Possible role of Kolaviron, a Garcinia kola bioflavonoid in inflammation associated COVID-19 infection

G. kola

G. kola is a largely cultivated tree and highly valued in West and Central Africa for its edible nuts.^[33] The seed, commonly known, as bitter kola is eaten by many and it is culturally acceptable in Nigeria. Extracts of the plant have been employed in the African herbal medicine for the treatment of ailments such as laryngitis, liver diseases,^[34] cough, and hoarseness of voice.^[35]

The seed of G. kola [Figure 2] is employed as a general tonic and it is believed to have aphrodisiac properties. The roots and stems are cut into short chew-sticks used for cleaning teeth. G. kola extract among ten common Nigerian chewing sticks examined for antibacterial properties, displayed good activity.^[36] G. kola seeds have also been indicated in the traditional African medicine in the treatment of inflammatory disorders. Extracts of the various parts of the plants have been employed in the treatment of laryngitis^[33] and liver disease.^[34] Extracts of the seeds gave a remarkable improvement of liver function in patients with chronic hepatitis and cholangitis after treatment for 14 days at a Nigeria herbal home.^[34,37] The seeds are used medicinally to treat parasitic, microbial, and viral infections as well as treatment of bronchitis, throat infections, chest colds, and coughs.

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Figure 2:

Garcinia Kola and Kolaviron: Chemical structure and pharmacological properties.

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Kolaviron as anti-inflammatory agent

In the pathogenesis of SARS-CoV-2 infection, the major molecular events are the release of cytokine storm which plays a key role in the exaggerated and multi organ damaging-inflammation of the disease [Figure 1]. The release of inflammatory mediators by the neutrophils, macrophages, and lymphocytes of the immune system activate adaptive immunity conferring protection against infection but induces inflammation at the same time. Subsequently, T helper cells activate other types of cells such as monocytes and B cells through the release of cytokines, such as TNF-α, IL-1β, and IL-2, promoting and elongating the process of inflammation.^[47] Kolaviron is reputed for its potent anti-inflammatory property as demonstrated in various in vitro and in vivo models.

The anti-inflammatory property of kolaviron through inhibition of IL-6 secretion in lipopolysaccharide stimulation of macrophages was demonstrated.^[48] Kolaviron suppressed the DSS-mediated increase in colonic nitric oxide concentration and myeloperoxidase activity, and significantly prevented the increase in inflammatory mediators, IL-1β and tumor necrosis factor alpha in the colon of DSS-treated rats.^[49] Kolaviron may therefore be a candidate to modulate the immune-inflammatory cascade presented in COVID-19.

Furthermore Olajide et al. (2021a)^[50] demonstrated that extract from G. kola seeds reduced the production of pro-inflammatory cytokines TNFα, IL-6, IL- 1β, and IL-8 in human PBMCs stimulated with SARS-CoV-2 spike protein S1. This observation has significant clinical implications and the potential ability of kolaviron to attenuate vicious cycle of inflammatory responses which are characterized by excessive release of pro-inflammatory cytokines resulting in the cytokine storm in COVID-19 which ultimately target lung and other tissues.^[51,52] It was reported that G. kola seed extract prevented toxicity to A549 lung epithelial cells which were co-cultured with SARS-CoV-2 spike protein S1-stimulated PBMCs, suggesting that the anti-inflammatory activity of G. kola promoted the prevention of cytokine-mediated damage to A549 lung epithelial cells suggesting that the anti-inflammatory effects of G. kola seed extract could represent a pragmatic strategy for the treatment of ARDS and lung damage in patients with severe COVID-19.^[50]

Several studies have implicated COVID-19 cytokine storm in lung damage and ARDS.^[53,54] In this regard, SARS-CoV-2 is known to activate immune cells leading to the production of a large number of pro-inflammatory cytokines which cause lung damage [Figure 3]. Acute respiratory distress, inducing severe lung injuries characterized, in particular, by elevated levels of inflammatory markers like IL-6 is shown in COVID-19 infected individuals. Several studies have correlated the released cytokines with the severity of the disease pathology, prognosis, and mortality.^[55] In COVID-19, acute lung injury presents itself with bilateral pulmonary infiltrates, alveolar-capillary vasculitis with neutrophil infiltration, accompanied by pro-inflammatory cytokine release. In rodents, significant release of IL-6 levels has also been related to acute lung injury,^[56] as reported in patients with severe acute respiratory syndrome and those with COVID-19.^[56,57] Similarly, in influenza virus infection, on recognition and infection of pulmonary epithelial cells, influenza virus stimulates early, and rapid secretion of cytokines, such as TNF-α, IL-6, and IFN-γ and inflammatory chemokines including MCP-1 and MIP. Together, these two constituents of innate immunity have been directly implicated in lung pathology as they occlude pulmonary parenchyma thus blocking its function.

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Figure 3:

Schematic diagram depicting the pathogenesis of COVID-19 and proposed mechanism of action of kolaviron. On infection of the lung cells by SARS-CoV-2, there is subsequent elevation of oxidative stress leading to uncontrolled inflammation and a dysregulation of immune response accumulating in exaggerated cytokine storm syndrome. ARDS-mediated multi organ damage results from the aberrant cytokine storm causing severe COVID-19. It is proposed that kolaviron may interfere with ROS mediated oxidative stress and uncontrolled inflammation, modulating the immune response, abrogating pro-inflammatory cytokines, and attenuating the resultant cytokine storm.

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Release of these chemokines and cytokines subsequently result in taxis-driven influx, accumulation and activation of innate inflammatory cells neutrophils, monocytes, dendritic cells, and macrophages, which if finely orchestrated, may lead to viral clearance and disease resolution, or when excessively unleashed, result in severe immunopathological damage, illness and fatality.^[58-60] Excessive pulmonary activation of neutrophils and macrophages has been reported in influenza virus treated mice.^[41] Kolaviron elicited significant modulatory effects on all these immunopathogenic responses as it elicited reduced cellular infiltration and less pathology in the pulmonary tissue. Reduced expression of MCP-1 and iNOS was also observed in kolaviron-treated influenza-challenged mice.^[42] In addition, kolaviron abated the high immunostaining signal for the innate cell derived pro-inflammatory cytokine IL-1β, IL-10, and chemokine RANTES in mice infected with influenza virus.^[42]

Kolaviron as anti-oxidative agent

ROS, derived from molecular oxygen but more reactive than oxygen itself, play an essential role in viral infections by inducing innate immune responses due to the opening of inter endothelial junctions that allow the migration of inflammatory cells through the endothelial barrier.^[61,62] The recruitment of inflammatory cells at the site of infection causes excessive ROS production, which is considered essential for the genesis and progression of inflammatory diseases.^[63,64] Furthermore, generation of ROS has been reported to enhance the gravity of viral infections due to their participation in inflammatory processes.^[65] Influenza virus and a number of respiratory viruses have been shown to generate ROS on account of overwhelming recruitment of inflammatory cells at the site of infection^[66] resulting in disturbance of redox imbalance and oxidative stress with consequent oxidative damage to the cells.^[67] In SARS-CoV-2 viral infection, studies have reported increased oxidative stress with attendant migration of neutrophils to the infected area.^[68]

Kolaviron has been investigated in a wide range of established in vitro assays involving ROS as a potent antioxidant and free radical scavenger. Thus, kolaviron elicited significant reducing power and a dose-dependent inhibition of oxidation of linoleic acid, inhibited H₂O₂, and was more effective than BHA and β-carotene.^[69] Kolaviron also significantly scavenged superoxide generated by phenazine methosulfate NADH and scavenged hydroxyl radicals as revealed by significant inhibition of the oxidation of deoxyribose. In vivo, kolaviron reduced background levels of protein oxidation marker (2-aminoadipic semialdehyde) in plasma and liver and γ-glutamyl semialdehyde (GGS) as well as malondialdehyde in liver.^[70] Furthermore, kolaviron reduced damage to proteins and lipids induced by Fe3+/ EDTA/ascorbate mixtures ex vivo.^[71] Moreover, kolaviron dose dependently inhibited the intracellular ROS production induced by H₂O₂ in HepG2 cells.^[72,73]

It has been hypothesized that endogenous GSH deficiency could be one of the causes of severe symptoms in COVID-19 patients. The reduction in GSH and the attendant increase in ROS have been found to correlate with the debilitating symptoms and the slowest recovery times in COVID-19 infected individuals.^[74] Likewise, it is known that GSH concentrations are decreased in the elderly,^[75] which could explain their susceptibility to the severity of COVID-19. Similarly, and interestingly, in influenza virus infection, intracellular redox imbalance has been shown to be a prominent feature.^[76] In addition, viral persistence and expression of late viral proteins have been found to be dependent on increased oxidative environment resulting from persisting depletion of the most prevalent and powerful intracellular antioxidant GSH. Kolaviron elevated GSH level in influenza virus treated mice. During viral infection, the rate of O₂ - coupling to generates OONO. as opposed to its dismutation by superoxide dismutase (SOD) has been reported to be approximately 3 times faster.^[77] In influenza virus treated mice, kolaviron was reported to reverse this redox reaction as revealed by few detections of viral particles in the treated mice.^[41]

Release of iron into the bloodstream generating ROS leading to oxidative stress has been reported in COVID-19. In SARSCoV-2 infection, free Fe (III) ions into the bloodstream following attack on the heme groups of hemoglobin in red blood cells, and through the Fenton and Haber–Weiss reactions, the level of ROS is raised.^[78] Furthermore, excess ROS oxidize hemoglobin to methemoglobin (iron in the ferric state, Fe (III)) which cannot bind oxygen effectively, resulting poor oxygen transport.^[68] The overall effect of oxidative stress on red blood cells results in hypoxia causing respiratory failure commonly seen in COVID-19.^[79] Kolaviron has been shown to be effective antioxidant reversing Haber-Weiss and Fenton generating radicals in various models including red blood cells.^[70] Thus, kolaviron inhibited the Cu²⁺-induced as well as Fe²⁺ mediated oxidation of rat serum lipoprotein. Overwhelming oxidative stress induces DNA damage, impairs macromolecules, and causes plasma membrane damage.^[47] In lymphocytes incubated with Fe³⁺/GSH, kolaviron was found to attenuate the oxidant-induced DNA strand breaks as well as base oxidation.^[71]

Respiratory viral infections have been associated with dysregulation of several signaling pathways, including the inhibition of Nrf2/Keap1nuclear erythroid factor 2-related factor 2 (Nrf2) and activation of NF-κB signaling, Komaravelli and Casola.^[67] For instance, lung biopsies collected from patients with COVID-19, the Nrf2 pathway was found to be inhibited, while agents that stimulate and enhance Nrf2 have been observed to suppress SARS-CoV-2 replication and the inflammatory response.^[80] Kolaviron was reported to enhance Nrf2 signaling and offer neuroprotection in BV2 microglia.^[81]

Activation of the NF-κB transcription factor signaling pathway has been linked to the pathogenesis of severe COVID-19.^[82] It has been recently shown that NF-κB activation is a mechanism for SARS-CoV-2 spike protein S1-induced exaggerated production of TNFα, IL-6, IL-1β, and IL-8 in PBMCs.^[83] On the other hand, inhibition of NF-κB activation, with the resultant reduction in exaggerated production of pro-inflammatory cytokines like TNFα has been associated with the reduction in cytokine storm in severe COVID-19.^[84] Kolaviron has been shown to modulate these signaling pathways in a number of disease conditions as well as viral infections. It was reported that the seed extract of G. kola inhibited both cytoplasmic activation, DNA binding, and transcriptional activity of NF-κB in SARS-CoV-2 spike protein S1-simulated PBMCs as well as in RAW264 macrophages and BV-2 microglia stimulated with bacterial LPS.^[48,50,81] Similarly, in influenza virus mice infection, kolaviron-related redox switch affected NF-κB, the master regulator of pro-inflammatory cytokines/chemokines, as its expression was markedly inhibited as well as its associated stress repines protein COX-2.^[42] Furthermore, kolaviron downregulated the expression of NF-κB and COX-2 in dimethyl nitrosamine (DMN)-treated rats.^[40]

Kolaviron as hepatoprotective agent

Liver injury is one of the common complications in patients with COVID-19. Liver injury, inflammatory cytokine storm generated by the excessive immune response induced by coronavirus infection has been implicated.^[85] Inflammatory cytokines and chemokines such as (IL-2, IL-7, IL-10, MCP1, and TNF-α) have been found to be elevated in COVID-19 patients.^[86] Liver ischemia reperfusion injury, a pathophysiological process commonly occurring after rapid recovery of blood circulation, has been ascribed to be an underlying mechanism of COVID-19-associated hepatic injury.^[87] Several therapeutics including antibiotics (macrolides and quinolones), antiviral drugs (ribavirin and lopinavir/ritonavir), and nonsteroidal anti-inflammatory widely used to treat COVID-19, have been reported to cause hepatotoxicity.^[88] For instance, a report showed that a pooled incidence of drug induced liver injury in patients with confirmed SARS-CoV-2 infection.^[89] In addition, a study revealed that a proportion of patients who received lopinavir/ ritonavir after admission had abnormal liver function.^[90]

The hepatoprotective effects of kolaviron have been demonstrated against hepatotoxicity induced by a wide range of drugs and hepatotoxins such as CCl₄,^[91] diclofenac,^[92] aflatoxin,^[93] 2-acetyl aminofluorene,^[94] and dimethylnitrosamine.^[95] The underlying mechanism of its heptoprotection involves antioxidant, anti-inflammation, and inhibition of lipid peroxidation.^[40,91,93] Kolaviron suppressed certain pro-inflammatory genes whose expression has been shown to be regulated by transcription factors. Thus, kolaviron abolished the expression of COX-2 and iNOS proteins in DMN-treated rat liver and abrogated the DNA binding activity of NF-kB and AP-1 induced by DMN.^[40] It also increased levels of antioxidants including glutathione, modulated altered lipid profiles, and restored liver function biomarkers in drug-induced liver toxicity in rodents.^[91,94]

Kolaviron as cardioprotective agent

Abounding evidence has identified oxidative stress and inflammation as relevant pathophysiological pathways in the development of cardiovascular diseases (CVDs) involving endothelial dysfunction and thrombosis.^[5,95] Several enzyme systems, including nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase (NOX), xanthine oxidase (XO), uncoupled endothelial nitric oxide synthase, and the mitochondrial electron transport chain, have been implicated in the generation of ROS in the cardiovascular system.^[96,97] Moreover, inflammation, occurs in the vascular tissue involving inflammatory immune cells, interactions between cell surfaces, and proinflammatory mediators.^[98,99] Oxidative stress and inflammation resulting from an increased production of adhesion molecules, following low-density lipoprotein (LDL) oxidation results in the migration and infiltration of inflammatory cells in the vascular tissue.^[100] Oxidation of LDL is generally believed to promote atherosclerosis and foam cell formation.^[39] Thus, kolaviron inhibited the process of LDL oxidation which may delay the progression of atherosclerosis. Lipoprotein resistance to copper-induced oxidation was highly improved in rats treated with kolaviron (100 mg/kg) for 7 days as demonstrated by significant increase in lag time, a decrease in area under the curve and slope of propagation.^[39] Furthermore, kolaviron inhibited the Cu²⁺-induced oxidation of rat serum lipoprotein and prevented microsomal lipid peroxidation induced by iron/ascorbate in a concentration dependent manner.^[39]

SARS-CoV-2 has been shown to alter the host redox balance and elicit a damaging inflammatory response, contributing to cardiovascular complications. Thus, in patients with SARS-CoV-2 an elevated level of cardiac biomarkers, such as homocysteine, troponins, myoglobin, C-reactive protein, IL-2, IL-6, and ferritin, result in myocardial injury.^[6,86] In rats, kolaviron attenuated homocysteine-induced atherosclerosis and oxidative stress by increasing the activity of the antioxidant enzymes and reducing the markers of oxidative stress and inflammation. Kolaviron also reduced the serum levels of LDL-C, triglycerides, and total cholesterol with attendant reduction in heart rate as revealed by electrocardiograph data.^[101] Antioxidant enzyme systems, such as SOD, catalase, glutathione peroxidases protect vasculature, and cardiomyocytes of cardiovascular system against ROS.^[96] Kolaviron elicited antioxidant property and cardioprotective effect on isolated rat heart. In ischemic reperfusion injury rat heart model, kolaviron elevated plasma levels of FRAP, TEAC, and ORAC in the rats and increased the levels of antioxidant enzymes and attenuated lipid peroxidation. Furthermore, kolaviron inhibited the activation of p38 MAPK, promoted the activation of Akt and promoted inhibition of cardiomyocytes apoptosis signal through reduction in the expressions of Caspase 3, cleaved Caspase 3, and cleaved PARP in rats subjected to reperfusion injury.^[102,103] Cardiovascular complications in patients with COVID-19 arise from altered sympathetic activity, coagulation, hemodynamics following infections, oxidative stress, and inflammation. On account of the cardio protective effect of kolaviron and its modulatory effects on biomarkers of CVD, it may have a therapeutic role in cardiovascular risk factors and squeal in COVID-19.

Kolaviron as potential antiviral agent

A plethora of plant-based natural products have been reported to have antiviral properties and are gaining attention of investigators for their therapeutic potential in COVID-19. Thus, phytochemicals isolated from medicinal plants have shown great potential in inhibiting replication and blocking entry of viruses, including coronaviruses.^[104,105] Specifically, several models including in vitro and animal models have demonstrated antiviral property against SARS-CoV-2.^{[104,106,107]} For instance, the antiviral activities of Limonene against HSV-1, influenza, yellow fever, and dengue virus have been reported.^[108-110] The mechanism of the antiviral activity of this compound is mainly through inhibition of viral replication via direct action on the virus.^[47] Kolaviron elicited immunomodulatory and immunorestorative properties in in vitro and in vivo immunocompetent and immunocompromised animal models. which could be harnessed for possible clinical benefits to immunodeficient patients.^[111] GB1, a biflavonoid and a component of kolaviron showed antibacterial activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci with minimal inhibitory concentrations (MIC) of 32 and 128 mg/ml, respectively.^[112] The antibacterial activities of methanol extracts, containing kolaviron, of G. kola evaluated using broth microdilution showed MIC of 256 to 1024 μg/mL against multidrug resistant Gram-negative bacteria overexpressing active efflux pumps.^[113] Bacteria and pathogens have been reported to exacerbate the effect of corona virus in COVID-19 patients. Examples are Streptococcus pneumoniae, S. aureus, Klebsiella pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumonia, Legionella pneumophila, and Acinetobacter baumannii, and viruses such as influenza, coronavirus, rhinovirus/enterovirus, parainfluenza, influenza B virus, and human immunodeficiency virus.^[114] The methanol extract and fractions of G. kola seed elicited protective effect against Streptococcus pyogenes, S. aureus, Plesiomonas shigelloides, and Salmonella typhimurium using agar-well diffusion method and MIC and minimum bactericidal concentration assays.^[115]

Influenza A viruses induce cytokine storm and host’s intracellular redox imbalance to ensure continuous replication and survival, leading to severe immunopathology and death. We reported the protective role of kolaviron, on BALB/c mice challenged with influenza A/Perth/ H3N2/16/09 (Pr/H3N2) virus. Kolaviron administered at 400 mg/kg orally to BALB/c mice for 3 days, 3 h, and 1 h before infection with 1LD50 or 3LD50 (14-day study) and 5LD50 (6-day study) of Pr/H3N2 improved lung aeration, parenchyma integrity with patchy cellular infiltration and reduced lung consolidation, inflammatory cells infiltration as well as overall improvement in morbidity and mortality.^[41] Furthermore, excessive pulmonary activation of neutrophils and macrophages in mice infected with Pr/H3N2 were attenuated by Kolaviron.^[41,42] While excessive CD4 T cells during influenza infection has been demonstrated to contribute to immunopathology, the pivotal role of CD4+ cells in influenza clearance through direct targeting and elimination of infected cells and activation of adaptive immune response has also been demonstrated^[116-118] and the IL-10 secreting prowess of CD4+ which has been implicated in viral infection. In mice infected with influenza virus, kolaviron suppresses IL-10 production, which could selectively fine tune host’s CD4+.^[42]