Endophytic fungi: a potential source for drugs against central nervous system disorders

Abstract

Neuroprotection is one of the important protection methods against neuronal cells and tissue damage caused by neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and multiple sclerosis. Various bioactive compounds produced by medicinal plants can potentially treat central nervous system (CNS) disorders. Apart from these resources, endophytes also produce diverse secondary metabolites capable of protecting the CNS. The bioactive compounds produced by endophytes play essential roles in enhancing the growth factors, antioxidant defence functions, diminishing neuroinflammatory, and apoptotic pathways. The efficacy of compounds produced by endophytic fungi was also evaluated by enzymes, cell lines, and in vivo models. Acetylcholine esterase (AChE) inhibition is frequently used to assess in vitro neuroprotective activity along with cytotoxicity-induced neuronal cell lines. Some of drugs, such as tacrine, donepezil, rivastigmine, galantamine, and other compounds, are generally used as reference standards. Furthermore, clinical trials are required to confirm the role of these natural compounds in neuroprotection efficacy and evaluate their safety profile. This review illustrates the production of various bioactive compounds produced by endophytic fungi and their role in preventing neurodegeneration.

Introduction

Neurodegeneration is a progressive disorder or damage of neurons in the nervous system. In neurodegeneration, brain neurons and spinal cord start deterioration, causing initial symptoms such as coordination and ability to think and move [1, 2]. Neurodegenerative diseases affect millions of people worldwide every year. Two most common types of neurodegenerative diseases are Parkinson’s disease (PD) and Alzheimer’s disease (AD). In 2016, about 5.4 million Americans were living with AD; by 2020, 930,000 people in the USA were living with PD. Neuroprotection is essential to restore the neurons from deterioration. Therefore, an approach is required to prevent or delay the progressive loss of neurons to avoid central nervous system (CNS) disorders [1].

The elevation of oxidative stress, excitotoxicity, protein aggregation, iron accumulation, mitochondrial dysfunction, and inflammation causes neurodegeneration [3]. Glutamate, an excitatory neurotransmitter of CNS, causes excitotoxicity by inhibiting cysteine uptake, reducing glutathione levels, and accumulating ROS (reactive oxygen species) inside the cell. Ultimately, the influx of calcium ions inside the cells increases and disrupts mitochondrial Ca²⁺ homeostasis and membrane permeability, resulting in neuronal cell death [4, 5]. Excess of glutamate cytotoxicity contributes to neurological disorders such as AD, PD, ischemic brain injuries, epilepsy, amyotrophic lateral sclerosis, and traumatic brain injuries. Although, many drugs such as tacrine, donepezil, rivastigmine, and galantamine approved by FDA to manage neuroprotection, their use is limited due to minute therapeutic accomplishments and various side effects. Therefore, developing and administering novel drugs with lesser side effects and better therapeutic outcomes are essential.

Various models (in vitro and in vivo) and enzymes have been used to study the neuroprotective efficacy of secondary metabolites produced by endophytic fungi. Endophytes are the microorganisms that reside within the host plant in symbiotic association without causing any harm to the host plant [6, 7]. The interaction of endophytes with higher plants makes them a reservoir of bioactive compounds with unique structures regarding human safety and concerns [8–11]. According to various research and data analyses, it has been reported that secondary metabolites produced by endophytic fungi could be an alternative approach for novel drug discovery and development and can be used as a precursor element for the pharmaceutical industry. Endophytes are preferred over plants as exploitation of plants results in degradation of the environment and loss of biodiversity. So, to overcome this problem, there is an urgent need to switch to bioactive compounds from fungal endophytes [12]. The diversity of the host plants induces endophytic fungi to produce various bioactive compounds with a wide range of activities like antibiotics, immunosuppressants, antioxidants, anticancer, and neuroprotectants [13–15]. Natural antioxidants and glutamate antagonists prevent neurological diseases from oxidative damage due to the high concentration of glutamate [2, 16], and therefore, they are identified as neuroprotective agents. This review aims to summarize all the research on various bioactive molecules obtained from endophytic fungi for neuroprotection.

Overview of different classes of endophytic fungi

There is a broad classification of endophytes, including latent pathogens and dormant saprophytes. However, phylogenetic analysis reveals that endophytes differ from parasites within the host. The primary groups in which endophytes have been divided and recognized so far are the clavicipitaceous endophytes (C-endophytes) and non-clavicipitaceous endophytes (NC-endophytes) [12]. C-endophytes belong to Clavicipitaceae family (Hypocreales; Ascomycota), and many species of these fungi produce bioactive molecules belong to the genera Cordyceps, Balansia, Epichloe, etc. The NC-endophytes are large, but not well defined taxonomically and belong to Ascomycota and Basidiomycota. C-endophytes infect grasses, and they are systemic and vertically transmitted through seeds [17]. The NC-endophytes are in healthy tissues of the host and non-systemic and horizontally transmitted from plants to plants and have classes such as Class II, III, and IV [18]. Endophytes have high bio-functional activities; therefore, their uses are highlighted in treating of various human diseases.

Neurodegenerative diseases

Neurodegenerative diseases refer to conditions due to neuronal cell death, particularly in CNS, which is the deterioration of neurons that progress to an advanced stage over time. These diseases are primarily incurable, but the progression of the conditions can be delayed. Neurodegenerative diseases are linked to various symptoms; the person may lose the ability to think, move, and coordinate, and a significant sign is loss of memory [2, 19]. There are several neurodegenerative diseases (NDs), but Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common disorders [20]. Other diseases include multiple sclerosis, amyotrophic lateral sclerosis (ALS), Lewy body dementia, and spongiform encephalopathy [21–23].

Alzheimer’s disease

This is the most common, devastating, and incurable disorder affecting social and occupational activity with a prevalence of 10–30% and an incidence of 1–3% in the people of over age 65 years [24]. Pathogenesis associated with Alzheimer’s is impaired learning and memory, loss of cholinergic neurons, and personality change. Memory deterioration starts in the late 60s, affecting 50 million people across the globe with the risk of doubling every 5 years, with an estimated 152 million cases caused by the year 2050 [20, 25, 26]. The two main hallmark signs of the disease are senile plaques and neurofibrils tangles (NFTs). Senile plaques are made up of fibrillar 𝛽-amyloid (A𝛽) peptides in the brain due to imperfect regulation of the 𝛽-amyloid precursor protein, whereas NFTs are associated with Tau protein. These signs are thought to induce oxidative damage and inflammation to cause AD [27, 28]. It is now well known that memory impairment in patients with senile plaques results from cholinergic deficiency. This is due to decreased neurotransmitter acetylcholine (ACh) release and reduced choline uptake. Acetylcholinesterase (AChE) is an enzyme which is responsible for the cleavage of acetylcholine, and reversible inhibition of AChE increases ACh within the synaptic cleft, which positively affects patients [29]. In several cases, it has been reported that mitochondrial cascade dysfunction would trigger AD by reducing the amount of glucose and oxygen in human’s brain [24].

Parkinson’s disease

This is the second most common long-term ND that affects every seven to 10 million people worldwide. PD is an age-related ND disease that adversely affects the quality of life, and ultimately the patient becomes their own prisoner [30]. The indications include rigidity, bradykinesia, resting tremors, loss of posture and coordination, difficulty in maintaining equilibrium, and experience severe depression. The disease manifests due to the loss of dopamine-generating cells in the mid-brain, but the reason for cell death is unknown. PD is not limited to basal ganglia; it is also interconnected with non-motor neurons disorder like dementia [31]. One of the reasons for PD is susceptibility to oxidative damage. First, there is dopamine breakdown by auto-oxidation, followed by the generation of superoxide anion, ROS, hydrogen peroxides, and many more. Other causes of PD are exposure to metals, pesticides, and environmental factors. Thus, these factors create an association between the disease and oxidative damage of neuronal cells [17].

Huntington’s disease (HD)

This is an autosomal dominant inherited disorder that is incurable and characterized by rapid deterioration and degeneration of the brain, leading to death. The symptoms associated with HD include changed behaviour, dementia, and involuntary movement [21]. The progressive brain disorder is due to CAG repeat expansion within coding of huntingtin (HTT) gene, along with an elongated polyglutamine tract. Sometimes, HD manifests in patients below the age of 18 years [32]. The age of Huntington’s manifestation may be predicted using CAG-repeat length. It can be calculated as: age * (CAG − L), where age is current age, L is a constant which is close to threshold of CAG length for onset of HD, and CAG is the repeat length [33]. HD is characterized by motor and cognitive dysfunction along with psychiatric changes, which ultimately leads to loss of independence and eventually causes death. Experiments have been done to demonstrate that the length of CAG is related with Huntington’s progression, and juvenile onset of HD also affects the pathophysiology of the brain [34, 35]. To determine the better therapeutic treatment, one has to study about the complexity of pathology of brain and selectively modulate the functioning of the neurons.

Amyotrophic lateral sclerosis (ALS)

This disease is also known as Lou Gehrig’s disease, a rare neuromuscular disease caused by the mutation and folding of the gene coding for superoxide dismutase (SOD) enzyme. Like other NDs, this is also incurable and ultimately leads to death, within 3–4 years after the onset of the symptoms. Symptoms associated with this disease are weight loss, fasciculation in the absence of muscle, weakness, depression, slurred speech, and emotional liability [22]. The world’s famous physicist Stephen Hawking is the exception case of the person surviving over 50 years after the diagnosis. ALS involves muscular atrophy (amyotrophic) and scarring of the spinal cord (lateral sclerosis) [36]. Majority of cases are sporadic, but familial forms (FALS) constitute around 10% of the cases which are associated with copper/zinc superoxide dismutase gene mutations [37]. The physiological basis involved in ALS are glutamate-induced cytotoxicity, autophagy, neuroinflammation, and axonal transport mechanisms disruptions [38]. Moreover, MRI is also not recommended to search abnormalities in ALS patient brain, but it can reveal the consequences of pathological changes, and MRS have the ability to detect metabolic abnormalities [39]. In ALS patients, CNS imaging with the purpose of non-invasive, accurate biomarkers of disease could aid early diagnosis and provides support in clinical trials. Cerebrospinal fluid (CFS) and blood levels are the most effective biofluid markers which can correlate with the disease progression and strongly associated with the involvement of the motor neuron system [40]. However, ALS follows multiple cascade pathways; therefore, there is a need of multidrug therapy which can target the cascade of pathways involved in the disease progression.

Cerebrovascular disease

This is the second major cause of death due to stroke, leading to acute degeneration of the CNS. Most strokes are of an ischemic or haemorrhagic type and differ from other NDs [23]. In stroke there is a dysfunction of the areas of the brain which are oxygenated blood deprived. Due to oxygen deficiency, neurons get damaged, and patients suffer from impairment of sensations, an aphasia, and after sometime movement and cognitive impairment [41]. Modern stroke diagnosis is eventually required to classify the disruption of blood supplies of CNS that leads to a cascade of events that ultimately causes permanent neuronal damage. At the time ischemic stroke energy-dependent process for cell survival fails. Furthermore, this leads to failure of mitochondrial membrane integrity which causes apoptosis. There are several studies that have been carried out to identify the basic mechanism behind cellular death in ischemic tissues [42]. Age and sex factors are the most common determinants of ischemic strokes. Cases which are reported for strokes are found to be higher in elderly females in comparison with males. In case of women, risk factors that are associated to strokes include use of oral contraceptives, menopause, hormone replacement therapy, and pregnancy. The risk factor associated with stroke increases with age; hence, it is an age-related factor [43]. To investigate potential therapeutic agent with less side effects and long-term effects, researches are more focused towards the detailed study behind the signalling pathway involved in post-ischemic cell death.

Neurodegenerative diseases are associated with various defects in different cellular processes, such as protein misfolding, mitochondrial dysfunction, oxidative stress, excitotoxicity, and inflammation. However, these defects cause neuronal cell death due to alterations in the physiological and biochemical processes in brain and peripheral nervous system [44] (Fig. 1).

Fig. 1.

Different pathways that are responsible for alterations in the physiological processes to cause neurodegenerative diseases. Reprinted from Bulck et al. [66] with permission from International Journal of Molecular Sciences

Physiological factors

Protein misfolding and aggregation

Protein folding and its 3-D structure are directly associated with protein functioning. When a protein adopts an abnormal 3-D configuration to cause protein misfolding, a change in the native conformation of protein changes its biological activity. It becomes deleterious and cannot return to its previous conformation. Initially, in a correctly folded protein, hydrophobic amino acids are present in the core. But, in misfolded proteins, these amino acids are exposed to the terminal, which leads to the formation of insoluble aggregates in the brain generally known as amyloid deposits. Accumulations of misfolded proteins are a sort of neurotoxins that cause apoptosis of neuronal cells. A class of proteins inhibits protein aggregation and prevents neuronal cells from being damaged. Such proteins are known as molecular chaperons; they bind to misfolded proteins and guide them to refold into their native conformation. But sometimes, this system fails, and aggregation takes place that subsequently causes NDs [45]. In AD, protein misfolding is associated with amyloid plaques and hyperphosphorylated Tau protein, whereas in some rare cases of early PD, mutations can be seen in a synaptic protein called 𝛼-synuclein. In addition, there is a mutation of Huntington in HD, and in ALS, it is SOD. All of the factors lead to protein aggregation and form inclusions in the brain. Therefore, the protein accumulation and aggregation may lead to inflammation and neurotoxicity, which at last give birth to neurodegenerative diseases [46].

Oxidative stress

Oxidative stress is another cause of CNS disorder that encourages ROS generation, nitric oxide, and lipid peroxidation in the body. Free radicals and ROS generate in the body when the antioxidant mechanism fails. These radicals have incomplete electrons and are more reactive and unstable [2, 4]. The class of ROS implies hydrogen peroxide (H₂O₂), superoxide radicals (O²⁻), hydroxyl radicals (OH), nitric oxide (NO), and many others [5]. The brain is susceptible to oxidative damage as 20% of the oxygen is utilized. Various studies revealed that cellular damage is due to free radicals and ROS, thus the pathophysiology of NDs and other diseases [47].

Inflammation

Inflammation is a complex body response to harmful stimuli. It aims to protect brain and spinal cord both from tissue damage or any pathogen invasion. Sometimes, this protective mechanism becomes deactivated or deleterious when the stimuli are excessive or out of control. There are various reasons for inflammation in NDs, such as aggregation of misfolded proteins, accumulation of proteins and modified cellular components, neuronal injuries, and defective anti-inflammatory mechanisms. Neuroinflammation does not usually trigger NDs; it is directly involved in neuronal dysfunctions which contributes to death of neurons and ultimately to neurodegenerative disease progression [48]. In AD, aggregation and accumulation of β-amyloid (Aβ) are also associated with inflammation, which leads to increased proinflammatory cytokines, complement system, and acute-phase proteins. In addition, microglia and astrocytes also stimulate cytokines and chemokines like TNF-𝛼 and IL-6 and IL-8, respectively [49]. Neurodegenerative diseases are associated with long lasting microglia activation and chronic inflammation. Therefore, chronic inflammation results in an abnormal increased level of cytokines, neurotoxin production, and oxidative stress which triggers a pro-inflammatory cycle and amplifies processes such as mitochondrial dysfunction, protein deposition, and impairment in blood-brain barrier (BBB) permeability [50]. TLRs (Toll-like receptors) activation sustains the chronic inflammation at the glial level. Amongst different classes of TLRs, TLR4 is most expressed in microglia. Inflammation is a crucial aspect of disease progression; therefore, targeted pathways responsible for neuroinflammation seem to be a promising strategy to counteract neurodegenerative diseases [51]. Compounds with different molecular targets for different pathways are the best candidates to fight against these conditions. Therefore, on this basis, study of secondary metabolites with varied structure and chemical composition can help to identify the most effective anti-inflammatory agents.

Peptides and mitochondrial dysfunction

Mitochondria are a powerhouse of the cell, responsible for energy metabolism, Ca²⁺ homeostasis, generation of free radicals, and cell apoptosis. These factors ultimately lead to neurodegenerative disorders like AD and PD. Studies showed that excessive intracellular calcium or accumulation of calcium by mitochondria progress to cell death. Thus, mitochondrial depolarization and dysfunction result in neurodegenerative disorders [3, 4].

Cell viability and cell cytotoxicity

Loss of particular subsets of neurons results in neuronal cell death and ultimately leads to neurodegenerative diseases. Other factors responsible for neuronal cell death follow the development of oxidative stress, excitotoxicity, and apoptosis [4].

Biochemical factors

Neurotransmitter level

Biochemical processes are associated with several neurotransmitters, such as ACh, dopamine (DA), and monoamine oxidase (MAO). The changes in the level of neurotransmitters are involved in the pathology of NDs. For example, low levels of ACh and DA characterize AD and PD, respectively. Acetylcholine is a neurotransmitter released from the presynaptic vesicles of cholinergic neurons via choline acetyltransferase (ChAT). A loss or downregulation of this neurotransmitter is associated with the development of Alzheimer’s disease (AD). Therefore, AChE and neurodegenerative diseases are linked together, and the strategies are based on the neuroprotective effect by inhibiting AChE [52]. MAO catalyzes a reaction to yield H₂O₂, aldehyde, ammonia, and amine; at high concentrations they are toxic and contribute to NDs. Thus, MAO inhibitors provide a mild effect on reducing PD and result in early ND treatment [53]. DA is a neurotransmitter that provides proper brain functioning, but reduced level and degradation of DA in neurons caused PD and HD, respectively.

Glutamate excitotoxicity

Excitotoxicity results in excessive stimulation of receptors, which is the crucial mechanism of neuronal cell death and causes central nervous system diseases such as stroke, brain trauma, neurodegenerative diseases [54]. Glutamate is one of the major excitatory neurotransmitters in the brain, associated with energy stabilization. Therefore, fluctuation of energy metabolism causes neuronal cell death. Overstimulation and accumulation of glutamate generate ROS, mitochondrial dysfunction, and excess calcium, all of which contribute to cell death [55]. Glutamate is an endogenous neurotoxin that causes neuronal cell death. So, when tested with bioactive compounds, the cell viability significantly improved glutamate-induced cells and showed a strong protective effect against neuronal cell lines [56].

Other factors

Ageing

Ageing is a complex process that changes both morphological and biochemical processes. These changes comprise changes in the CNS, skin, cardiovascular system, hormonal imbalance, and reproduction system. It is one of the additional factors that are responsible for neurodegenerative diseases. As neurons start to deteriorate after a certain age, this is associated with mitochondrial dysfunction, free radical production and oxidative stress, microglia dysfunction, reduced efficiency of molecular chaperones, reduced synaptic densities, and blood-brain barrier system disruption [43]. Brain ageing involves complex cellular and molecular processes that ultimately cause cognitive decline. Progressive accumulation of iron and deletion of mitochondrial DNA leads to age-related genetic changes. Furthermore, ageing caused by alteration in the cell membrane composition and inflammation which consequently increase abnormal deposition of proteins continues with the process of ageing. Hindrance in the cholinergic activities is the key area of brain ageing which in some forms causes neurodegeneration [57, 58]. Rapid ageing of the population is limited to therapeutic cure so there is an urgent need for new approaches to treat neurodegeneration. So, natural compound-based drugs could be more relevant and effective to find therapies related to neurodegenerative diseases.

Dietary habits and lifestyle factors

Increasing rate in neurodegenerative diseases is consequently related to modernization, change in lifestyle, diet plans, and sleeping habits. Modern lifestyle includes alcohol consumption, weight gain, and deprived sleep. Therefore, they are relatively at high risk to neurological disorders [59]. This may be due to stress, lack of healthy diet and physical exercise, high cholesterol, and many more. Diet rich in cholesterol and saturated fatty acid somehow alters the cell membrane composition leading to an increased risk of cerebrovascular diseases. Smoking, consumption of vitamin D, and coffee are significantly associated with AD as higher consumption of cigarettes and vitamin D gave lower odds for AD, but high consumption of coffee gave higher odds for AD. Certain changes in diet plan cause obesity, and overweight patients showed excess adiposity and eventually the risk of severe ischemic stroke increases [60]. Therefore, physical activities must be incorporated in lifestyle to reduce the risk of cerebrovascular damage and improve cognitive function. Thus, lifestyle with high sugar, alcohol, tobacco, and fat content runs parallel with ageing and neurodegeneration [59–61].

Genetic factor

Mitochondria is known to be a major site of free radical generation in cells, and any subsequent changes in mitochondrial DNA due to oxidative damage result in ageing and neurodegeneration. Mitochondrial genome is more susceptible to damage in comparison to nuclear DNA. Therefore, decline in defence mechanism in the cells due to oxidative stress damages nucleic acids. If DNA is damaged and not repaired, then it becomes highly mutagenic upon replication and is transferred to another generation leading to neurodegeneration at early stage [62]. Major mitochondrial genes involved in Parkinson’s are synuclein alpha (SNCA), Leucine rich repeat kinase 2 (LRRK2), Parkin protein gene (PARK2), and many others. These genes show direct relationship between pathogenesis of PD and mitochondria. The genes that are responsible to cause AD are APP gene, PSEN1, PSEN2, and APOE4. However, APOE gene that encodes protein apolipoprotein E has high risk of AD with apolipoprotein E ε4 allele [63]. Genetic mutation in HTT gene and then inheritance of damaged DNA to the offspring can cause Huntington’s disease [64]. Therefore, it can be concluded that any alteration in the gene due to ROS, mitochondrial dysfunction, and inflammation can damage the DNA, which lead to NDs [65].

Secondary metabolites from endophytic fungi

Various natural products could be obtained from endophytic fungi, which include alkaloids, cytochalasins, polyketides, terpenoids, flavonoids, quinones, peptides, xanthones, phenolic compounds, and many others [67, 68]. Bioactive compounds are also essential for fungal ecology as they mediate interactions between fungi and host plants in signalling, defence, symbiotic association, and many more [12]. These compounds show various biological activities such as antimicrobial, antiparasitic, anticancer, neuroprotective, antioxidant, insulin mimicking, enzyme inhibitive, immune-suppressive, etc. They can also produce effective cytotoxic metabolites. Research also revealed that these compounds treat neurological disorders due to their high functional activity [68, 69]. An array of secondary metabolites of diverse classes have been identified as alkaloids, steroids, quinones, phenols, terpenoids, and polyketides [70]. Secondary metabolites obtained from different endophytes such as Mucor sp., Aspergillus sp., Penicillium sp., Colletotrichum sp., and many others were identified to possess biological activities. Table 1 represents a diverse class of biologically active compounds obtained from endophytic fungi and their host plants.

Table 1.

Diverse classes of natural compounds isolated from endophytic fungi associated with different plant species

Diverse classes	Compounds	Host plants	Endophytic fungi
Alkaloids	Chaetoglobosins A, G, V, Vb, and C	Ginkgo biloba	Chaetomium globosum
	Camptothecin (CPT)	Nothapodytes foetida	Entrophosphora infrequens
	Vincristine(leurocristine)	Catharanthus roseus	Fusarium oxysporum
	Chaetoglobosin U	Imperata cylindrical	Chaetomium globosum
	Terezine E	Centaurea stoebe	Mucor sp.
	Aromatic butenolides, asperimides A–D	Suriana maritima L.	Aspergillus terreus
	Brasiliamide J-a, brasiliamide J-b, and peniciolidone	Panax notoginseng	Penicillium janthinellum
	Fusarithioamide B	Anvillea garcinii	Fusarium chlamydosporium
	Aflaquinolone H	Eichhornia crassipes	Aspergillus versicolor
Phenols	Tridepsides cytonic acids A and B	Quercus sp.	Cytonaema sp.
	Flavanoids	Juniperus cedre	Nodulisporium sp.
Steroids	Ergosterol, 3β,5α,6β-trihydroxyergosta-7,22-diene, and many more	Artemisia annua	Colletotrichum sp.
	Fusaristerol B, Fusaristerol C, and Fusaristerol D	Mentha longifolia L.	Fusarium sp.
Terpenoids	Eremophilane-type sesquiterpenes	Licuala spinosa	Xylaria sp.
	Bisabolane sesquiterpenoids	Xylocarpus moluccensis	Aspergillus sp. xy02
	Cyclonerane sesquiterpenes	Laminaria japonica	Trichoderma harzianum X-5
	Seiricardine D	Ceriops tagal	Cytospora sp.
	Fumagillene A and Fumagillene B	Ligusticum wallichii	Aspergillus fumigatus
	Bipolenins G and H	Fresh potatoes	Bipolaris eleusines
	14-nordrimane-type sesquiterpene, phomanolide	Aconitum vilmorinianum	Phoma sp.
	Eremophilane sesquiterpene, xylareremophil	Sophora tonkinensis	Xylaria sp. GDG-102
	Purpurolides	Edgeworthia chrysantha	Penicillium purpurogenum IMM003
	Norsesquiterpenoidal enantiomers and Preuisolactone A	Panax notoginseng	Preussia isomera XL-1326
	Drechmerins A–G	Panax notoginseng	Drechmeria sp
	Koninginols A–C	Morinda officinalis	Trichoderma koningiopsis A729
	Harziane diterpene and proharziane diterpened	Laminaria japonica	Trichoderma harzianum X-5
	Kadhenrischinins A–H and 7b-schinalactone C	Kadsura angustifolia	Penicillium sp. SWUKD4.1850
	Integracide E and Isointegracide E	Artemisia annua	Hypoxylon sp. 6269
	Fusariumin A and B	Santalum album	Fusarium sp. YD-2
	Isopenicins A– C	Isodon eriocalyx var. laxiflora	Penicillium sp. sh18
Quinones	Nigrosporone A and Nigrosporone B	Choerospondias axillaris	Nigrospora sp. BCC 47789
	Ethylnaphthoquinone derivatives	Kandelia candel	Neofusicoccum austral SYSU-SKS024
	Evariquinone	Morus alba L	Colletotrichum sp. JS0367
Polyketides	Pestalustaine B	Sinopodophyllum hexandrum	Pestalotiopsis adusta
	Pestalotiopisorin B	Rhizophora stylosa	Pestalotiopsis sp.
	Seven new dihydroisocoumarins	Przewalskia tangutica	Lachnum palmae
	Peniisocoumarins A–J	Kandelia candel	Penicillium commune QQF-3
	a-pyrone type derivatives	Distylium chinense	Xylariales sp. (HM-1)
	Wortmannine F and G	Tripterygium wilfordii	Talaromyces wortmannii LGT-4
	Isochromanes	Cordyceps sinensis	Aspergillus fumigatus
	Xanthoquinodin B9	Rhapis cochinchinensis	Chaetomium globosum 7s-1
	Asperetide and asperanthone	Hypericum perforatum L.	Aspergillus sp. TJ23
	Isoshamixanthone	Callistemon subulatus	Aspergillus sp. ASCLA
	Diaporthsins A–K	Dendrobium nobile	Diaporthe sp. JC-J7
	Butenolides and terrusnolides A–D	Tripterygium wilfordii	Aspergillus sp.
	β-lactone polonicin A	Camptotheca acuminate	Penicillum polonicum

Neuroprotective compounds from endophytic fungi

Globally millions of people have mental illnesses and neurological disorders. Modern treatments and medicines are preferred as a solution to treat the ailment, but those are expensive, out of reach to an ordinary person, and with several side effects. Therefore, natural products are extensively exploited, and their targeted impact on the treatment proves them as essential medicine source. Nowadays, a large number of metabolites that are derived from plants and organisms associated with them have great importance to humankind. Their positive results tend to incline medicinal practitioners more towards natural products for reliable treatment with lesser side effects and cost-effectiveness.

Various phytoconstituents show noticeable results and proved to be a potential source for treating neurological disorders. Endophytic fungi are associated with every possible source, like marine sediments, ocean, plant parts, deserts, etc., which are promising sources for these compounds. These bioactive compounds were known or novel to target neurodegenerative diseases. Chemical investigation of fungi led to the isolation of alkaloids (alternatine A, chrysogenamide A), liphatic polyketone (alternin A), terpenes (1R,5R,6R,7R,10S)-1,6-dihroxyeudesm-4(15)-ene) [71–73] quinones, cytochalasans [4, 74] pyrone derivatives, steroids, and many more [75–77].

The following sections describe endophytic fungi, their chemical constituents, and related pharmacology concerning neurodegenerative disorders. Bioactive molecules with prominent activity have also been discussed. Figure 2 represents several bioassays performed to check the activity of the compounds as a neuroprotectants. In vitro testing is employed for the identification of potentially hazardous chemicals or the confirmation of specific toxic properties in the early stages of the development of potential therapeutic drugs. The use of animal models for experimentations is considered under in vivo testing as they share exceptional anatomical and physiological similarities with human beings and initiate researchers to investigate different mechanisms and develop an approach before applying their discoveries to humans.

Fig. 2.

Various classes of secondary metabolites from endophytic fungi, their screening techniques, and inhibitory effect on neuronal cell

Acetylcholinesterase inhibitory activity

Acetylcholine is a neurotransmitter released from presynaptic vesicles of cholinergic neurons via choline acetyltransferase (ChAT). Alzheimer’s disease (AD) and the neurotoxicity of amyloid components are due to acetylcholinesterase (AChE) activity. AChE breaks down acetylcholine (Ach) into two derivatives choline and acetic acid, and an increase of acetylcholine level leads to loss of cholinergic functions. Therefore, there is a relationship between AChE and neurodegenerative diseases, and different strategies are adopted to inhibit AChE activity [52]. Various compounds are isolated from host plants and endophytes associated with them, and when tested in vitro for AChE inhibition, they provide prominent results in treating neurological disorders.

Huperzine-A (HupA) (1) (Fig. 3), a novel lycopodium alkaloid isolated from a traditional Chinese medicinal plant Huperzia serrata, can therapeutically treat myasthenia gravis and Alzheimer’s disease (AD) in the early stage. It selectively inhibits AChE and improves many cognitive functional defects. Some reports are available on extraction of Hup A from endophytic fungi associated with H. serrate. Wang et al. reported the AChE inhibitory activity from different endophytic fungi such as Ascomycota, Dothideomycetes, and Colletotrichum species with more than 80% inhibition [78]. Zhu et al. reported that endophytic fungus Shiraia sp. isolated from H. serrata showed AChE inhibitory activity [79]. The endophytic fungus Ceriporia lacerate HS-ZJUT-C13A isolated from H. serrate exhibited AChE inhibitory activity [80]. Hup A compound was isolated from Cladosporium cladosporioides LF70 from the leaves of H. serrate [81]. However, methanol extracted HupA exhibited more potent inhibition than authentic and purified HupA.

Fig. 3.

Chemical structures of bioactive compounds isolated from diverse range of endophytic fungi with neuroprotective activities

Lei et al. reported AChE inhibitory activity in vitro from the endophytic fungus Colletotrichum sp. F168 from H. serrata, which was about 18.2% [82]. Dong et al. investigated HupA producing endophytic fungus Trichoderma sp., which exhibited significant AChE inhibition of 81.89% [83]. Xia et al. reported the production of Huperzine A from endophytic fungi, Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V isolated from H. serrata exhibiting AChE inhibition activity [84].

Zaki et al. extracted Hup A from the endophytic fungus Alternaria brassicae with 75.5% AChE inhibition [85]. The endophytic fungus Fusarium sp. Rsp5.2 isolated from H. serrate produced Hup A and showed an IC₅₀ value of 2.849 ± 0.0026 μg/mL [86]. Xu et al. extracted novel bioactive compounds, orsellide A, orsellide C, and chetomin which showed moderate AChE activities. The compounds were isolated from the endophytic fungus Chaetomium sp. NF00754 associated with Pharbitis nil, with IC₅₀ values of 7.34, 5.19, and 7.67 μM, respectively [87]. Yu et al. isolated Chaetomium sp. M453 from H. serrate and investigated steroidal bioactive compounds against AChE inhibition. Compound 3β-hydroxy-5,9-epoxy-(22E,24R)-ergosta-7,22-dien-6-one showed weak activity (20–60%), while other new steroids exhibited no AChE inhibition [88].

The compounds, 1-O-methylemodin, 5-methoxy-2-methyl-3-tricosyl-1,4-benzoquinone, 6-epoxy-7-one-8, 22-dien-ergosta extracted from endophytic fungus Chaetomium sp. YMF432 from H. serrate showed moderate inhibitory activities with IC₅₀ values of 37.7 ± 1.5, 370.0 ± 2.9 μM and 67.8 ± 1.7 μM, respectively [89]. Ruan et al. isolated Chaetomium globosum in Chinese yam (Dioscorea opposita) capable of producing two novel compounds, 10,11-dihydroxylaureonitol and yamchaetoglobosin A. The compounds were tested against AChE, and yamchaetoglobosin A displayed inhibitory activity with a ratio 38.2% at a concentration of 50 μM [90]. Li et al. isolated 14 metabolites from C. globosum from seeds of Panax notoginseng and evaluated their AChE inhibitory activity. Compounds 3-methoxyepicoccone showed significant inhibition with IC₅₀ value of 5.55 μM, and epicoccolides B with 72.6% at 50 μM concentration. Therefore, these two compounds are novel AChE inhibitors that could be potential multi-target agents for AD [91].

Xu et al. showed that Paecilomyces sp. TE-540 associated with Tabacum produced 2-hydroxyalbrassitriol and 12-hydroxyalbrassitriol that resulted in moderate inhibitory activities against AChE with IC₅₀ values of 43.02 ± 6.01 and 35.97 ± 2.12 μM, respectively [92]. Oliveria et al. showed association of Xylaria sp. and Penicillium sp. with Piper aduncum and Alibertia macrophylla, respectively, and evaluated bioactive compounds by TLC-based acetylcholinesterase inhibitory assay. All three compounds showed AChE inhibition, while dihydroisocoumarin displayed moderate activity [93]. Gubiani et al. investigated AChE inhibition from Camarops sp. from Alibertia macrophylla (Rubiaceae)., and their results revealed that compounds (2E,4R)-2,4-dimethylnon-2-enoic acid, (2E,4S)-2,4-dimethyloct-2-enoic acid, and xylarenone D showed weak activity and xylarenone C had a minimum inhibitory concentration of 6.25mg/mL [94].

Zhang et al. isolated Cladosporium cladosporioides MA-299 from Bruguiera gymnorrhiza leaves and identified as ent-cladospolide F. This compound exhibited AChE inhibition with an IC₅₀ value of 40.26 μM [95]. Compounds, arisugacin B (2), terreulactone C (3), and territrem C (4) (Fig. 3) were isolated from Penicillium sp. SK5GW1L associated with mangrove plants. These compounds showed a strong AChE inhibitory effect with IC₅₀ 3.03, 0.23, and 0.028 μM, respectively [96]. Medina et al. investigated marine red alga Asparagopsis taxiformis and isolated Nemania bipapillata (AT-05), an endophytic fungus from it. Five new terpenes and a known compound were isolated and evaluated against anti-AChE inhibition. Compound (+) -(2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol showed inhibitory potential against both huBChE and huAChE, while compound nemenonediol A inhibited only huAChE [97].

Wu et al. isolated AChE inhibitors from marine fungus Talaromyces sp. strain LF458 associated with the sponge Axinella verrucosa. Three compounds talaromycesone A (5), talaroxanthenone (6) (Fig. 3), and AS-186c, out of eight compounds, exhibited potent inhibitory activities with IC₅₀ 7.49, 1.61, and 2.60 μM, respectively [98]. Cao et al. isolated the endophytic fungus Acrostalagmus luteoalbus TK-43 from Codium fragile, a marine green alga. Three compounds and their enantiomers were evaluated for AChE inhibitory activity, but only (±) acrozine A exhibited inhibition activity with IC₅₀ 9.5 μM. But none of the compounds showed stronger activity than tacrine, positive control (IC₅₀ = 0.14 μM) [99].

Acetylcholinesterase inhibitory activities of meroterpenoid compounds from an endophytic fungus Aspergillus sp. 16-5c isolated from mangrove were evaluated by Long et al. [100]. Out of ten compounds only three compounds, isoaustinol, dehydroaustin, and dehydroaustinol showed AChE inhibition with IC₅₀ values of 2.50, 0.40, and 3.00 μM, respectively.

Co-culture of microorganisms is recent advancement to cultivate two or more microorganisms in the same confined environment. Co-culturing has revealed that microorganisms have the potential to yield structurally diverse bioactive compounds. Zhou et al. reported five new metabolites of tremulane sesquiterpene and pulvilloric acid-type azaphilone by co-culturing endophytic fungus Nigrospora oryzae with Irpex lacteus. The compounds were tested against antifungal and acetylcholinesterase activity. Tacrine was used as a positive control for AChE inhibition. Nigrosirpexin A showed 35% AChE inhibition at 50μM [75].

Meng et al. extracted two compounds botrallin and palmariol B from Hyalodendriella sp. associated with Populus deltoides Marsh × P. nigra L (Neva hybrid), which showed stronger AChE activities with IC₅₀ values of 103.70 and 115.31 μg/mL, respectively [101]. Working with co-cultures, combinations, and genetic engineering to produce secondary metabolites is still unexplored; more extensive study and research need to be done. Bhagat et al. isolated endophytic fungus Alternaria alternata from Catharanthus roseus, which inhibited AChE and BuChE with 78% and 73%, respectively [102]. AChE inhibitory activity was recorded with cytochalasin H, a compound extracted from the Phomopsis sp. associated with Senna spectabilis [103].

Three compounds, fumitremorgin C, spirotryprostatin A, and cephalimysin A, were isolated from Alternaria alternata associated with Lamium amplexicaule, which showed a strong AChE inhibitory activity with IC₅₀ values of 24.37, 17.91, and 33.49 μg/mL, respectively [104]. Hou et al. reported that natural dibenzopyrone derivatives from A. alternata confer protective effects on PC12 cells from oxidative-stress mediated damages [105]. Polli et al. studied the extract of metabolites E-G6-32 and revealed that the major component of bioactive compounds was identified as asperpentyn (7) (Fig. 3) produced by an endophytic fungus, Curvularia sp. G6-32 associated with Sapindus saponaria L. The fungal extract showed an excellent BuChE inhibitory activity with IC₅₀ of 110 ± 0.05 μg mL-1 compared to donepezil as a positive control [106].

Qi et al. evaluated BACE1 and AChE inhibitory activities of sixteen bioactive compounds isolated from Aspergillus terreus from Tripterygium wilfordii. Notably, spiroterreusnoids A–F can potentially inhibit BACE1 and AChE with IC₅₀ range 5.86 to 27.16 μM and 22.18 to 32.51 μM, respectively [107]. The anticholinergic activity was evaluated in terms of AChE activity from the crude extract of endophytic fungus Fusarium sp. from Euphorbia sp. Fungal isolate OQ-Fus-2-F was selected to investigate AChE inhibition activity and revealed the reduction in enzyme activity with IC₅₀ = 177.0 ± 13.7 μg/mL, a concentration 32 times higher than positive control (Galantamine) [108]. Bioactive alkaloids were isolated from the fermentation broth of A. terreus associated with Artemisia annua. The novel alkaloid compound 16α-hydroxy-5-N-acetylardeemin displayed acetylcholinesterase inhibition. It shows an IC₅₀ value of 58.3 μM, while tacrine (positive control) was 37.9 μM [109]. Colletotrichine B isolated from Colletotrichum gloeosporioides GT-7 from Uncaria rhynchophylla showed AChE inhibition with IC₅₀ of 38.0 ± 2.67 μg/mL [110].

Three bioactive compounds, 2′-deoxyribolactone, hexylitaconic acid, and ergosterol (8) (Fig. 3), isolated from endophytic fungus Curvularia sp. from a Rauwolfia macrophyll showed AChE inhibition with IC₅₀ (μM) values of 1.93, 1.54, and 1.52 respectively. Galanthamine and tacrine were used as positive control with IC₅₀ (μM) 0.5 and 0.01, respectively [111]. Huang et al. [112] isolated endophytic fungus Phomopsis sp. 33. from mangrove plant Rhizophora stylosa, and five compounds, phomopsichin A–D and phomoxanthone A, were isolated and identified from the fungi. They all showed weak AChE inhibition compared with positive control Huperzine A (IC₅₀=45.2 nM). An unusual alkaloid compound, rhizovagine A, was isolated by Wang et al. [113] from endophytic fungi Rhizopycnis vagum Nitaf22 from Nicotiana tabacum and evaluated for its inhibitory activity against AChE and provides moderate inhibition (IC₅₀ 43.1 μM) in comparison with tacrine (IC₅₀ 6.1 μM). Some natural compounds can significantly inhibit colchicine-induced apoptosis of neuronal cells by the reduction of Ca²⁺ influx and inhibition of JNK and p38 phosphorylation and caspase-3 activation. Secalonic acid A is a compound obtained from marine fungus Aspergillus ochraceus and Paecilomyces sp. to protect neurons against MPTP/MPP+ by reversing mitochondrial apoptotic pathways [114–116].

Cell line testing

Human cell line models are used to test and identify novel mechanisms. Neural deterioration is due to increased levels of cytotoxicity and AChE in the hippocampus. Neuronal cell lines are integrative for testing compounds and understanding their mechanisms of neurodegeneration [77]. Neuronal cell lines such as HT22, PC12, SH-SY-5Y, and Neuro2a are used extensively to test and develop neuroprotective compounds from endophytic fungi [2, 71]. These cell lines were induced first with cytotoxicity and then treated with a bioactive compound to yield progressive results against cytotoxicity. Moreover, standards are used to identify the compounds’ capacity to resist neuronal dysfunction.

Yurchenko et al. isolated alkaloids and polyketides from the marine fungi Penicillium sp. KMM 4672, Aspergillus flocculosus, and Aspergillus sp. KMM 4676 [117–121]. 6-Hydroxy-N-acetyl-β-oxotryptamine, a melatonin-like compound, reduced ROS levels by 18% and 35% in 6-hydroxydopamine (6-OHDA)- and paraquat (PQ)-induced models. Similarly, candidusin A and 4-dehydroxycandidusin A can protect Neuro2A cells from 6-OHDA toxicity effects via reactive oxygen species scavenging [122, 123]. These results suggested that such compounds may be used to develop anti-PD leads. Two new auroglaucin derived compounds, niveoglaucins A and niveoglaucins B, and four known compounds, 5-hydroxy-6-(3-methylbut-2-enyl)-2- (pent-1-enyl) benzofuran-4-carbaldehyde, flavoglaucin, tetrahydroauroglaucin or aspergin, and isodihydroauroglaucin, by spectroscopic methods were identified by Yurchenko et al. [124]. These secondary metabolites were derived from extracts of marine sediment endophytic fungus Aspergillus niveoglaucus collected from Vietnam. All compounds were tested for neuroprotective activity in 6-OHDA induced neuroblastoma Neuro2a cells. Niveoglaucins A, an alkaloid isolated from Microsporum ap. and Aspergillus sp., is red algae-related fungus. The compound could protect PC 12 cells against neuronal cell death by altering the mitochondrial dysfunction mechanism. They could also prevent rotenone-induced cytotoxicity as Neoechinulin A has a mitochondrial-related cryoprotective mechanism [4, 125–127]. Smetanina et al. isolated compounds from Aspergillus niveoglaucus that can exhibit neuroprotective activity towards toxin-induced cell models of PD. The compound cryptoechinuline B (9) (Fig. 3) showed neuroprotective activity in all 6-OHDA, PQ, and rotenone-induced in vitro models, while neoechinulin C protected only PQ model and neoechinulin B and neoechinulin in rotenone and 6-OHDA induced model respectively [128]. Yang et al. investigated novel anti-neuroinflammatory natural secondary metabolites from Aspergillus terreus Y10, a marine-derived fungus. The study showed the anti-neuroinflammatory effect of open-ring butenolide and asperteretal F in LPS-induced microglia cells [129].

Girich et al. isolated low molecular weight bioactive compounds from three marine endophytic fungi, Aspergillus flocculosus, A. terreus, and Penicillium sp. KMM 4672. Asterriquinone B4 and asterriquinone C1 from A. terreus protected the cell model against all neurotoxins (6-OHDA, rotenone, and PQ) and increased cell viability. Further, these compounds showed significant neuroprotective activity with IC₅₀ values of 91.45 ± 1.87 and 42.32 ± 1.45 μM, respectively [130]. Lee et al. studied the endophytic fungal strain Gaeumannomyces sp. (JS0464) from the rhizome of marine halophyte Phragmites communis. They identified eleven compounds from spectroscopic analysis and stimulated them towards NO reduction activity in microglia BV-2 cells. Nine compounds significantly reduced NO production [131].

Zhang et al. extracted butryolactone-I (ZB5-1) from A. terreus and reported the anti-neuroinflammatory effect on LPS induced BV-2 microglia cells [132]. Two new compounds, (Z)-7,4′-dimethoxy-6-hydroxy-aurone-4-O-b-glucopyranoside and (1S,3R,4S)-1-(4′hydroxyl-phenyl)-3,4-dihydro-3,4,5-trimethyl-1H-2-benzopyran-6,8-diol, were isolated from Penicillium citrinum of mangrove plant (Bruguiera gymnorrhiza). Both the compounds were tested against MPP⁺ induced PC 12 cells and compared with NAC (N-acetyl-L-cysteine, positive control). But only (Z)-7,4′-dimethoxy-6-hydroxy-aurone-4-O-b-glucopyranoside significantly increased the cell viability and mitochondrial membrane potential. It also inhibited caspase-3 and caspase-9 activity in MPP⁺-treated PC12 cells [133]. Xyloketal B, from mangrove endophytic fungus Xylaria sp., can scavenge free radicals and thus protect PC 12 cells against ischemic-induced neurotoxicity. Moreover, it can also show neuroprotective effects as it inhibits NADPH-oxidase derived ROS production [134–136].

Two compounds (2R,3S)-pinobanksin-3-cinnamate and 15-hydroxy-(22E,24R)-ergosta-3,5,8(14),22-tetraen-7-one were isolated from Penicillium sp. FJ-1 associated with Acanthus ilicifolius Linn (mangrove plant). The neuroprotective effect was demonstrated on PC 12 cells. The result of the experiment showed that (2R,3S)-pinobanksin-3-cinnamate increased the viability of corticosterone-damaged PC12 cells to give neuroprotective activity [137]. Endophytic fungus Streptomyces sp. associated with mangrove plants produced various compounds. After certain modifications of the quinone rings (C-17 and C-19) of gelandamycin, geldanamycin and 19-O-methylgeldanamycin enhanced the survival of P19 cells at a low concentration of 1 mM and prevented neurotoxicity against paclitaxel and vinblastine [77].

Xu et al. isolated three new and twelve known compounds from fungal endophyte A. alternata associated with Psidium littorale. The neuroprotective activity of all fifteen isolated compounds was tested against glutamate-induced PC12 cells, and compounds showed significant improvement in the cell viability from 67.8 ± 5.1% to 84.8 ± 6.5 (40 and 80 μM, respectively). The three compounds that exhibited neuroprotective activities are isosclerone (10) (Fig. 3), indole-3-methylethanoate, and ergosta4,6,8(14),22-tetraen-3-one [71]. Song et al. isolated one novel anthraquinone and three known anthraquinones from endophyte Colletotrichum sp. JS-0367 of Morus alba and identified compounds with the help of different methods, including 1D/2D-NMR and HRESIMS. The cell viability significantly increased only with compound evariquinone as it inhibits intracellular ROS aggregation and Ca²⁺ influx, reduces the phosphorylation of MAPKs, and strongly attenuates cell apoptosis [4].

Lee et al. evaluated the effect of bioactive compounds isolated from endophyte Fusarium lateritium associated with Cornus officinalis fruits. Three tricyclic pyridone alkaloids were identified as 6-deoxyoysporidinone, 4,6′-anhydrooxysporidinone, and sambutoxin. All three compounds were tested for glutamate induced oxidative stress, excess Ca²⁺ influx, and apoptosis in HT22 hippocampal neuronal cell line. Sambutoxin decreases cell viability from 69.2 (25 μM) to 36%, while 6-deoxyoysporidinone exhibits no protective effect. The cell viability with the treatment of 4, 6′-anhydrooxysporidinone gradually increases by 63.1 to 104.8% at different concentrations. Thus, 4, 6′-anhydrooxysporidinone shows more protective effects than NAC (1 mM; 87.3 ± 4.1%, and at 10mM; 99.9 ± 3. 8%) [2].

A new meroterpenoid, sartorypyrone E, and eight other known compounds, sartorypyrone A, cyclotryprostatin B, fumitremorgin B, fumitremorgin A, aszonalenin, acetylaszonalenin, and fischerin (11) (Fig. 3) were isolated Neosartorya fischeri JS0553 from the leaves of Glehnia littoralis [138]. Further, the protective effects of these compounds against glutamate induced HT22 cell were determined, and fischerin showed neuroprotective effect. Bang et al. reported the glutamate excitotoxicity in the neuronal cell relates to CNS injury and neurodegenerative diseases. They were the first to report glycosylated cyclic depsipeptides with neuroprotective activity. Based on experimentation, five unique compounds, colletotrichamides A−E, were isolated from halotype Suaeda japonica associated with fungus Colletotrichum gloeosporioides JS419. As a result of glutamate toxicity tests on hippocampal HT22 cells, colletotrichamide C (12) (Fig. 3) exhibited strong neuroprotective activity. This indicates that colletotrichamide C and their derivatives have significant advantages over other molecules to reduce glutamate excitotoxicity [73]. Choi et al. tested neuroprotective activity of secondary metabolites isolated from Fusarium solani JS-0169 from Morus alba leaves and found that fusarubin (13) (Fig. 3) exhibited potent antioxidant and neuroprotective activities when tested against glutamate-induced HT22 cells [139].

A novel compound, chrysogenamide A was isolated from endophytic fungus Penicillium chrysogenum associated with Cistanche deserticola, which exhibited neuroprotective effect against H₂O₂ injured Human neuroblastoma SH-SY5Y cells [140]. This compound inhibits cell death by upgrading cell viability by 59.6% (at 1 × 10⁻⁴ μM). Shen et al. isolated various classes of cytochalasans from endophytic fungus Chaetomiun globosum WQ and Phomopsis sp. IFB-E060 associated with Imperata cylindrical and Vatica mangachapoi, respectively [74]. Chaetoglobosin F and isochaetoglobosin D from C. globosum WQ and cytochalasin H from Phomopsis sp. IFB-E060 showed neuroprotective activity against MPP⁺ induced damaged PC 12 cells, but amongst three, chaetoglobosin F showed the most substantial protective effect to protect cells from oxidative stress. Zhu et al. isolated thirteen compounds from the endophytic fungus Phyllosticta capitalensis associated with Loropetalum chinense. Compound citreoanthrasteroid A and linoleic acid showed potential activities with EC₅₀ value of 24.2 and 33.9 μ M on glutamate-induced PC12 cells [141].

Animal models

Mice models have been used to address on bioactive compounds isolated from endophytic fungi. The main aim was to address developing and accessing novel therapeutic drugs against neurodegeneration. The research was conducted with regulatory provisions, inspection, and licensing of animal use; therefore, it takes a long time for fundamental results and validation. It has been reported that biological products derived from microbial sources effectively improve cognitive dysfunction in rats and elevate memory impairment [142].

Vig et al. reported the neuroprotective efficacy of quercetin (14) (Fig. 3) isolated from endophytic fungus Nigrospora oryzae associated with Tinospora cordifolia, which showed significant decrease in AChE activity and restored SCO-provoke (scopolamine) alteration in cholinergic activity in hippocampus [143]. The experiment concluded that quercetin has enough potential to cure learning and memory shortfalls due to the AChE mechanism. Therefore, it plays a vital role in treating and managing AD. Wang et al. isolated natural products from endophytic fungus Galactomyces geotrichum associated with Laminaria japonica and evaluated secondary metabolites on D-galactose-induced AD mice [144]. Their results suggested that all seven compounds, 7,8-dimethyl-iso-alloxazine, 1-methyl-3-benzyl-6-(4-hydroxybenzyl)-2, 5-piperzainedione, cyclo-(Phe-Pro), cyclo-(Leu-Pro), cyclo-(Pro-Gly), cyclo-(Gly-Leu), and uracil (15) (Fig. 3), protected the brain against damages and decreased AChE content in AD mice. Table 2 lists several endophytic fungi from various host plants capable of producing bioactive compounds with neuroprotective activities.

Table 2.

List of some bioactive compounds produced by endophytic fungi possessing neuroprotective activities

Host plant	Endophytic fungi	Neuroprotective compounds	Screening method	Mechanism of action	References
Huperzia serrate	FL14 (Ascomycota spp.), FL7 (Dothideomycetes spp.), and JS4 (Colletotrichum spp.)	Huperzine-A	Ellman	Natural compounds are AChEIs and modify β-amyloid peptide, ↓ oxidative stress, inflammation, anti-apoptotic and regulate nerve growth factor. ↑α-secretase activity, ↓ Aβ levels. Inhibits nitric oxide production in LPS-activated macrophages	[78]
	Shiraia sp.				[79]
	Cladosporium cladosporioides LF70				[81]
	Trichoderma species				[83]
	Mucor racemosus NSH-D, Mucor fragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum NSW-V				[84]
	Alternaria brassicae				[85]
	Fusarium sp. Rsp5.2				[86]
Pharbitis nil	Chaetomium sp. NF00754	Orsellide A, orsellide C, and chetomin	Ellman		[87]
Huperzia serrate	Chaetomium sp.	1-O-methylemodin, 5-methoxy-2-methyl-3-tricosyl-1,4-benzoquinone, 6-epoxy-7-one-8(14),22-dien-ergosta	Ellman		[88]
Chinese yam, Dioscorea opposite	Chaetomium globosum	Yamchaetoglobosin A	Ellman		[90]
Panax notoginseng	Chaetomium globosum	3-methoxyepicoccone and epicoccolides B	Ellman		[91]
Tabacum L.	Paecilomyces sp. TE-540	12-hydroxyalbrassitriol and 2-hydroxyalbrassitriol	Ellman		[92]
Piper aduncum and Alibertia macrophylla	Xylaria sp. and Penicillium sp.	Dihydroisocoumarin	TLC-based acetylcholinesterase inhibitory assay		[93]
Bruguiera gymnorrhiza	Cladosporium cladosporioides MA-299	Ent-cladospolide F	Ellman		[95]
Mangrove Plant	Penicillium sp. SK5GW1L	Arisugacin B, territrem C, and terreulactone C	Ellman		[96]
Asparagopsis taxiformis (Marine red alga)	Nemania bipapillata (AT-05)	(+)-(2R,4R,5R,8S)-4-deacetyl-5-hydroxy-botryenalol, and nemenonediol A	Ellman		[97]
Sponge Axinella verrucosa	Talaromyces sp. strain LF458	Talaromycesone A, talaroxanthenone, and AS-186c	Ellman		[98]
Codium fragile (Marine green alga)	Acrostalagmus luteoalbus TK-43	Acrozine A	Ellman		[99]
Mangrove tree	Aspergillus sp. 16-5c	Isoaustinol, dehydroaustin, dehydroaustinol	Ellman		[100]
Coculturing of fungi	Nigrospora oryzae with Irpex lacteus	Nigrosirpexin A	Ellman		[75]
Hybrid “Neva” (Populus deltoides Marsh × P. nigra L.)	Hyalodendriella sp. Ponipodef12	Botrallin and palmariol B	Ellman		[101]
Catharanthus roseus	Alternaria alternate	Altenuene	Ellman		[102]
Senna spectabilis	Phomopsis sp.	Cytochalasin H	Ellman		[103]
Lamium amplexicaule	Alternaria alternate	Fumitremorgin C, spirotryprostatin A, and cephalimysin A	Ellman		[104]
Sapindus saponaria L.	Curvularia sp. G6-32	Asperpentyn	Ellman		[106]
Tripterygium wilfordii Hook.f.	Aspergillus terreus Thom	Spiroterreusnoids A–F	Ellman		[107]
Uncaria rhynchophylla	Colletotrichum gloeosporioides GT-7	Colletotrichine B	Ellman		[110]
Rauwolfia macrophyll	Curvularia sp. T12	2′-deoxyribolactone, hexylitaconic acid, and ergosterol	Ellman		[111]
Nicotiana tabacum	Rhizopycnis vagum Nitaf22	Rhizovagine A	Ellman		[113]
Marine sediments	Aspergillus flocculosus	6β,9α,14-trihydroxycinnamolide and insulicolide A	Murine neuroblastoma Neuro-2a cells	Protection against 6-OHDA-induced neuronal death ↓ oxidative stress, ↓ ROS, AChEIs	[118]
	Penicillium sp. KMM 4672	6-Hydroxy-N-acetyl-β-oxotryptamine	Neuro2A cells		[117]
	Aspergillus niveoglaucus	Flavoglaucin	Neuro2A cells		[119]
	Aspergillus niveoglaucus	Cryptoechinuline B, neoechinulin C, neoechinulin B, and neoechinulin	Neuro2A cells		[128]
	Aspergillus terreus Y10	Asperteretal F	LPS-induced microglia cell		[129]
Mangrove plant	Aspergillus terreus	Asterriquinone B4 and asterriquinone C1	Neuro2A cells		[130]
Rhizome of marine halophyte Phragmites communis	Gaeumannomyces sp. (JS0464)	Stemphol C, stemphol D, 1-O-methyl-6-O-(α-D-ribofuranosyl)-emodin, stemphol, 1-O-methylemodin, 5α,8α-epidioxyergosta-6,9,(11),22-trien-3-ol, ergosterol peroxide, 5α,8α-epidioxy-(22E,24 R)- 23-methylergosta-6,22-dien-3β-ol, and β-sitosterol	Microglia BV-2 cells	↓ NO and anti-inflammatory	[131]
Bruguiera gymnorrhiza	Penicillium citrinum	(Z)-7,4′-dimethoxy-6-hydroxy-aurone-4-O-b-glucopyranoside	PC 12	↓oxidative stress, ↓calcium influx, ↓phosphorylation (in vitro); PC12 cell line ↓NO, ↓iNOS (in vitro); PC12 cell line	[133]
Acanthus ilicifolius Linn.	Penicillium sp. FJ-1	(2R,3S)-pinobanksin-3-cinnamate and 15-hydroxy-(22E,24R)-ergosta-3,5,8(14),22-tetraen-7-one	PC 12		[137]
Imperata cylindrical	Chaetomiun globosum WQ	Chaetoglobosin F and isochaetoglobosin D	PC 12		[74]
Vatica mangachapoi	Phomopsis sp. IFB-E060	Cytochalasin H	PC 12		[74]
Loropetalum chinense var. rubrum	Phyllosticta capitalensis	Citreoanthrasteroid A and linoleic acid	PC 12		[141]
Psidium littorale	Alterneria alternate	Isosclerone, indole-3-methylethanoate, and ergosta4,6,8(14),22-tetraen-3-one	PC 12		[71]
Mangrove plant	Streptomyces sp.	Gelandamycin and 19-O-methylgeldanamycin	P19 cells	↓ neurotoxicity and cytotoxicity	[77]
Morus alba (mulberry)	Colletotrichum sp. JS-0367	Evariquinone	Murine hippocampal HT22 cells	Antioxidant (in vitro); HT22 cell line	[4]
Cornus officinalis	Fusarium lateritium	4, 6′-anhydrooxysporidinone	HT22 hippocampal neuronal cell line		[2]
Glehnia littoralis	Neosartorya fischeri JS0553	Fischerin	HT22 cell		[138]
Suaeda japonica	Colletotrichum gloeosporioides JS419	Colletotrichamide C	HT22 cell		[73]
Morus alba	Fusarium solani JS-0169	Fusarubin	HT22 cell		[139]
Cistanche deserticola	Penicillium chrysogenum No. 005	Chrysogenamide A	SH-SY5Y cells	Induction of PGK1 (in vitro); SH-SY5Y cell line. ↓ Ca2+ influx and inhibit JNK and p38 phosphorylationCaspase-3 activation	[140]
Tinospora cordifolia	Nigrospora oryzae	Quercitin	Animal model (mice)	SCO-provoked alteration restored and AChE activity ↓	[143]
Laminaria japonica	Galactomyces geotrichum	7, 8-dimethyl-iso-alloxazine, 1-methyl-3-benzyl-6-(4-hydroxybenzyl)-2, 5-piperzainedione, Cyclo-(Phe-Pro), Cyclo-(Leu-Pro), Cyclo-(Pro-Gly), Cyclo-(Gly-Leu), and Uracil	Animal model (mice)	↑ antioxidant capacity, ↓ AChE activity and ↑ choline acetyltransferase activity.	[144]

Challenges and future prospectives

Bioactive compounds isolated from endophytic fungi have great potential in therapeutics, but very few bioactive compounds from endophytic fungi with good quality and quantity have been explored in managing neurodegenerative diseases. Some studies reveal that endophytic fungi’s distribution and population structure depend on taxonomy, genetic background, physiology, and environmental conditions. Therefore, these findings conclude that there is a scope for investigating bioactive compounds that certain medicinal plants produce under specific ecological conditions. Endophytic fungi occur within all known host plants in various ecosystems, but due to differences in their geographical areas there is a difference in diversity and host-pathogen interactions. These preferences have not been well documented; hence, data needs to be compiled from regions to continents to encompass the whole ecosystem. Host-plant interactions benefit each other in three different ways: (1) enhancement in the growth of the host plant, (2) resistance from biotic and abiotic stress of the host plant, and (3) accumulation of secondary metabolites that can be used as drugs and initially the medicinal plants produced them. Thus, these aspects have practical implications for obtaining and producing drugs with improved quality and higher quantities. The exploration of endophytic fungi with neuroprotective compounds is one of the insightful topics for many researchers. Some of the rare and endangered medicinal plants that are extremely difficult to germinate can be facilitated with the application of endophytic fungi. Targeted endophytic fungi can promote seed germination of medicinal host plants, increasing the opportunity for seed germination that cannot be germinated under normal conditions. Various host plants are still researching to produce a secondary metabolite that can be further developed for potential and targeted drug candidates against neurodegeneration. However, endophytic fungi in the genus Mycena have facilitated the artificial culture of Dendrobium nobile and D. chrysanthum [145]. This method is beneficial for rare and endangered plant species used in breeding programs where seed germination is crucial.

Although notable progress has been made in fungal endophytes, the work reported on the beneficial strains related to neuroprotection is confined to experimental studies. Much more effort should be made into cell line trials and applications to obtain higher-quality secondary metabolites, so that further in vivo studies can be done effortlessly. Multiple studies have been done in vitro and in vivo to quantify the neuroprotective effects of bioactive compounds isolated from endophytes. However, most of the promising neuroprotective compounds isolated from endophytes have a low yield. This problem might be overcome by co-culturing and better optimizing the culturing conditions for producing neuroprotective compounds. This current article upgrades the information regarding the diverse range of endophytic fungi used to isolate neuroprotective compounds. There is an urgent need for deep research on endophytic fungi producing neuroprotective compounds, which can aid the treatment of different neurodegenerative diseases and fulfil the market demand as a potential drug. Further, a systemic approach is needed as it will contribute to identifying therapeutic compounds and describing their need for neuroprotection. There are ethical issues with using animal models in the early stages of drug discovery. Thus, in vitro studies help us to study the cellular response in a closed system where the experimental conditions can be maintained. Endophytes can promote the accumulation of bioactive compounds produced initially by the host plant to utilize this valuable advantage. Therefore, we can increase the synthesis and accumulation of secondary metabolites of the medicinal plants for higher-quality crude drugs by adding particular endophytic fungi to the host plants. In this way, a new range can be developed for producing natural medicines effectively and efficiently because the relationship between host-pathogen is completely understood.

Here, we summarize endophyte diversity and different host plant community compositions to explore secondary metabolites using different techniques, but the vast majority of endophytes have yet to be adequately characterized. Therefore, studying the methods that can obtain a complete qualitative picture of the endophyte community is suggested. High-throughput sequencing has several advantages over traditional techniques in terms of cost, time consumption, sample size, and recovery of more species. It should be employed more widely in future exploration of endophytes to open up the “black box” of the fungal community. There is a need for bioinformatics data and whole-genome sequencing to predict the gene (s) responsible for secondary metabolite production. Investigation should be done to trigger specific genes responsible for the overproduction of beneficial secondary metabolites. Other strategies include genetic engineering, strain improvement, and gene cluster amplification for the overproduction of secondary metabolites. Another important difficulty in increasing the yield of desired secondary metabolites could be overcome with the help of mutagenesis, optimizing culture conditions, using combinatorial chemical synthesis with inducers or elicitors for the targeted biosynthetic pathways. Introduction to modern techniques such as genome mining and metagenomics can help in the optimized production of secondary metabolites from the fungal community. One of the genome editing tools, CRISPR/Cas, promotes the identification of novel metabolites. These techniques are limited to the identification of imperative gene cluster (s). However, several silent gene clusters can be activated with the help of molecular and epigenetic based protocols as endophytes imitate the natural habitat of their host plants [68]. Growth conditions also trigger the performance of endophytic fungi. Therefore, co-cultivation and adding inducers in the culture media can stimulate the production of novel secondary metabolites.

Conclusions

This review gives an insight into various endophytes and their bioactive compounds as neuroprotective agents. Different methods have been used extensively such as enzymatic methods to evaluate the in vitro neuroprotective activity of the bioactive compounds, while some preferred cell lines and in vivo models to evaluate neurodegenerative disorders. Studies are followed by the characterization and identification of bioactive compounds to evaluate the real potential of endophytic fungi as a source of neuroprotective agent. Currently, the available neuroprotective drugs are ineffective, toxic, and expensive. They also have side effects. Therefore, the compounds from endophytes have endless possibilities and biological properties to treat or slow neurodegenerative disorders. Researchers tend to shift towards recent advancements in biotechnology for screening novel biomolecules for treating life-threatening disorders. The bioactive compounds mentioned play their protective role via decreasing AChE, ROS, cytotoxicity, and restoration of neurons. Endophytic fungi have been studied for many years, but some endophytes remain unexplored. Phytochemicals such as alkaloids, terpenoids, tannins, anthraquinones, and flavonoids can be used as potential drug candidates to treat neurological disorders. Endophytic fungi have produced a wide array of bioactive compounds, but their importance to treat neurodegeneration is barely explored. The bioactive compounds are required to be isolated from various sources. Therefore, there is a need to exploit secondary metabolites from fungal endophytes in terrestrial, mangrove, and marine habitats. They may provide high-value neuroprotective compounds for large-scale production of drug candidates. Subsequently, the process of optimization, co-cultivation, and strain improvement could be explored to upgrade the potential of these endophytic fungi. Thus, the review arouses attention towards the compounds isolated from endophytic fungi with good neuroprotective activities and further investigation of the bioactive compounds in vitro and in vivo to create a potential and targeted drug by utilizing fungal endophyte resources that can create a better life for the mankind and greatly enhance the human pharmaceutical sector.

Declarations

Consent for publication

We declare that this manuscript, fully or partially, was not published before and is not currently being considered for publication elsewhere.

Conflict of interest

The authors declare no competing interests.