Biological Activities of Some New Secondary Metabolites Isolated from Endophytic Fungi: A Review Study

Secondary metabolites isolated from plant endophytic fungi have been getting more and more attention. Some secondary metabolites exhibit high biological activities, hence, they have potential to be used for promising lead compounds in drug discovery. In this review, a total of 134 journal articles (from 2017 to 2019) were reviewed and the chemical structures of 449 new metabolites, including polyketides, terpenoids, steroids and so on, were summarized. Besides, various biological activities and structure-activity relationship of some compounds were aslo described.


Introduction
During the growth of microorganisms, some secondary metabolites biologically active are produced to make their lives better. Using chemical and biological methods, Elshafie et al. displayed that the cell-free culture filtrate of Burkholderia gladioli pv. agaricicola (Bga) Yabuuchi has a promising antibacterial activity against the two microorganisms B. megaterium and E. coli [1]. Camele et al. reported that the tested isolate of an endophytic bacterium Bacillus mojavensis showed antagonistic bacterial and fungal activities against several strains as well as biofilm formation ability [2]. Endophytes refer to the microorganisms that exist in various organs, tissues or intercellular space of plants, while the host plants generally do not show any symptoms of infection. Generally speaking, endophytes include endophytic fungi, endophytic bacterium and endophytic actinomycetes [3]. As a very important microbial resource, endophytes exist widely in nature. It is ubiquitous in various terrestrial and aquatic plants. Endophytes have been isolated from bryophytes, ferns, pteridophytes, hornworts, herbaceous plants and various woody plants. The region also ranges from tropical to arctic, from natural wild to agricultural industry ecosystem [4]. They have unique physiological and metabolic mechanisms, which enable them to adapt to the special environment inside plants, and at the same time, they can encode a variety of bioactive substances. In addition, endophytes coevolved with the host plants for a long time to produce some metabolic substances similar or identical to the host plants with medicinal value [5]. Some endophytes can even assist the host of medicinal plants to synthesize effective active compounds, the ground-breaking discovery provides a new method to produce the effective compounds which have similar effects with natural medicines isolated from plant tissues directly. At the same time, it has solved the problem of resource shortage and ecological destruction caused by slow growth of some natural plants and large amount of artificial exploitation [3]. The more beneficial thing is that some of them are environmentally friendly. Elshafie et al. have studied the fungus Trichoderma harzianum strain T22 (Th-T22) and indicated that Th-T22 showed significant mycoremediation ability in diesel-contaminated sand, suggesting that it can be used as a bioremediation agent for diesel spills in polluted sites [6]. Among the common endophytes, the endophytic fungi are most often isolated [4]. The first endophytic fungus was isolated from Perennial study on endophytic fungi has a long history of more than 100 years, but the research on endophytic fungi of medicinal plants has not been formally carried out until the last 30 years, which has gradually attracted the attention of domestic and foreign scholars.
The multiformity of endophytes enable they can produce a variety of secondary metabolites. In recent years, the metabolites isolated from the endophytic fungi include alkaloids, steroids, terpenes, anthraquinones, cyclic peptides, flavonoids commonly [5]. Some secondary metabolites exhibit high biological activities. The antitumor, antibacterial, antiinflammatory, antiviral, antifungal and other compounds have been produced by different endophytic fungi. Therefore, the chemical variety of secondary metabolites produced by endophytic fungi has advantage for new drug development [8].
In this review, 449 new secondary metabolites, together with their chemical structures and biological activities were summarized. The structure-activity relationships and absolute configureuration of some compounds have also been described. Among all new compounds, terpenoids account for the largest proportion (75%), followed by polyketones (36%). The proportion of different types of compounds in all new compounds is shown in Figure 1. These new compounds were isolated from various fungi associated with different tissues from different plants. As a result, their structures varied a lot, which leads to their multitudinous biological activities. In addition to common antimicrobial activity and anti-tumor activity, some compounds also showed anti-enzyme activity and inhibition of biofilm formation, inhibition of phytoplankton growth, and so on.
Compounds  [16]. The antifungal assay displayed that 1 and 5 exhibited pronounced biological effects against F. oxysporum with MIC (minimum inhibitory concentration) value of 8 g/mL, whereas 5 can potently inhibited F. gramineum at concentration of 8 g/mL, compared with the positive control ketoconazole (MIC value of 8 g/mL) [9].
Compounds 68-74 were assayed for their antifungal activities against C. albicans. Geneticin (G418), was used as positive control with the MIC value of 6.3 µg/mL. Compound 69 displayed inhibitory effect against C. albicans with an MIC value of 12.5 µg/mL, while compounds 68 and 74 exhibited weak inhibitory effect against C. albicans with MIC values of 100 µg/mL and 150 µg/mL [32].
Antifungal activities (Minimum inhibitory concentrations; MICs) of the isolated metabolite 170 were determined using a serial dilution assay against Mucor hiemalis DSM 2656. Compound 170 showed moderate to weak antifungal activity against Mucor hiemalis DSM 2656 with a MIC value of 33.33 µg/mL [52].
One fungus Candida albicans (ATCC 10231) was used for antifungal tests, the results showed that compound 177 exhibited significant antifungal activity against C. albicans with the MIC value of 2.62 µg/mL. The positive control for antifungal tests was used by ketoconazole with MIC value of 0.10 µg/mL [54].
The methylated dihydropyrone 189 and compound 274 were tested for in vitro antifungal activity using the Oxford diffusion assay against M. violaceum (Microbotryum violaceum) and S. cerevisiae (Saccharomyces cerevisiae), 189 and 274 exhibited moderate antifungal activity, inhibiting the growth of S. cerevisiae and M. violaceum at 25 µg/mL. Nystatin was the positive control for antifungal assays, previous studies had shown the MIC values of nystatin in the S. cerevisiae culture used was 4 µg/mL and for M. violaceum was 2 µg/mL [58].
Prochaetoviridin A 230 was evaluated for its antifungal activities against 5 pathogenic fungi S. sclerotiorum, B. cinerea, F. graminearum, P. capsici and F. moniliforme at the concentration of 20 µg/mL. It showed moderate antifungal activity with inhibition rates ranging from 13.7% to 39.0% [69].
Compounds 244 and 245 were evaluated against phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum (Cladosporium sphaerospermum) using direct bioautography. The results showed that 244 exhibited antifungal activity, with a detection limit of 5 µg, for both fungi, while compound 245 displayed weak activity (detection limit > 5 µg), with a detection limit of 25 µg. Nystatin was used as a positive control, showing a detection limit of 1 µg [80].
Compound 266 was tested for antimicrobial activities against two plant-pathogenic fungi Fusarium oxysporum f. sp. momordicae nov. f. and Colletotrichum gloeosporioides, and exhibited potent activity against both strains with MIC values of 5 µM, which was close to that of the positive control, amphotericin B (MIC = 0.5 µM) [77].
The purified metabolite 293 was tested for antimicrobial activity against selected pathogens namely C. albicans. Funiculosone (293) displayed antimicrobial activity inhibiting fungal pathogens. Funiculosone was able to inhibit the growth of C. albicans with an IC 50 (50% inhibitory concentration) of 35 µg/mL [95].
Antifungal activity was determined against C. neoformans ATCC90113. The results showed that globosuxanthone E 294 displayed antifungal activity against Cryptococcus neoformans ATCC90113 with the MIC value of 32 µg/mL. Amphotericin B was used as a positive control for antifungal activity and exhibited an MIC value of 0.5 µg/mL [96].
The isolated compound 349 was evaluated for antifungal activities against C. neoformans and P. marneffei, it displayed weak antifungal activity against C. neoformans with MIC value of 32 µg/mL. Amphotericin B was used as positive control for fungi, displayed the MIC values of 1.0 µg/mL and 2.0 µg/mL against C. neoformans and P. marneffei [63].
Three fungi (Aspergillus flavus, Fusarium oxysporum and Candida albicans) were used in antifungal activity tests by disk diffusion method, the antifungal activity was recorded as clear zones of inhibition surrounding the disc (mm). Compound 362 showed antifungal activity against F. oxysporum (zone of inhibition was 6 mm) and variable activities against A. flavus and the yeast C. albicans (zone of inhibition was 5 mm). Nystatin (10 mg/disc) was used as standard antifungal (zone of inhibition against A. flavus and F. oxysporum were 12 mm and 17 mm) [116].
In search for novel antifungal compounds, 368 and 369 were tested against C. neoformans and C. gattii. Compounds 368 and 369 exhibited moderate antifungal activities against Cryptococcus neoformans and Cryptococcus gattii, each with minimum inhibitory concentration values of 50.0 µg/mL and 250.0 µg/mL, respectively [120].
The antifungal activity of the compound 374 were evaluated against fungal strains Phyllosticta citricarpa LGMF06 and Colletotrichum abscissum LGMF1268 in order to select the best culture conditions to produce bioactive secondary metabolites. The isolated compound 374 displayed antifungal activity against the citrus phytopathogen Phyllosticta citricarpa with the inhibition zone of 30 mm. Amphotericin B was used as positive control with the inhibition zone of 37 mm [123].
Compound 418 was tested for antimicrobial activities against five plant-pathogenic fungi A. brassicae, Colletotrichum gloeosprioides, Fusarium oxysporum, Gaeumannomyces graminis, and P. piricola. It exhibited inhibitory activity against A. brassicae and P. piricola with the same MIC value of 64 µg/mL. The positive control against A. brassicae and P. piricola was amphotericin B with MIC values of 4 µg/mL and 8 µg/mL respectively [136].
Antifungal activity was determined against C. neoformans ATCC90113. Simplicildone K 430 and globosuxanthone E 431 displayed weak antifungal activity against Cryptococcus neoformans ATCC90113 with the same MIC values of 32 µg/mL. Amphotericin B was used as a positive control for antifungal activity and exhibited an MIC value of 0.5 µg/mL against C. neoformans ATCC90113 [96].
The antimicrobial activity was determined by the paper disk diffusion method (100 µg compound in 8 mm paper disk), using meat peptone agar for Staphylococcus aureus and Pseudomonas aeruginosa, peptone yeast agar for Candida albicans, and potato dextrose agar for Aspergillus clavatus. 164 showed moderate antibacterial activity against Staphylococcus aureus NBRC 13276 (5: 24 mm) at a concentration of 100 µg/disk (MIC value: 3.2 µg/mL). Chloramphenicol was used for positive control against S. aureus (1 µg/mL) [50].
Antimicrobial activities (Minimum inhibitory concentrations; MICs) of the isolated metabolite 170 was determined using a serial dilution assay against Bacillus subtilis DSM [52].
Antimicrobial tests were used for the disc diffusion method. Two Gram-positive methicillin-resistent Staphylococcus aureus, Bacillus subtilis (ATCC 6633), two Gram-negative pseudomonas aeruginosa (ATCC 9027), Salmonella typhimurium (ATCC 6539), were used. Compound 176 showed strong antibacterial activity against the P. aeruginosa and MRSA with the MIC values of 1.67 µg/mL and 3.36µg/mL, respectively. Compound 177 exhibited significant antibacterial activity against B. subtilis with the MIC value of 5.25 µg/mL.
Positive control for antifungal tests were used by Ampicillin with the MIC values of 0.15 µg/mL, 0.15 µg/mL and 0.07 µg/mL against P. aeruginosa, MRSA (Methicillin-resistant Staphylococcus aureus) and B. subtilis, respetively. The results indicated that the methylester displayed improved biological activity and showed a selective antibacterial activity against P. aeruginosa and MRSA. Compound 176 exhibited more strong antimicrobial activity than compound 177 [54].
Antimicrobial activity testing of the compound 212 was carried out against a set of microorganisms using paper-disk diffusion assay. 212 exerted moderate-high activities (13 mm, 16 mm, 15 mm, 10 mm, 11 mm and 14 mm) against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Saccharomyces cerevisiae, Bacillus cereus and Bacillus subtilis ATCC 6633. Gentamycin was used as positive control with the diameter of agar diffusion of 22 mm, 18 mm, 17 mm, 23 mm, 20 mm and 18 mm against the 5 bacteria as mentioned above [68].
Minimum Inhibitory Concentration (MIC) assays were used to assess antibacterial activity of the isolated compounds 227-228 against human pathogens (Escherichia coli, Micrococcus luteus, and Pseudomonas aeruginosa) and plant pathogen (Ralstonia solanacearum). Chloromycetin was used as a positive antibacterial control. Notably, compound 227 demonstrated potent activity against P. aeruginosa with an MIC value of 1 µg/mL, which was better than that of the positive control chloromycetin (MIC = 4 µg/mL). Compound 228 displayed activity against Micrococcus luteus and Pseudomonas aeruginosa with the same MIC value of 8 µg/mL (2 µg/mL and 4 µg/mL against Micrococcus luteus and Pseudomonas aeruginosa for Chloromycetin). In contrary to compounds 228 and the known compound A (Figure 13), B ( Figure 13) showed stronger antibacterial activity (MIC values of 4, 4, 8, and 8 µg/mL against E. coli, M. luteus, P. aeruginosa, and R. solanacearum, respectively), indicating that hydroxylation at C-10 can augment antibacterial activity [74].  [66].
Antimicrobial activity testing of the compound 212 was carried out against a set of microorganisms using paper-disk diffusion assay. 212 exerted moderate-high activities   Compound 229 was tested for in vitro antimicrobial activity against 2 bacteria B. subtilis (ATCC 23857), and E. coli (ATCC 67878). Chloramphenicol was the antibacterial positive control. 229 showed modest antibiotic activity to E. coli with an MIC value of 100 µg/mL [58].
Antimicrobial activities were determined against four terrestrial pathogenic bacteria, including Pseudomonas aeruginosa, Methicillinresistant Staphylococcus aureus, Bacillus subtilis and Escherichia coli by the microplate assay method. Compound 231 exhibited modest antibacterial activity against Escherichia coli and Pseudomonas aeruginosa with 12.5 µg/mL, 50 µg/mL, respectively [75].
Antimicrobial activity was estimated by the inhibitory zone to five indicator microorganisms (Bacillus subtilis CMCC 63501, Candida albicans CMCC 98001, Escherichia coli CMCC 44102, Pseudomonas aeruginosa CMCC 10104 and Staphylococcus aureus CMCC 26003). Compounds 237 and 238 exhibited growth inhibitory activity against E. coli with MIC values of 32 µg/mL. Chloramphenicol was used as positive control with an MIC value of 4 µg/mL against E. coli [76].
Compounds 253, 289-291 were assayed for their antibacterial activities against Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium. All of the four compounds exhibited antibacterial activities against Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus with the same MIC values of 25 µg/mL, 50 µg/mL and 25 µg/mL, respectively. Ampicillin was the positive control against bacteria, the MIC of ampicillin was lower than 0.78 µg/mL against Salmonella typhimurium, and Staphylococcus aureus, while the MIC value against Escherichia coli was 100 µg/mL [83].
The antimicrobial activity was determined by the paper disk diffusion method (100 µg compound in 8 mm paper disk), using meat peptone agar for Staphylococcus aureus and Pseudomonas aeruginosa. Comound 287 exhibited antibacterial activity against S. aureus and P. aeruginosa with MIC values (µg/mL) of >50 and 6.25. Chloramphenicol and kanamycin were used for positive control against S. aureus and P. aeruginosa (each 1 µg/mL), respectively [31].
Compound 293 was tested for antimicrobial activity against selected pathogens namely S. aureus, E. coli and Pseudomonas aeruginosa C. Gessard. Funiculosone (293) displayed antimicrobial activity inhibiting the bacterial pathogens. Funiculosone was able to inhibit the growth of E. coli, S. aureus and C. albicans with IC 50 of 25 µg/mL and 58 µg/mL and 35 µg/mL respectively [95].
The antibacterial activities of pure compound 309 was evaluated against Grampositive bacteria Staphylococcus aureus and Bacillus subtilis and Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli using the disk diffusion assay. The new com-pound 309 showed inhibitory activity against S. aureus at 0.04 µg/paper disk, and the diameter of inhibition zone was 0.71 cm. The MIC for compound 309 against S. aureus was 100 µg/mL using the broth microdilution method, while streptomycin was employed as the positive control with an MIC of around 50 µg/mL [102].
Two Gram-positive bacteria Bacillus subtilis (ATCC6633) and Staphylococcus aureus ATCC (25923) were used. The antibacterial assay and the determination of the minimum inhibitory concentration (MIC) were determined according to continuous dilution method in the 96-well plates. Compound 313 showed antibacterial activity against Bacillus subtilis with an MIC value of 12.5 µg/mL. Ciprofloxacin was the positive control [104].
Compound 318 was tested for antibacterial activity against Mycobacterium marinum ATCCBAA-535. Although rifampin as positive control showed significantly in vitro antibacterial activity against Mycobacterium marinum ATCCBAA-535 with IC 50 of 2.1 µM, compound 318 also exhibited potential inhibitory activity with IC 50 of 64 µM [106].
The antibacterial activities of the isolated compounds 325-329 were evaluated against the soil bacterium Acinetobacter sp. BD4 (Gram-negative), the environmental strain of Escherichia coli (Gram-negative), as well as human pathogenic strains of Staphylococcus aureus Antibacterial activity of the new compound 330 against Vibrio parahaemolyticus and Vibrio anguillarum was determined by the conventional broth dilution assay. 330 showed moderate inhibitory effects on Vibrio parahaemolyticus with an MIC value of 10 µg/mL. Ciprofloxacin was used as a positive control [110].
Antibacterial efficacies of the metabolite 339 were determined by serial dilution assay. Compound 339 showed strong activity against Bacillus subtilis and Micrococcus luteus with MIC values of 8.33 µg/mL and 16.66 µg/mL, respectively, while the MIC values of Oxytetracyclin used as the positive control against Bacillus subtilis and Micrococcus luteus were 4.16 µg/mL and 0.40 µg/mL, respectively. While the MIC value of compound C ( Figure 13) against Mucor hiemalis (16.66 µg/mL) was the same as that of nystatin used as positive control. The two active metabolites are anthranilic acid derivatives with a phenylethyl core. Since metabolite 340, which contains a phenylmethyl group instead of a phenylethyl residue, was not active, it was concluded that the phenylethyl moiety in compounds 339 and C is essential for their antimicrobial activity [60].
The isolated compound 347, which was obtained in sufficient amounts, was evaluated for antimicrobial activities against S. aureus ATCC25923 and methicillin-resistant S. aureus. Simplicildone A 347 displayed weak antibacterial against Staphylococcus aureus with MIC value of 32 µg/mL. Vancomycin which was used as positive control for bacteria, displayed the MIC values of 0.5 µg/mL and 1.0 µg/mL against both S. aureus and methicillin-resistant S. aureus [63].
The antimicrobial activity of compound 367 was evaluated using the strains of methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bacillus subtilis, and Escherichia coli. Compound 367 exhibited weaker activity in comparison to the positive control tetracycline against methicillin-resistant S. aureus (MRSA) with the MIC value of 128 µg/mL, and against K. pneumoniae and P. aeruginosa with equal MIC values of 32 µg/mL [119].
The antimicrobial activity of the compound 374 was evaluated against the Grampositive bacteria Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA) (BACHC-MRSA). The resulting inhibition zones were measured in millimeters. 374 displayed antibacterial activity against sensitive and resistant S. aureus, the diameter of inhibition zone was 14 mm, Ampicillin was antibacterial control with the diameter of inhibition zone of 30 mm [123].
Antibacterial activity was evaluated against S. aureus and methicillin-resistant S. aureus. Simplicildone K 430 exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus with equal MIC values of 128 µg/mL. Vancomycin was used as a positive control for antibacterial activity and displayed equal MIC values of 0.5 µg/mL against both S. aureus and methicillin-resistant S. aureus [96].

Antiviral Activity
Anti-enterovirus 71 (EV71) was assayed on Vero cells with the CCK-8 (DOjinDo, Kumamoto, Japan) method. The 50% inhibitory concentration (IC 50 ) of the testing compound was calculated using the GraphPad Prism software. Ribavirin was used as the positive control with an IC 50 value of 177.0 µM. Vaccinol J 125 exhibited in vitro anti-EV71 with IC 50 value of 30.7 µM, and the inhibition effect was stronger than positive control ribavirin [44].
Anti-HIV activities of compound 150 was tested in vitro by HIV-I virus-transfected 293 T cells. At the concentration of 20 µM, 150 showed a weak inhibitory rate of 16.48 ± 6.67%. Efavirenz was used as the positive control, with an inhibitory rate of 88.54 ± 0.45% at the same concentration [47].

Cytotoxic Activity or Anticancer
Nectrianolins A-C 11, 12, and 13 were evaluated for their in vitro cytotoxicity against HL60 (human leukemia 60) and HeLa cell lines by the MTT method using a published protocol. Compounds 11, 12, and 13 exhibited cytotoxic activity against the HL60 cell line with IC 50 values of 1.7 µM, 1.5 µM and 10.1 µM, respectively. Additionally, compounds 11, 12, and 13 exhibited cytotoxicity against the HeLa cell line with IC 50 values of 34.7 µM, 16.6 µM and 52.1 µM, respectively [13].
Compounds 29 and 236 were evaluated for their cytotoxic activities against three human tumor cell lines HeLa, HCT116 (Human colon cancer tumor cells), and A549 (Human lung cancer cells), both of them exhibited weak to moderate cytotoxic activities with IC 50 [19].
The cytotoxic activity of the isolated compounds 78-79 and 113-114 were tested against Hela cells. Compound 79 showed weak cytotoxic activities against Hela cells with IC 50 value of 43.7 ± 0.43 µM. Compound 78 did not show significant cytotoxic activity. As the oxoindoloditerepene epimers, the 3α-epimer 79 was clearly more cytotoxic than the 3β-epimer 78, suggesting that their cytotoxic activity depended on their stereochemistry. The acetoxy derivatives 113 and 114 showed weak cytotoxic activities against Hela cells with IC 50 values of 83.8 ± 5.2 µM and 53.5 ± 2.1 µM respectively [35].
Since many triterpenoids isolated from plants of the family Schisandraceae are reported to reduce the risk of liver diseases and cancer, compounds 93-100 were evaluated for in vitro cytotoxicity against human hepaticellular liver carcinoma cell (HepG2), according to the MTT method, with cisplatin as the positive control (IC 50 value of 9.8 ± 0.21 µM). It should be noted that those metabolites 93-100 produced during fermentation showed stronger cytotoxicity to HepG2 cell line than that of nigranoic acid, the main component of non-fermented K. angustifolia [39].
The in vitro cytotoxicity of compound 119 against the human acute monocytic leukemia cell line (THP-1) was evaluated using a resazurin-based assay and an ATPlite assay. Compound 119 demonstrated marked cytotoxicity against the human acute monocytic leukemia cell line (THP-1) with the IC 50 value of 8.0 µM [42].
Standard MTT assays employing MDA-MB-435 and A549 cell lines were performed. The IC 50 was determined by a 50% reduction of the absorbance in the control assay. would play an important role in cytotoxic activity. Additionally, the activity profile reflected that the hydroxyl-substituted position had a different impact on cytotoxic activity. 2-Pyrones were more active as cytotoxic agents if the alkyl chain at C-6 was oxygenated but the addition of the hydroxyl subunit to C-8 and C-9 significantly decreased the activity [59].
The isolated compound 202 was preliminary evaluated for its cytotoxicities against MCF-7, NCI-H460, HepG-2, and SF-268 cell lines with cisplatin as the positive control. The new compound 202 exhibited weak growth inhibitory activity against the tumor cell lines MCF-7 and HepG-2 with IC 50 values of 70 and 60 µM, respectively [64].
Cytotoxic activities of compound 209 against HeLa, MCF-7 and A549 cell lines were evaluated by the MTT method. Adriamycin was used as a positive control. The results showed that 209 displayed cytotoxic activity against A549 cell lines with IC 50 value of 15.7 µg/mL [66].
Compound 221 was assessed for its antiproliferative activities against the mouse lymphoma (L5178Y) cell line using the in vitro cytotoxicity (MTT) assay and kahalalide F as a standard antiproliferative agent (IC 50 (Figure 13) was found to be inactive (>50 µM), which suggested -NH 2 group might play a very important role for their cytotoxicity. Doxorubicin (Adriamycin) was used as positive control in this assay (IC 50 values against the 4 human cancer cell lines: 0.43 ± 0.12 µM, 0.61 ± 0.09 µM, 0.41 ± 0.11 µM and 0.25 ± 0.08 µM respectively) [72].
The isolated compound 287 was examined for cytotoxic activity by MTT assay. Camptothecin was used as positive control for HL60 with IC 50 = 23.6 nM. 287 exhibited cytotoxicity against human promyelocytic leukemia HL60 cells with IC 50 value of 1.33 µM. The higher cytotoxicity of 287 and E (Figure 13) compared to that of the related compounds F ( Figure 13) and G (Figure 13) was attributed to their increased cell membrane permeability due to the presence of the hydroxyl group [69].
The cytotoxicities of compound 297 were tested by using human promyelocytic leukemia HL-60, human hepatoma SMMC-7721, non-small cell lung cancer A-549, breast cancer MCF-7 and human colorectal carcinoma SW4801 cell lines, 297 showed cytotoxicity against MCF-7 with the ratio of inhibition at 72% for a concentration at 40 µM (IC 50 of positive control Taxol < 0.008 µM) [98].
The cytotoxicities of compound 311 were evaluated against the A549 and HepG2 cell lines by the MTT method. Newly isolated compound 311 showed weak activities with IC 50 values of 11.05 µM and 19.15 µM, respectively, against the tested cell lines. Doxorubicin was used as a reference (0.94 µM and 1.16 µM) [103].
The obtained compound 320 was evaluated for its cytotoxic activities against A549 human lung cancer cells and HepG2 human liver cancer cells. Compound 320 exhibited potent cytotoxic activities towards A549 human lung cancer cells and HepG2 human liver cancer cells with IC 50 values of 23.73 ± 3.61 µM and 35.73 ± 2.15 µM, respectively [90].
The anti-tumor activities of compounds 336-337 were evaluated against Ramos and H1975 cell lines. 337 displayed the most promising anti-tumor activity against both Ramos and H1975 cell lines with IC 50 values of 0.018 µM and 0.252 µM, respectively. Compound 337 may be more effective in anti-tumor activity against Ramos and H1975 than stand drug Ibrutinib and afatinib, with IC 50 values of 28.7 µM and 1.97 µM. These findings suggest that compound 337 might be promising lead for leukemia and lung cancer treatments. In addition, 336 also displayed anti-tumor activity against both Ramos and H1975 cell lines with IC 50 values of 17.98 and 7.3 µM, respectively [113].
Compound 363 was evaluated for its cytototoxicity against different cancer cell lines MOLT-4, A549, MDA-MB-231and MIA PaCa-2 by MTT assay. Interestingly, compound 363 showed considerable cytotoxic potential against the human leukaemia cancer cell line (MOLT-4) with IC 50 value of 20 µmol/L, it was not as active as the positive control flavopiridol (IC 50 value of 0.2 µmol/L) [117].
The in vitro cytotoxicity assay was performed according to the MTS method in 96-well microplates. Five human tumor cell lines were used: human myeloid leukemia HL-60, human hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7, and human colon cancer SW480, which were obtained from ATCC (Manassas, VA, USA). Cisplatin was used as the positive control for the cancer cell lines (IC 50

Other Activities
α-Glucosidase inhibitors are helpful to prevent deterioration of type 2 diabetes and for the treatment of the disease in the early stage, so the α-glucosidase inhibitory effects of the isolated compounds were evaluated. As a result, compounds 247, 248 exhibited potent α-glucosidase inhibitory activity with IC 50 values of 25.8 µM, 54.6 µM, respectively, which were much better than acarbose (IC 50 (Figure 13), compounds 247 and I showed potent α-glucosidase inhibitory effects, whereas J and K were inactive, which attested that the position of the hydroxyl group had a significant impact on the activity [10].
AChE inhibitory activities of the compound 14 were assayed by the spectrophotometric method. Compound 14 indicated anti-AChE activity with inhibition ratio at 35% in the concentration of 50 µM. Tacrine (Sigma, purity > 99%) was used as a positive control of inhibition ratio at 52.63% with the concentration of 0.333 µM [14].
The inhibition of the marine phytoplankton Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense by 31-37 were assayed. The results showed that 32-34 were more active to C. marina, K. veneficum, and P. donghaiense 3.7, 6.9, 9.4 and 12 µg/mL). A structure-activity relationship analysis revealed that the phenyl group in 32-34 may contribute to their inhibitory ability, but the isomerization at C-9 and/or C-11 of 32-37 only has slight influences on their activities. K2Cr2O7 was used as positive control with IC 50 values of 0.46, 0.98, 0.89 and 1.9 µg/mL, respectively [21].
The biological effects of compound 38 were evaluated on the seedling growth of Arabidopsis thaliana, and 38 displayed an effect on the root growth but no remarkable inhibition of leaf growth in Arabidopsis thaliana [22].
Nuclear transcription factor (PXR) can regulate a suite of genes involved in the metabolism, transport, and elimination of their substances, such as CYP3A4 and MRP, therefore, it is regarded as an important target to treat cholestatic liver disorders. So compound 76 was assayed for agonistic effects on PXR. Compound 76 displayed the significant agonistic effect on PXR with EC 50 value of 134.91 ± 2.01 nM [33].
Brine shrimp inhibiting assay was assayed. Compound 80 displayed brine shrimp inhibiting activities with IC 50 value of 10.1 µmol/mL. The SDS (sodium dodecyl sulfate) was employed as positive control and its inhibiting ratio was 95% for brine shrimp and LC 50 0.6 µmol/mL [36].
Monitoring the NO level in LPS-activated cells has become a common approach for evaluating the potential anti-inflammatory activities of compounds. Isolates 82-92 were evaluated for their inhibitory activity against NO production in LPS-activated RAW 264.7 marcrophages, while indomethacin was used as a positive control. Compounds 89-91 exhibited inhibitory effects with IC 50 values of 21, 24 and 16 µM, respectively, which are lower than that of the positive control indomethacin (IC 50 = 38 ± 1 µM), while compound 85 exhibited moderate inhibition with an IC 50 value of 42 µM. Preliminary structure-activity relationships revealed that the analogues with the S absolute configureuration at C-18 (e.g., The tested compounds 200, 276, 344-346 were investigated for their capacity to inhibit biofilm formation in the reference strains of S. aureus, E. faecalis and E. coli. Aacetylquestinol 276, 345 and 200 were found to cause a significant reduction inbiofilm production by E. coli ATCC 25922 with the percentage of biofilm formation: 50.6 ± 17.6%, 23.7 ± 24.8% and 57.6 ± 8.1%, respectively. On the other hand, emodin 344 and 345 showed inhibition of biofilm production in S. aureus ATCC 25923 (21.1 ± 11.5% and 21.8 ± 18.9%). Interestingly, 345, which is the most effective in inhibiting biofilm formation in E. coli ATCC 25922, also caused nearly 80% reduction of the biofilm production in S. aureus ATCC 25923 [62].
Compound 207 was evaluated for its acetylcholinesterase (AChE) inhibitory activity using the Ellman colorimetric method, it showed weak AChE inhibitory activity with the inhibition ratio of 11.9% at the concentration of 50 µmol/mL [65].
The anti-inflammatory activities of the isolated compounds 210-211 were evaluated by measuring the inhibitory activity of nitric oxide (NO) production levels in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. 210-211 exhibited moderate inhibitory activities on NO production in LPS-stimulated RAW264.7 cells without cell cytotoxicities [67].
The transformed products 224-225 and the parent compound L ( Figure 13) were evaluated for the neuroprotective activity using the LPS-induced neuro-inflammation injury assay. 224-225 exhibited moderate neuroprotective activity by increasing the viability of U251 cell lines with EC 50 values of 35.3 ± 0.9 nM and 32.1 ± 0.9 nM, respectively, while L (EC 50 = 8.3 ± 0.4 nM) exhibited comparable activity with the positive control ibuprofen (EC 50 = 19.4 ± 0.7 nM). The transformed products 224-225 and L all exhibited considerable neuroprotective activity in the invitro LPS-induced neuro-inflammation injury assay, suggesting that the hupA moiety shared by these compounds may be used as a lead structure for the development of neuroprotective drugs [73].
The artificial insect mixed drug method was used to determine the insecticidal activities of compound 228. Compound 228 displayed remarkable insecticidal activities against first instar larvae of the cotton bollworm Helicoverpa armigera with mortality rates of 70.2%. Commercially-available matrine was used as positive control, causing 87.4% mortality rate under the same conditions. Acute cytotoxicity towards hatching rate, malformation and mortality of zebrafish embryos or larvae were also performed. Compounds 227 and 228 significantly decreased the hatching rate of zebrafish embryos, compound 228, used at concentrations of 5-100 µg/L, decreased the hatching rate of zebrafish embryos to below 20% [74].
The potential phytotoxicity of 246 against lettuce seedlings (Lactuca sativa L.) was studied. Aqueous solutions of 246 ranging between 25 and 200 µg mL −1 , were assayed for its effects on seed germination, root length, and shoot length of the lettuce. Compound 246 showed the most robust inhibitory effect on root growth. Compound 246 inhibited root growth by 50% at a concentration of 25 µg/mL. In addition, the highest concentration of 246 (200 µg/mL) strongly exerted an inhibitory effect on seed germination (90% inhibition) [81].
Compounds 256-257 were investigated for their inhibitory activities against the LPSactivated production of NO in RAW264.7 cells using the Griess assay with indomethacin as a positive control (IC 50 = 37.5 ± 1.6 µM). The effects of compounds on cell proliferation/viability were determined using MTT method, and none of the test compounds exhibited cytotoxicity at their effective concentrations. Compounds 256 and 257 showed strong inhibitory effects on the production of NO, with IC 50 values of 0.78 ± 0.06 and 1.26 ± 0.11 µM, respectively [84].
The Indoleamine 2,3-dioxygenase (IDO) inhibitory activity assay of compounds 284-286 were carried out. The results showed that compound 285 possessed significant inhibitory activity against IDO with IC 50 value of 0.11 µM. Epacadostat, as the positive control, was one of the most potent IDO inhibitors with IC 50 value of 0.05 µM. For compounds 284 and 286, they showed relatively strong inhibitory activity with IC 50 values of 1.47 µM and 6.36 µM, respectively [92].
NF-κB has been considered as an attractive therapeutic target for the cancer research. Compound 288 was investigated for its effects on NF-κB pathway by reporter gene assay. The results showed that it could activate the NF-κB pathway with increments in the relative luciferase activity at a concentration of 50 µM [93].
The phytotoxic activities of 295 and 296 were investigated by seed germination test on lettuce (Lactuca sativa L.) with 2,4-dichlorophenoxyacetic acid (0.3 µg/mL) as the positive control. Compounds 295 and 296 each inhibited the growth of both roots and hypocotyls at 30 µg/mL. Furthermore, 295 suppressed seed germination at 100 µg/mL [97].
Acetylcholinesterase (AChE) inhibitory activities of the compound 302 were assayed by the spectrophotometric method developed by Ellman with modification. 302 showed weak AChE inhibitory activity (The percentage inhibition was at 20%~60% in 50 µM) [99].
The 5-lipoxygenase (5-LOX) inhibitory potential of 306-308 from Fusarium sp. was assessed in an attempt to explore their activity against 5-LOX. It is noteworthy that 306 displayed prominent 5-LOX inhibitory activity with IC 50 value of 3.61 µM, compared to that of indomethacin (IC 50 = 1.17 µM), while 307 and 308 had moderate activity with IC 50 values of 7.01 µM and 4.79 µM, respectively [101].
α-Glucosidase inhibitory activity was performed in the 96-well plates and acarbose was used as the positive compound. In the inhibitory assay against α-glucosidase, compound 313 displayed moderate activities [104].
The anti-inflammatory activities of selected isolated 4 compounds 314-317 were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Compound 317 showed the most NO inhibitory effects, with the inhibition of 17.4% NO production in LPS stimulated RAW264.7 cells at 10 µM. At the same concentration, compound 315 significantly inhibited the NO production, with 11.2% inhibitory rate. Compound 314 showed weak NO inhibitory effects at 10 µM, with inhibitory rates of 6.5%. At the same concentration, quercetin, the positive control, inhibited NO production to 12.9% [105].
The Superoxide anion radical scavenging activity of compound 331 was investigated. It displayed strong antioxidant activity with EC 50 value of 1.08 mg/mL on superoxide anion racdicals. Ascorbic acid (Vc) was used as positive control with EC 50 value of 0.33 mg/mL [111].
Human carboxylesterases (hCE 1 and hCE 2) are the important enzymes that hydrolyze chemicals with functional groups, such as a carboxylic acid ester and amide, and they are known to play vital roles in drug metabolism and insecticide detoxication. The isolated compounds 379-385 were assayed for their inhibitory activities against hCE 2. Loperamide was used as a positive control with IC 50 value of 1.31 ± 0.09 µM. Compounds 379, and 383-385 displayed significant inhibitory activities against hCE 2 with IC 50 values of 10.43 ± 0.51, 6.69 ± 0.85, 12.36 ± 1.27, 18.25 ± 1.78 µM, respectively [94].
The inhibitory effects on human carboxylesterases (hCE1, hCE2) of compound 386 were evaluated. The results demonstrated that bysspectin A 386 was a novel and highly selective inhibitor against hCE2 with the IC 50 value of 2.01 µM. Docking simulation also demonstrated that active compound 386 created interaction with the Ser-288 (the catalytic amino-acid in the catalytic cavity) of hCE2 via hydrogen bonding, revealing its highly selective inhibition toward hCE2 [124].
Compounds 392-393 were also evaluated for growth inhibition activity against newly hatched larvae of H. armigera Hubner. Compounds 392 and 393 showed growth inhibition activities against newly hatched larvae of H. armigera Hubner with the IC 50 values of 150 and 100 µg/mL, respectively. Azadirachtin was used as positive control with the IC 50 value of 25 µg/mL [128].
Antioxidant activity of the compound 403 was determined by DPPH assay and compared with the positive control BHT. Compound 403 showed moderate antioxidant activities with IC 50 value of 120.1 ± 11.7 µg/mL [131].
The new compounds 406-407 were subjected for determination of the xanthine oxidase (XO) inhibitory activity using microtiter plate based NBT assay. Allopurinol was used as a positive control with IC 50 value of 0.18 ± 0.02 µg/mL. 406 and 407 showed XO inhibitory activity with IC 50 values of 2.81 ± 0.71 and 0.41 ± 0.1 µg/mL, respectively. The oxidized form of 406 also showed high XO inhibition with IC 50 value of 0.35 ± 0.13 µg/mL [133].
Compound 421 was tested for osteoclastic differentiation activity using murine macrophage derived RAW264.7 cells. 421 significantly increased the number of mature osteoclasts at the comparable levels to the positive control of kenpaullone, compared to the negative control (DMSO), suggesting that 421 activated a signaling pathway in osteoclastic differentiation [139].
Phtotoxicity assay against lettuce seedlings of compound 432 was carried out using a published protocol. The new compound (−)-dihydrovertinolide 432 exhibited phytotoxicity against lettuce seedlings at a concentration of 50 mg/L [140].
All new compounds were tested for in vitro anti-inflammatory activities against nitric oxide production in liposaccharide (LPS)-induced RAW264.7 cells, and dexamethasone was used as the positive control. Compound 436 showed significant inhibitory activity against NO production in LPS-induced RAW264.7 cells with an IC 50 value of 1.9 µM. They were also evaluated for in vitro antidiabetic activities based on the inhibition of alpha-glucosidase, PTP1b, and XOD. Compounds 437 and 441 showed moderate inhibitory activities toward XOD and PTP1b, respectively, at 10 µM with inhibition rates of 67% and 76% [87].
New compound 447 was tested for acetylcholinesterase (AChE) inhibitory activities using the Ellman method with tacrine as the positive control. The results revealed that compound 447 showed weak AChE inhibitory activity wth IC 50 value of 23.85 ± 0.20 µM. Tacrine are the positive control used to estimate AChE inhibitory activity with IC 50 value of 0.26 ± 0.02 µM [27].
All information about the new compounds are briefly summarized in the Table 1 below.

Conclusions
From 2017-2019, a total of 449 new secondary metabolites isolated from plant endophytic fungi using different culture method like common culture, co-culture with bacteria, addition of metal ions and so on, were summarized in this review. These compounds have a variety of unique structures, the difference in structure leads to various biological activities of these compounds. Some of these metabolites display significant antimicrobial effects, cytotoxic activities, antioxidant activities and other biological activities, which indicate that they have potential to be agents to treat some diseases. In this review, structure-activity relationships of some compounds were also reviewed.
According to genome sequencing, a lot of microorganisms have the potential to produce secondary metabolites with novel structures. However, many fungal gene clusters may be silent under standard laboratory growth conditions. As a result, some pathways to yield secondary metabolites cannot be expressed. Therefore, activating these pathways means that we can get more novel compounds. The approach of microorganism co-culture, involving the cultivation of two or more microorganisms in the same lab environment can do a favour for us. Interestingly, 29 new compounds summarized above were obtained through co-culture of bacteria and fungi or two fungi. Besides, by adding CuCl 2 into fermentation medium of an endophytic fungus P. citrinum 46, two compounds were isolated. The results showed that adding Cu 2+ into medium to activate silent fungal metabolic pathways can increase the discovery of new compounds.
Because the compounds mentioned above were isolated from endophytic fungi in different parts of different plants in different regions, they have a variety of structures and biological activities. In addition to anti-tumor and anti-microbial activities, some compounds also exhibit unique biological activities. Among them, 7 compounds showed weak to moderate AChE inhibitory activity. Some compounds exhibited moderate to potent α-glucosidase inhibitory activity compared with those of positive control. By using adapted 2,2 -diphenyl-b-picrylhydrazyl (DPPH) method, a few of compounds were found to show moderate to remarkable antioxidant activity. Some of them also showed weak to significant inhibitory activity against NO production in LPS-induced RAW264.7 cells. The biological activity properties of 18 compounds were evaluated for inhibitory activity against some enzymes like pancreatic lipase, the 5-lipoxygenase (5-LOX), the Indoleamine 2,3-dioxygenase (IDO), Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB), the xanthine oxidase (XO) and so on, they showed weak to high inhibition.
Endophytic fungi isolated from different parts of plants are a huge treasure house on account of the discovery of novel secondary metabolites with biological activities and unique structures. Since the endophyte resources were discovered, more and more researches have been conducted on them. Just from my review article, the new secondary metabolites isolated from plant endophytes during the three years from 2017 to 2019 were counted. Among them, 38 articles were published in 2017, 136 new compounds were obtained; 39 articles were published in 2018, 117 new compounds were obtained; 57 articles were published in 2019, and 196 new compounds were obtained. It can be discovered that in the past three years, the research trend of plant endophytes and their metabolites have increased year by year. The more new compounds obtained, the greater the possibility of screening compounds with excellent biological activity. This is also an important significance for researchers to study plant endophytes. Through this review, i hope to arouse more people's interest and attention in this field and screen out compounds with good biological activities to create a better life for mankind by utilizing endophytes resources.
Author Contributions: C.Z. was responsible for the ideation of the whole article; R.Z. performed the review writing, data collection and data analysis, as well as post-revision work; S.L. contributed to determine the title of the review and made suggestions for the revision of the review; X.Z. helped to revise and proofread the manuscript. All authors have read and agreed to the published version of the manuscript.