Leafy greens, algal metabolites as novel drugs against COVID-19

•Study shows potential inhibition of SARS-CoV-2 main protease by natural phytochemicals
•Ultra violet-LED lights can kill coronaviruses, HIV with flip of switch, research finds
•Deadly venom from poisonous sea snails could hold key to developing new painkillers

Scientists have validated more natural ‘cures’ for Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) that causes COVID-19. In recent researches, scientists found cruciferous vegetables such as broccoli, Brussels sprouts, and cabbages, algae metabolites and natural metabolites inhibit the virus.

Another study also found that the same light bulbs used in offices and public spaces can destroy coronaviruses and Human Immuno-deficiency Virus (HIV).

Indeed, despite the success recorded with vaccination against COVID-19, scientists emphasise the need for the development of effective treatments against the virus. In a recent study published in the journal Communications Biology, researchers from John Hopkins University, United States, discuss the use of the isothiocyanate sulphoraphane (SFN) for the treatment of both Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) that causes COVID-19 and seasonal human coronavirus (HCoV)-OC43 infections.

Sulphoraphane is a compound within the isothiocyanate group of organosulphur compounds. It is obtained from cruciferous vegetables such as broccoli, Brussels sprouts, and cabbages. It is produced when the enzyme myrosinase transforms glucoraphanin, a glucosinolate, into sulforaphane upon damage to the plant (such as from chewing or boiling during food preparation), which allows the two compounds to mix and react. Young sprouts of broccoli and cauliflower are particularly rich in glucoraphanin.

Sulphoraphane occurs in broccoli sprouts, which, among cruciferous vegetables, have the highest concentration of glucoraphanin, the precursor to sulphoraphane. It is also found in cabbage, cauliflower, Brussels sprouts, bok choy, kale, collards, mustard greens, and watercress.

Although there has been some basic research on how sulphoraphane might exert beneficial effects in vivo, there is no high-quality evidence for its efficacy against human diseases.

In the current study, VeroC1008 cells were incubated with SFN for one to two hours before they were inoculated with either the HCoV-OC43 or the wild-type strain of SARS-CoV-2. SFN was found to effectively inhibit virus-associated cell death in a dose-dependent manner for both coronaviruses. The median inhibitory concentrations (IC50) were similar for both HCoV-OC43 and SARS-CoV-2 at 10 micromolar (µM) and 12 µM, respectively.

To confirm these results, the same assay was performed in human diploid fibroblast cell line MRC-5 using only HCoV-OC43. This assay revealed highly similar results, albeit with a slightly higher IC50 of 18µM. The cytotoxicity was similarly dose-dependent, with the median cytotoxic dose (TD50) between 73 µM and 89 µM.

The inhibitory effect of the SFN was similar when added before or during infection, thereby leading researchers to speculate that it has an effect on both extracellular entry and viral processes within the cell. Following this, a potential synergistic effect of SFN combined with remdesivir was explored, which revealed several effective combination ratios below the corresponding IC50 values for each individual drug.

The researchers then sought to evaluate the antiviral effects of SFN treatment in mouse models. Transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) were inoculated with 8.4 x 106 TCID50 of SARS-CoV-2. Daily doses of SFN were administered to the mice orally, starting one day after viral inoculation.

All infected mice showed significant weight loss by day four; however, by day six, it was evident that SFN-treated mice were losing significantly less weight than controls. When examining the alveolar fluid and lung viral titers of infected mice, those treated with SFN had significantly lower viral burdens.

SFN-treated mice also showed significantly reduced pulmonary pathology than non-treated mice, with lower levels of alveolar and peribronchiolar inflammation. Immunostaining for the spike protein also revealed a wider distribution of infection within the lungs of infected animals.

High-dimensional flow cytometry was then used to evaluate any changes that might have arisen in the mice treated with the immunomodulatory drug as compared to controls. Changes in the spleen showed small differences; however, these findings were not significant.

When the changes in the lungs were observed, it was revealed that SFN treatment significantly reduced the recruitment of myeloid cells in the lungs, which is often seen in COVID-19. This effect likely reduced inflammation and immune-related damage.

The activation of lung alveolar and interstitial macrophages, which can introduce further cytokines, was also significantly reduced. CD8+ and CD4+ T-cells isolated from the lungs of mice exhibited significantly reduced expression of activation and proliferation markers.

SFN was found to significantly inhibit SARS-CoV-2 infection both in vivo and in vitro. The significant evidence described here thus supports further research, especially given the synergistic effects of SFN with the already-approved remdesivir.

SFN could help developing nations struggling with low vaccination rates, as well as support developed nations if new variants emerge with an enhanced ability to evade vaccine-induced immunity.

Also, a recent study posted to the Research Square preprint server, and currently under consideration at the Journal of Molecular Modeling, assessed the molecular dynamics of potentially active natural phytochemicals to target the severe acute SARS-CoV-2 main protease (Mpro).

SARS-CoV-2 vaccines have significantly curbed the morbidity and mortality caused by COVID-19 pandemic. However, there is considerable skepticism regarding vaccine-related health complications post-administration.

The present study investigated the effectiveness of phytochemical constituents, present in four medicinal herbs, in neutralizing SARS-CoV-2 Mpro and their mechanism of action.

The team employed the SARS-CoV-2 Mpro receptor that consisted of domains I and II having β-barrels while domain III had α-helices. Docking and visualization were performed on this receptor. Aegle marmelos, Coleus amboinicus, Aerva lanta, and Biophytum sensitivum constituting 20, 24, 17, and 26 phytochemicals, respectively, were used to obtain the phytochemicals.

The study then assessed the drug-likeness of the screened drugs, ensuring that each compound had 500 g/mol or less molecular weight, five or fewer hydrogen donors, less than 10 hydrogen acceptors, less than 10 rotatable bonds, less than 140 polar surface area (PSA), and less than 12 total hydrogen bond donors and acceptors.

Furthermore, the five pharmacokinetic properties, namely absorption, distribution, metabolism, excretion, and toxicity (ADME/T) of the drugs were also evaluated. A quantitative structure-activity relationship (QSAR) was used to calculate the ADME/T properties like aqueous solubility, cytochrome P450 inhibition (CYP250), blood-brain barrier penetration (BBB), hepatotoxicity, plasma protein binding (PPB), and human intestinal absorption.

The team chose the binding site of the Mpro according to the location of peptide inhibitor N3. Also, a hotspot was formed at the interaction site of the polar and nonpolar receptors using high throughput virtual screening (HTVS). Moreover, the binding energies (BEs) of the phytochemicals were calculated and molecular dynamics (MD) simulations of protein-ligand complexes with high docking values were performed.

The study assessed 87 phytochemicals obtained from the four herbs. The binding energy for Aegle Marmelos phytochemicals ranged from −8.55 kcal/mol to -7.14 kcal/mol while the LibDock score ranged between 142.00 and 63.00. Notably, tigogenin, aegelinoside, and dehydromarmeli had the highest binding energies of -8.55 kcal/mol, -8.54 kcal/mol, and -8.53 kcal/mol, respectively.

Also, imperatorin, O-prenylhalfordinol, skimmianine, xanthotoxin, N-[2-ethoxy-2-(4-methoxyphenyl)ethyl]cinnamide, aeglemarmaelosine, aegeline, and anhydromarmeline had binding energies ranging from − 8.41 kcal/mol to -8.06 kcal/mol.

An alpha-glucosidase inhibitor found in Aegle Marmelos called aegelinoside B, interacted with glutamic acid-166 (GLU166), glutamine-192 (GLN192), threonine-190 (THR190), arginine-188 (ARG188), and GLN189.

Out of the 17 phytochemicals present in Aerva lanata, a biologically active canthin-6-one alkaloid, called ervoside, had a LibDock score of 129.69 kcal/mol. Amino acids residues like tyrosine-54 (TYR54), histidine-172 (HIS172), cysteine-145 (CYS145), serine-144 (SER144), methionine-165 (MET165), and MET49 interacted with ervoside.

Another phytochemical called feruloyltyramine had a LibDock score of 123.22 kcal/mol and had three hydrogen bonds with the viral protein. Quercetin, with a LibDock score between 110 to 130 kcal/mol, exhibited protective effects against COVID-19-related kidney injuries as a result of its five hydrogen bonds with glycine-143 (GLY143), SER144, and MET165 amino acid residues.

Epicatechin, present in Biophytum sensitivum, had binding energy of -7.69 kcal/mol. Residues including HIS164, HIS163, SER144, phenylalanine-140 (PHE140), and asparagine-142 (ASN142) interacted with this ligand. Also, all the phytochemicals present in Coleus amboinicus had LibDock scores of less than 100.

All compounds except 3-hydroxy-4-methoxybenzoic acid had sufficient oral bioavailability to function efficiently as potential oral drugs. Either low, medium, high, or very high levels of BBB penetration was observed for most of the drugs except gamma sitosterol, 4′,7-Dimethoxykaempferol, stigmast-4-en-3-one, dl-Phenylephrine, 3-hydroxy-4-methoxybenzoic acid, ervoside, and methoxykaempferol. Also, gamma sitosterol, anhydromarmeline, and stigmast-4-en-3-one showed significant levels of plasma protein binding.

The study findings showed the potency of biochemical phytochemicals obtained from herbs in targeting the SARS-CoV-2 Mpro receptor.

According to this study, six of the active phytochemicals can be further assessed as potential Mpro inhibitors based on their significant docking scores. These efficient phytochemicals are ervoside and feruloyltyramine obtained from Aerva lanata, epicatechin from Biophytum sensitivum, and epoxyaurapten, marmin, and aegelinoside B from Aegle Marmelos.

The researchers believed that these results could form the foundation for further studies of medicinal herbs and their usage as a therapeutic device against SARS-CoV-2.

Also, bioactive metabolites extracted from natural resources serve as drugs for the treatment of various diseases. A new review article published in the Journal of Biotechnology has provided information on various nutraceutical metabolites extracted from algae. The authors also discussed the effectiveness of these bioactive metabolites to treat several diseases, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causal agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic.

Algae, eukaryotic plants belonging to the kingdom Protista, originated around a billion years ago. Based on their size, these photosynthetic organisms are classified as macroalgae (multicellular) and microalgae (unicellular).

Additionally to maintaining carbon dioxide levels on earth and preventing climate change, algae contain various proteins and dietary fibers that can serve as anti-inflammatories, anti-microbes, and disease prevention agents. One of the key advantages of deriving drugs from microalgae is their metabolic plasticity. Pharmacologically significant algae can be cultivated on a large scale using photo-bioreactors.

In 1950, marine organisms were explored for the first time to obtain novel drugs. Among red, green, and brown algae, the red algae contain the highest number of bioactive compounds, e.g., polysaccharides, lipids, polyphenols, steroids, glycosides, flavonoids, tannins, saponins, alkaloids, triterpenoids, anthraquinones, and cardiac glycosides. Many of these metabolites have antimicrobial, anti-inflammatory, and antioxidant properties. In addition, in microalgae, many chemical substances are deposited in their cell walls, making them thick and resilient to environmental changes. This is the reason why algae can withstand harsh conditions.

The edible form of marine algae contains high-value compounds, like carotenoids and astaxanthin, which exhibit exceptional antioxidant properties. This is why algae have been widely used as a health supplement for humans, with its market value reaching $3.4 billion. Marine microalgae (seaweed) naturally produce carrageenans, sulphated polysaccharides composed of fucoidan, fucosterol, sodium alginate, and protein. In addition, spirulina, known as a superfood, is extracted from the algae named, Spirulina platensis. Furthermore, several metabolites, such as cyanovirin, scytovirin, and microvirin, isolated from cyanobacteria, are effective against various viral, bacterial, and fungal diseases.

Many algae contain lipids, low-value compounds present in various forms such as fatty acids, sterols, glycerides, fat-soluble vitamins, and phospholipids. Several studies have revealed that both microalgae and macroalgae can synthesize a therapeutically relevant class of fatty acids, example, polyunsaturated fatty acids (PUFAs). Algae-based sterols, such as fucosterol, ergosterol, and chondrillasterol, exhibit anti-inflammatory reactions. Various medically important lipids and fatty acids are obtained from Chlorella vulgaris, Undaria pinnatifida, Haematococcus pluvialis, and Cladophora rupestris. Several types of vitamins, example, Vitamin C, Vitamin E, Vitamin B2, B3, B9, B12, Vitamin K, etc., are synthesized by algae, example, Skeletonema marinoi, Tetraselmis suecica, and Chaetoceros sp.

Both microalgae and macroalgae contain all the essential amino acids, which are not synthesized by the human body. These essential amino acids protect cells from damage and attack of free radicals. Palmaria palmata comprises amino acids, such as leucine, valine, methionine, isoleucine, and threonine that resemble the protein content present in egg white protein and ovalbumin.

The majority of microalgae contain PUFAs, including eicosapentaenoic acid and docosahexaenoic acid, i.e., the two most important omega-3-fatty acids. These compounds significantly reduce the risks of cardiac diseases. Griffithsin (GRFT), a protein that has been extracted from macroalgae Griffithsia sp, exhibits anti-Human Immunodeficiency virus (HIV)-1 property. This antiviral agent has also shown efficacy against Hantaviruses and Coronaviruses (e.g., SARS-CoV-2 and MERS). Scientists revealed that GRFT binds with the glycosylation sites in the S1 subunit of the Spike protein of coronavirus, possibly, a receptor-binding domain (RBD), and inhibits viral infection. Carrageenans obtained from red algae exhibit antiviral activity. Several studies have revealed that they can inhibit the replication of viruses, such as HIV, Hepatitis-A, Human papillomaviruses, Dengue virus, Japanese encephalitis virus, and murine cytomegalovirus.

Fucoidans are extracted from the cell walls of brown seaweeds, which contain L-fucose and sulfate residue groups. This compound has exhibited various therapeutic activities, including effectiveness against coronavirus, HIV, human cytomegalovirus, coronavirus, Influenza virus, and murine norovirus. The primary mechanism of action has been associated with the sulfate group blocking the virus entry into the host via competing for the attachment of positively charged virus glycoprotein envelope onto the host cell. Recently, fucoidan isolated from brown seaweed Saccharina japonica has shown effectiveness against SARS-CoV-2. Ulvans, which are sulfur polysaccharides (SPS) obtained from green algae seaweed, example, Ulva sp. revealed antiviral, immune-modulating, antioxidant, antihyperlipidemic, and anticancer properties. Additionally, this metabolite helps reduce chronic diseases related to gastrointestinal health.

Algal metabolites extracted from Hydroclathrus clathratus, Ulva prolifera, Gracilaria lemaneiformis, Laurencia papillosa, Sargassum fusiforme, etc., also exhibit anti-bacterial and anti-fungal properties.

Through continuous research and development of marine resources, algae and their metabolites increasingly play a significant role in functional foods and nutrition. Clinical and experimental trials are now being conducted on many algal metabolite-derived drugs. Macro and microalgae contain carotenoids, sulfur polysaccharides, lipids, proteins, and vitamins essential for health and can also be used to treat and prevent many life-threatening infections and diseases, such as SARS-CoV-2. Algae represent a vast reservoir and treasure trove for high-value-added biocompounds widely used in food, nutraceuticals, pharmaceuticals, cosmetics, and other industries.

Meanwhile, the same light bulbs used in offices and public spaces can destroy coronaviruses and HIV, according to a new study from U of T Scarborough.

Researchers killed both viruses using UV-LED lights, which can alternate between white light and decontaminating ultraviolet (UV) light. With a cheap retrofit, they could also be used in many standard lighting fixtures, giving them a “unique appeal” for public spaces, says Christina Guzzo, senior author of the study.

“We’re at a critical time where we need to use every single possible stop to get us out of this pandemic,” says Guzzo, an assistant professor in the department of biological sciences. “Every mitigation strategy that can be easily implemented should be used.”

UV lights kill viruses through radiation. Guzzo, alongside PhD students Arvin T. Persaud and Jonathan Burnie, first tested the lights on bacterial spores notorious for their resistance to this radiation (known as Bacillus pumilus spores).

“If you’re able to kill these spores, then you can reasonably say you should be able to kill most other viruses that you would commonly encounter in the environment,” says Guzzo, principal investigator at the Guzzo Lab.

Within 20 seconds of UV exposure, the spores’ growth dropped by 99 per cent.

The researchers then created droplets containing coronaviruses or HIV, to mimic typical ways people encounter viruses in public, such as from coughing, sneezing and bleeding. The droplets were then exposed to UV light and placed in a culture to see if any of the virus remained active. With just 30 seconds of exposure, the virus’ ability to infect dropped by 93 per cent.

Upon testing the viruses at different concentrations, they found samples with more viral particles were more resistant to the UV lights. But even with a viral load so high Guzzo calls it “the worst-case scenario,” infectivity dropped 88 per cent.

Though it wasn’t included in the study, Guzzo and her students also compared UV light to two heavy duty disinfectants used in lab research. They found the lights were similarly effective in their ability to deactivate viruses.

“I was really surprised that UV could perform on the same level of those commonly used lab chemicals, which we regard as the gold standard,” she said. “That made me think, ‘Oh, my gosh, this is a legitimate tool that’s really underutilised.’”

While the lights still left a small percentage of the virus viable, Guzzo references the “Swiss cheese model” of defence against COVID. Every strategy to fight the spread has its holes, but every layer is another chance to stop straggling virus particles.

Repeated exposure to UV light is key to catching those missed particles — fortunately, it’s as easy as flipping a switch. It’s also simpler to change a light bulb than an air filtration system. Guzzo noted that UV-LEDs are cheap and could be easy to retrofit in existing light fixtures, and that the bulbs are long-lasting and simple to maintain.

“You could disinfect in a way that wouldn’t be infringing on people’s enjoyment of that everyday ‘normal’ life that they long for,” Guzzo said.

The lights also benefit from automation. A standardized, germicidal dose of light can be delivered each time, while the process of wiping down spaces with disinfectants leaves room for human error. Chemicals and waste from these disinfectants also end up in watersheds and landfills as hands are washed and wipes thrown away.

But the lights aren’t harmless, and there’s a reason for wearing sunscreen and sunglasses — UV radiation damages nucleic acid, and repeated, prolonged exposure is harmful. That’s why Guzzo said the lights should be used when public spaces are empty, such as vacated buses that have finished their routes, or empty elevators travelling between floors. Escalator handrails could be continuously disinfected by putting UV lights in the underground part of the track, cleaning it with each rotation.

Safe Antivirus Technologies, Inc., a Toronto-based start-up company that partnered with Guzzo for the study, is developing unique UV-LED lighting modules. With motion sensors, the lights automatically switch to UV light when a room is empty, then turn back to regular light with movement.

Funded by the Natural Sciences and Engineering Research Council (NSERC) Alliance COVID-19 Grant and published in the Virology Journal, this study highlights UV-LEDs as a tool that could be used beyond the pandemic, ideally to help prevent another.

“Worldwide events like the COVID-19 pandemic, as terrible as they are, hopefully can still be learned from,” Guzzo says. “One thing we learned is that this is an underutilised tool we should think more about implementing.”

Also, study claims deadly venom from poisonous sea snails could hold the key to developing new painkillers.

Scientists from the University of Glasgow have revealed plans to harness their venom to develop new painkillers that are more effective and less addictive than current options.

Dr. Andrew Jamieson, who is leading the project, said: “The cone snail might seem like an unlikely prospect for breakthroughs in drug discovery, but the conotoxins it produces have a lot of intriguing properties which have already shown promise in medicine.”

What are cone snails? Cone snails are marine gastropods characterized by a conical shell and beautiful colour patterns.

Cone snails possess a harpoonlike tooth capable of injecting a potent neurotoxin that can be dangerous to humans. There are about 600 species of cone snails, all of which are poisonous.

They administer stings by extending a long flexible tube called a proboscis, before firing a venomous, harpoonlike tooth at their victim. Their venom contains chemicals called conotoxins – highly potent neurotoxic peptides, which cause paralysis by blocking parts of the nervous system.

While this can prove fatal for anyone standing in the cone snail’s way, researchers believe that modified versions of their peptides could be used to safely block pain receptors in humans.

The University of Glasgow group is teaming up with machine learning and artificial intelligence experts from the University of Southampton to research how the cone snail’s venom affects human muscles.

Firstly, the team will investigate how conotoxin peptides are structured at a molecular level. They will then build on that knowledge to synthesise new peptides which show promise for interacting with a particular type of receptors in the human nervous system known as nicotinic acetylcholine receptors, ornAChRs.

Finally, the team will run simulations to determine how effective the synthesised peptides are at binding with muscle receptors.

Jamieson said: “This project brings together some of the UK’s leading researchers across a wide range of disciplines to learn about how conotoxins work.

“Then we’ll look at ways we can engineer new analogues to investigate how effective they might be as novel drugs for a range of medical applications.

“Those new molecules’ ability to interact with nicotinic acetylcholine receptors could lead to new forms muscle relaxants for anaesthesia, or painkillers which are just as effective as opioids but don’t have the same associated potential for addiction.

“It’s an exciting project and we’re looking forward to getting started.”

While humans are rarely seriously injured by cone snails, as it stands, there is currently no anti-toxin available. This means that serious stings cannot be effectively treated.

The researchers hope that their project could also help lead to the development of the first-ever treatments for conotoxin poisoning in the future