Harmine and its derivatives : Biological activities and therapeutic potential in human diseases

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Introduction
Natural products have served humanity as a fundamental source of medicine throughout the history of civilization.The term natural product is usually defined as chemical entities that are mainly originated from the living species such as microorganisms, plants, terrestrial vertebrates, marine organisms, and invertebrates (Rasul et al., 2013).Plants offer an extensive reservoir of natural products providing a far-reaching diversity of novel chemical entities in drug industries (Newman et al., 2003).
Plants have established the basis of refined traditional medicinal systems including Chinese, Unani, Ayurvedic, and some others.These traditional systems have a great potential regarding the discovery of many beneficial drugs (Gurib-Fakim, 2006).Since past decades, natural products have been an affluent source of many chemical entities for drug discovery (Harvey et al., 2015).To date, 61% anti-cancer agents and approximately 49% anti-infective compounds are directly inspired from nature (Luo et al., 2014).
Alkaloids have been recently described as the most significant group of natural products with basic nitrogen atoms, playing an important role in the ecological aspects of organisms (Bouayad et al., 2012).Additionally, β-carbolines are the major class of alkaloids with a tricyclic pyrido [3,4-b]indole ring (Filali et al., 2015).They have been proved as natural constituents of human body fluids and tissues.They also display a wide range of behavioral, psychopharmacological, and biochemical effects in both humans and animals (Patel et al., 2012).This review will emphasize the most recent researches on harmine focusing on its pharmacological and biological properties.

Structure Activity Relationship
Harmine (C13H12ON2) is commonly distributed among the animals, marine creatures, plants, and insects (Zhang et al., 2015).Harmine derivatives are gifted with pharmacological profiles (Zhang et al., 2016).Chemical structures of biologically active harmine and its derivatives are represented in Figure 1.
Structure-activity relationship of harmine derivatives has been established by TD-DFT study (Lamchouri et al., 2013) and this study showed that position-3 and 9 played a crucial role in antitumor as well as neurotoxicity of harmine derivatives.Structure activity relationship analysis indicated that replacement of proton presents at position-9 with short alkyl or aryl group increased the cytotoxicity while replacement of proton presents at position-3 with long alkyl or aryl group reduced the antitumor activity.Without any substitution of the proton at position-3 in harmine derivatives led to enhanced antitumor activity.SAR analysis demonstrated that by introducing alkoxy substituents into position-7 of harmine led to enhanced cytotoxic activities, the length of alkoxy chain affected both cytotoxicity and cell line specificity, N9-alkylated harmine derivatives displayed specific cytotoxic effects, N2-alkylated.Furthermore, SARs studies suggested that substitution of proton of position-9 of harmine enhanced the antitumor activity, 7-methoxy of harmine very important in determining the neurotoxic effects, replacement of methoxy at position-7 with a bulky alkoxy group led to eliminating neurotoxic effects and enhanced antitumor activity (Cao et al., 2013).
Structure-activity relationship of N-9 substituted harmine analog 11d demonstrated that by the introduction of a haloalkyl or benzene-sulfonyl group in the N9position of harmine could significantly increase the anticancer activity (Du et al., 2016).Harmine derivative 2 and HRMS (harmine derive isoxazole) acting as anti-Alzheimer, anti-cancer, and anti-inflammatory agent showed good activity due to N9-substitution (Filali et al., 2015).

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Moreover, in vivo and in vitro results demonstrated that substitutions at the position-2, 7 and 9 of harmine led to excellent enhancement in antitumor activity and remarkable reduction in the adverse effects of the drugs because the structural modification at the said positions results in reduced uptake of drug by normal cells and increase the cancer cell specificity (Li et al., 2015).

Plant Sources
Harmine was first discovered and isolated from the Peganum harmala which is generally utilized as potent herbal medicine due to its abortifacient, emmenagogue, hallucinogenic, lactagogue, and hypothermic properties (Filali et al., 2015).The seed extracts of P. harmala have been traditionally used in Northwest China to cure malaria and alimentary tract cancers for hundreds of years (Cao et al., 2013).P. harmala is a perennial herbaceous plant originally of family Zygophyllaceae but has been recently updated as a member of family Nitrariaceae (Filali et al., 2015).Besides P. harmala, harmine was identified in P. nigellastrum (Ma et al., 2000), and P. multisectum (Liu, 2011).
Harmine was also isolated from the roots and leaves of A. paniculata.It is useful in Chinese Traditional Medicine as an anti-code for snakebite, and to treat dysentery, dyspepsia, malaria, influenza, and respiratory diseases (Kurzawa et al., 2015).Moreover, the stalks of B. caapi vine (Dos Santos and Hallak, 2017), and the roots of O. tuberosa (Bais et al., 2003) are found to be a rich source of harmine.Quantitative analysis on harmine showed that it is present in major quantity in P. caerulea while in minor quantity in P. incarnata (Fyre and Haustein, 2007).

Biological Activities
The biologically active compound harmine has been demonstrated for its broad spectrum of pharmacological and biological traits such as anticancer, anti-microbial, anti-oxidant, neuroprotective, anti-inflammatory, and anti-depressant (Figure 2).Several in vitro and in vivo investigations have elucidated its medicinal characteristics and mechanism of actions.

Anti-cancer activity
Programmed cell death or apoptosis is a natural process of eliminating old cells from our body.Approximately all the anti-cancer agents stimulate apoptosis to remove malignant cells.Nevertheless, in cancer, de-regulation of signaling pathways provide gateway towards cellular proliferation which is uncontrolled resulting in the survival of tumor, cancer recurrence and therapeutic resistance (Mohammad et al., 2015).Previous investiga-  Ma et al., 2000Bangladesh J Pharmacol 2018;13: 203-213 tions have reported that AMPK (adenosine-5'-monophosphate-activated protein kinase) and MAPK (mitogen activated protein kinase) signaling pathways are noteworthy to activate apoptosis and autophagy (Li et al., 2017).Cumulative data by the researchers strongly commend that various chemopreventive agents prompt apoptosis in cancerous cells (Rasul et al., 2012;Rasul et al., 2014).
About 9 derivatives of harmine are reported to have anti-cancer effects in lung and liver cancers (Chen et al., 2005).Overexpression of dual specificity tyrosinephosphorylated and regulated kinase 1A (DYRK1A) has been implicated in an uncontrolled cell proliferation and tumorigenesis.Harmine is an effective inhibitor of protein kinase DYRK1A.Harmine-associated prohibition of DYRK1A triggered apoptosis in human oligodendroglioma (Hs683) cancer cells along with caspase-9 activation (Frederick et al., 2012;Atteya et al., 2017).A harmine derivative, harmine hydrochloride (Har-hc) has been well-known for its anti-cancer potential against glioblastoma C6, U87, and U373 cells with different inhibitory concentrations.Enhanced levels of p21, and Bax while reduced levels of Bcl-2, and Bcl-xl were examined after Har-hc treatment (Liu et al., 2013).Harmine also showed anti-proliferative activity towards thyroid TPC-1 cells via down-regulation of Bcl-2 dose-dependently (Ruan et al., 2017).cytotoxicity in gastric cells by up-regulating apoptosisrelated proteins (Li et al., 2017).Harmine together with paclitaxel have been reported as the novel drug candidate in numerous cancer types.Harmine and paclitaxel leads to cellular growth inhibition dose-dependently and apoptosis induction in SGC-7901 cells.The cell death mechanism involves activation of Bax while reduced expression of Bcl-2, COX-2, MMP-2, and PCNA expressions (Yu et al., 2016).In BGC-823 gastric cells, harmine 3c compound (benzylidene substituted βcarboline) exhibited strong apoptotic effect via intracellular ROS production, and suppression of PI3K/Akt pathway (Zhang et al., 2016).
Harmine derivative B-9-3 is an angiogenesis inhibitor.It has been reported to possess strong antitumor and angiogenic effects against Lewis lung cancerous cells (LLC).B-9-3 significantly inhibited the growth of vascular fibroblasts, and endothelial cells, and stimulated regression of LLC, human ovary (SKOV-3), prostate (22RV1), and mouse fore-stomach tumor cells.
A hybrid of hydroxamic acid and β-carboline alkaloid harmine exhibited cytotoxicity against lung carcinoma (H460) cells through apoptosis induction associated with the activation of Bax, and down-regulating the Bcl-2 expression (Xu et al., 2016), and also known to have protective effects against A549 cancer cells (Filali et al., 2015;Du et al., 2016).
Harmine has the competency to prevent cancer progression in liver Hep3B and Huh-7 cells by inhibiting DYRK1A activity with effective inhibitory concentrations of 10 and 0.25 µM, respectively (Bruel et al., 2014;Zhang et al., 2015).Harmine has also been proved to be effective against liver HepG2, SMMC-7221, and Bel-7402 cells as it can stimulate cellular apoptosis with the activation of Bax, Fas, and caspase-3/-9 along with the down-regulating the expression of Bcl-2, and Mcl-1 (Cao et al., 2011).
Harmine and its derivatives have been investigated for their anti-cancer activities against colon HCT-116 cells (Filali et al., 2015;Filali et al., 2016;Xu et al., 2016;Zhang et al., 2016)(Figure 3).In another study, harmine exhibited anti-proliferative effect against colon (Caco2) cancer cells through inhibition of protein kinase DYRK1A (Bruel et al., 2014).Increased expression of Bax, caspase-3 along with PARP cleavage while the decreased activity of Bcl-2, CDK5/p25 and GSK3ᵅ / ᵝ were observed against colorectal RKO, and DLDI cells by harmine treatment (Zhang et al., 2016).Harmine prevented the growth of human SW620 cells in a doserelated manner.The percentage of apoptotic cells was enhanced from 12.0 to 26.4% when treated with harmine.Results have shown that harmine has the capability to cause cell cycle arrest at G2/M and S phases associated with the increased expression of cyclin A/B1/E2, CDK1/cdc2, p-cdc2 (Tyr15), and Myt-1 while decreased expression of cyclin D1.Apoptotic cellular death is accompanied by activation of Bax, PARP, and caspase-3/-9 along with down-regulation of Bcl-2 and Mcl-1 levels.Harmine also inhibited ERK, and Akt pathways in SW620 cells (Liu et al., 2016).
Harmine exerted antitumor effects against bladder EJ cells via up-regulating the expression of Bax and downregulating the expression of Bcl-2 levels (Xu et al., 2016).Harmine derivatives carrying several substituents at position-2, 7 with other β-carboline rings have played effectual roles in modulating cytotoxic activity against mouth KB cells (Zhang et al., 2016).Harmine together with G-CSF and ATRA inhibited proliferation of leukemia HL-60 cells in the time-and dose-related concentra-tions (Zhang et al., 2015).
It can be concluded that harmine derivative 11d arrest cell cycle at G2/M phase but whether in G2 or M phase should be investigated.Moreover, it would be interesting to interrogate the mechanism by which harmine derivative JKA97 arrest cell cycle at G0/G1 phase in breast adenocarcinoma cells as in case of other cancer cell lines it is generally arresting cells at G2/M or S phase.So, extensive studies are still obligatory to fully understand the molecular mechanism by which harmine and its derivatives regulate the cell cycle.Furthermore, the exact mechanism of action of harmine in colon (HCT-116), ovary (OVCAR-3), prostate (22RV1), and leukemia (HL-60) cancers has not been fully investigated.

Anti-inflammatory activity
Various studies have determined that anti-inflammatory activity of natural products are linked with the prohibition of pro-inflammatory mediators and cytokines (TNF-α, iNOS, COX-2), ROS, and transcription factors (NF-κB) (Debnath et al., 2013).
P. harmala alkaloids including harmine have the competency to block myeloperoxidase activity (MPO), and MPO-mediated LDL-oxidation with IC50 of 0.26 µM.Molecular docking analysis indicated that all P. harmala alkaloids have great selectivity for the active site of MPO (Bensalem et al., 2014).Harmine has also been reported to suppress TNF-α, IL-6, and NO production in LPS-induced RAW264 macrophages and THP-1 cells of humans in a dose-dependent manner (Yamazaki and Kawano, 2011;Liu et al., 2017) (Table II).
There are only preliminary studies regarding antiinflammatory efficacy of harmine.

Anti-oxidant activity
Harmine from P. harmala has the capacity to inhibit CuSO4-mediated LDL oxidation and free radical formation.The anti-oxidant effect of harmine was determined by an increased lag phase time of conjugated dienes, and MDA production.It showed free radical scavenging activity at 10 µM concentration (Berrougui et al., 2006).In another study, harmine was found to prohibit lipid peroxidation in enzymatic Fe 3+ ADP-NADPH and non-enzymatic Fe 3+ ADP-DHF oxygen radical generating systems in a concentrationdependent way (Tse et al., 1991).Furthermore, harmine along with other β-carbolines possess protective activity against H2O2-stimulated oxidative injury in both yeast and mammalian cells (Moura et al., 2007).
1-Methylated β-carboline (harmine) also has a potential to block SlN-1-induced mitochondrial injury in PC12 cells.The molecular mechanism involves inhibition of apoptotic cell death, and caspase-3/-9 activity via decreasing ROS, and GSH levels (Choi et al., 2004) (Table III).To date, there have been a limited number of studies regarding anti-oxidant, and free radical scavenging activities of harmine.Also, no studies have been done on the anti-oxidant effect of harmine derivatives.

Neuroprotective activity
The multifarious array of bioactive compounds abundantly found in nature plays an important role in the treatment and prevention of neurodegenerative diseases such as Alzheimer's, Huntington's and other neuronal dysfunctions (Essa et al., 2012).Alkaloids that are isolated from the seeds of TAPH such as harmine exerts the cerebroprotective effect on ethanol-stimulated neurodegeneration and sodium nitrite-stimulated hypoxia in young mice.TAPH eloquently protects the brain from sodium nitrite-induced memory deterioration and retention by reducing the transverse latency time (TLT), and enhancing step down latency (SDL) in a dose-related manner.It also inhibits acetylcholinesterase activity, protects DNA fragmentation, upregulates GSH level, and down-regulates TBARS expression in brain (Biradar et al., 2013).
Preclinical studies on harmine have suggested that it exhibits the neuroprotective effect in a rat model of sclerosis via increasing the activity of glutamate transporter-1 (GLT-1), and decreasing TNF-α, IL-1β levels in the hippocampus.Further, post-GCl harmine administration has the ability to debilitate cerebral infarct volume, and to reduce neuronal cell death in vivo (Li et al., 2011;Sun et al., 2014).Some recent experimentation showed that continuous usage of ayahuasca (harminerich hallucinogen) is linked with improved neuropsychological functioning.Moreover, harmine treatment reduced inflammation, excitotoxicity and, oxidative stress along with enhanced levels of neurotrophic factors (BDNF), and glutamate transporters in the hippocampus (Dos Santos and Hallak, 2017).

Antidepressant activity
Various studies in animals have shown that ayahuasca and harmine possess antidepressant-like effects in the behavioral animal model of depression.Harmine and tetrahydroharmine encouraged adult neurogenesis and stimulated neural stem cell growth, migration, and differentiation in vitro (Morales-Garcia et al., 2017).Furthermore, acute and chronic administration of harmine at high dosage (5, 10, 15 mg/kg) demonstrated behavioral and physiological effects by elevating SOD, CAT, BDNF and ACTH circulation levels in a rat model of hippocampus and prefrontal cortex (Fortunato et al., 2009;Fortunato et al., 2010a;Fortunato et al., 2010b;Reus et al., 2010).In another investigation, harmine significantly increased the proliferation in human neural progenitor cells and blocked DYRK1A activity, respectively (Dakic et al., 2016).

Anti-microbial activity
Harmine its various synthetic derivatives are known to possess anti-microbial effects against different fungal species such as Fusarium oxysporum, Colletotrichum gloeosporioides (Salman et al., 2016).Harmine exerted a noticeable prohibitory effect on germination of conidia at the concentration between 0.5 to 1 mM.In another study, harmine showed fungicidal activity (>60%) against Physalospora piricola at 50 mg/kg concentration (Olmedo et al., 2017).
Furthermore, harmine and its derivatives have antiviral activities against tobacco mosaic virus as well as anti-fungal effects against Puccinia sorghi (Lu et al., 2015).Harmine has been found to prohibit HSV infection at CC50 value around 337.1 µM and, EC50 value of 1.5 µM in a dose-dependent way.Harmine significantly down-regulated HSV-2-mediated activation of NF-κB along with p65 nuclear translocation, and IκB-α degeneration.It also blocked HSV-2-induced JNK phosphorylation and p38 MAPK kinase activity, respectively (Chen et al., 2016).Moreover, a synthetic derivative of harmine, 9N-methylharmine demonstrates a strong inhibitory effect on DENV-2 generation.The quantification of extracellular and intracellular viral genomes designated that 9N-methylharmine has capability to debilitate maturation time and discharge of viral entities to the extracellular medium influencing the transmission of the disease (Quintana et al., 2016).
The study of harmine's anti-viral mode of action indicated that it eloquently prohibits enterovirus (EV71) via targeting NF-κB signaling pathway with CC50 value of 500 µM and, EC50 value of 20 µM in vitro.The associated mechanism involves decreased ROS produc- tion, and suppressed EV71-prompted NF-κB activation.Additionally, harmine treatment has a potential to defend AG129 mice against EV71 replication in vivo (Quintana et al., 2016;Chen et al., 2018).

Other biological activities of harmine
Harmine has also been known to possess several other biological activities.Primitive studies have declared that harmine is an auspicious anti-malarial agent selectively targeting P. falciparum PfHsp90.The unique and non-toxic harmine analogues 17A and 21A have the affinity to bind with heat shock protein-90, inhibits P. falciparum at concentration of 4.2 ± 1.3 µM and 5.7 ± 1.7 µM during in vitro investigation, decreases parasitaemia and extends survival of P. berghei-affected BALB/c mice (100 mg/kg) in vivo (Bayih et al., 2016).

Conclusion
Harmine is a potent drug candidate and its synthetic derivatives exhibit pharmacological and biological effects in various ailments through diversified mechanisms of action.Pharmacodynamically and pharmacokinetically up-graded harmine and its derivatives may also boost up further advances.This review has emphasized on recent researches from various in vivo and in vitro investigations on the ability of harmine and its derivatives to cure different pathological conditions.
Figure 1: Hermine and its derivatives Figure 2: Biological activities of harmine