Nitrogen is an important component of living cells present in nucleic acids, proteins, enzymes, cofactors, signalling molecules, numerous plant secondary products and necessary metabolic processes involved in the synthesis and transfer of energy. Plants require nitrogen throughout their development. Plants containing large amount of nitrogen produces nitrogenous secondary metabolites forming a part of their structure. Plants obtain nitrogen from roots by nitrogen fixing bacteria and deterioration of dead tissues by micro-organisms, thus promoting plant growth.1 Nitrogen combines chemically with oxygen or hydrogen to form various nitrogenous compounds which can be used by plants. The organic nitrogenous compounds are formed from inorganic nitrogen compounds present in the environment by nitrogen assimilation. These nitrogenous compounds are added to the soil as fertilizers in the form of ammonium (NH4+) and nitrate (NO3+). Nitrogenous metabolites are widely distributed throughout the plant kingdom.
Nitrogen is a major constituent in almost drugs used in medicine. Nitrogen containing compounds have an important role in research through decades as a history in the development of organic synthesis. Nitrogen containing medicinal compounds has been used since centuries, as they form the basis for many drugs such as taxol, campothecin, vincristine, etc. These compounds occur in a diverse natural products and drugs showing importance in many aspects of life and commercial processes, from the industrial production of fertilizers to the building blocks of life.2 Endophyte is a microorganism that lives inside of a plant and colonise the intercellular spaces of the plant tissues. They include organisms such as fungi, bacteria and actinomycetes which spend its lifespan inside cellular tissues for healthy host without producing any symptoms.3 They are chemical synthesizers producing novel compounds that show numerous pharmacological and biological activities.4 Mutualistic relation between endophytes and host plants results in beneficiary for both,5 thus mutual relationship, between taxonomy and ecology are being studied. Majority of the secondary metabolites already discovered in plants, but microorganisms are also capable of producing more than 20000 biologically active compounds, influencing the performance and survival of other organisms.6 Many of these microbes producing active secondary metabolites are involved in a host endophyte relationship and thus exhibit various biological activities as antibiotic, antitumor, anti-inflammatory, antioxidant, etc. Numerous secondary metabolites produced by various endophytes possess unique structures belonging to alkaloids, glycosides, benzopyranones, flavonoids, phenolic acids, quinones, steroids, xanthones, terpenoids, tetralones and others and bioactivities, potentially useful.7 Alkaloids, non-protein amino acids, cyanogenetic glycosides, amines, glucosinolates, alkamides, lecithins, peptides and polypeptides are important group of secondary metabolites having nitrogen produced by endophytes.
Secondary nitrogen is a commonly used in endophyte/host and plant interactions for host plant growth, uptake by endophytes and demand for synthesis of highly nitrogen containing compounds. Many of these compounds are required for the growth of the plants in the environment. Taxol, Campothecin, Vincristine, Phomoenamide, Aspergillusol A, Helvolic acid are some of the nitrogenous secondary metabolites obtained from the different endophytes exhibiting various pharmacological activities.
Many attempts were made to isolate and identify various secondary metabolites from endophytes to produce inexhaustible supply of bioactive compounds which are used commercially. Changes in the conditions of the culture can be explored by optimizing various biosynthetic pathways leading to the production of derivatives and analogues of novel compounds.8 The increasing awareness of the secondary metabolites of medicinal plants can be influenced by endophytic infection. These endophytes may produce them on their own as that of plant or they may also alter the metabolite produced by the plant. Hence, there can be variability in the type of bioactive compounds which shows the possibilities to produce some novel type of medicines that may give results against some incurable diseases.9 In recent studies, attention has been given to the biotransformation process of endophytes which acts as biocatalysts in the chemical transformation of natural products and drugs are due to their ability to modify chemical structures with a high degree of stereo specificity and to produce known or novel enzymes that increase the production of compounds of interest.10
This review article covers detailed information about nitrogen containing secondary metabolites isolated from endophytic microbes associated with medicinal plants during the years 2008-2016. Chemical diversity of these compounds and their potential biological/ pharmacological activities are also discussed.
NITROGEN CONTAINING ANTICANCER METABOLITES
Cancer is an uncontrolled growth of abnormal cells and invasion into normal tissue of various organs. In 2007, it was estimated that 7.6 million people were killed around the world due to cancer. By 2050, the annual death is may rise to 17.5 million, due to unavoidable reasons such as population growth, aging, lifestyle, environmental factors and carcinogenic agents. The existing drugs for treatment of cancer shows no specific toxicity to proliferating normal cells, produce various side effects and ineffective against many types of cancer.11 No availability of new drugs or technologies for treating the disease, the cure of cancer has been increased by diagnosing earlier and more precise treatments.12 Some of the examples of anticancer drugs obtained from medicinal plants are Campothecin from Campotheca acuminate (Nyssaceae), vinblastine, vincristine from Catharanthus roseus (Apocynaceae), burseran from Bursera microphylla (Burseraceae), colchicines, demecolcine from Colchicum luteum (Liliaceae). Recently several secondary metabolites from endophytes showing anticancer properties also have been investigated. Following are some examples of the nitrogen containing secondary metabolites from endophytes cited, on the production of anticancer agents and are shown in Table 1.
Vincristine (1) was the first reported metabolite of the endophytic fungus Alternaria sp. from the phloem of Catharanthus roseus. Compound 1 was also produced from an endophytic Fusarium oxysporum from the pholem of Catharanthus roseus (Apocynaceae). It is used as chemotherapeutic agent in acute nephroblastoma and lymphoblastic leukemia. The primary action of compound 1 is by interfering with microtubule formation and mitotic spindle dynamics, disruption of intracellular transport, decreasing tumour blood flow, with consequence of anti-angiogenesis.13 New modified dipeptides, Trichodermamides A-C (2-4), were obtained from the endophytic fungus Eupenicillium sp. from bark of Glochidion ferdinandi (Phyllanthaceae). Compound 2 showed cytotoxic activity against the human colorectal carcinoma HCT116 and human lung carcinoma A-549 with IC50 values of 0.68 and 4.28 μg/ml, respectively. Six new tetramic acid derivatives, Penicillenols A1, A2, B1, B2, C1, and C2 (5-10) were identified from Penicillium sp. GQ-7, an endophytic fungus associated with Aegiceras corniculatum (Primulaceae). All the compounds were screened for their cytotoxic effects on four cell lines by the MTT method. Penicillenols A1 and B1 showed cytotoxicities against HL-60 cell line with IC50 values of 0.76 µM and 3.20 µM. Cycloaspeptide A (11) was isolated for the first time from the endophytic fungus Penicillium janczewskii KM Zalessky, from the plant Prumnopitys andina (Podocarpaceae). It exhibited low cytotoxicity towards human lung fibroblasts with IC50 ≥ 1000 µM. Penicidones A-C (12-14) were isolated from the culture of Penicillium species, an endophytic fungal strain residing in the stem of Quercus variabilis (Fagaceae). Compounds 12-14 exhibited in vitro cytotoxicity against four human cancer cell lines SW1116, K562,
KB and Hela indicating that they are moderately cytotoxic with IC50 values between 21.1 and 90.8 μM.17 An ergoline alkaloid, 9 deacetoxy fumigaclavine (15) was isolated from an endophytic fungus, Aspergillus gumigatus obtained from the stem of cynodon dactylon (Poaceae). It showed cytotoxicity against K562 cell lines with an IC50 of 3.1 mM.18 Two indole alkaloids fumitremorgins B (16) and C (17) were produced by the endophytic fungus Alternaria sp. FL25 from Ficus carica (Moraceae).19 Cochliodinol (18) and isocochliodinol (19) were isolated from an endophytic fungus Chaetomium species which resides in the stem of Salvia officinalis (Lamiaceae). Compound 18 showed potent cytotoxicity with an EC5020 of 7.0 mg/ml. Campothecin (20) a pentacyclic quinoline alkaloid was isolated from endophytic fungi entrophosphora associated with the inner bark of the plant Nothapodytes foeatida (Icacinaceae). This is followed by the isolation of campothecin from seed endophyte Neurospora crusa of Campothecta accumalata (Cornaceae). Campothecin and its two analogues viz 9 methoxy campothecin (21) and 10 hydroxy campothecin (22) were found to be isolated from an endophytic fungus, Fusarium solane of campthecta accumalata.21 The mechanism involved in cytotoxocity was found to be by inhibition of DNA topo isomerase I enzyme.22 They were tested for cytotoxic activity against humans in lung cancer (A549), liver cancer (hep 2), ovarion cancer (OVCAR-5) cell lines. Commonly used anticancer drug, Taxol (23) (Paclitaxel), a diterpene taxane, was isolated from the bark of Pacific yew tree Taxus brevifolia for the first time. It showed activity against a series of human solid tumor xenografts including CX-1 colon and MX-1 breast xenografts. Taxol was also isolated from the fungal endophyte A. alternata var. monosporus obtained from the inner bark of Taxus yunnanensis (Taxaceae). Compound 23 was also produced by an endophytic fungus, Fusarium redolens, isolated from Himalayan yew.23 Till now, 20 genera of endophytic fungi were screened to produce paclitaxel. Some of them are Alternaria alternate TPF6 from Taxus chinensis var. mairei, Aspergillus fumigatus EPTP-1 Podocarpus sp,24 Aspergillus niger var. taxi HD86-9 Taxus cuspidate,25 Botryodiplodia theobromae BT115 Taxus baccata,26 Botrytis sp. XT Taxus chinensis var. mairei. Four new quinazolinone alkaloids, Aniquinazolines A-D (24-27), were isolated and identified from the culture of Aspergillus nidulans MA-143, an endophytic fungus obtained from the leaves of marine mangrove plant Rhizophora stylosa (Rhizophoraceae). They exhibited potent brine shrimp toxicity with LD50 values of 1.27, 2.11, 4.95 and 3.42 μΜ, respectively.27 The structures of potent anticancer metabolites from endophytes are depicted in Figure 1.
NITROGEN CONTAINING ANTI-BACTERIAL METABOLITES
Infectious diseases caused by microbes are major health problems in society. The impact is large in developing countries due to the unavailability of medicines and widespread drug resistance. The incidence of drug-resistant pathogens has drawn the attention of the pharmaceutical and scientific communities towards studies on the potential antimicrobial activity of plant derived substances. The increasing problem of microbial resistance has become severe and the search for the use of antimicrobial drugs in the future is still uncertain. Antimicrobial resistance has been a major health issue and still presents threat to health care system globally. Studies have shown that microbes have developed resistance to antibiotics through various molecular mechanisms such as prevention of access to drug targets and modification of the drug.28 Antimicrobial metabolites are low-molecular-weight organic compounds made by microorganisms to protect plant from outer invade, that are active at low concentrations against other microorganisms, and are the most bioactive natural products isolated from endophytes.8 Search for new antimicrobial agents is needed due to the infections and diseases are global problems causing due to drug-resistant pathogens. Endophytes, by producing the secondary metabolites show resistance mechanism to overcome pathogenic invasion. Hence, studies demonstrated isolation of large number of anti-microbial compounds from endophytes, belonging to several structural classes like alkaloids, peptides, quinines and terpenoids. Asperfumoid (28) a new alkaloid, was isolated from an endophytic fungus Aspergillus fumigatus CY018, associated with Cynodon dactylon (Poaceae). Compound 28 inhibited the growth of Candida albicans (CA) with MIC value of 75 μg/ml. It was also isolated from the endophytic fungus Penicillium species, from the leaves of Hopea hainanensis (Dipterocarpaceae) as it inhibited the growth of CA with IC50 values of 20 and 25 μg/ml respectively.29 A bioactive compound, 7-amino-4-methyl coumarin (29) was isolated from endophyte Xylaria sp isolated from Ginkgo biloba (Ginkgoaceae). Compound 29 showed strong antibacterial and antifungal activities in vitro against Staphylococcus aureus with MIC value of 16 μg/ml. It was the first coumarin metabolite to show antimicrobial activity.30 Phomoenamide (30) and Phomonitroester (31) were obtained by an endophytic fungi Phomopsis species (PSU-D15), isolated from leaves of Garcinia dulcis (Cluciaseae). Compound 30 showed anti-microbial activities against Mycobacterium tuberculosis (MT) H37Ra with a MIC value of 6.25 μg/ml.31 Two new metabolites, Cyclo (Pro-Thr) (32) and cyclo (Pro-Tyr) (33) produced by the fermentation broth of endophytic fungus Penicillium species isolated from mangrove plant Acrostichum aureurm showing antibacterial activity.32 Polyketide amino acids derived antibiotics, Pyrrocidines A (34) and B (35), were obtained from endophytic fungi Acremonium zeae residing in Zea maize (maize kernals). They exhibited antimicrobial activity against microbial pathogens causing seedling blights and stalk rots. Compound 34 showed anti-bacterial activity against Bacillus mojavensis and Pseudomonas fluorescens (PF) with MIC values of 1-2 µg/ml.33 Three new indolosesquiterpenes namely Xiamycin B (36), Indosespene (37) and Sespenine (38) were isolated from the culture broth of Streptomyces sp. HKI0595, a bacterial endophyte isolated from mangrove tree, Kandelia candel (Rhizophoraceae). Both exhibited antimicrobial activities against several Gram-positive and Gram-negative bacteria reveling multi-resistance34. Tenuazonic acid (39) was isolated from Alternaria alternata SVJM015, residing in the leaves of Indigofera enneaphylla (Poaceae). It showed activity against MT with MIC value for 250 μg/ml.35 (3,1″-didehydro-3[2″(3″,3″-dimethylprop-2-enyl)-3″-indolylmethylene]-6-methyl piperazine-2,5-dione) (40) is tryp-alanine derived cyclic dipeptide containing an indole and a diketopiperazine moiety which was isolated from the culture medium of Penicillium chrysogenum, an endophytic fungus of the mangrove plant Porteresia coarctata (Poaceae). Compound 40 showed significant antibacterial activity against Vibrio cholera (VC) (MCM B-322), a pathogen causing cholera in humans.36 Four ansa macrolides or ansamycins, Divergolides A–D (41-44), were isolated from a bacterial endophyte Streptomyces sp. HKI0576 of the mangrove tree Bruguiera gymnorrhizain (Rhizophoraceae). Compound 41 showed the strongest activity against Mycobacterium vaccae (MV) with IC50 values ranging from 1.0-2.0 µM.37 The structures of potent antibacterial metabolites are depicted in Figure 2.
NITROGEN CONTAINING ANTIFUNGAL METABOLITES
The functioning of invasive fungal infections increases during cancer chemotherapy, organ transplantation and allogeneic bone marrow transplantation. The use of synthetic antifungal agents against plant and human diseases has created awareness among the researchers worldwide, and thus much of the research areas are focused on development of novel, potent and green principle based antifungal agents using medicinal plants, owing to their different mode of action, different target sites with minimized side effects than the conventional available drugs.38 However, the availability of antifungal agents for the treatment of various life threatening fungal infections is less so the new drugs should be made available in the market; the development of protection to anti-fungal drugs has become increasingly superficial, especially in patients with long term treatment. Microbial natural products have been an alternative natural cause for the isolation of unique molecules for various therapeutic applications.39 Many nitrogen containing metabolites from endophytes are reported to possess antifungal activity.
Curvularides A–E (45-49), are the five-hybrid peptide-polyketides, which were obtained from the endophytic fungus Curvularia geniculata, isolated from the limbs of Catunaregam tomentosa (Rubiaceae). Compound 45 showed most significant antifungal activity against Candia albicans than other compounds.40 3-methylcarbazole (50) and 1-methoxy-3-methylcarbazole (51) were obtained from Streptomyces sp. LJK109 from Alpinia galanga (Zingiberaceae). Both the compounds showed antifungal activities with MIC of 30 to 240 µg/ml.41 A chlorinated benzophenone alkaloid, Pestalachloride A (52) was obtained from Pestalotiopsis adusta, an endophytic fungus of the unknown Xinglong Chinese tree stem. The Compound 52 showed antifungal activity against the plant pathogens such as Gibberella zeae (anamorph F. graminearum), Verticillium alboatrum and Fusarium culmorum.42 A new compound KL-4 (53) was isolated from the fungal endophytic Aspergillus species of seeds of Gloriosa superba (Colchicaceae) and was subjected to antimicrobial and anticancer activities. It showed broad spectrum as antifungal with IC50 value of 30 μg/ml.43 Chaetoglobosin A (54), Chaetoglobosin D (55), Chaetoglobosin G (56), Chaetoglobosin R (57) were isolated from fungal endophyte Chaetomium globosum No. 04 inhabited in medicinal plant Ginkgo biloba (Ginkgoaceae). These antifungal metabolites were active against the phytopathogenic fungi Rhizopus stolonifer (RS) and Coniothyrium diplodiella (CD)0.44 12 β-hydroxy-13α-methoxy verruculogen TR-2 (58) and 3-hydroxy fumiquinazoline A (59), were isolated from the fermentation broth of Aspergillus fumigatus LN-4, an endophytic fungus isolated from the stem bark of Melia azedarach (Meliaceae). Compounds 58 and 59 exhibited antifungal activities against some phytopathogenic fungi (Botrytis cinerea, Alternaria solani, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, Fusarium oxysporum f. sp. niveum, Fusarium oxysporum f. sp. vasinfectum, and Gibberella saubinettii). Compound 58 exhibited antifungal activities with MIC values of 6.25–50 μg/ ml.45 All the compounds showed good antifungal activities as shown in Table 1 and the structures are listed in Figure 3.
NITROGEN CONTAINING ANTIVIRAL METABOLITES
The emergence of resistance and multi-resistance against available drugs, the side effects and high cost of current therapies as well as the Hepatitis Immuno Virus/AIDS epidemic and AIDS associated opportunistic infections, such as cytomegalovirus and polyoma virus, made the development of novel antiviral drugs a central priority. Antiviral metabolites are the fascinating compounds from endophytes for inhibition of viruses. Some of these metabolites found to be promising against H1N1, influenza and other virus infections (Table 1).
Xiamycin A (60), a novel indole sesquiterpene, produced by a bacterial endophyte Streptomyces sp. GT2002/1503, isolated from the stem of mangrove plant Bruguiera gymnorrhiza (Rhizophoraceae). Compound 60 exhibited moderate antiviral activities against HIV. It specifically blocked CCR5 (R5) tropic HIV-1 while it showed no effect on CXCR4 (X4) tropic HIV-1.46 Tricyclic sesquiterpenoids, Brasilamides A-D (61-64) were isolated from cultures of the plant endophytic fungus Para coniothyrium brasiliense. Verkley (M3–3341), isolated from branches of Acer truncatum (Sapindaceae). Compound 61-64 showed modest inhibitory effects on HIV-1 replication in C8166 cells.47 Eight isoindolone derivatives, Merimidine A (65), Emerimidine B (66), Emeriphenolicin A-F (67-72) were isolated from endophytic fungus Emericella sp. (HK-ZJ) associated with the inner bark of the mangrove plant Aegiceras corniculatum (Primulaceae). Compound 66 exhibited anti-influenza and antiviral (H N) activities using the cytopathic effect (CPE) inhibition assay.48 Perlolyrine (73) and 1-hydroxy-β-carboline (74) were isolated from the fermentation broth of an endophytic actinomycetes Jishengella endophytica 16111, present in the root of the mangrove plant, Xylocarpus granatum (Meliaceae). Compound 74 exhibited moderate anti-H1N1 virus activity .they showed anti-influenza A (H1N1) virus activity with the half maximal inhibitory concentration (IC50) and selectivity index (SI) value of 38.3μg/ml.49 Figure 4 shows important antiviral metabolites from endophytes.
NITROGEN CONTAINING ANTIMALARIAL METABOLITES
Malaria has a major health threat in developing countries and yearly more than 200 million new malaria cases are reported,50 emphasising the urgent need for new drugs against malaria. In India, its management is challenging due to the huge population and large geographical area by rapid growth51 and the resistance developed by the malaria parasite.52 Antimalarial metabolites obtained from endophytes may come up with the need. A new tryptophan polyketide hybrid, Codinaeopsin (75) was obtained from endophytic fungi Codinaeopsis gonytrichoides present in Vochysia guatemalensis, (Vochysiaceae) a white yemeri tree collected in Costa Rica. Compound 75 is active against 3D7 strain of Plasmodium falciparum, with an IC50 value of 2.3 μg/ml. Fermentation culture from the endophytic fungus Pestalotiopsis sp. (BRIP 39872) from Melaleuca quinquenervia (Myrtaceae) yielded three novel caprolactams, Pestalactams A-C (76-78). Compounds 76 and 78 were tested against two different strains of the malaria parasite Plasmodium falciparum (3D7 and Dd2) and both the compounds displayed antimalarial activity, with 16-41% parasite growth inhibition achieved at 25 µM.54 The structures of antiviral metabolites from endophytes are depicted in Figure 5.
NITROGEN CONTAINING IMMUNOSUPPRESSIVE METABOLITES
The immunomodulatory compounds are divided into immunosuppressive and immunoregulatory drugs. Many of them have been obtained from endophytes from plants. Emerging of new diseases as autoimmune disorders, use of immunosuppressive drugs in the market has been increased. Immunosupressive drugs are used to prevent allograft rejection in transplant patients and to treat auto immune diseases such as rhematoid arthritis and insulin dependent diabetes. As a result, researchers are focusing on the production of such drugs from the alternative source as endophytes.
Trichomides A (79) and B (80), two new cyclodepsipeptides were isolated from the endophytic fungus Trichothecium roseum. Trichomide A has immunosuppressive effect more selectively than cyclosporine A. It was found that trichomide A decreases the expression of Bcl-2, increases the expression of Bax, and has a small or negligible effect on the expressions of p-Akt, CD25, and CD69 as depicted in Table 1.55 The structures are listed in Figure 7.
NITROGEN CONTAINING METABOLITES WITH MISCELLANEOUS ACTIVITIES
Compounds bearing antioxidant activity are effective against damage caused by reactive oxygen species and oxygen-derived free radicals, contributing to numerous pathological effects, such as DNA damages, carcinogenesis, and cellular degeneration. Chrysogenamide A (81), a new member of the macfortine group of alkaloids, was identified from Penicillium chrysogenum No. 005, an endophytic fungus associated with Cistanche deserticola (Orobanchaceae). It exhibited a neurocyte protection effect against oxidative stress-induced cell death in SH-SY5Y cells.56 Other activities such as molluscicidal activity of endophyte JJ18 from Pseudolarix kaempferi Gord was also reported.57
The objective of this paper is to review the diversity of nitrogenous secondary metabolites from endophytes with various biological/pharmacological activities such as anticancer, antimicrobial, antifungal, antiviral, antimalarial, etc in the plant tissues. This review contains 81 nitrogenous compounds with various activities against plant and human pathogens, produced by endophytes inhabiting in various medicinal plants. Endophytes have the evidence of producing same or novel secondary metabolites with a wide-range of pharmacological activities which shows potential use in agricultural, industrial and medical areas. Thus, nitrogenous secondary metabolites produced by endophytes originate from various biosynthetic pathways belonging to different structural groups such as alkaloids, amines, peptides, etc. Endophytes producing bioactive natural compounds have shown promising potential and usefulness concerns for human health and safety. Modern biotechnology, as genetic engineering, metabolic technology and microbial fermentation process, showed advantages to understand and manipulate the importance of microorganism resources making beneficial for the mankind. However, there is need to understand and discover about the host/entophyte relationship before utilizing endophytes in the discovery of medicinally important compounds. In conclusion, a more comprehensive understanding of the biochemistry, genetics and biology of endophyte and host, may lead to new opportunities for developing medicinally important products to alleviate various human ailments.