Endophytic Mycoflora as a Source of Biotherapeutic Compounds for Disease Treatment

Article history: Received on: 27/04/2016 Revised on: 14/06/2016 Accepted on: 29/07/2016 Available online: 29/10/2016 Endophytic mycoflora are ubiquitous organisms residing in the internal tissues of the plants, at least for a portion of their lives without causing apparent symptoms of infection. Endophytes serve as rich sources of novel natural compounds with a wide-spectrum of biologically active agents. This review reveals the significance of endophytic mycoflora from plants as sources of bioactive organic compounds. The bioactive compounds produced by endophytic fungi originate by various biosynthetic pathways like PKS/NRPS. These compounds belong to diverse structural groups such as alkaloids, benzopyranones, chinones, cytochalasines, depsipeptides, enniatines, flavonoids, furandiones, isocumarines, peptides, polyketones, phenols, quinols, terpenoids, tetralones and xanthones were characterized by NMR, mass spectrometry, X-ray crystallography etc. Therefore, endophytes, represent a chemical reservoir for array of new compounds which are anti-cancerous, antimicrobial, anti-diabetic, anti-oxidant, anti-parasitical, anti-viral, anti-mycobacterium, anti-insecticidal, antimalarial, anti-biotic, immunosuppressive & immunomodulatory agents, also in addition, other compounds were used in pharmaceutical and agrochemical industries. This paper mainly focuses on the exploration of novel and useful compounds from endophytic mycoflora, and study of their roles in cure of diseases, the recent scenario of screening approach for novel drugs and their pharmacological interest.


INTRODUCTION
Plants produce bioactive constituents capable of preventing or curing illnesses. They also provide a unique environment for endophytes (Rebecca et al., 2011). Endophytes are microorganisms that inhabit the healthy tissues of living plants without causing any apparent symptoms of disease . The majority of endophytes are fungi (Kharwar et al., 2008).  added that endophytic fungi have a mutualistic relationship with the host, protecting the host against pathogen and in some cases may be an opportunistic pathogen. Most of the endophytes are known to possess biosynthetic capabilities greater than the host plant due to their long co-evolution and genetic recombination (Fernandes et al., 2009). Endophytic fungi have been recognized as important and novel resources of natural bioactive products with potential application in agriculture, medicine and food industry . According to Hussain et al. (2009) ;Nithya et al. (2011) plants have been recognized as a repository of fungal endophytes with novel metabolites of pharmaceutical importance. Many endophytes have the potential to synthesize various bioactive metabolites that may directly or indirectly be used as therapeutic agents against numerous diseases (Kusari and Spiteller, 2012). Endophytes contain different bioactive compounds for commercial exploitation of vital therapeutic drugs, which mainly include different types of secondary metabolites and were reported to elicit a number of pharmacological effects (Xu et al., 2008;Joseph and Priya, 2011). Dompeipen et al.,(2011); Tenguria et al.,(2011) pointed out that above bio-therapeutic compounds are selected on the basis of their role in the treatment of various infectious diseases.
This review focuses particularly on the role of endophytic fungi in the production of natural products, the importance of endophytic fungi in the screening approach for novel drugs as a novel alternative method to obtain such compounds. The above bio-therapeutic compounds were selected on the basis of their roles in the treatment of various infectious diseases.

REQUIREMENTS FOR NEW MEDICINES AND PHARMACEUTICAL AGENTS
There are many diseases and health problems that people deal with every day. Because of the development and spread of drug-resistant pathogens, infectious diseases, diabetes mellitus, rheumatoid arthritis, ischemia, cardiovascular diseases and neurodegenerative diseases remain global problems (Espinel et al., 2001;Valko et al., 2007). Worldwide, these diseases cause millions of deaths annually. According to Tran et al.,(2010) global human health is threatened by cancers and various infectious diseases, where cancer is one of the major health problems in both developed and developing countries. After cancer cardiovascular diseases is the second leading cause of death (Patnaik et al., 2011). Because of high death rate associated with cancer, serious side effects of chemotherapy and radiation therapy, many cancer patients seek alternative complementary methods for treatment (Kaur et al., 2011). Novel anti-cancer drugs are also required due to the high worldwide mortality. Acquired immune deficiency syndrome (AIDS) is a disease of the human immune system caused by infection with human immune deficiency virus (HIV) (Sepkowitz, 2001). The ingress to the human population of diseases like AIDS and severe acute respiratory syndrome requires the discovery and development of new drugs to combat them. Sandhu et al.,(2014) stated that tuberculosis and malaria represents infectious diseases which were known since extreme antiquity. These diseases remain large-scale problems not only from medical but also from social point of view. Annually, owing to tuberculosis, about 3 million people die all over the world and approximately 8 million events of first registered tuberculosis are observed every year. The endophytes have been identified as promising sources of new pharmacologically active secondary metabolites that might be suitable for medicinal and agrochemical applications (Strobel and Daisy, 2003).

BIOACTIVE NATURAL PRODUCTS FROM ENDOPHYTIC MYCOFLORA
Bioactive natural compounds produced by endophytes are promising potential tools useful in safety and human health concerns. However, there is still a significant demand of drug industry for synthetic products due to economic and timeconsuming reasons . According to Strobel, (2003); Zhang et al. (2005) problems related to human health such as the development of drug resistance in pathogenic bacteria, fungal infections and life threatening virus, claims for new therapeutic agents for effective treatment of diseases in human, plants and animals are currently unmet. Natural products are rich sources of therapeutic agents as they inspire the advancement on synthetic methodologies and to the possibility of making analogues of original bioactive compounds with improved pharmaceutical properties. Endophytic fungi are thus rich sources of novel organic compounds with interesting biological activities and high level of biodiversity .
The production of bioactive compounds by endophytes, especially those exclusive to their host plants, is not only important from an ecological perspective but also from biochemical and molecular standpoints. There exist many exciting possibilities for the exploitation of endophytic fungi for the generation of a pleothera of novel biologically active secondary metabolites. The key challenge for the establishment and sustainence of in vitro biosynthetic potential of endophytes involves the task of repeated subculturing under auxenic monoculture conditions, which leads to the reduction of secondary metabolites production capabilities. This led to focus on the rediscovery of known secondary metabolites (Walsh and Fischbach, 2010;Kusari and Spiteller, 2012).
The discovery and production of secondary metabolites from endophytic fungi has emerged as an exciting field in biotechnology. Aly et al.,(2011);Sandhu et al.,(2014) stated that in the past two decades, many valuable bioactive compounds with anti-microbial, anti-insecticidal, cytotoxic & anticancer, antioxidant, anti-malaria, anti-viral, immunosuppressive, antituberculosis etc. activities have been successfully discovered from the endophytic mycoflora. Some of these bioactive compounds are discussed with their structures and functions.

Anti-microbial Bioactive Compounds from Endophytic Mycoflora
The emergence of antibiotic-resistant microorganisms calls for inventive research and development strategies. Inhibition of these pathogenic microorganisms may be a promising therapeutic approach (Sadrati et al., 2013). Plants and fungi are the chief sources of natural compounds used for medicine, where medicinal plants and endophytes have attracted considerable interest for their wide variety of bioactive metabolites (Newman and Cragg, 2007). Demain and Sanchez, (2009) reported that production of bioactive secondary metabolites by medicinal plants and their endophytes have provided countless therapeutic applications. Many of these compounds are being used for the treatment of a number of diseases (Sandhu et al., 2014). The antimicrobial compounds could be used as drugs and as food preservatives in the control of food spoilage and food-borne diseases . So far, Tan and Zou, (2001) reported large number of anti-microbial compounds isolated from endophytes, belonging to several structural classes such as; alkaloids, peptides, steroids, terpenoids, phenols, quinines and flavonoids. Yu et al. (2010) isolated three compounds namely; melleolides K, L and M from Armillaria mellea which showed anti-microbial activities against Grampositive bacteria, yeast and fungi.
Chaetomugilin A and D with anti-fungal activities were also isolated from an endophytic fungus Chaetosphaeridium globosum collected from Ginkgo biloba plant . Kjer et al. (2009) added that two new anti-microbial compounds; 10-oxo-10H-phenaleno[1,2,3-de]chromene-2-carboxylic acids, xanalteric acids I and II and 11 were known as secondary metabolites. These were obtained from extracts of the endophytic fungus Alternaria sp., isolated from the mangrove plant Sonneratia alba collected in China (Table 1). They showed antibacterial activities against Enterococcus faecalis, Pseudomonas aeroginosa and Staphylococcus epidermidis.

Anti-cancerous Bioactive Compounds from Endophytic Mycoflora
Novel anti-cancer drugs are also required due to high worldwide mortality rate (Pisani et al., 1999). Cancer is a disease characterized by unregulated cell proliferation and leads to the spread of abnormal cells and uncontrolled tissue growth (American Cancer Society, 2009). It has been considered as one of the major causes of death worldwide (about 13% of all deaths) in 2004 (WHO, 2009). ; Debbab et al. (2011) reported some evidences that bioactive compounds produced by endophytes could be used as alternative approach for the discovery of novel anti-cancer drugs. Thus, the cure of cancer has been enhanced mainly due to diagnosis improvements which allow earlier and more precise treatments (Pasut and Veronese, 2009). Endophytic fungi are rich sources of novel organic compounds with interesting biological activities and high levels of biodiversity (Krohn et al., 2007). Taxol, a diterpenoid, also called paclitaxel have gained interest, possibly due to its unique mode of action compared to other anti-cancer agents (Gangadevi and Muthumary, 2008). This compound interferes with the multiplication of cancer cells, reduces or interrupts their growth (Firakova et al., 2007).  previously reported that Taxol (C 47 H 51 NO 14 ) was firstly isolated from the bark of trees belonging to Taxus family (Taxus brevifolia), which was its most common source. Several reports about Taxol anti-cancer properties were published since its discovery (Lu et al., 2006), in addition, other sources for production of Taxol have been investigated in the last decade. The isolation of Taxol producing endophyte Taxomyces andreanae (Table 2) has provided an alternative approach to obtain cheaper and more available products via microbial fermentation (Stierle and Strobel, 1993). Pestalotiopsis terminaliae fungus isolated from the Terminalia arjuna plant produced the highest amount of Taxol (Gangadevi and Muthumary, 2009). Camptothecin is another important alkaloid anticancer compound (Fig 2), a potent anti-neoplastic agent which was firstly isolated from the wood of Camptotheca acuminata Decaisne (Nyssaceae) in China (Wall et al., 1966). The important precursors for clinically useful anticancer drugs, such as topotecan, irinotecan, camptothecin and 10-hydroxycamptothecin (Uma et al., 2008). The products obtained from the endophytic fungus Fusarium solani, recovered from Camptotheca acuminata were camptothecin and two analogues (9-methoxycamptothecin and 10-hydroxycamptothecin) which Kusari et al.,(2009) reported to have anticancer properties.
The anticancer drugs like etoposide and etopophos phosphate have precursor's podophyllotoxin and their analogues, due to their properties of cytotoxicity and antiviral activities . These podophyllotoxin are aryl tetralin lignans which were naturally synthesized by Podophyllum spp. A novel anticancer agent Ergoflavin, a dimeric xanthenes linked in position 2 was isolated from endophytic fungi recovered from the leaves of an Indian medicinal plant Mimusops elengi (Sapotaceae) (Deshmukh et al., 2009). Secalonic acid D, a mycotoxin, isolated from mangrove endophytic fungus ZSU44, belongs to ergochrome class known to have potent anti-cancer activities and induction of leukemia cell apoptosis. Moreover, it showed high cytotoxicity on HLA60 and K562 cells

Anti-diabetes Bioactive Compounds from Endophytic Mycoflora
Diabetes mellitus (DM) or simple diabetes is a metabolic disorder resulting from a defect in insulin secretion, insulin action or both. Insulin deficiency in turn leads to chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism. It is the most common endocrine disorder and by the year 2010, it was estimated that it affected more than 200 million people worldwide (ADA, 2009). This disease can cause wide range of heterogenous complications such as retinopathy, neuropathy, nephropathy, cardiovascular complications and ulcerations due to tissues or vascular damages (Bastaki, 2005). Dompeipen et al. (2011), pointed that endophytic microbe's ability to produce bioactive compounds in association with its host plants is an opportunity to get sources of anti-diabetic drugs, which as they are natural, inexpensive and ecofriendly.

Anti-tuberculosis Bioactive Compounds from Endophytic Mycoflora
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis which remains a major public health problem and cause ill-health among millions of people each year. The resistance of bacteria to different drugs is still increasing. Therefore, findings of new anti-tuberculosis agents are an important issue (Sittikornpaiboon et al., 2014). From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites named as phomoenamide, phomonitroester and deacetyl phomoxanthone B, were isolated together with three known compounds, dicerandrol A, (1S,2S,4S)-p-menthane-1,2,4-triol and uridine. Phomoenamide exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra .

Anti-viral Bioactive Compounds from Endophytic Mycoflora
Another charming use of antimicrobial products from endophytic fungi is the inhibition of viruses. Many reports demonstrated the importance of endophytic fungi in the production of anti-viral agents. Two novel human cytomegalovirus (hCMV) protease inhibitors, cytonic acids A and B (Fig 5) have been isolated from solid-state fermentation of the endophytic fungus Cytonaema sp. Their structures isomers were elucidated by mass spectrometry and NMR methods as p-tridepside (Guo et al., 2000). Exploration of endophytes associated with leaves of Quercus coccifera led to the isolation of endophyte with the ability to synthesize hinnuliquinone, a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) protease (Singh et al., 2004). Moreover, Mellisol and 1,8-dihydroxynaphthol 1-O-aglucopyranoside were isolated from the fungus Xylaria mellisii (BCC 1005) and showed inhibitory activities against herpes simplex virus-type 1 (Pittayakhajonwut et al., 2005).
Moreover, Zhang et al. (2011) reported the isolation and structure elucidation of Emerimidine A and B from culture of the endophytic fungus Emericella sp. Both of them showed moderate inhibition of Influenza virus H1 N1 with IC 50 values of 42.07 mg/mL and 62.05 mg/mL, respectively.

CONCLUSION
In the present scenario, human beings are suffering from various health problems due to infectious diseases, drug resistance, neurodegenerative diseases, cardiovascular diseases etc in their daily life. There is an urge to investigate novel compounds for the treatment of these diseases. Therefore, endophytic fungi provide broad variety of secondary metabolites with their unique structures like flavonoids, terpenoids, alkaloids, phenolic acid etc. Such bioactive metabolites find wide-range of application against infectious diseases, autoimmune, enteric, cardiovascular, and other diseases. The potential of finding new drugs that may be effective candidates for treating newly developing diseases in humans is remarkable. Hence, we concluded that the endophytic mycoflora are novel and important microbial resources for producing bioactive compounds, and have attracted attention of many researchers for their potential applications and studies. However, future studies include various biosynthetic pathways responsible for the production of novel bioactive metabolites from the endophytic mycoflora. Also, molecular biology based studies can be used to isolate and identify the different types of genes found in biosynthetic pathways and used for the large scale production of novel bioactive compounds in laboratory as well as at commercial level. However, genetic engineering techniques can be carried out further for the gene transfer leading to the development of more efficient species.