Neuroprotective Metabolites from Endophytic Microbes: A Review

Introduction

Alteration of normal activity of nervous system due to exposure to natural or manmade toxic substances which kill or even discompose neurons that transmit and process signals in the brain and other parts of nervous system leads to neurotoxicity. Hence, neurotoxicity refers to direct or indirect effect of chemicals that disrupt the nervous system. Human beings are grieving from nervous related ailments due increase in the population and aging.¹ This is due to occurrence of oxidative stress, especially the remarkable aggregation of reactive oxygen species (ROS), damage of mitochondria and nucleic acid.² The increase in the concentration of ROS is due to high concentration of glutamate leading to neuron death.³ Exposure to lead (Pb) and mercury (Hg), consumption of additives added in certain foods, use of substandard cosmetics, chemotherapy given after cancer surgery, treatment involving radiation, organ transplant procedures, and side effects due to drug therapy are some of the main causes leading to neurotoxicity. Symptoms of neurotoxicity include cognitive and behavioral changes, sexual infirmity, numbness or weakness in limbs, headache, amnesia or dementia etc. Individuals with certain disorders may be especially vulnerable to neurotoxicants.

Endophytes could be any organism, either bacteria, fungi, actinomycetes or mycoplasma, which reside inside the tissues of plants showing mutualistic relationships without infecting any of the plant cells. Variety of novel secondary metabolites and known analogues of plant metabolites are produced by endophytic microbes. Structurally distinctive and therapeutically active natural products, such as flavonoids, phenolic acids, polyketides, terpenoids, benzopyranones, quinines, steroids, alkaloids, etc., are obtained from endophytes for their potential use in medicine, agriculture or industry.⁴ Further, these are recognized as prospective sources of new compounds for exploitation in medicine industry with more and more bioactive natural products isolated from the endophytes.⁵ Recently, secondary metabolites from plant-associated fungi have drawn attention from chemists and pharmacologists due to their novel structures and significant biological activities such as antimicrobial, antiviral, anticancer, antioxidant, neuroprotective, and antifungal activities^6-10 in drug discovery and development.

Availability of drugs for neurodegenerative diseases is still limited. Although many new drug candidates have been explored recently, adverse effects like kidney infections, irregular heartbeat, diarrhea, loss of appetite, weight loss, depression, hindered their clinical applications. Natural compounds have been widely studied as potential neuroprotective agents because of their characteristics of multiple targets and low cytotoxicity. If the focus is narrowed to neuroprotective compounds, research data is available on bioactive compounds obtained from endophytes for their neuroprotective effects. Hence, this review provides an update on scientific literature available related to various endophytes isolated from medicinal plants, their secondary metabolites, therapeutic potential and molecular mechanism of action with special reference to neuroprotection.

Neuroprotective Metabolites from Endophytic Microbes

The occurrence of Penicillium citrinum, a filamentous fungus in a medicinal plant, Ocimum tenuiflorum L was first time explored by Wu et al. ⁹ Recent studies found the inhabitance of this fungus in various parts of plants like Ceratonia siliqua, Codonopsi spilosula, Digitaria bicornis (Lam.) Roem. & Schult. Boswellia sacra. Many chemically diversified bioactive metabolites such as polyketides, alkaloids, bisabolane-type sesquiterpenes, benzopyran derivatives etc., are isolated from Penicillium citrinum. ^10-16 Wu et al., isolated Penicillium citrinum from mangrove Bruguiera gymnorrhiza. Ethanolic extract of mycelia was used to isolate novel secondary metabolites, (Z) -7,4′-dimethoxy-6-hydroxyaurone-4-O-β-glucopyranoside (1) and acitrinin derivative (1S,3R,4S)-1-(4′-hydroxyl-phenyl)-3,4-dihydro -3,4,5-trimethyl-1H-2-benzopyran-6,8-diol (2). Neuroprotective activity of isolated compounds was studied against MPP-induced oxidative stress in PC12 cells. Compound (1) exhibited potent neuroprotective activity.¹⁷

An endophytic fungus Colletotrichum sp. JS-0367 inhabited in the leaves of Morus alba Linn. was isolated by Song et al. Chemical investigations on ethyl acetate fraction of the fungus found to contain one novel anthraquinone derivative 1,3-Dihydroxy-2,8- dimethoxy-6-methylanthraquinone (3) and three known anthaquinone compounds, 1-hydroxy-2,3,8- trimethoxy-6-methylanthraquinone (4), 1,2-dihydroxy-3,8-dimethoxy-6-methylanthraquinone (5), and evariquinone (6). Neuroprotective activity of all the isolated compounds was studied against glutamate induced murine hippocampal HT22 cell death. Compound (6) exhibited potent neuroprotective activity by attenuating glutamate-mediated apoptotic cytotoxicity in HT22 cells.¹⁸

Penicillium chrysogenum is common fungus in temperate and subtropical regions and can be found on salted food products. Many researchers demonstrated the medicinal importance of this fungus by isolating bioactive secondary metabolites usually from marine organisms and in plants like Huperzia serrata, Ephedra pachyclada, Oryza sativa etc. Metabolites like polyketide derivatives, polyoxygenated steroids and tetracyclic diterpenes, diterpenoids were obtained from different species of Penicillium chrysogenum with potential biological activities.^19-25 A new bioactive alkaloid Chrysogenamide A (7) and four established metabolites, Circumdatin G (8), 2-[(2-hydroxypropionyl) amino] benzamide (9), 2’,3’-dihydrosorbicillin (10) and (9Z,12Z)-2,3-dihydroxypropyl octadeca-9,12-dienoate (11) were isolated by Lin et al. from EtOAc extract of an endophytic fungus Penicillium chrysogenum No. 005 inhabiting in roots of Cistanchede serticola (Y. C. Ma). Compound (7) exhibited neuroprotective activity against oxidative stress induced cell death in SH-SY5Y human neuroblastoma cells by decreasing the cell viability and decreasing the cell death induced by hydrogen peroxide. This suggests the neurocyte protection activity of compound (7).²⁶ Phyllosticta capitalensis exists as a leaf inhabited endophyte present in plants growing in both temperate and tropical regions.²⁷ This fungus was found to be dominant in plants like Vaccinium dunalianum and Citrus, Tea etc.28-30 Zhu et al. isolated endophytic fungus Phyllostica capitalensis from the foliar part of Loropetalum chinensevar rubrum. From the ethanolic fraction of the fungus, one new dioxolanone derivative, Guignandionone G (12) and twelve established metabolites including Citreoanthrasteroid A (13) and linoleic acid (14) were isolated and identified. Compounds were screened for neuroprotective activity in glutamate injured PC12 cell model. Compound (13) and (14) exhibited significant neuroprotective activities with EC50 of 24.2 and 33.9 μM, respectively.³¹ Alternaria alternata is one of the most common pathogens found in a variety of natural food products including fruits and vegetables, cereal plants, seeds, and other plant organs. As an endophytic organism, it is found to be good source of cephalotaxine-type alkaloids, biflavonoids and Vinblastin.^32-34 Xu et al., isolated fungal endophyte Alternaria alternata from leaves of Psidium littorale. Chemical investigations of EtOAc fraction of fungus gave a novel liphaticpolyketone namedalternin A (15), a new indole alkaloidal derivative, alternatine A (16), a new sesquiterpene designated as (1R,5R, 6R,7R,10S) 1,6-Dihroxyeudesm-4(15)-ene (17), and 12 known compounds including isosclerone (18), indole-3- methylethanoate (19) and ergosta4,6,8(14),22-tetraen-3-one (20). Neuroprotective activity of all the isolated compounds was studied in glutamate induced PC12 injured cell model. Among the compounds tested, (18), (19) and (20) showed potent neuroprotective activity.³⁵ Two new 2,5-diketopiperazine derivatives, nigrosporamide A (21) and B (22) and eight known analogues were obtained from EtOAc extract of culture broth of Nigrospora camelliae–sinensis S30 present in mangrove Lumnitzera littorea by Huang et al. Neuroprotective activity of all the isolated compounds was studied by determining cell viability on H2 O2 - mediated cytotoxicity for HT22 cell. None of the compounds tested showed neuroprotective activity.³⁶ Thawai et al., isolated an endophyte belonging to genus Microbispora from soil surrounding the roots of Zingiber montanum. Ethyl acetate fraction of fermentation broth of M. hainanensis DSM 45428 gave 2α-hydroxy-8 (14), 15-pimaradien-17,18-dioic acid, a new diterpene compound (23), along with nine known compounds. Compound (23) possessed anti-AChE activity with 52.81±1.24% inhibitions. Further in docking studies, it displayed the suppressive effect on the recombinant human acetylcholinesterase (rhAChE) enzyme with IC50 value of 96.87±2.31 μg/mL by π-alkyl interaction with Trp86 residue of rhAChE. It was also concluded that Compound (23) protected neuronal cells without neurotoxicity from oxidative stress at 1 ng/mL.³⁷ Shen et al., isolated 10-indolyl cytochalasans namely, chaetoglobosin F (24), chaetoglobosin F (25), chaetoglobosin E (26), cytoglobosin A (27), penochalasin C (28), and isochaetoglobosin D (29) and two 10-phenyl cytochalasans viz. cytochalasin H (30) and 18-methoxycytochalasin J (31) from two plant endophytes, Chaetomiun globosum WQ present in Imperata cylindrical and Phomopsis sp. IFB-E060 inhabited in Vatika mangachapai plants. Amongst compounds tested for neuroprotective potential, compounds (24), (29) and (30) exhibited strongest neuroprotective effect on H2 O2 /MPP-induced PC12 cell models estimated by radical scavenging assay by increasing cell viability and decreasing lactate dehydrogenase release.38 In a later study, Westerdyk ellanigra, a mangrove endophyte was separated from the roots of Avicennia marina (Forssk.) Vierh. Westalsan (32), a new cytochalasan alkaloid, along with phomacin B (33) and 19-hydroxy-19,20-dihydrophomacin C (34), were attained from the endophytic fraction. Compounds (32) and (34) showed potent inhibitory activities with IC50 of 0.088 μM and 0.056 μM respectively.³⁹ Bang et al. isolated endophytic fungus Neosartorya fischeri JS0553 from the roots of Glehnia littoralis. Chromatographic investigations on ethyl acetate fraction of the endophyte gave a new meroditerpenoid named sartorypyrone E (35) and eight known compounds, sartorypyrone A (36), cyclotryprostatin B (37), fumitremorgin B (38), fumitremorgin A (39), aszonalenin (40), acetylaszonalenin (41), fischerin (42) and pyripyropene A (43). Among the isolates, compound (42) inhibited ROS, influx of Ca²⁺, mitogen-activated protein kinase phosphorylation in glutamate induced HT22 cell death model and thus exhibited neuroprotective activity.⁴⁰ Vig et al., isolated endophytic fungus Nigrospora oryzae from mature leaves of Tinospora cordiofolia. Ethyl acetate extract of the fungus exhibited AChE inhibitory activity of about 91.4% at 1000 µg/mL. Further, ethyl acetate fraction at 5 mg/kg also showed anti-dementia activity against scopolamine induced neurotoxicity in mice by restoring the AChE concentration in hippocampus. Spectroscopic analysis of ethyl acetate fraction identified the neuroprotective metabolite as Quercetin (44).⁴¹ Neuroprotective activity of azaphilone type polyketides isolated from endophytic Penicillium sp. JVF17 inhabited in the foliar part of Vitexrotundifolia was evaluated against glutamate-induced neurotoxicity by Bang et al. Among the fourteen compounds tested, penazaphilone E (45), isochromophilone VI (46) and peniazaphilone (47) showed potent neuroprotective activity at 25 μM with 100% protection.⁴²

Huperzia serrata (Tunb. ex Murray) Trev. (Lycopodiaceae), a Chinese perennial medicinal fern has potential pharmacological properties, used mainly in the treatment of brain related disorders like Alzheimer disease (AD), schizophrenia and removal of blood stasis.^43-46 Therapeutically useful constituents belonging to the group of flavonoids, alkaloids and triterpenoids have been isolated from the whole plant of H. serrata. ⁴⁷ Lycopodium alkaloids present in the plant are of high importance because of their therapeutic activities.⁴⁸ A highly selective and reversible acetylcholinesterase inhibitor compound is Huperzine A (HupA, 48), which is strongly recommended in the management of AD and Myasthenia gravis in China and USA.^49,50 Due to high demand of HupA, H. serrata has been overexploited and fragmented which made the plant an endangered species in China. Hence, alternate ways like tissue culture technology and endophytic microbes have been identified and studied as a significant source of HupA. Many researchers reported endophytic fungi, which produce HupA. Cladosporium cladosporioides LF70,⁵¹ Shiraia sp. Slf14,⁵² Paecilomyces tenuis YS-13,⁵³ Fusarium sp. Rsp5.2,⁵⁴ Alternaria brassicae AGF041,⁵⁵ Mucorrace mosus NSH-D, Mucorfragilis NSY-1, Fusarium verticillioides NSH-5, Fusarium oxysporum NSG-1, and Trichoderma harzianum⁵⁶ were found to be important sources of HupA. Another potential alkaloidal metabolite isolated from Huperzia serrata (THUNB.) TREVIS is Huperzine B (HupB 49). HupB successfully demonstrated AChE inhibitory activity. Studies on hupB proved a higher therapeutic index in AD models.^57-62 Considering this, an attempt has been made by Zhan et al., for microbial transformation of HupB by Bjerkandera adusta CCTCC M 2017159, a fungal endophyte previously isolated from H. serrata. The study found that Bjerkandera adusta CCTCC M 2017159 was capable of transforming HupB in to its oxygenated derivatives. Chromatographic and spectroscopic methods identified the compounds as 8α,15α-epoxyhuperzine B (50), 16-hydroxyhuperzine B (51) and carinatumin B (52). A moderate neuroprotective activity was exhibited by compound (50) in LPS-induced neuroinflammation injury assay with EC₅₀ of 40.1 nM. This may be due to increase in the viability of U251 cell lines.⁶³ Li et al., isolated a mangrove endophytic fungus Phomopsis sp.xy 21 from leaves of Thai Xylocarpus granatum. Polyketide-derived alkaloids phomopsol A (53), phomopsol B (54), and 3-(2,6-dihydroxyphenyl)-4- hydroxy6-methylisobenzofuran-1(3H)-one (55) were isolated from endophytic fraction. Neuroprotective activity of all the compounds was evaluated against corticosterone-induced injury in PC12 cells. A concentration dependent (5.0−40.0 μM) neuroprotective activity was observed for compounds (53) and (55) with cell viabilities of 76% and 96%, respectively.64 Küçüksolak et al., carried out microbial biotransformation of Cyclocephagenol (56), a novel cycloartane-type sapogenin with tetrahydropyran unit present only in Astragalus sps. Alternaria eureka, an endophytic fungus separated from the leaves of Astragalus angustifolius was used for biotransformation. About 21 metabolites were obtained after biotransformation along with (57). H₂ O₂ induced cell injury method was used to study the neuroprotective activities of parent compound and metabolites. Further, 6-OHDA induced in vitro Parkinson’s disease neurotoxicity model was also carried out for the selected compounds. Results showed that in H₂ O₂ -induced cell death, both (56) and (57) exhibited good activity in a dose dependent manner. It was further concluded that, compounds obtained with oxidation at C-12 improved neuroprotective activity.⁶5 Dahae et al., isolated endophytic fungal strain Fusarium lateritium SSF2 from fruits of Cornus officinalis. Chromatographic and spectroscopic studies of methanolic fraction of endophyte gave tricyclic pyridone alkaloids viz. 6-deoxyoysporidinone (58), 4,6′-anhydrooxysporidinone (59), and sambutoxin (60). Compound (59) demonstrated neuroprotective effect against glutamate-induced HT22 cell death, attenuated the ROS generation, inhibited increased levels of Ca²⁺ and depolarization of mitochondrial membrane potential in a dose dependent manner. In addition, it also enhanced the expressions of Nrf2 and HO-1.⁶⁶ Fusarium solani and its species are the most common fungal pathogens of chondrichthyans. Endophytic nature of this fungus was observed in medicinal plants like Cassia alata, Glycyrrhiza glabra, Catharanthus roseus, Chloranthus multistachys etc. This fungus was found to harbor bioactive secondary metabolites belonging to the class of napthaquinone and aza-anthraquinone derivatives, 7-desmethyl fusarin C derivatives, alkaloids and Polyhydroxysterols.^67-71 Choi et al. isolated an endophytic fungus Fusarium solani JS-0169 from the leaves of Morus alba. Six bioactive metabolites were obtained from ethyl acetate fraction of culture filtrate of fungi. Chemical investigation identified one new gamma pyrone, and four established compounds, fusarester D (61), karuquinone B (62), javanicin (63), solaniol (64) and fusarubin (65). Neuroprotective activity of all isolated compounds was evaluated against glutamate-induced cytotoxicity in HT22 cells murine hippocampal neuronal cell death. Compound (65) significantly increased the cell viability to 90.7±4.5% at the concentration of 12.5 µM. This is also supported by its DPPH radical scavenging activity. Further, to identify the possible mechanism of action target genes of (65) were predicted using the Bioinformatics Analysis Tool for Molecular mechanism of Traditional Chinese Medicine (BATMAN-TCM) platform and related biological pathways were investigated using Gene Set Enrichment Analysis (GSEA). One of the mechanism predicted by this study was increase in the levels of ubiquinone, a marker antioxidant co-enzyme observed during the biochemical investigations of neurodegenerative diseases by fusarubin. Molecular docking studies revealed a strong binding affinity to NQO1 by fusarubin which may be due to the formation of ubiquinol from ubiquinone to depict antioxidant activity.⁷²

Glycosylated cyclic lipodepsipeptides, collerotrichamides (A-E, 66-70) were isolated from fungus Colletotrichum gloeosporioides JS419 inhabited in the leaves of Suaeda japonica by Bang et al. Neuroprotective activity of all the compounds was evaluated against glutamate in hippocampal HT22 cells. Compounds (67), (68) and (70) showed neuroprotective activity against glutamate in hippocampal HT22 cells. Among the three, (68) exhibited almost 100% cell viability at 100 μM.⁷³ Liu et al., isolated an endophytic fungus Penicillium sp. FJ-1 from mangrove Acanthus ilicifolius Linn. Chemical investigation showed presence of new flavanone, (2R,3S)- pinobanksin-3-cinnamate (71) and new steroid, (22E,24R)-ergosta-3,5,8(14),22-tetraen -7-one (72) from crude fraction of the fungus. Compound (71) exhibited strongest neuroprotective effects against corticosterone-damaged PC12 cells.⁷⁴ A halophytic fungus Penicillium chermesinum (ZH4-E2) was isolated from the stem of Kandelia candel. Ethyl acetate fraction of the fungus gave eight compounds, of which 3ʺ-deoxy-6′-O-desmethylcandidusin B (73) and 6′-O-desmethylcandidusin B (74) inhibited acetylcholinesterase with IC50 values of 7.8 and 5.2 μM, ^{respectively.75} A new α-pyronemeroterpene, Arigsugacin I (75), and two known compounds, arigsugacins F (76) and territrem B (77) were isolated from the methanolic extract of the endophytic Penicillium sp. SK5GW1L inhabited in the leaves of a mangrove plant Kandelia candel. Compounds (75), (76) and (77) exhibited AchE inhibition with IC50 of 0.64 µM, 0.37 µM, and 7.03 nM, respectively.⁷⁶ Continuing investigations on Penicillium sp. SK5GW1L, Ding et al., isolated α-pyronemero terpenoids namely, 3-epiarigsugacin E (78), arisugacin D (79), arisugacin B (80), territrem C (81), and terreulactone C (82). Among all the compounds tested, (80), (81) and (82) showed AChE inhibitory action with IC50 of 3.03, 0.23 and 0.028 μM, respectively.⁷⁷ Curvularia sp. G6-32, an endophytic fungus was isolated from the leaves of Sapindus saponaria L. Spectroscopic and mass spectrometry analyses of ethyl acetate concentrate of the fungi was found to contain (-)-asperpentyn (83), an epoxyquinone derivative. The ethyl acetate fraction inhibited activity of butyrylcholinesterase with IC50 of 110 µg /mL.⁷8 Singh et al. isolated fungal endophytes having AChE inhibitory potential from methanolic extract of foliar endophytes inhabited in Withania somifera, Tinospora cordifolia and Ficus religiosa. Diethyl ether extract of fungal mycelia Cladosporium uredinicola isolated from T. cordifolia showed maximum AChE inhibitory activity.⁷⁹ AlQaralleh et al. isolated an endophytic Fusarium sp. from the stem of Euphorbia sp. Fraction prepared with ethyl acetate of the fungus was screened for in vitro AChE inhibitory activity. The ethyl acetate fraction of Fusarium sp. inhibited the AChE with IC⁵⁰ of 177.0±13.7 µg/mL. It was concluded that the AChE inhibitory potential may be due to alkaloidal constituents in the endophytic fraction.⁸⁰ Aspergillus niger, an endophytic fungus inhabited in the leaves of Centella asiatica was isolated by Shastry et al. Fractions of the fungus prepared with ethyl acetate and n-butanol (50 and 100 mg/kg) were screened for nootropic activity in young and aged mice using elevated plus maze and Morris water maze tests. After the study, brain homogenate was used to estimate AChE and biogenic amines. Ethyl acetate fraction of Aspergillus niger significantly improved memory of both young and older mice by increasing the acetylcholine concentration. Further, the content of dopamine and nor-adrenaline was found to be decreased in the brain homogenate. This is also supported by histopathological studies of hippocampal region of mice brain.⁸¹ Details of neuroprotective constituents from the endophytes are mentioned in Table 1. The structures of these metabolites are presented in supplementary Figure 1.

Since endosymbiotic microbes must interact biochemically with host tissues, it is likely that many endophytes produce secondary metabolites with specific biological activity. Recent interest has focused on pharmaceutical and therapeutic applications of endophytic microbes. As far as concerned to the neuroprotection, there is a separate list of herbs in Indian system of medicine (Medyarasayanas) which are used as brain tonics. The ecological and environmental factors made it difficult to get these drugs in all seasons. Hence, identification of a specific endophytic microbe producing neuroprotective metabolite is a potential area where researchers can focus. Biotechnological approach can be utilized to increase the yield of the metabolite. Endophytic microbes can also be utilized as catalytic mediators for the green synthesis of neuroprotective metal nanoparticles with reduced toxicity.

Conclusion

Neurodegeneration is observed when there is an alteration in the functional and structural integrity of the central nervous system. Since there is a limited ability of regeneration of neurons in neurodegenerative disorders, a reliable and successful therapy is a major issue. Current therapeutic approaches rely mainly on abrogation of symptoms and leave the dying neurons to their fate. Drugs like tacrine, donepezil, rivastigmine, and galantamine were approved as AChE inhibitors for the treatment of neurodegenerative diseases. However, they are non-selective and have adverse health side effects. Therefore, neuroprotective compounds from natural microbial sources represent an interesting alternative. Microbes are used to produce alternative fuels to meet growing energy demands, new crops to feed our rapidly growing population, and medicines to fight emerging human diseases. The promising use of 16 endophytic microbes in agriculture, pharmaceuticals and medicine has attracted researches to find alternative source of therapeutically useful compounds. The multiple applications of endophytic organisms have been intensively exploited over the last two decades not only as a treasure of therapeutic metabolites against a wide array of disease, but also as means to reduce environmental pollution and improve agriculture. Chemical moieties from known endophytes have been used against many disease models including neurodegenerative diseases. Many endophytic constituents like HupA, HupB have been commercially exploited for neuroprotective formulation development. Hence, there is great hope from endophytic microbes and their novel metabolites for their potential use in the alleviation of many diseases.

Conflict of Interest

None