Bioactive Alkaloids as Secondary Metabolites from Plant Endophytic Aspergillus Genus

Alkaloids represent a large family of natural products with diverse structures and bioactivities. These compounds and their derivatives have been widely used in clinics to treat various diseases. The endophytic Aspergillus is a filamentous fungus renowned for its extraordinary ability to produce active natural products of high therapeutic value and economic importance. This review is the first to focus on Aspergillus-derived alkaloids. Through an extensive literature review and data analysis, 263 alkaloids are categorized according to their structural features into those containing cytochalasans, diketopiperazine alkaloids, quinazoline alkaloids, quinoline alkaloids, indole alkaloids, pyrrolidine alkaloids, and others. These metabolites exhibited diverse biological activities, such as antibacterial activity, cytotoxicity, anti-inflammatory activity, and α-glucosidase, ACE, and DPPH inhibitory activities. The bioactivity, structural diversity, and occurrence of these alkaloids are reviewed in detail.


Introduction
Endophytic fungi are an important class of plant-associated microorganisms that have provided a bountiful source of bioactive metabolites which benefit human health and have for decades attracted increasing attention from researchers [1][2][3].Among them, the genus Aspergillus is one of the most widely studied filamentous fungi and renowned for its extraordinary productivity when it comes to active natural products with therapeutic values, making it of economic importance [4][5][6].At present, the genus Aspergillus is known to comprise more than 340 species, such as the common A. terreus, A. flavipes, A. fumigatus, and A. ochraceus species [7].These species have been reported to produce a large and chemodiverse range of metabolites, including polyketides, steroids, alkaloids, and terpenoids.These have been shown to exhibit significant anticancer, antibacterial, antifungal, and anti-inflammatory activity properties [6,8].
Alkaloids represent a large family of low-molecular-weight organic compounds containing at least one nitrogen atom.They are mainly derived from amino acids and incorporated in complex cyclic structures.To date, dozens of alkaloids have been separated from endophytic fungi and have been shown to display biodiversity [9].Some of them have been widely applied to treat a variety of diseases [10].Examples include vinblastine and vincristine from Talaromyces radicus CrP20 of Catharanthus roseus [11]; 9-methoxycamptothecin and 10-hydroxycamptothecin from Fusarium solani of Apodytes dimidiata E. Mey.ex Arn (Icacinaceae) [12]; camptothecin from Entrophospora infrequens of Nothapodytes foetida (wellknown anticancer agents) [13], huperzine A from various endophytic fungi collected from Huperzia sp., and Phlegmariurus sp.(used as a neuroprotective agent) [14].Thus, the alkaloids have great therapeutic and application value in clinics.It is worthy to continue to explore the alkaloids with novel structures and potent biological activities or new mechanism of action.
Alkaloids are also one of the major types of metabolites produced by Aspergillus species.These alkaloids possess diverse structures with significant physiological effects, including anti-inflammatory activity, antimicrobial activity, cytotoxicity, and α-glucosidase inhibition activity.According to structural features, alkaloids from Aspergillus are mainly divided into cytochalasans, diketopiperazine alkaloids, quinazoline alkaloids, quinoline alkaloids, indole alkaloids, and pyrrolidine alkaloids, though there are others.A number of excellent reviews on the chemical structures and biological activities of alkaloids have been published in recent years [9,10,[15][16][17][18][19][20][21][22][23].Two of these reviews are on alkaloids from Aspergillus genus.In 2020, Xu K., et al. summarized the chemistry and bioactivity of heterocyclic alkaloids from marine-derived Aspergillus species [22].In 2021, Youssef FS et al. reviewed structures and activities of alkaloids from Aspergillus derived from marine organisms [23].At present, comprehensive literature with special focus on the alkaloids derived from the plant endophytic fungi Aspergillus have not been retrieved.Herein, this review focuses on structural diversity and bioactivity, as well as source information of alkaloids to fill the research gap.A total of 263 alkaloids (1-263) were comprehensively summarized, including the name of the fungus from which it is derived and its host plant, as well as the compound names, chemical structures, and bioactivity of isolated metabolites.We hope that the review can provide a valuable reference for drug discovery and development of alkaloids derived from plant endophytic fungi Aspergillus species.

Methodology
Preparation for the present study began in May 2023, thus this review mainly presents the literature published from January 2004 to May 2023 using the PubMed and Web of Science databases.The literature search was performed using keywords endophytic fungi, Aspergillus, and alkaloids to retrieve information focused on the discovery of natural products.The research papers written in English, and the abstracts in English and full text in Chinese were included in this review.
A chemical study of A. fumigatus associated with Diphylleia sinensis L. generated a new compound, fumitremorgin D (60), which exhibited thin cytotoxicity on HepG2 with an IC 50 value of 47.5 µM [34].
The known compounds okaramine A (68) and JBIR 75 (69) were isolated from the endophyte A. aculeatus associated with leaves of the papaya plant Carica papaya.None of them showed cytotoxicity against the L5178Y mouse lymphoma cell line at 10 µg/mL [36].
An investigation into endophyte Aspergillus sp.Y-2 harbored on needles of Abies beshanzuensis led to the identification of a new compound, beshanzuamide A (98), together with five known isolates: 72, 85, 89, 91, and asperochramide A (99).None of the metabolites displayed any obvious activity against A549 or HeLa cells with IC 50 values beyond 50 µM [45].
A new quinazoline derivative, versicomide E (128), was identified from the moss endophytic fungus Aspergillus sp.This compound was not found to exhibit anti-inflammatory activity to suppress NO production induced by LPS in RAW 264.7 cells [44].
The endophyte A. fumigatus CY018 obtained from the leaf of Cynodon dactylon produced new compound, asperfumoid (186), which acted as an antifungal against C. albicans, with an MIC of 75 µg/mL [62].
A chemical study of A. fumigatus LN-4 obtained from Melia azedarach led to the discovery of 244 and pseurotin A1 (245), which demonstrated nontoxicity toward brine shrimps [31].
A chemical study of A. fumigatus LN-4 obtained from Melia azedarach led to the discovery of 244 and pseurotin A1 (245), which demonstrated nontoxicity toward brine shrimps [31].

Summary and Discussion
Endophytic fungi are a promising source for novel secondary metabolites.The genus Aspergillus is a major reservoir of alkaloids with various structures and diverse bioactivities.In this review, a total of 263 alkaloids derived from endophytic Aspergillus (Figure 8), containing 22 cytochalasans, 104 diketopiperazines, 46 quinazolines, 14 quinolines, 54 indoles, 7 pyrrolidines, and 16 others, were acquired from studies on Aspergillus genus in the past decades (Figure 8).Among them, diketopiperazine and indole compounds were the main metabolites derived from plant endophytic fungi of the genus Aspergillus.All these metabolites were identified from 46 Aspergillus strains (Figure 9), of which A. fumigatus accounted for 28.26% (13 strains), followed by A. versicolor (5, 10.87%), A. flavipes (4, 8.70%), A. terreus (2, 4.35%), A. nidulans (2, 4.35%), other species (6, 13.04%) including A. aculeatus (1), A. amstelodami (1), A. cristatus (1), A. creber (1), A. micronesiensis (1), and A. ochraceus (1), and Aspergillus unknown species (14, 30.43%).Detailed analysis revealed that the discovery probability of known alkaloids is high (61.98%)(Figure 10).The microbials inhibited in a special bioenvironment have unique metabolic pathways and potent potential to produce novel bioactive natural products [72].Therefore, the research of new biological resources is more conducive to the discovery of new biologically active alkaloids.Furthermore, the growing number of Aspergillus genome sequences proved that the potential of biosynthetic metabolites is far from having been mined, and bioinformatics analysis revealed that many biosynthetic gene clusters are silent or have low expression under standard laboratory conditions [4,5].With the development of new research strategies, such as heterologous expression, epigenetic modifiers, and OSMAC, silent and low-expression biosynthetic gene clusters encoding alkaloids in Aspergillus might be discovered, and more structurally diverse alkaloids with potent pharmaceutical applications will be found for drug research.
Molecules 2023, 28, x FOR PEER REVIEW 31 of 36 such as heterologous expression, epigenetic modifiers, and OSMAC, silent and low-expression biosynthetic gene clusters encoding alkaloids in Aspergillus might be discovered, and more structurally diverse alkaloids with potent pharmaceutical applications will be found for drug research.The alkaloids summarized in this literature exhibited antibacterial activity; cytotoxicity; anti-inflammatory activity; and α-glucosidase, ACE, and DPPH inhibitory activities.Many of these metabolites demonstrated potent biological activity.For example, gartryprostatin C (82) displayed potent inhibitory activity against the human FLT3-ITD mutant AML cell line MV4-11, with an IC 50 value of 0.22 µM [39].Asperpyridone A (252) improved glucose uptake and is a potential hypoglycemic agent [71].However, it is noteworthy that most compounds (114, 43.35%) were inactive in the assays or untested.Further studies for these isolated compounds are necessary to discover their different bioactivity.In addition, some potent active compounds have only been studied in vitro, without further research in vivo and mechanisms of action, which may be limited by the yield of compounds.As we know, some metabolites are generated by endophytic fungi in low quantities under laboratory culture conditions, which make separation difficult and hinder further investigation.Therefore, it requires the interdisciplinary cooperation of chemists, pharmacologists, and biologists to conduct in-depth research on chemical synthesis and modification, as well as genetic regulation to increase the production of active compounds and new analogues, providing chemical research foundation for drug discovery.

Conclusions
Plant endophytic fungi have provided abundant resources of natural products with unique structural features and diverse biological activities, which play a critical role for drug development.The plant endophytic Aspergillus is a dominant community in natural products exploration.In this literature, the bioactivity, structural diversity, and biosources of alkaloids derived from plant endophytic Aspergillus species during January 2004 to May 2023 were described.Approximately 263 alkaloids isolated from 46 strains of Aspergillus species were reviewed according to their structural features, including cytochalasans, diketopiperazine alkaloids, quinazoline alkaloids, quinoline alkaloids, indole alkaloids, pyrrolidine alkaloids, and others.Among them, 149 alkaloids have significant physiological activities, such as antibacterial activity, cytotoxicity, anti-inflammatory activity, and α-glucosidase, ACE, and DPPH inhibitory activities.Therefore, these active alkaloids have tremendous potential as lead compounds for the exploitation of new drugs.The interdisciplinary research of chemistry, biology, and pharmacology for alkaloids derived from plant endophytic Aspergillus sp. has attributed to driving the application of alkaloids in the drug discovery and development.

Figure 8 .
Figure 8. Different classes of Alkaloids from plant endophytic fungi Aspergillus.

Figure 9 .Figure 9 .
Figure 9.The proportions of Aspergillus species reviewed in this paper.

Figure 8 .
Figure 8. Different classes of Alkaloids from plant endophytic fungi Aspergillus.

Figure 9 .Figure 10 .
Figure 9.The proportions of Aspergillus species reviewed in this paper.

Table 1 .
Cytochalasans from endophytic fungi of Aspergillus genus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 2 .
Diketopiperazine Alkaloids from endophytic fungi of Aspergillus genus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 3 .
Quinazoline Alkaloids from endophytic fungi of Aspergillus genus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 4 .
Quinoline alkaloids from endophytic fungi of Aspergillus genus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 4 .
Quinoline alkaloids from endophytic fungi of Aspergillus genus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 5 .
Indole Alkaloids from endophytic fungi Aspergillus genus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 6 .
Pyrrolidine Alkaloids from endophytic fungi of Aspergillus and their biological activities, metabolite class, fungus, host plant(s), reference.

Table 7 .
Other Alkaloids from endophytic fungi of Aspergillus and their biological activities, metabolite class, fungus, host plant(s), reference.