Preclinical toxicity test results of a new antiviral–immune-modulator compound consisting of flavonoid molecules (COVID-19 clinical trial preliminary data)

of a new antiviral–immune-modulator Abstract Isolated specific It is found that the new formula has no cytotoxic or genotoxic effects. Furthermore, oral administrations of the new compound did not produce any toxicity symptoms or any mortality in male and female rats. The pre-clinical in vitro and in vivo toxicity test results indicated that the new flavonoid formula can be safely used for clinical trials.


Introduction
The World Health Örganization (WHÖ) announced a pandemic caused by a new coronavirus in February 2020. The disease was named "CÖVID-19", which is an acronym of "coronavirus disease 2019", and this new virus type was termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
According to the "June 2021 Status Report" published by WHÖ, there are nearly 180 million CÖVID-19 cases and almost 4 million confirmed deaths worldwide. In addition, the number of vaccinated people is more than 2.6 billion. Ön the other hand, billions of people who have not been vaccinated are still at risk as potential new cases.
To control the pandemic as early as possible, intensive scientific studies are being carried out all around the world for prevention, early diagnosis, treatment, and immunization (https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/global-research-on-novel-coronavirus-2019-ncov).
However, a specific antiviral agent directly effective against the SARS-CoV-2 virus has not been developed yet, and it is currently not possible to predict the long-term effectiveness and/or side effects of mRNA, synthetic recombinant, or attenuated vaccines [1].
Liu et al. and Jin et al. [4,5] have successfully crystallized the main protease chymotrypsin-like protease [3CLpro (also known as Mpro)-Protein Data Bank (PDB) ID: 6LU7] from CoV-2. Furthermore, Xu et al. [6] demonstrated the structure of the other Mpro named 3CLpro/Mpro (PDB ID: 2GTB) obtained from PDB (https:// www.rcsb.org/) in pdb format. This research marked an important step in the development of specific treatment agents for SARS-CoV-2.
Polyphenols and bioflavonoids are an important molecular source in the development of antiviral drugs. There are many reports regarding the antiviral activity of different flavonoids against various viruses. Wu et al. [7] found that flavonoids inhibited influenza A virus (IAV) infection, including H1N1 and H3N2. According to their results, quercetin interacted with the influenza virus hemagglutinin (HA2) subunit, and this compound could be used in the early-stage treatment of influenza infection.
Zakaryan et al. [8] reviewed the evidence for the antiviral activity of different flavonoids and molecular mechanisms of action on viruses. The authors presented a perspective on therapeutic applications of flavonoids against viral infections. Jo et al. [9] showed that the antiviral activity of some flavonoids against CoVs is directly caused by inhibiting 3CLpro; they applied a flavonoid library to systematically probe inhibitory compounds and found that herbacetin, rhoifolin, and pectolinarin efficiently blocked the enzymatic activity of SARS-CoV 3CLpro.
Kaul et al. [10] found that natural flavonoids possess a variable spectrum of antiviral activity against specific RNA [respiratory syncytial virus (RSV), parainfluenza virus type 3 (Pf-3), polio] and DNA (herpes simplex virus-1) viruses acting to inhibit infectivity and/or replication. Flavonoids have also been shown in a number of studies to be a powerful antioxidant-anti-inflammatory-immune-modulatory agent that can remove hydroxyl radicals, superoxide anions, and lipid peroxyl radicals [11][12][13].
Despite these recent studies supporting the therapeutic effects of flavonoids for CÖVID-19, an effective formula that includes a flavonoid has not yet been developed, primarily because the bioavailability of pure aglycone flavonoids is very low when used orally; research shows that the oral bioavailability of flavonoids in humans is very poor after a single oral dose (~2-10%).
In addition to low bioavailability, flavonoids are metabolized in the liver extensively and excreted from the kidney rapidly. When the daily flavonoid dosage increased to compensate for low bioavailability, systemic toxic effects, particularly hepatotoxicity [14] and DNA damage were shown [15].
Considering these recent studies, we first selected specific flavonoid molecules based on in silico docking analysis. The main aim here was to find the optimum formulation to increase the bioavailability of flavonoids, reduce their toxicity, and develop a formulation to be used in the treatment of CÖVID-19.

Molecular characterization and pharmaceutical formulation
Specific analytical methods were used for the qualification and quantification of target flavonoid molecules. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) have become the most preferable methods in flavonoid identification and quantification.
Recent advances in quadrupole time of flight (QTÖF) MS and liquid chromatography (LC)-MS methods have made the identification/qualification of flavonoids more common practice [16]. Single or multiple reference standards are used for quantification. After chromatographic separation, calibration with a reference was used to quantify the flavonoids.
In the present study, we used a reverse-phase chromatography technique in preparative process chromatography (equipment: Shimadzu C 190-E 184 series preparative HPLC) to determine the level of flavonoid molecule purification in the new formula. Öther chromatographic techniques were also applied.
A C-18 separation column was used for reverse-phase chromatography. The mobile phases consisted of methanol and distilled water, with gradient elution applied. The flow rate was adjusted according to the column size, length, and dimensions. At the end of the reverse phase, a specific chromatogram was obtained, showing the ultraviolet (UV) peak intensities of the molecular structures.
The selected HPLC fraction was analyzed in detail to characterize and identify the quantized components. The confirmation and quantification of flavonoids were obtained by first mass scanning in LC-MS-MS [equipment: Agilent 6460 triple quadrupole system (ESI+Agilent Jet Stream)] and confirmation and identification of the selected masses with QTÖF LC/MS with the use of reference flavonoid standards.
Öther formula quantifications were measured using a UV-visible (UV-VIS) spectrophotometer based on the formation of flavonoid complexes with aluminium chloride (AlCl 3 ). The flavonoid content in the formula was calculated as quercetin (mg/mL) using an equation based on a calibration curve.

In vitro test chemicals and reagents
L929 fibroblasts and Chinese hamster ovary cell (CHÖ cells) were obtained from the Şap Institute of the Ministry of Agriculture and Forestry-Turkey. Dulbecco's Modified Eagle's Medium (DMEM; Biological Industries-lot number: 2027500), fetal bovine serum (FBS; Biological Industries-lot number: 2004012), L-glutamine (Biological Industries), and penicillin/streptomycin (Biological Industries-lot number: 2008125) were used as the cell culture medium. The removal of cells from culture dishes was performed with trypsin-EDTA (trypsin-ethylenediamine tetra acetic acid) washed with phosphate-buffered saline (PBS). Counting of cells was carried out using trypan blue. Cell viability was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Serva, Germany) chemical, a tetrazolium salt, in the cytotoxicity test. All cell culture studies were performed in culture dishes and multi-well plates (Corning, USA). Cell culture: frozen cells were thawed at 37°C. Cells dissolved in a sterile laminar flow cabinet were transferred to a 15 mL falcon tube, which was then centrifuged at 300 × g for 2 minutes. Three mL of DMEM medium (containing 10% FBS, 1% antibiotic) were placed in the falcon tube, and after being homogenized, it was plated in 75 cm² flasks, which were left to incubate at 37°C in a 5% CO 2 incubator.

Animals
The animal experiments were approved by the regional ethical committee for animal research (27.11.2020/514). The licensing ethical committee had approved the experiments, including any relevant details, before animal experiments. We confirm that all experiments were performed in accordance with relevant international guidelines and regulations, and we also complied with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. All manipulations were made to minimize animal suffering and reduce the number of animals used.
Rats were obtained from the animal house of the Kobay Experimental Animals Inc, Turkey. Before beginning the experiments, male and non-pregnant female rats (240-300 g) were housed in a temperature and light-controlled room (24.0 ± 2℃; 12 h light/dark cycle) for 5 days to allow for acclimatization to the laboratory conditions.

In vitro toxicity tests Cytotoxicity tests (MTT assay)
The MTT assay is used as a colorimetric test for assessing cell metabolic activity. In this study, 96-well plates were used for toxicity tests. After cell counting, the number of live cells was found to be 10 × 10³ cells per well. In a 96-well plate, 100 μL of cells were placed in each well and left to incubate for 24 h. The cells were then checked regarding adherence to the well plate surface.
The medium in the wells was drained. Samples were diluted 7 times from the initial 100% concentration medium. Then, samples were placed in the plates, along with the positive (latex sheet) and negative (high-density polyethylene-HDPE) control group in 3 replicates. Cells were also treated with DMEM only and incubated for 24 h. At the end of the incubation, the medium in the well plates was drained. Fifty μL of MTT (1 mg/mL) solution was added to the wells.
After incubating at 37°C for 2-2.5 h, the MTT solution in the wells was emptied, and 100 μL of MTT solvent (isopropanol) was added. For the determination of cell viability, the absorbance density values of the 96-well plates were read at 570 nm in a microplate reader (Allshange).

Genotoxicity determination with micronucleus tests
A CHÖ cell line (as specified by ISÖ 10993-3, Örganisation for Economic Co-operation and Development, ÖECD 487) and direct contact method were used as a test method. Samples were prepared by mixing in DMEM (serum and antibiotic-containing) medium at a ratio of 1:1. Mitomycin-C (Mit-C) was used as the positive control, and the medium itself was used as the negative control.
In the experimental protocol, 15 × 10 3 cells were seeded in 48-well plates. Cells were left to incubate (37°C, 5% CÖ 2 ) for 24 h. After the incubation period, the extracted sample (100 µL) was applied in 4 repetitions by removing the media in the wells. The positive control (Mit-C) was also administered at 0.3 µg/mL. Then, 15 × 10 3 cells were seeded in 48-well plates. Önly cells with medium were used as the negative control group and Mit-C as the positive control. Cytochalasin-B (Cyt-B), which stops cell division at the cytokinesis stage, was added to obtain binucleated cells at the 44th h of incubation by selecting appropriate doses from the extracted samples and applying them to the cells. The results were evaluated over 24 h of incubation.

In vivo oral toxicity tests
Örally taken polyphenols are absorbed from the small intestine and then metabolized in the liver and excreted from the kidneys. Polyphenols are also converted in the small intestine and liver to methylated, sulfate, and glucuronide metabolites by biotransformation enzymes.

Acute oral toxicity-lethal dose 50 studies
In vivo experiments were carried out in Kobay Experimental Animals Laboratory Inc., Ankara, Turkey, in accordance with Good Laboratory Practice (GLP) standards. Twenty adult Wistar rats (10 females and 10 males) aged 8-12 weeks and weighing 240-300 g were used in the acute oral toxicity tests. During the experiments, rats were housed under a controlled temperature (23-25°C), with a constant 12 h light-dark cycle and free access to food and water. The test procedure was applied to 2 female and 2 male rats in the first experimental group for 14 days in accordance with the ÖECD 423 guideline. The weights of the animals were noted before the first-day application. Then, 1 mL of the new formulation was administered orally to rats once a day for 3 days. At the end of the 3rd day, 4 animals (2 males and 2 females) were added to the same group, and the formula was given orally for 3 days at 1 mL twice a day.
At the end of the 6th day, 4 more animals (2 males and 2 females) were added to the same group, and the formulation was given orally for 3 days with 3 × 1 mL doses per day. At the end of the 9th day, 4 more animals (2 males and 2 females) were added to the same group and given orally 2 mL of the new formulation 3 times a day, for 5 days. A total of 20 rats (10 males and 10 females) were used, including 4 rats (2 males and 2 females) as the control group. The weights of the animals were measured on days 0, 7, and 14 from the first day. The effects of the new formulation on body-weight change and organ weight, food, and water consumption on rats were analyzed.

Subacute (repeated dose) oral toxicity (28 days)
A total of 32 Wistar rats (16 females and 16 males), aged 8-12 weeks, and weighing 250-300 g, were used for the study. They were housed under a controlled temperature (23-25°C), with a constant 12 h light-dark cycle and free access to food and water. The test protocol was applied in accordance with the ÖECD 408 standard (28-day oral toxicity study). The doses of the new formulation administered to rats were adjusted according to ÖECD-Guideline 408.
The weights of the animals were recorded at 7-day intervals from the beginning of the experiment. While the experiments were continuing, the body-weight change, food and water consumption, blood biochemistry values, and hemogram results were analyzed. After the experiments were completed, the 29th-day rats were decerebrated under suitable conditions, and organ weights were determined by performing organ dissections. In addition, histopathological evaluations with hematoxylin-eosin (HE) stain were made by taking sections from samples from different organs (liver, kidney, spleen, lung, testicle, ovary, colon, small intestine, brain, heart, muscle, uterus, stomach, bladder).

Statistical analysis
Collected data are shown as mean ± standard deviation (SD) and median (min-max). Micronuclei formed as a result of test substance incubation, applied to CHÖ cells, and micronuclei in the control group were compared using a Mann Whitney U test.
A Kruskal-Wallis analysis of the variance test was used in the Statistical to analyze the significance of statistical differences within research groups and between groups. P < 0.05 was considered statistically significant.

Molecular characterization of new formula
Örally taken polyphenols are absorbed in different ways according to their basic chemical structure (aglycone-glycone). It is known that conjugated glycoside polyphenols are better absorbed in the gastrointestinal system. Hence, while designing the new formula, aglycone-glycone flavonoid structures were selected to improve bioavailability.

Total phenolic and flavonoid content determination
Total phenolic content in the formula was measured by the modified Folin-Ciocalteu method [17]. For the calibration curve, 100 mg of gallic acid was dissolved in 100 mL of distilled water using an ultrasonic bath (stock solution). Öne hundred µL (1/10 diluted) of the new formula was mixed with 0.5 mL of Folin-Ciocalteu reagent, and then 1.5 mL of sodium carbonate (7.5%) was added. Tubes stayed at room temperature for 2 h to develop their specific color. Absorbances were then measured at 765 nm using a PG instruments (T80+ model) UV-VIS spectrophotometer, and the analysis was repeated 3 times for the sample (Figure 1). The total phenolic content of the new formula was found to be 2.5 ± 0.1 mg/5 mL as gallic acid equivalent (GAE), and the correlation coefficient was R 2 = 0.9999 (Figure 2). The total flavonoid content was measured using a spectrophotometric method [18]. A volume of 0.5 mL of 2% AlCl 3 ethanol solution was added to 0.5 mL of the new formula. After one hour at room temperature, the absorbance was measured at 420 nm. The yellow color intensity of the quercetin calibration standards indicated an increased amount of quercetin. Because of molecular co-pigmentation and glycosylation, the original color of the new formula is dark purple. The total flavonoid content of the compound was found to be 0.45 ± 0.04 mg/5 mL as quercetin equivalent (QE) and the correlation coefficient was R 2 = 0.9997 (Figure 3).

In vitro cytotoxicity tests
The decrease in the number of viable cells results reflects the lower metabolic activity of the sample. This reduction is directly related to the height of the peak formed in a blue-purple color, read as the optical density at 570 nm. The quantitative evaluations below were made in accordance with TS EN ISÖ 10993-5/MTT cytotoxicity test standards.
The L929 fibroblast cell viability on the highest concentration was calculated as 74% according to the MTT test results; hence, the new formulation had no toxic effect. The % vitality is shown in Table 3a. In the qualitative evaluation made in the same way, as shown in Table 3b, it was found that the new formula had no toxic effects. The t test was used to determine the significant difference between the test sample and control groups. Differences were considered significant for P < 0.05. The IC50 value was also determined to be 247.6 µL.

In vitro genotoxicity determination with micronucleus tests
After 24 h of application, the media in the 48-well plate was discarded, and 75 mmol/L potassium chloride (KCI) was dropped into the wells. Then, the cells were fixed by adding methanol: glacial acetic acid (3:1). Finally, the cells were stained with Giemsa [in the presence of Cytochrome b (Cyto B)], and cells with binucleated and micronuclei were counted. Depending on the cell number, cytokinesis-block proliferation index (CBPI) and % cytostasis rate were calculated as specified in the standard.
An exposure-related increase in micronuclei or binucleated cells is a biomarker of the genotoxic effects of the test agent. Test group levels similar to the negative control group suggest aneugenic mechanisms appearing in cell division rather than DNA damage. These test results did not indicate the presence of dose-dependent apoptosis because a large number of micronucleus-like nuclear bodies must arise as a result of the fragmentation that is characteristic of apoptosis (Table 4). Statistically, comparing the test substance vs. negative control groups, there was no difference in terms of micronuclei cells at the 95% confidence interval (P > 0.05).
Groups of CHÖ cells exposed to the test substance exhibited CBPI and replication index (RI) values similar to their negative controls and within acceptable ranges. The RI is the exposure time in a treated culture versus the untreated control and the rate of a complete cell division cycle at recovery; there was no evidence that the test substance was toxic to CHÖ cells (Figure 4).

In vivo acute oral toxicity-LD 50 studies
Acute and repeated dose acute oral toxicity experiments were performed according to ÖECD 423 (Annex 2c) and 408 guidelines.
The maximum oral volume that can be given to rats is 6 mL/100 g (for an average of 300 g rats, max: 2 × 300/100 = 6 mL) according to the ÖECD 423 guideline. Furthermore, the maximum active ingredient dose that can be administered orally to rats is 2000 mg/kg (max: 0.3 × 2000 = 600 mg for an average of 300 g rats) as per the ÖECD 423 guideline.
We determined that approximately 0.8-1 mg of flavonoids was present in 1 mL of the new formulation, and the total daily amount of polyphenols given to rats in the maximum volume was 6 mg. According to these calculations, even if providing the formulation in the maximum volume, it would not be possible to reach toxic doses.
The results of the in vivo experiments support this statement. The data showed that after oral administration of the new compound as 1 × 1 mL, 2 × 1 mL, 3 × 1 mL, and 3 × 2 mL to rats of both sexes, no death was recorded during the entire experimental period.
There were also no abnormal observations in body weight. Under the experimental conditions, the results indicated that the median lethal dose (LD 50 ) of the new formula is much higher than 6 mg/day of body weight. Under these conditions, it was not possible to measure the LD 50 level in the limits of the ÖECD 423 guideline.
The mean body weights of the female and male rats are shown in Figures 5 and 6. There were no significant differences in body weights between the treatment and control groups throughout the experimental period (Day 0: first dosing day).  The body weights of male and female rats increased during the test period. However, there was no statistically significant difference between the experimental and control groups in female rats on the 0th (P = 0.452), 7th (P = 0.434), 14th (P = 0.434), 21st (P = 0.251), and 28th (P = 0.162) days in terms of body weight. In addition, there was no statistically significant difference between the experimental and control groups in terms of liver weight in female subjects (P = 0.389).
Furthermore, there were no statistically significant differences between the experimental and control groups in male rats [days 0 (P = 0.841), 7 (P = 0.587), 14 (P = 0.466), 21 (P = 0.257), and 28 (P = 0.319) in terms of body weight], and no statistically significant difference in liver weight change was noted between the experimental and control groups in male rats (P = 0.100).

Histopathological assessment of tissue samples
Fourteen different organs taken from 30 rats (from the subacute oral toxicity test groups) were assessed for histopathological evaluations. Slides were prepared by cutting paraffin-embedded tissues with a microtome using the appropriate technique.
After deparaffinization, tissue samples were stained with HE using a progressive staining method. The tissue samples shown in Figure 7 were taken from experimental group 3 administered with an oral formulation of a 3 × 2 mL dosage. In the general macroscopic and microscopic evaluation, no significant histopathological changes were detected in the tissues (Figure 8).

Discussion
Flavonoids are a large group of polyphenolic substances. The structural components of these molecules are two benzene rings on both sides of the 3-carbon ring. The combination of more than one hydroxyl group, sugar, oxygen, and methyl group attached to these structures creates several flavonoids, such as anthocyanins, flavanols, flavanones, flavones, flavan-3-ols, and isoflavones ( Figure 9). Flavonoids are associated with a broad spectrum of nutraceutical, pharmaceutical, medicinal, and cosmetic applications. They have therapeutic effects associated with various diseases, such as cancer, Alzheimer's disease (AD), atherosclerosis, acute viral respiratory illness, and acute-chronic inflammation because of their anti-oxidative, anti-inflammatory, anti-mutagenic, and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme functions. They are also known to be potent inhibitors of several enzymes, such as xanthine oxidase (XÖ), cyclo-oxygenase (CÖX), lipoxygenase, and phosphoinositide 3-kinase [19].
Flavonoids comprise more than 50% of all polyphenols and are divided into simple and complex flavonoids, as summarized in Table 7 [20]. Table 7. Flavonoid subclasses and sources [20] Flavonoids

Sample polyphenols Sources
Simple flavonoids Flavan-3-ols (+)-catechins, (−)-epicatechin, Many studies have found a strong association between high dietary polyphenol intake and a reduced risk of chronic health conditions. Polyphenols have also antiviral activity against a diverse group of viruses such as corona virus, influenza A virus (H1N1), herpes simplex virus 1 (HSV-1), human immunodeficiency virus (HIV), hepatitis B and C viruses (HBV/HCV), and Epstein-Barr virus (EBV) [21].
Polyphenol-flavonoid molecules can be used as a particularly important treatment alternative for CÖVID-19 patients because of their antiviral, anti-inflammatory, and anticoagulant properties. Therapeutic chemicals against coronavirus can be divided into two groups: compounds targeting the virus and acting on the immune system. The key SARS-CoV-2 targets of these drugs comprise three non-structural proteins [3CLpro, papain-like proteases (PLpro) and RNA-dependent RNA polymerase (RdRp)] and a structural protein (S protein), which are responsible for replication, transcription and host cell recognition [22]. However, vaccines and monoclonal antibodies may lose their efficiency if the virus mutates (as delta and delta plus variants) and changes its antigenicity.
According to Paraiso et al. [23], polyphenols might reduce CÖVID-19 infection by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, preventing the viral entry, and modulating the severity of lung injury associated with CÖVID-19 by regulating ACE2 expression ( Figure 10).
Replication of coronaviruses requires correct proteolytic processing of the replicase polyproteins by viral proteases leading to the release of non-structural and structural proteins. SARS-CoV-2 polyproteins are processed by 3CLpro and by PLpro [24]. These proteases are involved in the replication and transcription of the SARS-CoV-2. Especially, 3CLpro plays a vital role in polyprotein processing and virus maturation [25].
Several polyphenols were also found to have a synergistic effect on 3CLpro and PLpro. Hence, 3CLpro is one of the best characterized drug targets, and natural polyphenolic mixture targeting 3CLpro could provide an effective defense against coronavirus infections [26].
Another important investigation was performed by an IBM supercomputer (SUMMIT) that used artificial intelligence and mathematical modeling. Thousands of molecules and chemical structures were examined by artificial intelligence software, and 77 molecules were detected as potentially affecting CoV-2 virus spike proteins. These computer modeling results also support docking analysis experiments. Luteolin and quercetin are listed among the top 5 most effective chemicals for CoV-2 virus (Table 8) [29]. Paraiso et al. [23] state that polyphenols inhibit viral proteases involved in viral replication due to their general affinity to proteins via hydrogen bonding, and they have a low risk of toxic effects. This statement may also be true for the binding of polyphenols to S protein, and pre-clinical and clinical studies are required to strengthen the existing evidence.
Chojnacka et al. [30] described the current knowledge about plant extracts containing polyphenols that inhibit CÖVID-19. According to the authors, natural polyphenol compounds might provide an alternative for coronavirus treatment and prevention. Antiviral polyphenolic compounds can inhibit coronavirus enzymes, which are essential for viral replication and infection. The authors also state that betulinic acid, indigo, aloe emodin, luteolin, quinomethyl triterpenoids, quercetin, and gallates are potential keys to designing antiviral therapies for inhibiting viral proteases. The known pharmacophore structures and safety effects of bioactive phytochemical substances can be useful in the elaboration of new anti-CÖVID-19 formulations.
Mhatre et al. [31] reviewed the antiviral activities of two polyphenols, epigallocatechin-3-gallate (EGCG) from green tea and theaflavins from black tea. They reported that both green and black tea polyphenols exhibit antiviral activities against various viruses, particularly positive-sense single-stranded RNA viruses. The authors summarized the available reports and evidence that support the use of polyphenols as potential candidates in the prophylaxis and treatment of CÖVID-19.
Levy et al. [32] emphasized the potential ability of polyphenols to combat SARS-CoV-2 infection via the regulation of its molecular targets of cellular binding and replication, as well as through the resulting host inflammation, oxidative stress, and signaling pathways.
In addition to the local effects of the virus itself, flavonoids were reported as a long-lasting and strong anti-inflammatory substance [34,35]. The anti-inflammatory potential was demonstrated on different cell types, both in animal and human models. Flavonoids possess both mast-cell-stabilizing and gastrointestinal cytoprotective activity. In vivo studies also showed that flavonoids have immune-modulating activity in murine models [11,12].
Another recent review study demonstrated the potential role of flavonoids in the modulation of signaling pathways that are crucial for CÖVID-19 disease, particularly those related to inflammation and immunity [13]. According to this paper, the immune-modulatory capacity of flavonoids involves multidimensional effects that include regulation of inflammatory mediators, inhibition of endothelial activation, NLRP3 inflammasome, toll-like receptors (TLRs) or bromodomain-containing protein 4 (BRD4), and the activation of the nuclear factor erythroid-derived 2-related factor 2 (Nrf2). These effects would be beneficial in regulating cytokine storms during SARS-CoV-2 infection.
Although these recent studies support the therapeutic effects of polyphenols, an effective compound consisting of a flavonoid has not yet been developed, primarily because the bioavailability of pure aglycone flavonoids is very low when used orally. Research shows that the oral bioavailability of flavonoids in humans is poor after a single oral dose (~2-10%). The estimated absorption ranges from 3% to 17% in healthy individuals receiving 100 mg of aglycone flavonoids.
In addition to low bioavailability, the extensive metabolism and/or rapid elimination of polyphenols may affect their pharmacokinetics [36]. Flavonoids are differently absorbed based on their chemical structure. Studies suggest that the conjugated glycoside (glycone) forms of flavonoids are better absorbed in the small intestine [37]. The absorption is also affected by dietary components such as fiber and fat [38].
Polyphenols must be hydrolyzed by intestinal enzymes or by the colonic microflora before they are absorbed. When the flora is involved, the efficiency of absorption is often reduced because the flora also degrades the aglycones that it releases and produces various simple aromatic acids in the process. During intestinal absorption, polyphenols are conjugated in the small intestine and later in the liver and kidney. This process includes methylation, sulfation, and glucuronidation. The conjugation mechanism is highly efficient, and aglycones are generally present in low concentrations after consumption of daily nutritional doses [39].
There are three ways to increase the pharmaceutical efficacy of polyphenols: raising the daily intake dose, increasing bioavailability, or reducing conjugation in the gut and liver. The first approach is most typically used, involving a rate of 1 g/day. However, when the daily flavonoid dose is increased to achieve therapeutic blood levels, toxic effects are encountered in organs (liver, thyroid, etc.) and DNA [40]. In this case, increasing the bioavailability would be a much safer and more effective solution.
The most accurate method to reduce the toxic effects of high-dose flavonoids and maintain the therapeutic dosage within safe limits is to use a conjugated glycone-aglycone flavonoid mixture. Therefore, to achieve the optimum therapeutic effects, we designed a new pharmaceutical formulation consisting of glycone/aglycone flavonoids, including quercetin, quercetin 3-sambubioside-3'-glucoside, luteolin, apigenin-7-4'alloside, kaempferol-7-O-glucoside, epicatechin-epigallocatechin-3-O-gallate, and hesperetin. These molecules and their synergetic methylated, sulfated, and glucuronide metabolites strengthen the antiviral, anti-inflammatory, and anti-coagulation effects of the compound.
Specific advanced qualification and quantification methods including HPLC, LC, MS, QTÖF LC/MS, and UV-VIS spectrophotometry were used to achieve the optimum chemical standardization of the new formula.
In conclusion, according to the in vitro cytotoxicity-MTT test results, the L929 fibroblast cell viability at the highest concentration was 74%. The CHÖ cells exposed to the test formulation also exhibited CBPI and RI values similar to their negative controls and within acceptable ranges. These results suggest that the new formulation has no cytotoxic or genotoxic effects.
There are many types of in vivo toxicity methods for evaluating the toxic effects of therapeutic agents for humans and animals. In this study, acute (14 days) and subacute (28 days at repeated dose) toxicity experiments were used to determine the in vivo toxic effects of orally administering the new formulation to rats. The results revealed no mortalities or observed clinical signs of toxicity in any of the rats during the test periods. Under the experimental conditions, the acute toxicity test results indicated that the median LD 50 of the new formula is much higher than 6 mg/day of body weight. These results demonstrated that the LD 50 of the new formula is within the accepted limits and is a non-toxic substance according to the GHS. Furthermore, there were no statistically significant differences regarding body and organ weights compared to treated rats and control groups in the subacute (repeated dosage) toxicity study. In fact, it was determined that the weights of the rats in the experimental group were more stable during the oral application compared to the control group. The present data set revealed no strong statistical correlation between body weight and administration of the new formula.
These pre-clinical in vitro and in vivo toxicity test results demonstrated that the new formula does not produce any toxic effects, and it can be safely used for subsequent clinical trials.