Bioactivity and In Silico Studies of Isoquinoline and Related Alkaloids as Promising Antiviral Agents: An Insight

Viruses are widely recognized as the primary cause of infectious diseases around the world. The ongoing global pandemic due to the emergence of SARS-CoV-2 further added fuel to the fire. The development of therapeutics becomes very difficult as viruses can mutate their genome to become more complex and resistant. Medicinal plants and phytocompounds could be alternative options. Isoquinoline and their related alkaloids are naturally occurring compounds that interfere with multiple pathways including nuclear factor-κB, mitogen-activated protein kinase/extracellular-signal-regulated kinase, and inhibition of Ca2+-mediated fusion. These pathways play a crucial role in viral replication. Thus, the major goal of this study is to comprehend the function of various isoquinoline and related alkaloids in viral infections by examining their potential mechanisms of action, structure-activity relationships (SAR), in silico (particularly for SARS-CoV-2), in vitro and in vivo studies. The current advancements in isoquinoline and related alkaloids as discussed in the present review could facilitate an in-depth understanding of their role in the drug discovery process.


Introduction
Human health has been impacted for decades by a variety of life-threatening viruses such as the hepatitis C virus (HCV), influenza virus, herpes simplex virus (HSV), hepatitis B virus (HBV), dengue virus (DENV), human immunodeficiency virus (HIV), human coronaviruses (HCoV), human cytomegalovirus (HCMV), and Zika virus. Viral infections have become a serious global concern for health workers due to their uncontrollable morbidity and death rate. The recent SARS-CoV-2 (severe acute respiratory syndromecorona virus-2) has affected approximately 631 million people with 65 lakh deaths as per the WHO dashboard, on 14 November 2022. COVID-19 or SARS-CoV-2 is a novel coronavirus that shares 79 percent of its DNA with the SARS-CoV (severe acute respiratory syndromecorona virus) and 50% of its DNA with the Middle East Respiratory Syndrome (MERS) virus [1][2][3]. Similarly, HIV is also a major health concern worldwide. In 2021, approximately 6 lakh deaths occurred due to HIV-related causes, and approx. 1.5 million people have been diagnosed with HIV viral infection [4]. It is estimated that at the end of 2021, people living with HIV infection reached up to 38.4 million. The global prevalence of HBV infection was 296 million in 2019 with 1.5 million new infections each year. Furthermore, it is reported primary precursor for biosynthesizing the other varieties of isoquinoline alkaloids in plants is the benzyltetrahydroisoquinoline type alkaloid (Figure 1). These alkaloids possess numerous biological activities including antifungal, antidiabetic, anticancer, anti-inflammatory, antibacterial, and antiviral. The possible mechanism by which isoquinolinerelated alkaloids could show antiviral activity is illustrated in Figure 2 [26,40,41].  Recent research on viral infections has confirmed the potential role of various categories of isoquinoline and related alkaloids in the prevention of diseases such as SARS-CoV and SARS-CoV-2 [42][43][44].

Therapeutic Targets for SARS-CoV-2 Inhibition: In Silico Approaches
One of the main advantages of in silico drug designing process is its cost-effective Recent research on viral infections has confirmed the potential role of various categories of isoquinoline and related alkaloids in the prevention of diseases such as SARS-CoV and SARS-CoV-2 [42][43][44].

Therapeutic Targets for SARS-CoV-2 Inhibition: In Silico Approaches
One of the main advantages of in silico drug designing process is its cost-effective nature in the research and development process. Molecular modeling and in silico methodologies have gained lots of attention nowadays. Thus, these approaches have been extremely useful in identifying targets and predicting the efficacy of new drugs in recent pandemics. In the recent SARS-CoV-2 infection cycle, angiotensin-converting enzyme (ACE2) fuses with SARS-CoV-2 spike protein to promote and facilitate the virus's entry. Natural compounds inhibiting ACE2 will directly help in the management of COVID-19 as it restricts the entry of SARS-CoV-2 to the host cell. Similarly, inhibition of other target proteins such as spike protein, main proteases (M pro /3CL pro ), RNA-dependent RNA polymerase (RdRp), and non-structural proteins (NS) may suppress the SARS-CoV-2 infection [45]. Isoquinoline and their related alkaloids effectively bind with all these above-mentioned targets of SARS-CoV-2 enlisted in Table 1.

Main Protease (M pro ) or 3-Chemotrypsin-Like Protease (3CL pro )
Apart from ACE2, the bis-benzylisoquinoline alkaloidal category shows potential binding affinity against M pro /3CL pro , PL pro , RNA-dependent RNA polymerase (RdRp), and other NS proteins. Berbamine and oxyacanthine are also bis-benzylisoquinoline alkaloids isolated from Berberis asiatica Roxb. ex DC. Docking of these compounds using AutoDock Vina demonstrated maximum binding affinity with the binding free energy of −20.79 kcal/mol and −33.35 kcal/mol against M pro [48]. CEP obtained from Stephania cepharantha Hayata has been discovered to have a critical role in the prevention and management of COVID-19 [56]. This phytoconstituent and its natural analogues showed satisfactory results when evaluated collectively by in silico and in vitro methods. Moreover, tetrandrine was also assessed in this research but showed slightly lower activity than CEP [57]. CEP showed strong binding interaction against 3CL pro (−8.5 kcal/mol) and TMPRSS-2 (−7.4 kcal/mol) when docked with the help of Swiss PDB viewer, PyRx, and PyMol software

Main Protease (M pro ) or 3-Chemotrypsin-like Protease (3CL pro )
Apart from ACE2, the bis-benzylisoquinoline alkaloidal category shows potential binding affinity against M pro /3CL pro , PL pro , RNA-dependent RNA polymerase (RdRp), and other NS proteins. Berbamine and oxyacanthine are also bis-benzylisoquinoline alkaloids isolated from Berberis asiatica Roxb. ex DC. Docking of these compounds using AutoDock Vina demonstrated maximum binding affinity with the binding free energy of −20.79 kcal/mol and −33.35 kcal/mol against M pro [48]. CEP obtained from Stephania cepharantha Hayata has been discovered to have a critical role in the prevention and management of COVID-19 [56]. This phytoconstituent and its natural analogues showed satisfactory results when evaluated collectively by in silico and in vitro methods. Moreover, tetrandrine was also assessed in this research but showed slightly lower activity than CEP [57]. CEP showed strong binding interaction against 3CL pro (−8.5 kcal/mol) and TMPRSS-2 (−7.4 kcal/mol) when docked with the help of Swiss PDB viewer, PyRx, and PyMol software [52]. These studies indicate that bis-benzylisoquinoline alkaloids should be evaluated more for the prevention of SARS-CoV-2 infection.
An in silico and molecular dynamic investigation on three primary alkaloids namely BBR, choline, and tetrahydropalmatine from Tinospora cordifolia (Willd.) Miers was conducted with the help of Auto Dock and MG Tools of Auto DockVina software. BBR (−7.3 kcal/mole) had the highest affinity for the SARS-CoV-2 protein 3CL pro [47]. Natural compound noscapine mainly present in the opium poppy plant also possesses high M pro inhibition activity exhibiting a very high docking score (−292.42 kJ/mol) [49]. Further molecular dynamic (MD) simulation studies also confirmed the stability of noscapine with M pro [58]. The M pro inhibition of palmatine obtained from T. cordifolia was also performed with the help of the SwissDock server. The binding score of palmatine and other natural compounds such as gingerol, and BBR with SwissDock-based docking software EADock DSS showed greater than −8 kcal/mol binding affinity toward the M pro [50]. Molecular docking of a bis-benzylisoquinoline alkaloid oxycanthine showed a binding affinity of −10.99 kcal/mol via Auto Dock which ultimately suggested that it could be a successful candidate in the treatment or prevention of SARS-CoV-2. BBR and capsaicin also showed slightly good binding energy (−7.910 kcal/mol and −5.510 kcal/mol respectively) than caffeine [59]. A recent in silico study, using Autodock (version 4.2) on selected isoquinoline alkaloids including cephaeline, coptisine, galanthamine, glaucine, drotaverine, chelidonine, hydrastine, boldine, fumaricin stated that coptisine had the best binding affinity (−9.15 kcal/mol) toward M pro . MD simulation study of coptisine depicted a stable complex of N3-M pro with a binding energy of −8.17 kcal/mol [54].

RNA-Dependent RNA Polymerase (RdRp)
RdRp is an important target to cure RNA-based viral diseases such as MERS, SARS, and SARS-CoV-2 as it is responsible for viral RNA replication. In SARS-CoV-2 infection, RNA genome replication is a prominent step to spread the infection; however, RdRp inhibitors prevent this step and treat the infection. Molecular docking of 38 Chinese patent drugs including phytocompounds, flavonoids and alkaloids, was studied for a molecular docking study against RdRp, ACE2, and M pro . The in silico study, with the help of AutoDock Vina, the findings demonstrated that morphine, codeine, indirubin, and BBR showed effective binding capacity (>−6.0 kcal/mol) against M pro ; however, their binding affinity against RdRp was much higher (>−8.1 kcal/mol) than that of M pro [51]. CEP also showed binding with RdRp protein to inhibit viral replication [43]. A study includes 143 isoquinoline compounds for in silico screening using Molegro Virtual Docker (version 6.0, Molegro ApS, Aarhus, Denmark) against RdRp of the Zika virus. Molecular docking studies also demonstrated that Cassiarin D possessed a high binding score against RdRp (−150.7 kJ/mol) [53].

Antiviral Actions of Isoquinoline Related Alkaloids
Natural isoquinoline and its related alkaloids have been widely explored in recent years for their antiviral potential. For instance, BBR is a protoberberine alkaloid extracted from various species of the Berberis genus exerting antiviral activity against a diverse range of viruses including HIV, HSV, HCMV, and human papillomavirus (HPV) [27]. BBR interferes with certain pathways such as nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase/extracellular-signal-regulated kinase (MEK/ERK), and mammalian target of rapamycin and the adenosine monophosphate-activated protein kinase (AMPK/mTOR) to resist viral replication, thereby, providing antiviral action. Other antiviral activities of isoquinoline and related alkaloids are summarized in Table 2.

Protoberberine Alkaloids
Protoberberine alkaloids contain a tetracycline ring system and are mainly derived from the benzyltetrahydroisoquinoline by oxidation of the phenolic group and coupling with the N-methyl group of isoquinoline [36]. Natural protoberberine alkaloids have multiple pharmacological actions such as antiseptic, sedative, stomachic, analgesic action, and antiviral action [112].
Protoberberine alkaloids show prominent effects against HSV, HBV, SARS-CoV, SARS-CoV-2, and HCMV. A protoberberine alkaloid berberine (BBR) isolated from various species of Berberis represents antiviral against HIV by inhibiting tumor necrosis factor (TNF-α) and IL-6 in macrophage of cultured mouse J774A [61]. Inhibition of the NF-κB pathway and c-Jun N-terminal kinase (JNK) phosphorylation is the main mechanism behind the anti-HSV action of BBR [60]. A complex of ZnO/BBR complex was also tested against SARS-CoV-2 by performing a plaque reduction assay and Vero E6 cell anti-viral assay. Molecular docking of this complex indicates the potential to inhibit papain-like protease (PL pro ), spike protein, and spike-receptor-binding domain (RBD). Further, an in vitro study concludes that ZnO/BBR at various concentrations showed PL pro inhibition in the range of 70-98% and thus, suggested its role in SARS-CoV-2 infection. The complex inhibits S protein-ACE2 binding as compared to single BBR and ZnO-NP [64]. A review also showed the antiviral potential of BBR in different strains of coronaviruses [27].
Recently, a nanomedicine containing BBR (NIT-X) was investigated against SARS-CoV and SARS-CoV-2. This nano-oral formulation increased CD8 + cells and interferongamma (IFN-γ), thus showing immunomodulatory activity [62]. A study on Vero E6 cells evaluated the effectiveness of BBR and chloroquine along with synthetic drugs like lopinavir, remdesivir, and cyclosporine. IC 50 values of chloroquine (1.38 µM) and BBR (10.58 µM) suggested their anti-SARS-CoV-2 activity [63]. Moreover, in vitro studies on calu-3 cells demonstrated the potential of this nanoformulation to inhibit ACE2, TMPRSS2, IL-1α, IL-8, and IL-6 [62]. Furthermore, inhibiting pro-inflammatory mediators and improving immunomodulation would be an excellent therapy against SARS-CoV-2. A recent study included BBR and obatoclox for the assessment of anti-SARS-CoV-2 activity in Vero E6 cells and nasal epithelial cells, which showed EC 50 values of BBR is 9.1 µM, thus confirming the role of BBR in reducing the viral replication that justifies its promising effects against SARS-CoV-2 [65]. BBR was also found to be effective against severe post-COVID conditions such as pulmonary fibrosis and reduced inflammation during COVID-19 pneumonia [113]. The in vivo study of BBR shows activity against influenza viral pneumonia. It can cause reduction in pulmonary edema and lung index in mice and ultimately suppress lung hemorrhage [114]. BBR also showed effectiveness against chikungunya virus through decreasing viral load in wild-type C57BL6/J mice. This isoquinoline alkaloid significantly reduced joint swelling in mice [67]. Moreover, BBR also shows another anti-influenza activity by decreasing viral titers in the mouse lungs and thus eventually cause reduction in the mortality rate of mice [115].
Other protoberberine alkaloids such as dehydrocavidine, BBR, dehydroapocavidine, dehydroisoapocavidine, and dehydroisocorypalmine were derived from Corydalis saxicola Bunting plant and proved to possess anti-HBV activity. The in vitro assay performed on the 2.2.15 cell lines showed that dehydrocavidine and dehydroapocavidine represent 51% and 54% inhibition against HBeAg respectively [71,116]. Similarly, a study performed on the isoquinoline alkaloids namely saxicolalines A and N-methylnarceimicine and other alkaloids of C. saxicola showed anti-HBV activity. Along with these two isoquinoline alkaloids, dihydrochelerythzrine (IC 50 < 0.05 µM) also demonstrated potent activity against HBV [71]. An anti-HBV study conducted on 18 isoquinoline alkaloids obtained from the various plant species also demonstrated potent in vitro activity against HepG2.2.15 cell lines [117]. A few alkaloids such as columbamine iodide and jatorrhizine chloride also showed activity against HIV with IC 50 values of 58 and 71 µg/mL respectively [118].
BBR can inhibit Toll-like receptor-7 (TLR7), NF-κB, and myeloid differentiation primary response 88 (MyD88) in the TLR7 signaling pathway and thus restrict viral copies in mice, which is attributed to the treatment of influenza viral infection (H1N1) [69]. A study indicates that BBR can inhibit DNA polymerase and immediate-early (IE-3) proteins. BBR has also been evaluated against different strains of HCMV and exerted broad-spectrum anti-HCMV activity due to its suppressive nature against murine CMV (MCMV) showing EC 50 [68]. BBR is able to suppress IL-6, IL-1β, IL-1α, and TNF-α by inhibiting Nod-like receptor protein 3 (NLRP-3) pathway [119].
The structure-activity relationship of protoberberine alkaloids indicates that linkage between C2 and C3, a quaternary nitrogen atom and substitution with hexyl and methyl is important for anti-HIV, anti-HBV, and anti-polio activities [26,71,116,120,121] (Figure 3).

Aporphine Alkaloids
Aporphine alkaloids have been known for their antioxidant, anticancer, anthelmintic, antibacterial, antimalarial, and antiviral activity [122]. The in vitro activities of these alkaloids show potential action against HIV, HCV, and HSV viruses. A study of ixoratannin A-2 and aporphine alkaloid boldine on CEM-GXR cells showed anti-HIV activity with EC50 values of 34.4 and 50.2 µ M. This in vitro study demonstrated the anti-HCV effect of ixoratannin A-2 and boldine [73]. Similarly, another category such as aporphine alkaloids also exerted anti-HIV action. For example, twelve compounds were isolated from the plant Dasymaschalon rostratum Merr. and Chun from which raymarine A, 3-methoxyoxoputerine-N-oxide, and dasymaroine B showed EC50 values in the range of 1.93 to 9.70 µ M and exerted potent activity against HIV [75]. Moreover, marine alkaloids such as lamellarins alkaloids also exhibited anti-HIV action [123,124]. A natural steroidal alkaloid

Aporphine Alkaloids
Aporphine alkaloids have been known for their antioxidant, anticancer, anthelmintic, antibacterial, antimalarial, and antiviral activity [122]. The in vitro activities of these alkaloids show potential action against HIV, HCV, and HSV viruses. A study of ixoratannin A-2 and aporphine alkaloid boldine on CEM-GXR cells showed anti-HIV activity with EC 50 values of 34.4 and 50.2 µM. This in vitro study demonstrated the anti-HCV effect of ixoratannin A-2 and boldine [73]. Similarly, another category such as aporphine alkaloids also exerted anti-HIV action. For example, twelve compounds were isolated from the plant Dasymaschalon rostratum Merr. and Chun from which raymarine A, 3-methoxyoxoputerine-N-oxide, and dasymaroine B showed EC 50 values in the range of 1.93 to 9.70 µM and exerted potent activity against HIV [75]. Moreover, marine alkaloids such as lamellarins alkaloids also exhibited anti-HIV action [123,124]. A natural steroidal alkaloid obtained from marine sponge didehydro-cortistatin has shown its potential against HIV-mediated inflammation [125]. An antiviral study included didehydro-cortistatin and highlighted "Block-and-lock" approach for the treatment of HIV. Didehydro-cortistatin A causes a reduction in HIV transcription and suppression of viral rebound in bone marrow-liverthymus mice [126].
Two aporphine alkaloids namely magnoflorine and langinosine from the plant Magnolia grandiflora L. possessed strong HIV inhibition potential. Moreover, further cytotoxic assays were carried out using tumor cell lines to determine anti-HSV activity. The results showed anti-HSV action of M. lanuginosine methanolic extract with 76.7% inhibition and 47% inhibition against polio-virus. Both the alkaloids magnoflorine and lanuginosine represent potent cytotoxic activity also with IC 50 values of 0.4 and 2.5 µg/mL [127]. Nineteen aporphine alkaloids were also investigated on Vero cells of monkey kidneys against HSV, and results indicated the selection of three alkaloids namely oliverine HCl, pachystaudine, and oxostephanine as potent inhibitors of HSV [77]. The structure-activity relationship of aporphine alkaloids is demonstrated in Figure 4.
The in vitro examination of papaverine hydrochloride against HIV with the help of human T-cell line such as MT4 cells showed effective ED50 values of 5.8 µ M at a dose of 30 µ M [79]. A similar in vitro study conducted on papaverine using H9 cells in peripheral
The in vitro examination of papaverine hydrochloride against HIV with the help of human T-cell line such as MT4 cells showed effective ED 50 values of 5.8 µM at a dose of 30 µM [79]. A similar in vitro study conducted on papaverine using H9 cells in peripheral blood mononuclear cell (PBMC) culture indicated potent antiviral activity against HIV at a concentration of 10 µg/mL [137]. The anti-HIV activity of salsolinol, tetrahydropapaveroline, and N, N-dimethylsalsolinol was performed on Raji cells. These three isoquinoline alkaloids also showed activity against Epstein-Barr virus early antigen (EBV-EA) [138]. Dimethoxy-3,4-dihydro isoquinoline and dihydroxyisoquinolinium salts were also reported effective against HIV with IC 50 values of 2.07 µg/mL and 23.6 µg/mL respectively. EC 50 values of both the compounds were found to be >0.10 µg/mL [139]. Thirty-three isoquinoline alkaloids selected from Corydalis and Fumaria species represented antiviral activity against herpes simplex and para-influenza virus. The in vitro study of alkaloids against anti-HSV and anti-PI3 activity using Vero and madine-darby bovine kidney (MDBK) cell lines represents effective results [76].

Bisbenzylisoquinoline (BBI)
BBI comprises two benzyltetrahydroisoquinoline that is combined via phenolic oxidation to form bisbenzylisoquinoline [36]. However, the main precursor for the BBI alkaloids is tyrosine. These alkaloids are known to have antibacterial, antifungal, antiviral, and antimalarial activity [140].
This famous subcategory of isoquinoline alkaloids shows a potential role in viral infections. In a study, methanolic extract from Indonesian plants was investigated against HSV-1. Later the same researcher evaluated subtypes of isoquinoline alkaloids obtained from water and methanolic extract of Chinese medicinal plants by subjecting them to plaque reduction assay to investigate the anti-HSV activity. All BBI alkaloids included in the study were found to be effective and showed IC 50 values in the range of 14.8-43.2 µg/mL. Results confirmed that homoarmoline, isotetrandrine, berbamine, thalrugosine, and obamegine demonstrated anti-HSV-1 and HSV-2 potential with IC 50 in the range of 16.3 to 24.9 µg/mL [93,102].
CEP, a plant alkaloid found in Stephania cepharantha from the family Menispermaeceae showed antiviral activity against HIV in U1 cells through inhibition of the NF-κB pathway [94]. A study revealed another mechanism of CEP against HIV by decreasing plasma membrane fluidity [96]. CEP also inhibits pro-inflammatory mediators to prove its anti-inflammatory action [95,99]. The synthetic analog of CEP demonstrated effectiveness against HIV-1 [99]. Two alkaloids from the plant S. cepharantha roots namely aromoline and di-O-acetylsinococuline indicate HIV inhibitory activity with CC 0 values of 62.5 and 15.6 µg/mL, and IC 100 value of 31.3 and 7.8 µg/mL [80]. Synthetic derivatives of cepharanoline were investigated against HIV in U1 cells. Out of all the analogues of cepharanoline, five compounds were selected and showed more potent inhibition than CEP [97]. Furthermore, CEP in combination with the 8-difluoromethoxy-1-ethyl-6-fluoro-1,4-dihydro-7-[4-(2-methoxyphenyl)-1-piperazinyl]-4-oxoquinoline-3-carboxylic acid (K-12) also reduced viral infection by inhibiting NF-κB and TNF-α in U1 cells [141]. In vitro activity of CEP showed potent activity against HSV also with an IC 50 value of 0.835 µg/mL [142]. The antiviral activity of some important alkaloids including tetrandrine, CEP, neferine, and hernandezine on HEK 293T cells expressing ACE2 (293T-ACE2 cells) exhibited halfmaximal effective concentrations (EC 50 ) less than 10 µM [85], indicating inhibition of viral entry against SARS-CoV-2. Another in vitro assay was performed to identify CEP and nelfinavir (NFV) as anti-viral agents. N-protein expression and RNA levels were significantly decreased by NFV and CEP which suggested them to be potent drugs for COVID-19 [101].
It has been reported that CEP possessed anti-inflammatory and immunomodulatory action by suppressing cytokine release, NF-κB, nitric oxide (NO), and cyclooxygenase (COX). A newly established cell-culture model GX-P2V (pangolin coronavirus) such as SARS-CoV-2 model is being used to identify therapeutic drugs against COVID-19. CEP has been evaluated by this novel cell culture model and showed the highest inhibition against GX-P2V with an EC 50 value of 0.98 µmol/L. In addition, CEP also inhibited viral entry and multiplication in the host cell and exerted maximum antiviral activity [143]. Similar research performed on the same cell culture model along with transcriptome analysis showed the potential of CEP in inhibiting viruses through various pathways including cellular stress responses and autophagy [144]. Thus, considering the above facts, CEP could be beneficial for treating COVID-19. The in vivo study of CEP demonstrated inhibition of porcine epidemic diarrhea virus (PEDV) through reduction in viral load at a dose of 11.1 mg/kg of b.w. [145]. Another bisbenzylisoquinoline alkaloid dauricine, isolated from Menispermum dauricum in combination with clindamycin could inhibit NF-κB activation in mice to treat influenza viral H5N1 infection [146].
Two bis-benzyl isoquinoline alkaloids including tetrandrine and isotetrandrine isolated from the roots of Mahonia bealei (Fortune) Pynaert possessed anti-influenza activity at a concentration of 0.25 mg/mL [147]. Tetrandrine (30 mg/kg, i.p.) in combination with acyclovir (120 mg/kg i.p.) proved to have anti-HSV activity in the Bragg albino mouse model (BALB). This BBI alkaloid was found responsible to inhibit IL-6 in the cornea of mice and thus exerts inhibition against HSV [84]. It was observed that tetrandrine suppressed delayed-type hypersensitivity and produces inflammatory responses to the virus [148]. In addition to tetrandrine two other alkaloids including fangchinoline, and CEP were investigated against human coronavirus OC43 (HCoV-OC43) using a Medical Research Council cell strain 5 (MRC-5) fibroblast lung cell line. Results demonstrated dose-dependent inhibition of tetrandrine, fangchinoline and CEP with IC 50 values of 0.33, 1.01, and 0.83 µM respectively [83].
BBI alkaloid neferine was also assessed for anti-SARS-CoV-2 activity along with other 29 compounds. huh7, HEK293/hACE2 cell lines were used to check the potential of molecules toward SARS-CoV-2. This bis-benzylisoquinoline alkaloid possessed significant inhibition (approx. 75%) against SARS-CoV-2 pseudovirus and showed an EC 50 value of 0.36 µM. It blocked the viral entry by inhibiting Ca 2+ -mediated fusion and thus could be a promising agent to treat viral infections including newly emerged SARS-SoV-2 [88]. Cycleanine, a BBI alkaloid, possessed potent anti-HIV efficacy with an EC 50 value of 1.83 µg/mL [81]. Another BBI alkaloid, fangchinoline, also showed its effectiveness against various strains of HIV, showing EC 50 value in the range of 0.8 to 1.7 µM [82].
Berbamine, isolated from Berberis amurensis Rupr., demonstrated its anti-COVID-19 activity by causing a reduction in ACE2 and dipeptidyl peptidase-4 (DPP-IV) levels in the plasma membrane. A study conducted on Huh7-cells indicates that berbamine caused inhibition of Ca 2+ influx, which ultimately led to the inhibition of TRPMLs, which later resulted in the intervention of virus entry [91]. Moreover, berbamine also showed inhibition of the 2-E channel with an IC 50 value of 111.50 µM, thus preventing SARS-CoV-2 infection. The EC 50 value of this BBI alkaloid confirmed its inhibitory action against other flaviviruses as well as Japanese encephalitis virus (JEV) (1.62 mM) and Zika virus (2.17 mM) [91]. The β-carboline and isoquinoline alkaloids such as dictamine, cinchonine, and skimmianine showed inhibition against SARS-CoV. The quinine derivative, chloroquine also showed potent inhibition toward SARS-CoV-2 [149]. Multiple compounds were studied in vitro to investigate action against SARS-CoV-2 on the human epithelial colorectal adenocarcinoma cell line (Caco-2 cells). Top hits included synthetic drugs as well as phytocompound present in plants namely lopinavir, camostat, nafamostat, mefloquine, papaverine, and cetylpridinium [78].
The BBI alkaloids category seems to have an important role in the treatment and prevention of SARS-CoV-2 and other viral infections. Some alkaloids categorized under this division were evaluated for various viral targets and produced effective results. The structure-activity relationship of this category is depicted in Figure 6. nocarcinoma cell line (Caco-2 cells). Top hits included synthetic drugs as well as phytocompound present in plants namely lopinavir, camostat, nafamostat, mefloquine, papaverine, and cetylpridinium [78].
The BBI alkaloids category seems to have an important role in the treatment and prevention of SARS-CoV-2 and other viral infections. Some alkaloids categorized under this division were evaluated for various viral targets and produced effective results. The structure-activity relationship of this category is depicted in Figure 6.

Ipecac Alkaloids
A natural alkaloid, emetine present in the plant Psychotria ipecacuanhae (Brot.) Standl. has been reported against HIV with 80% inhibition [104]. Emetine showed inhibition of Zika virus nonstructural protein-5 (NS5) polymerase activity and ebolavirus (EBOV) with effective IC 50 values. They disrupted the entry of lysosomal function and exerted anti-viral action [150]. The semi-synthetic derivative of emetine known as emetine dihydrochloride hydrate is found to be effective against SARS and MERS with EC 50 values of 0.051 and 0.014 respectively [103]. A study identified four alkaloids including emetine as potential antiviral agents against echovirus (EV-1), and herpes simplex virus (HSV-2) on retinal pigment epithelial (RPE) cells [151].
The salts of ipecac alkaloids such as psychotrine dihydrogen oxalate and o-methylpsychotrine sulfate heptahydrate also exhibited potential inhibitory activity towards reverse transcriptase of HIV [152]. Some other alkaloids such as o-methylpsychotrine sulfate and papaverine also demonstrated anti-HIV activity [153]. One study investigated the effect of seven compounds including lycorine and emetine on HCoV-OC43 and MERS-CoV. Findings stated that lycorine suppressed the viral load in the BALB mice model and represented anti-HCoV-OC43 potential. However, emetine was found effective against MERS-CoV [111].

Naphythylisoquinoline Alkaloids
Michellamines alkaloids from Ancistrocladus congolensis J. were identified for their anti-viral action and found effective against HIV-1 and HIV-2 [104,105,[154][155][156]. A study indicated that michellamine A2, A3, A4, and michellamine B showed IC 50 values of 29.6, 15.2, 35.9, and 20.4 µM respectively, and thus exerted anti-HIV action [106]. Another study isolated korupensamine E along with michellamine B, D, E, and F from the same plant and was investigated for in vitro HIV activity. Results confirmed that all michellamine possessed antiviral activity against HIV with EC 50 values in the range of 17-188 µM [157]. A bis-benzylisoquinoline alkaloid neferine and their desmethyl and didesmethyl analogues such as isoliensinine and liensinine also showed effective activity against HIV [89].

Benzophenanthridines
The higher plants mainly consist of benzophenanthridine isoquinoline compounds exhibit a wide range of pharmacological activities. A study investigated 2000 synthetic and natural compound which include sanguinarine as a HIV-protease inhibitor with IC 50 value of 4.3 µg/mL [160]. An anti-HIV-1 and HIV-2 reverse transcriptase (RT) study included fangronine chloride and nitidine chloride to determine the antiviral potential. The IC 50 value of fangronine chloride (8.5 µg/mL) and nitidine chloride (7.4 µg/mL) suggest the efficacy of these two compounds against HIV-RT. Similar results were shown in the case of HIV-2 RT where fangronine chloride displayed a 9.5 µg/mL IC 50 value, whereas, nitidine chloride displayed a 7.1 µg/mL IC 50 value [109]. Anti-HCV and PI-3 (para-influenza) activity of norsanguinarine displayed moderate action against HCV and PI-3 with minimum inhibitory concentration (MIC) range from 16 to 32 µg/mL [76].

Inflammation Inhibition
Anti-inflammatory phytochemicals might be viable therapeutic candidates against various viruses, including SARS-CoV2, as they have direct antiviral effects and can alleviate the status of inflammatory diseases [22,23]. Moreover, it is evident from recent reports that severe COVID-19 patients cause elevation of inflammatory pro-markers which eventually lead to multiorgan failure [161]. Thus, researchers are now focusing on natural products such as medicinal plants that possess anti-inflammatory as well as anti-viral potential. Tetrandrine from the category of bis-benzylisoquinoline alkaloids of isoquinoline alkaloids has been reported for immunomodulation and anti-inflammatory activities. It has anti-inflammatory action against croton oil-induced ear edema in mice and demonstrates 95% inhibition at 12.5 µM [162]. This isoquinoline alkaloid also showed inhibition against proinflammatory mediators of cytokines, iNOS, and cyclooxygenase (COX-2) in human monocytic cells [163]. In vitro lipopolysaccharide assay suggested the potential of tetrandrine to inhibit TNF-α, IL-6, and NO release in lipopolysaccharide (LPS)-induced microglial activation [164]. Similarly, in vivo studies performed on BBR, which is another isoquinoline alkaloid at the dose of 50 mg/kg, indicated a reduction of IL-6, IL-10, IL-1β, and IFN-γ caused by thioacetamide injection [165]. Noscapine is also found to be responsible for showing inhibitory action against cytokine storm in COVID-19 [166]. Recently, an in vitro study indicated that litcubanine, an isoquinoline alkaloid greatly reduced the activation of inflammatory macrophages by LPS via the NF-κB pathway, which would reduce the levels of inflammatory mediators such iNOS, TNF-α, and IL-1β [167].

Clinical Findings
The efficacy of isoquinoline and related alkaloids in various scientific reports including in silico, in vitro, and in vivo evaluation justified a slew of human clinical trials to assess the safety, pharmacokinetics, and pharmacodynamic effectiveness in a variety of viral pathologies. Few recent clinical trials also justify the role of isoquinoline and related alkaloids in viral infections. For example, clinicians investigated the effects of BBR (NCT04479202) on intestinal function, serum concentrations of inflammatory biomarkers, and organ function in severe SARS-CoV-2-infected patients in a prospective randomized controlled clinical trial supported by the Chinese medical association. An herbal formulation of COVIDEX also contains BBR (NCT05228626) targeting SARS-CoV-2 in a clinical trial sponsored by Makerere University in Uganda. Tetrandrine (NCT04308317) in combination with standard drug improves prognosis and reduces the incidence of pulmonary fibrosis during rehabilitation in SARS-CoV-2 patients. Even though there are only a few clinical trials (enlisted in Table 3) in the literature that include the use of isoquinoline and related alkaloids with various viral infections, these moieties are expected to have a greater impact on viral load, making these molecules the alternative option for improving the limited clinical efficacy and expanding the field of use to other viral pathologies.

Conclusions and Future Perspective
The emerging variants of powerful viruses have made the task of clinical practitioners and scientists more challenging to find an effective solution against viral infections. However, naturally occurring isoquinoline and related alkaloids could be an alternative option for treating these rapidly mutating viruses. From research findings, it can be concluded that these alkaloids exert their antiviral action majorly by interfering with the signaling pathways such as NF-κB, and MEK/ERK that eventually restricts the entry and replication of the virus. Furthermore, inhibition of Ca 2+ -mediated fusion and SandN protein expression could also facilitate the antiviral actions. Isoquinoline and their related alkaloids also resist pro-inflammatory markers such as IL-6, IL-10, and IL-1β and exert anti-inflammatory action. Additionally, an increase in CD8+ cells and IFN-γ production by some of the isoquinoline alkaloids represent immunomodulatory effects.
The structure-activity relationship has clearly shown that the quaternary nitrogen atom is essential for the antiviral activity of protoberberine and benzophenanthridine alkaloids. However, substitution with a methyl group at benzyltetrahydroisoquinoline and BBI moieties potentiate the antiviral action. From the in vitro and in vivo studies, it is concluded that isoquinoline and related alkaloids including berbamine, CEP, tetrandrine, neferine and lycorine from the BBI category of isoquinoline alkaloids represent broad-spectrum activities against HSV, HIV, SARS-CoV, and SARS-CoV-2 infection. Thus, BBIalkaloids should be further structurally explored for the treatment of newly emerged viral strains.