	Adenine	73-24-5		Penicillium sp. YY-22	Dietary supplement	[29]
5	Adenosine	58-61-7		Penicillium sp. YY-20	Analgesic, antiarrhythmic	[29]
6	Anthranilamide	88-68-6		Penicillium No. 97	Fluorescent dyes	[54]
7	Anthranilic acid	118-92-3		Penicillium No. 97	Anticonvulsants	[55]
8	Cyclopaldic acid	477-99-6		Penicillium commune (TMSF169)		[56]
9	Ferulic acid	1135-24-6		Penicillium No. 97	Free radical scavengers, anti-inflammatory agents, antihypertensive agents, anticoagulants	[55]
10	Fructigenine A	144606-96-2		Penicillium No. 97	Inhibits the growth of leukemia cells	[55]
11	Indole-3-acetic acid	87-51-4		Penicillium No. 97	Used for preventing, destroying or mitigating pests	[55]
12	Methyl β-d-ribofuranoside	7473-45-2		Penicillium sp. YY-21	Used to synthesize novel alpha-amino acid esters against herpes simplex virus 1 (hsv-1) and hepatitis b virus	[29]
13	Orsellinic acid	480-64-8		Penicillium No. 97		[29]
14	p-Hydroxybenzoic acid	99-96-7		Penicillium No. 97		[55]
15	β-sitosterol	83-46-5		Penicillium No. 97	Hypolipidemic agents	[55]
16	Quercetin glycoside (orange pigment)	3520-72-7		Penicillium sp.		[34]

The compounds adenosine, deoxyadenosine and adenine which were isolated from the fermentation product of Penicillium sp. YY-20 have a strong scavenging capacity for DPPH free radical [34]. Wu isolated Penicillium cataractum SYPF 7131 from 58 endophytic fungi obtained from the leaves, stems and roots of G. biloba. This strain displayed the strongest antibacterial activity [35].

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.

Table 5

Secondary metabolite of Xylaria in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	7-Amino-4-methylcoumarin	26093-31-2		Xylaria sp. YX-28	A fluorescent dye used to stain biological specimens	[57]
2	Pentadecane	629-62-9		Xylaria sp. YX-28	Treatment of plantar keratosis with medicinal plant in diabetic patients	[57]
3	Quercetin	117-39-5		Xylaria Colletotrichum	Chemotherapy induced oral mucositis; treatment of erosive and atrophic oral lichen planus; chronic obstructive pulmonary disease; gastroesophageal reflux disease	[57]
4	Tetradecane	629-59-4		Xylaria sp.YX-28		[57]
5	Tridecane	629-50-5		Xylaria sp. YX-28		[57]
6	Dibutyl phthalate	84-74-2		Xylaria sp. YX-28	Against the larval trombiculid mite; preventing scrub typhus of topical application in troops	[57]
7	1,3-Diphenyl-2-pyrazoline	2538-52-5		Xylaria sp. YX-28		[57]
8	1-Acetyl-1,2,3,4-tetrahydropyridine	19615-27-1		Xylaria sp. YX-28		[57]
9	Z,Z-7,11-Hexadecadien-1-ol	53963-06-7		Xylaria sp. YX-28		[57]
10	Isosorbide	652-67-5		Xylaria sp. YX-28	Prevention of angina pectoris due to coronary artery disease; short-term reduction of intraocular pressure	[57]
11	Dimethoxy-phenol	91-10-1		Xylaria sp.YX-28	Food Flavoring Agents	[57]
12	1-hydroxymethyl-1,2,3,4,-tetrahydro-naphthalen-2-ol	872824-43-6		Xylaria sp. YX-28		[57]
13	(1,4-Dimethylpent-2-enyl)benzene	951288-80-5		Xylaria sp. YX-28		[57]
14	2,4-Bis(1,1-dimethylethyl)phenol	96-76-4		Xylaria sp. YX-28		[57]
15	3-Phenyl-4-methyl-isoxazol-5(4)-one	875244-90-9		Xylaria sp. YX-28		[57]
16	3,4-Dihydro-8-hydroxy-3-methyl-isocoumarin	1200-93-7		Xylaria sp. YX-28		[57]
17	[l(3-butenylthio)-2-nitroethyl]-benzene	128869-50-1		Xylaria sp. YX-28		[57]
18	Pentadecanoic acid, methyl ester	7132-64-1		Xylaria sp. YX-28	pesticide	[57]
19	14-Octadecenal	56554-89-3		Xylaria sp. YX-28		[57]
20	E-11,13-Dimethyl-12-tetradecen-1-ol acetate	400037-00-5		Xylaria sp. YX-28		[57]
21	Hexadecanoic acid, methyl ester	112-39-0		Xylaria sp. YX-28	Food flavoring agents	[57]
22	n-Hexadecanoic acid	57-10-3		Xylaria sp. YX-28	Inhibits HIV-1 infection; a potential candidate for specifically attack multiple myeloma cells	[57]
23	2-Undecenal	2463-77-6		Xylaria sp. YX-28		[57]
24	Hexadecanoic acid, 14-methyl-methyl ester	2490-49-5		Xylaria sp. YX-28		[57]
25	9,12-Octadecadienoic acid(Z,Z)-methyl ester	112-63-0		Xylaria sp. YX-28	Flavoring agent or adjuvant	[57]
26	9-Octadecenoic acid (Z)-,methyl ester	112-62-9		Xylaria sp. YX-28	Solvents	[57]
27	3,7,11-trimethyl-2,6,10-Dodecatrien-1-ol	4602-84-0		Xylaria sp. YX-28	Inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines	[57]
28	9,12-Octadecadienoic acid (Z,Z)	2197-37-7		Xylaria sp. YX-28	Treats the prevention of preeclampsia;	[57]
29	9-Octadecenamide (Z)	3322-62-1		Xylaria sp. YX-28	Induce drowsiness or sleep or to reduce psychological excitement or anxiety	[57]
30	Pentadecanoic acid,2-hydroxymethy l ester	98863-01-5		Xylaria sp. YX-28	Emulsifier	[57]
31	Ferruginol	514-62-5		Xylaria sp. YX-28	An antineoplastic agent; antibacterial agent; protective agent	[57]
32	9,12-Octadecadienoic acid(Z,Z)-,2-hydroxy-1-(hydroxy methyl)ethyl ester	544-35-4		Xylaria sp. YX-28	Flavoring agents	[57]
33	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	23470-00-0		Xylaria sp. YX-28	Lipid maps classification	[57]
34	Bis(2-ethylhexyl)phthalate	117-81-7		Xylaria sp.YX-28		[57]
35	5,6,8,9,10,11-Hexahydrobenz[A]anthracene	67064-61-3		Xylaria sp. YX-28		[57]
36	1,2,3,4-Tetrahydro-Triphenylene	5981-10-2		Xylaria sp. YX-28		[57]

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].

Table 6

Secondary metabolite of Fusarium in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	Adenosine	58-61-7		Fusarium solani GBT07 GBT07	Terminate paroxysmal supraventricular tachycardia; terminating stable and narrow-complex supraventricular tachycardias; adjunct to thallous chloride TI 201 myocardial perfusion scintigraphy and vagal maneuvers and clinical assessment	[11]
2	Benzeneethanol/Phenylethyl alcohol	60-12-8		Fusarium sp. G1024	Anti-infective agents, local; disinfectants; preservatives, pharmaceutical	[11]
3	Enniatin B	917-13-5		Fusarium sp.		[58]
4	Ginkgolide B	15291-77-7		Fusarium oxysporum		[59, 60]
5	Hexadecane	544-76-3		Fusarium sp. G1024		[11]
6	Kaempferide	491-54-3		Fusarium solani	An antihypertensive agent	[61]
7	Kaempferol	520-18-3		Fusarium oxysporum	A possible cancer treatment; antibacterial agent	[61]
8	Quercetin	117-39-5		Fusarium oxysporum		[57]
9	Rutin	153-18-4		Fusarium oxysporum	A role as an antioxidant; antiallergic; anti-inflammatory; antiproliferative; and anticarcinogenic properties	[61]
10	Soyasapogenol B	595-15-3		Fusarium oxysporum Schlecht GB-1(3)		[61]
11	Tetradecane	629-59-4		Fusarium sp. G1024		[11]
12	β-Sitosterol	83-46-5		Fusarium oxysporum Schlecht GB-1(3)	As anticholesteremic drug; antioxidant; treats hyperlipidemia.	[61]
13	Isorhamnetin	480-19-3		Fusarium sp	Warning; (tyrosinase inhibitor; an anticoagulant)	[62]
14	Decane	124-18-5		Fusarium sp. G1024		[11]
15	2-Ethyl-1-hexanol	104-76-7		Fusarium sp. G1024		[11]
16	2-Butanol,3,3′-oxybis-4-ethylphenol	123-07-9		Fusarium sp. G1024	Flavoring Agents	[11]
17	Dodecane	112-40-3		Fusarium sp. G1024	Increase the risk of neoplasms in humans or animals	[11]
18	1,2-benzisothiazole	272-16-2		Fusarium sp. G1024		[11]
19	4-Ethyl-2-methoxyphenol	2785-89-9		Fusarium sp. G1024	Flavoring agents	[11]
20	p-Nitroacetophenone	100-19-6		Fusarium sp. G1024	Potentiate the effectiveness of radiation therapy in destroying unwanted cells	[11]
21	2,3,5,6-Tetramethyl-p-benzoquinone	527-17-3		Fusarium sp. G1024	product quinones duroquinone	[11]
22	Eicosane	112-95-8		Fusarium sp. G1024	Flavoring Agents.	[11]
23	1,2-Benzenedicarboxylic acid bis(2-methylpropyl)ester	88-99-3		Fusarium sp. G1024		[11]
24	Dibutyl phthalate	84-74-2		Fusarium sp. G1024	Against the larval trombiculid mite; preventing scrub typhus of topical application in troops	[11]

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. Bacillus amyloliquefaciens can produce 8 kinds of compounds [35, 37] which have some biological activities. Two compounds, apigenin-8-C-glucoside and 2-(Hydroxymethylthio) ethanol, were isolated from Colletotrichum sp. NTB-2., in which apigenin-8-C-glucoside has strong inhibitory activity against Bacillus subtilis, Salmonella typhimurium and Pseudomonas cepacia [38]. Moreover, Colletotrichum sp. could produce flavones which exhibited potent anti-cancer, anti-HIV [39] and antioxidant activities [40].

Table 7

Secondary metabolite of other endophytics in Ginkgo biloba

No.	Metabolites	CAS number	Molecular structure	Endophytes	Application	References
1	2-(Hydroxymethylthio)ethanol	876503-58-1		Colletotrichum sp. NTB-2	Platelet aggregation inhibitor, an alpha-glucosidase inhibitor, an antineoplastic agent	[63]
2	Apigenin-8-C-β-d-glucopyranoside	3681-93-4		Colletotrichum sp.		[63, 64]
3	6-Ethoxyl-2,4-amide lactone			Bacillus amyloliquefaciens CGMCC 5569		[64]
4	6-Hydroxylbutyl-2,4-amide lactone			Bacillus amyloliquefaciens CGMCC 5569		[64]
5	6-Hydroxypropyl-2,4-amide lactone			Bacillus amyloliquefaciens CGMCC 5569		[64]
6	Biuret	108-19-0		Bacillus amyloliquefaciens CGMCC 5569	Used for preventing, destroying or mitigating pests	[64]
7	Ginkgolide B	15291-77-7		Oospora wallr. G10	Fibrinolytic agents	[65]
8	2′-Deoxyuridine/uracil deoxyriboside	951-78-0		Unidentified	Antimetabolites	[65]
9	3-Methylpiperazine-2,5-dione	6062-46-0		Unidentified		[65]
10	Adenine	73-24-5		Unidentified		[65]
11	Adenine deoxyriboside			Unidentified		[65]
12	Adenosine	58-61-7		Unidentified	Used as an initial treatment for the termination of paroxysmal Supraventricular tachycardia	[65]
13	Quercetin	117-39-5		Stemphylium sp. Act inomyces	Antioxidants	[37, 66]
				Nodulisporium hyalosporum Schizophyllum commune Fr.		[67]
				Fusella Sacc Alternaria sp Sphacelia sp. Anpelomyces humuli		[37]
				Phoma glomerate		[30, 61]
				Trichothecium		[53]
				Mucor circinelloides		[40]
				Sphaeropsis sp. B301		[68]
14	Kaempferol	520-18-3		Fusella Sacc Alternaria sp. Gibberella sp. Sphacelia sp. Dematium Pers	As a selective estrogen receptor modulator	[66]
				Trichothecium		[53]
				Sphaeropsis sp.		[68]
15	Cerebroside B	88642-46-0		Phyllosticta sp. TP78, (GenBank ID: KC445736)	An antimicrobial compound	[20, 21]
16	Cerebroside C	98677-33-9		Phyllosticta sp. TP78 (GenBank ID: KC445736)	Increases tolerance to chilling injury and alters lipid composition in wheat roots	[20, 21]
17	Enniatin B1	19914-20-6		Tuberculariaceae F1-3	Fusarium mycotoxins	[69]
18	Enniatin D	19893-21-1		Tuberculariaceae sp. F1-3	Inhibition of Botrytis cinerea spore germination	[69]
19	Benzeneethanol/Phenylethyl alcohol	60-12-8		Muscodor albus strain GBA	Anti-bacterial agents and antioxidants. Anti-Infective Agents	[69]
20	Ginkgolide C	15291-76-6		Gloeosporium; Tolura; Phacodium	Reduced lipid accumulation and suppresses adipogenesis	[32]
21	Kaempferide	491-54-3		Phoma glomerata	Reverse bacterial resistance to amoxicillin in AREC	[61]
				Anpelomyces humuli		[61]
22	Rutin	153-18-4		Mucor circinelloides GF521	Used therapeutically to decrease capillary fragility	[61]
				Nodulisporium hyalosporum		[67]
23	Sporothriolide	154799-92-5		Nodulisporium sp. A21	Used to treat the infection caused by candida albicans and cryptococcus neoformans	[55]
24	Isorhamnetin	480-19-3		Stemphylium sp. Alternaria sp Gibberella sp. Trichothecium	prevents endothelial dysfunction, superoxide production, Isorhamnetin appears to be a potent drug against esophageal cancer	[62]
				sphaeropsis		[68]
				Plantactinospora sp. NEAU-gxj3		[20, 21]
25	Antibiotic U-62162	82516-67-4		Plantactinospora sp. NEAU-gxj3	Inhibited the growth of Gram-positive bacteria	[20, 21]
26	Salternamide C	1662688-81-4		sphaeropsis		[68]
27	Abscisic acid	21293-29-8		Phoma betae	Plant Growth Regulator	[69]
28	Taxol	33069-62-4		Phomopsis sp. 2 strain BKH 30 (BSL No. 72)	An antineoplastic agent, tubulin modulators	[70]
				Muscodor albus strain GBA		[69]
29	Acetic acid, methyl ester	79-20-9		Muscodor albus strain GBA		[69]
30	2-Butanone	78-93-3		Muscodor albus strain GBA	Polar aprotic solvent	[69]
31	Acetic acid, 2-methylpropyl ester	110-19-0		Muscodor albus strain GBA	An antifungal agent	[71]
32	1-Propanol, 2-methyl	78-83-1		Muscodor albus strain GBA	Possesses nicotine-like synaptotropic actions on the nervous systems	[71]
33	1-Butanol, 3-methyl-,acetate	123-92-2		Muscodor albus strain GBA		[71]
34	Cyclohexane,1-methyl-4-methylene	2808-80-2		Muscodor albus strain GBA		[69]
35	2,3-Dimethyl-3-isopropyl-cyclopentene	73331-73-4		Muscodor albus strain GBA		[69]
36	1-Butanol, 3-methyl	123-51-3		Muscodor albus strain GBA		[69]
37	Pyrrolidine	123-75-1		Muscodor albus strain GBA		[72]
38	Germacrene B	15423-57-1		Muscodor albus strain GBA		[72]
39	α-Sinensal	17909-77-2		Muscodor albus strain GBA		[69]
40	Propanoic acid, 2-methyl	79-31-2		Muscodor albus strain GBA		[73]
41	Trans-caryophyllene	87-44-5		Muscodor albus strain GBA	Anti-inflammatory agents	[73]
42	4-Piperidinone, 1-methyl	1445-73-4		Muscodor albus strain GBA		[73]
43	Acetic acid, 2-phenylethyl ester	103-45-7		Muscodor albus strain GBA		[73]
44	(+)-Vitrene	90250-82-1		Muscodor albus strain GBA		[73]

In recent years, some new ginkgo endophytes and secondary metabolites have been discovered. Guo et al. [20, 21] discovered a new amide compound from Plantactinospora sp. NEAU-gxj3, Cao et al. [22] found the metabolite sporothriolide from the Nodulisporium of G. biloba, which has anti-phytopathogenic activity.

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.

Fig. 7

The production of Ginkgo extracts in China and its proportion in the world market. a The production of Ginkgo extracts in China from 2015 to 2019; b the proportion of China Ginkgo products in the world market from 2014 to 2018

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.