Although there are now multiple vaccines for SARS-CoV-2, their efficacy may be limited by current and future viral mutations.
Therefore, effective antiviral compounds are an essential component to win the battle against the family of coronaviruses. Ginkgolic Acid (GA) is a pan-antiviral molecule with proven effective in vitro and in vivo activity.
We previously demonstrated that GA inhibits Herpes Simplex Virus 1 (HSV-1) by disrupting viral structure, blocking fusion, and inhibiting viral protein synthesis. Additionally, we reported that GA displays broad-spectrum fusion inhibition encompassing all three classes of fusion proteins, including those of HIV, Ebola, influenza A, and Epstein Barr virus. Here, we report that GA exhibited potent antiviral activity against Human Coronavirus strain 229E (HCoV-229E) infection of human epithelial lung cells (MRC-5).
GA significantly reduced progeny virus production, expression of viral proteins, and cytopathic effects (CPE). Furthermore, GA significantly inhibited HCoV-229E even when added post-infection. In light of our findings and the similarities of this family of viruses, GA holds promising potential as an effective antiviral treatment for SARS-CoV-2.
Ginkgo biloba is a gymnosperm species used in Chinese herbal medicine since the 16th century. The seeds of G. Biloba were used as medicinal materials in traditional Chinese medicine for skin-based ailments as documented in ‘The Compendium of Materia Medica Ben Cao Gang Mu’ [10].
In the recent century, pharmaceutical interest has focused on the properties of G. Biloba extracts for the treatment of vascular disorders, vertigo, tinnitus, and various cognitive disorders [11,12].
Nowadays, G. Biloba is mainly present in East Asian countries such as China, Korea, and Japan [13]. G. Biloba is comprised of terpene trilactones (ginkgolides A, B, C, J), flavonols, glycosides, biflavones, proanthocyanidins, simple phenolic acids, 6-hydroxykynurenic acid, 4-O-methylpyridoxine, polyprenols, and alkylphenols [11,12,13]. Ginkgolic acid (GA) is an alkylphenol constituent extracted from the leaves and seed coats of G. biloba [10,14,15,16].
Previous literature has shown that GA demonstrates pleiotropic bioactive properties, including anti-inflammatory [17], antitumor [15,18], and antibacterial [19] effects. Our lab has recently shown that GA has antiviral activity against enveloped viruses such as Herpes Simplex Virus 1 (HSV-1), Human Cytomegalovirus (HCMV), and Zika Virus (ZIKV) through inhibition of viral fusion.
GA has also shown a broad spectrum of fusion inhibition encompassing all three classes of fusion proteins, indicating that GA inhibits virion entry by blocking the initial fusion event [20,21].
reference link :https://www.mdpi.com/1424-8247/14/10/980/htm
Ginkgo Biloba with mechanisms to reduce the microbial infections
Plants are an appreciated source of ingredients with an antiviral activity, which could be defended against attacking pathogens. Ginkgo Biloba Extracts (GBEs) are green dried leaves corresponds to the more common phytotherapeutics in the world. GBE had been used widely in respiratory and heart disease treatments, and it could also have been used to improved memory function [10].
Commercially, GBE presented as one of the best marketing herbal complements [11]. However, the beneficial influence of GBE on CNS and the cardiovascular system (CVS) related to it has scientific effectiveness. It is well-known that it highly had been used in different neurodegenerative sicknesses like Parkinson’s disease [12].
Scientifically data demonstrated the therapeutic properties of GBE through the polysaccharides of Gingko Biloba (GB) fruits have anti-PEDV (porcine epidemic diarrhea virus) activity [13]. Ginkgolic acid (GA) from Ginkgo Biloba can inhibit the activity of Human Immunodeficiency Virus protease (HIV) [14]. Also, GBE exhibits activity against both influenza as anti-influenza (H1N1and H3N2) and Hepatitis B virus as anti-HBV [15], [16].
In vitro has been shown that Ginkgolic Acid has pleiotropic effects include: antitumor activity during lipogenesis inhibition; activate of AMPK by reducing the expressions of invasion-related proteins; capable to liberate of Amyloid β (Aβ) which has been caused synaptic damage; and inhibits both the activity of protease and replications of HIV [14], [17], [18], [19]. So the GA has efficient activity against Staphylococcus aureus and Escherichia coli [20].
It works by several methods summarized as inhibition of the SUMOylation activity; hindering the intermediate E1-SUMO formation [21]; inhibiting the fatty acid synthase [22]; the inhibiting nonspecific SIRT gene [23]; and activating the protein phosphatase type- 2C [24].
Moreover, this reporting has shown that the GA has antiviral activity against Human Cytomegalovirus (HCMV), Zika Virus (ZIKV) and Herpes Simplex Virus1 (HSV-1) mainly by inhibiting the fusion in viral infections. Also, it can be inhibited the endocytic entry of non-enveloped adenovirus [25].
The antiviral actions of GA usually had been detected below the level of threshold cytotoxicity; these wide-spectrum activities of GA are accomplished during its inhibiting the viral entry and could be medicinally converted to systematic in severe cases of acute viral infections.
The cardio tropic virus causes inflammatory disease in cardiac muscle or myocardium which called Viral Myocarditis (VMC), this virus considered as the main reason for heart failure (HF) and sudden death [26], [27].
VMC also had been caused by Coxsackievirus B3 (CVB3) which acts as an enterovirus of the picorona virus family [28], [29].
Despite the extensive effort in previous decades, but until now, there is no specific virus preventive measure to CVB3-induced viral myocarditis in the clinics [30], [31].
The therapeutic activity of GBE investigated on Viral Myocarditis and revealed the feasible mechanism, these effects appeared on the other cardiovascular disease (CVD) such as Myocardial Infarction MI [32], [33], [34]. Additionally, the treatment with Ginkgo Biloba extracts significantly has been reduced the serum creatinine kinase isoenzyme level (CK-MB), heart weight, a score of histopathological, collagen volume fraction (CVF) and mortality.
Previous studies had confirmed the treatment with Ginkgo Biloba significantly had decreased the mRNA and the level of Matrix Metallo Proteinase (MMP-2 and MMP-9) expressions [35]. GBE also expressively had lowered the production of MMP-3 through damage to the inflammatory chondrocyte [36]. Thus, MMP-3 inhibition may be a new mechanism of GBE for the treatment of VMC.
In concerning S100 calcium-binding protein A4 (S100 A4) upregulation in viral myocarditis, It may be the principal target of GBE. As expected, the mRNA and the S100 A4 protein level significantly reduced after dealing with GBE. This result was not described previously and hence may remain as a novel theory. Moreover, the introducing factor of CVB3 influenced myocarditis is viral replication. So, inhibiting the replication of the virus act as the first line for the treatment of Viral Myocarditis [37], [38].
Role of Ginkgolic Biloba in inflammation and immunity
Ginkgo Biloba Extract mainly consists of flavonoids as flavone glycosides in percent such as (22–27%) quercetin, (2.6–3.2%) bilobalide, isorhamnetin, kaempferol, (2.8–3.4%) terpene trilactones (ginkgolides A, B, C) and less than 5 ppm GA during its cytotoxic effectiveness [10], [39].
Quercetin has high anti-inflammatory capacities that it reported as a long-term anti-inflammatory compound [40], [41]. It has an anti-inflammatory activity can be expressed in different cells in both the human and animal models [42], [43], [44], [45]. Additionally, it has gastrointestinal cytoprotective activity and can stabilize the mast cell [45].
It also can play essential roles in inflammation and immunity as a modulating, biphasic and regulatory achievement [43]. Quercetin possesses an immunosuppressive activity that acts on the functions of a special type of cell as dendritic cells [45].
However, several studies by using various cell outlines in vitro had confirmed that quercetin prevents lipopolysaccharide-induced Tumor Necrosis Factors-α (LPS induced-TNF-α), which had been produced from macrophage [42] and lipopolysaccharide (LPS)- induced-interleukin-8 (IL-8) in specific type A549 cells in the lung [43].
Moreover, the quercetin capable to inhibit the lipopolysaccharide-induced the level of mRNA in TNF-α and Interleukin-1α (IL-1 α) in glial cells, that had caused a reduction in apoptotic death of neuronal cells through the induction of microglial [44]. It also inhibits the production of enzymes in inflammation sites as cyclooxygenase (COX) and lipoxygenase (LOX) [45], [46].
Quercetin has restricted LPS-induced inflammation by inhibiting the (Src and Syk) that induced phosphatidylinositol-3-Kinase (PI3K-p85) phosphorylation and consequent formation of complex Toll-like receptor 4 (TLR4) MyD88/P13K, which limits the stimulation pathway in RAW 264.7 cells of downstream signaling [47].
It probably inhibits the production of inflammatory mediators (e.g., histamine, tryptase and cytokines) from the human umbilical cord in blood cultured of mast cells (HCBMCs); this way may cause inhibition of both calcium influx and phosphor protein kinase C (PKC) [48].
Its protective effects against Human Umbilical Vein Endothelial Cells (HUVECs) inflammations have shown clearly from the study of quercetin against hydrogen peroxide (H2O2) influenced inflammation, these effects indicated by vascular cell adhesion molecules-1(VCAM-1) downregulation and CD80 expression [49]. It also significantly can be induced the Th-1-Interferon-γ (IFN-γ) production and Th-2-Interleukin 4 (IL-4) down regulation as well as influence the gene expression via normal Peripheral Blood Mononuclear Cells (PBMC).
Additionally, quercetin management had elevated the phenotypic of INF-γ cell expression and lowered the IL-4 in positive cells during the flow analysis of cytometry, which substantiates with gene expression studies and protein secretion. According to these results, the potential activity of quercetin in immune-stimulatory effects may be influenced by the IFN-γ, Th-1-derived cytokines inductions and IL-4, Th-2 derived cytokines inhibition [50].
Quercetin can inhibit the matrix metallo proteinase, which has been inhibited by Plasminogen Activator Inhibitor-1(PAI-1) normally in dermal fibroblasts in humans [51]. Wherever, the interleukin-1(IL-1) stimulates the creation of IL-6 in mast cells of the human body that regulated through IgE-induced degranulation within serious biochemical pathways, the use of quercetin blocks the secretion on IL-6 and inactivate the two key signal transduction steps in the involvement pathways [52].
Quercetin is one of the few molecules that can stabilize the mast cell and protect the gastrointestinal tract (GIT) against the invading agent. It possesses a direct effect to regulate the useful properties of the immune system as a result of a signal pathway in human mitogen-activation PBMC and sanitized the T-lymphocyte cells, which is induced by Extracellular Regulated Kinase-2 (ErK2) Mitogen-Activated Protein (MAP)kinase [53].
The properties of quercetin had confirmed that it inhibits a large number of targeting molecules in micro-molar concentration levels either by down regulation or by destroying the pathway and functions of the inflammation. However, quercetin within Nano-molar doses has a biphasic behavior on basophil cell, thus effects may seem in the allergic inflammatory process.
The immunity and inflammatory effects of quercetin had been induced mainly by its effects on leukocytes and several intracellular signaling mediators as a kinase, phosphatase, enzymes and proteins in the cell membrane, which is vital for specific functions of cells.
Furthermore, the large range of intracellular objects and high number of possibly effective natural compounds act as therapeutic agents with anti-inflammatory effects need more observations and evidence to understand the critical role of these materials in animal [54]. In vitro has shown that the treated actuated T cell with quercetin can block IL-12 Tyrosine Phosphorylation (TYK2, STAT3, STAT4 and JAK2) that decreased the IL-12 level, which influenced T helper cell (Th1) proliferation and differentiation [55].
Clinical and epidemiological finding in COVID-19
The knowledge of the epidemiological and clinical findings of any disease considered as the first step in the evolving hypothesis regarding the disease’s attention. Covid-19 grasps the world and the recent research confirmed [56]. According to recent studies in the journal literature, it noticed the COVID-19 infection is correlated with elevated the production of pro-inflammatory cytokines and c-reactive protein [57], it can also increase the risk of pneumonia [56], sepsis, acute respiratory distress syndrome and caused heart failure [58].
In China, the Case Fatality Ratio (CFR) in coronavirus is approximately 6–10% especially for patients with diabetes, chronic respiratory tract disease, hypertension and cardiovascular disease [7]. COVID-19 spreads in two countries that are high air pollution regions as in China [59] and north of Italy [60].
The potential roles of Ginkgo Biloba for epidemiological characteristics of disease have been related for increasing the incidence of corona COVID-19 and CFR which are given in Table 1, most of the beneficial effects of Ginkgo Biloba given in Table 1 are from observational studies of the incidence and prevalence of the disease (Table 2).
Table 1:Role of Ginkgo Biloba in clinical, epidemiological finding for incidenece and case fatality rates.
| Characteristics | Relationship to Gingko Biloba | References |
|---|---|---|
| In pneumonia | Potentially useful drugs for the treatment of and prophylaxis against P. carnii pneumonia | [61] |
| In pulmonary sepsis | Suppress the response to inflammatory in acute lung injury (ALI) by preventing pathways of NF-kB and COX-2 | [62] |
| In asthmatic patient | The effect of GBE on both PKCα expressions in cell inflammatory and levels of IL-5 in asthmatic patients influenced sputum | [63] |
| Cigarette smoke | GBE protects the human lung endothelial cells from cigarette smoke extracts (CSE), which has been induced the apoptosis: this effect of heme oxygenase-1 (HO-1) | [64] |
| In bacterial infections: Escherichia coli (E. coli) and Staphylococcus aureus (St-au) | GBE inhibits the growth of these bacterial types (E-Coli and St-au) | [14] |
| In influenza virus | Ginkgo Biloba leaf extracts have an anti-influenza virus activity | [65] |
| In herpes virus | Hampering HHV-1 and HHV-2 infections by GBE and its phytochemical constituents | [66] |
| In different types of viruses | GA inhibits fusion of enveloped viruses | [25] |
| In myocardial virus | GBE may relieve viral myocarditis by suppressing S100A4 and MMP-3 | [67] |
| In inflammation and immunity | Quercetin effects in inflammation and immunity | [8] |
| CFR increase in diabetes mellitus | GBE protects the β-cell in the pancreas of diabetic type-2 rats and improves both their expression of insulin and antioxidant station | [68] |
| CFR increase cardio vascular disease (CVD) with age | Inversion as antioxidant, antiplatelet | [69] |
| Increase C-RP in DM, and CVD diseases | Inversion as antioxidant | [69] |
- CFR, case fatality rate; CVD, cardiovascular disease; GBE, Gingko Biloba extract; DM, Diabetes mellitus; NF-kB, nuclear factor kabba-B; COX-2, cyclo-oxygenase x-2; MMP-3, Matrix Metallo Proteinase.
Table 2:Role of Ginkgo Biloba as antivirus against several types of viral infections.
| Type of viruses | Antiviruses of Ginkgo Biloba | References |
|---|---|---|
| Herpes simplex virus type-1,2 (HSV-1,2) | Inhibition of the fusions and synthesis of proteins in the viruses. | [25] |
| Human cytomegalovirus (HCMV) | Inhibition of the genome replication | [70] |
| Zika virus (ZIKV) infection | Inhibition of the genome replication | [71] |
| Human immunodeficiency virus (HIV) | Inhibition of viral fusion proteins | [71] |
| Ebola virus (EBOV) | Inhibition of viral fusion proteins | [72] |
| Influenza A virus (IAV) | Inhibition of viral fusion proteins | [72] |
| Epstein Barr virus (EBV) | inhibition of viral fusion proteins | [72] |
| Measles virus | Inhibition of the targeting protein and DNA | [72] |
| Varicella-zoster virus (VZV) | Inhibition of the targeting protein and DNA | [72] |
| Coronavirus | Inhibition of the targeting protein and DNA | [72] |
Throughout the production of standardized ginkgolic biloba extract, some constituents enriched flavonoids and terpenoids), While further removed as (bi flavones, Ginkgolic acids. Variable manufacturing procedures lead to the extract of different substances with different therapeutic effects [73], [74], [75].
The effect of Ginkgo Biloba extracts (GBE) on influenza virus as anti-influenza activity
Influenza Virus is one of the Orthomyxoviridae families that had initiated the epidemics in the world. Although, effective vaccine is available, most people are infected every year and in severe cases, many people died by viral contamination. According to the properties of the antigen of two surface glycoproteins, the influenza viruses are classified as two types: NeurAminidase (NA) and HemAgglutinin (HA). NA has nine subtypes (N1–N9) while HA has 16 subtypes (H1–H16).
The infection of the Influenza virus has been created by an interaction between hemagglutinin and sialic acid moieties of glycoconjugates on the crowd cells [76]. The studies about the activity of the anti-influenza virus of the original products increased dramatically in the past several years [77].
Ginkgetin, a biflavone naturally isolated from leaves of Ginkgo Biloba that can inhibit the activity of influenza sialidase in the virus [78]. Previous results revealed that GBE prevented the adsorption of the virus on the surface of the host cell in the first step of influenza virus contamination.
The other component(s) of GBE than Ginkgetin may have activity against influenza viral infections. Ordinarily, The activity of GBE examined against three types of different influenza viruses, which includes the influenza A/PR/8/34 (H1N1), B/Lee/40 viruses and A/Udorn/72 (H3N2). So the sensitivity of Ginkgo Biloba extracts toward these viruses had a little difference. However, this discovery proposes that GBE may have a high variation of inhibitor toward influenza viral infections.
In conclusion, we have found that Ginkgo Biloba extracts interact directly with different types of influenza viruses, and prominently reduce their infection via prevented the adsorption of viruses on host cells. Likewise, the anti-influenza viral activity of GBE seems not to be limited to a specific influenza virus subtype.
In the past studies, some candidates overlooked the antiviral activity of GBE because of the presence of the transmission model of the influenza virus from cells to other cells. However, other previous studies had confirmed that necessity for caution to the investigators trying to find the compounds with anti-influenza virus activity [65].
Additionally, to knowledge the inhibitory effect of GBE in influenza virus infection, we clarified the exciting and attention insights in the screening system for anti-influenza effects. In considering the results have been shown, the exhausting of GBE, as an anti-influenza viral agent needs more supplementary studies to approve its effects in vivo.
Proposed antiviral action of Ginkgolic Acid (GA) by inhibiting the enveloped virus fusion as antivirus mechanism
Ginkgo leaves involve Ginkgolic acid and other constituents, GA commercially available as C13:0, C15:0 and C17:0. GA has multiple therapeutic effects resulted from its antioxidant activity. These effects include the treatment of some diseases such as cardiovascular illness, tumors, HIV contamination and bacterial infection (E. coli and Staphylococcus aureus [St-au]) [14], [134].
The GA works through some pathways involve fatty acid synthase inhibition [22]; protein phosphatase type-2C activation; inhibition of nonspecific (SIRT11) [24]; suppress the activation of STAT3 by inducing of the (PTEN and SHP-1) tyrosine phosphatase [135] and defense against Aβ-caused synaptic impairment in the hippocampus [19].
The study by Ronen et al. had revealed that GA has fusion inhibitory effects on the enveloped virus, that act the fusion of three classes of proteins and it impedes the nonenveloped adenovirus in human.
It also was observed the activity of GA by a potential secondary mechanism that including synthesis of proteins and DNA in virus [25].
These results are similar to the reporting of previous studies about the GA inhibitory effect on protein synthesis and DNA, so till now, the inhibitory mechanisms are not clearly understood [136]. It may be connected to receptors in the horde cells and activates several signaling pathways that cause the detention of the cells cycle. These may describe the effects of GA on cancer cells by inhibiting the rapid dividing of its. Additionally, GA may directly be acting on DNA and protein synthesis through entering the cell.
The inhibitory effects of GA had been tested on different types of the cell as Human Epithelial Carcinoma (HEC-2), Human Embryonic in the kidney and normal human astrocytes [137]. However, all tested cells appeared that GA has viral inhibitory effects without cytotoxicity within the range of inhibitory effects [138].
The general inhibition of viral protein-induced cell-cell fusion by GA reflected its inhibitory mechanisms for fusion. In addition, Lyso-PhosphatidylCholine (LPC) uniformly blocks fusion in membrane by convening positive impulsive curvature, which inhibits hemi fusion. However, this block can be resolved by adding the negative spontaneous curvature agent as Oleic Acid (OA), regardless of the fusion protein [139].
On the other hand, the reporting that Oleic Acid alleviates the conditions of the Gingkolic Acid-induced inhibition of (EBOV GP-refereed fusions) suggests that GA works like Lysophosphatidylcholine via generating positive impulsive curvature, it inhibits hemifusion. Unrelated viral fusions proteins induced the several Rigid Amphipathic Fusion Inhibitors (RAFI) associated with positive impulsive curvature, it would be indicated to the effect of the endocytic entry by nonenveloped virus as adenovirus [138].
Furthermore, the secondary mechanisms of GA due for inhibiting the DNA and protein synthesis in the virus would be anticipated to become targeting on both envelope and nonenveloped viruses.
In conclusion, we observed consistent and strong inhibitory effects of GA on various enveloped viruses fusion, including the major significant infected virus such as HIV, ZIKA, HSV-1, EBOV, EBV and IAV, besides its inhibitory effects on nonenveloped adenoviruses in human. Additionally, we found that GA could be potentially inhibited the DNA of HCMV virus and protein synthesis of HSV-1 via a secondary inhibitory mechanism.
Consequently, in the fact of the antiviral activity of GA on conventional permissive cells in infections, GA could be possibly treated the acute viral contaminations for example, coronavirus COVID-19, ZIKV, IAV, EBOV and measles, and may be considered convenient for effective management of vigorous lesions in topical applications as VZV, HSV-1 and HSV-2 [72].
Finally, these approaches for using ginkgolic acid to constrain the enveloped virus infections differ essentially from outdated strategies for microbicidal, that directing on genome replication of viruses. Expectedly, the GA from Ginkgolic Biloba had complement further antiviral agents and proposal anew kinds of enveloped and nonenveloped viruses inhibitors [72].
reference link : https://www.degruyter.com/document/doi/10.1515/jbcpp-2020-0310/html
