Humulus lupus extract rich in Xanthohumol reduces the risk of a fatal clinical course in critically ill patients treated for COVID-19

Background. The systemic in�ammatory response following severe coronavirus infection (COVID-19) is associated with poor outcome. Several anti-inammatory medications were studied in COVID-19 patients. Xanthohumol (Xn), a natural extract from hop cones, possesses strong anti-inammatory and antioxidative properties. The aim of this study was to analyse the effect of Xn on the in�ammatory response and the clinical outcome of COVID-19 patients. Methods Adult patients treated for acute respiratory failure (PaO 2 /FiO 2 less than 150) were studied. Patients were randomized into two groups: Xn – patients receiving adjuvant treatment with Xn at a daily dose of 4.5 mg/kg body weight for 7 days, and C – controls (patients receiving placebo – NaCl 0.9%). Observations were performed at four time points: immediately after admission to the ICU and on the 3rd, 5th and 7th days of treatment. The in�ammatory response was assessed based on the plasma IL-6 concentration, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), C-reactive protein (CRP) and D-dimer levels. The mortality rate was determined 28 days after admission to the ICU.


Introduction
Since December 2019, when a new type of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was described, many studies have been performed to identify an effective treatment to alleviate signs and symptoms.This infection is caused by β-coronavirus 2 (CoV-2) and is associated with multiorgan dysfunction resulting mainly from endothelial damage complicated by massive in ammatory response syndrome [1][2][3][4].Coronavirus-induced symptoms are referred to COVID-19 disease.
Several studies on COVID-19 documented a strong relationship between the severity of the clinical condition and the degree of in ammatory response to viral infection [5][6][7][8].Immune dysregulation is a trigger for cytokine storms.An increase in the release of in ammatory cytokines, especially interleukin-6 (IL-6), associated with T-

Ethical considerations
This prospective, observational study was conducted in accordance with the Declaration of Helsinki and applicable regulatory requirements.The local Institutional Review Board and the Bioethics Committee of the Medical University in Lublin, Poland approved the protocol (KE-0254/201/2020).Written informed consent was obtained from all patients just after admission to the ICU prior to randomization.Additionally, legal representatives were informed about the main purpose of this study.cell lymphopenia is correlated with poor outcome and death [5,6,8].A rapid increase in the in ammatory response is associated with uncontrolled production of reactive oxygen species and free radicals, which signi cantly impair cellular metabolism [9,10].Several authors have studied the e ciency of anti-in ammatory and antioxidant treatment to reduce COVID-19-related complications and death [5,[9][10][11][12][13][14][15][16][17].Interestingly, natural compounds have also been suggested as effective adjuvants in COVID-19 therapy [18,19].These compounds possess different anti-COVID-19 activities.Some of them compounds block IL-6 release, which could reduce the need for mechanical ventilation and thus also admission to the intensive care unit (ICU) [13][14][15].Other compounds directly inhibit viral replication by binding to speci c receptors [20,21].Interestingly, some compounds present similar chemical structures.In silico studies suggested a high e ciency of naturally occurring prenylated chalcones for the treatment against coronavirus infection [15,22,23].
Experimental studies documented a strong effect of Xn against many DNA and RNA viruses, such as herpes simplex virus types 1 and 2, cytomegaloviruses and porcine reproductive and respiratory syndrome virus (PRRSV) [22,[25][26][27][28]. Interestingly, PRRSV infection is similar to the course of coronavirus infection with main symptoms consisting of high fever, signi cant morbidity and severe respiratory disease resulting in high mortality.In vivo experiments showed that the PRRSV-infected piglets treated with Xn at a dose of 20 mg/kg exhibited only moderate clinical signs and low viral loads, whereas 25 mg/kg Xn practically reduced all clinical symptoms [28].Another experimental study documented that Xn inhibited many viral diseases, including SARS-CoV-2 and other fatal diseases caused by alpha-or beta-coronavirus [29].Notably, Xn is safe and well tolerated in healthy humans and is available as a dietary supplement.Based on its antiviral and anti-in ammatory properties, we hypothesized that administration of Xn could improve the clinical course and outcome in critically ill COVID-19 patients requiring mechanical ventilation.
Therefore, the aim of this study was to analyse the effect of Xn supplementation on the clinical course, in ammatory response and outcome in patients admitted to the ICU due to COVID-related acute respiratory failure with an oxygenation index (PaO 2 /FiO 2 ) less than 150.

Study drug treatment
All patients were treated following current guidelines at the time of admission.After admission to the hospital, remdesivir (Veclury, Ireland) was administered at an initial dose of 200 mg/day followed by 100 mg/day for 5 to 7 days.Additionally, vitamin D3 at a dose of 4000 U per day was supplemented in all patients.Corticosteroid therapy with dexamethasone (Dexaven, GmbH Arzneimittel, Germany) at a dose of 8 mg per day for 10 days and anticoagulant therapy with endoxaparinum natricum (Clexane, Sano -Aventis, France) were started upon admission to the ICU.All patients received continuous infusion of insulin to maintain plasma glucose concentrations between 100 and 160 mg/dL.

Monitoring
In all patients, systolic diastolic and mean arterial blood pressures (MAP), heart rate (HR) and expiratory CO 2 tension were monitored continuously.Additionally, haemodynamic variables, such as cardiac output/index (CO/CI), stroke volume variation (SVV), systemic vascular resistance index (SVRI) and central venous pressure (CVP), were monitored using the EV 1000 platform (Edwards Lifescience, Irvine, CA, USA).Masimo Root monitor (USA) with SEDLine was used for continuous measurement of regional cerebral oxygen saturation (SrO 2 ), frontotemporal electroencephalography, peripheral oxygen saturation (SpO 2 ) with haemoglobin level and oxygen reserve index (ORI).Fluid administration with balanced crystalloids and vasopressors (norepinephrine) were titrated to obtain MAP higher than 65 mmHg.

Patients selection and inclusion criteria
This study was performed between October 2020 and January 2021.Adult patients aged 18 years or older admitted to the ICU who were treated for severe COVID-19 with acute respiratory failure (PaO 2 /FiO 2 below than 150) due to bilateral and multifocal ground-glass opacities involving greater than half of the lungs were included in the study.The quantitative computed tomography (CT) with thoracic VCAR software and the parenchymal analysis option was used to assess the degree of parenchymal impairment.Patients who were treated for COVID-19 for more than one week were excluded.Other exclusion criteria were chronic renal failure, history of illness affecting the human immunologic defence system (modulated immunologic system, such as transplant patients) and/or prolonged in ammatory response such as malignancies, rheumatologic diseases and chronic in ammatory disease.Pregnant or lactating women were also excluded.Patients, who did not respond to prone ventilation strategy were screened for eligibility for extracorporeal oxygenation (ECMO) and were excluded from this study.Patients, who died within 7 days were also excluded due to uncompleted data.
Patients were randomized in a double-blind, placebo-controlled fashion into two groups using sealed envelopes.
Group Xn includes patients who received extract from Humulus lupus L rich in Xanthohumol (Hop-RXn™, BioActive-Tech Ltd, Lublin, Poland; http://xanthohumol.com.pl/) as an adjuvant therapy, and group C includes patients who received 0.9% NaCl formed the control group.Based on pharmacokinetics and bioactivity, Xn was administered enterally three times a day every 8 hours at a dose of 1.5 mg/kg body weight (4.5 mg/kg body weight/day) for 7 days [30].The rst dose of Xn was administered within 4 hours after admission to the ICU.In the control group, 3 mL of NaCl 0.9% was administered enterally three times a day.

Biochemical analysis
Routine biochemical examination with full blood count and morphology, including erythrocyte, platelet, leukocyte, neutrophil and lymphocyte counts, serum interleukin 6 (IL-6) concentration, C-reactive protein (CRP) and D-dimers, were performed immediately before admission to the ICU and on the 3rd, 5th and 7th days of treatment.All biochemical analyses were performed at the laboratory of University Hospital No 4 in Lublin, Poland using commercial reagents.Arterial blood gas analysis was performed a minimum of 4-6 times per day using GEM 5000 (Werfen, Barcelona, Spain).The PaO 2 /FiO 2 ratio was calculated as the ratio between the oxygen tension obtained from routine blood gas's analysis and the fraction of inhaled oxygen (FiO 2 ).The following formulas were used for calculation of NLR and PLR: -NLR: number of neutrophils divided by number of lymphocytes, -PLR: the number of platelets divided by the number of lymphocytes.

Pulmonary disease evaluation
The ventilator settings were determined in accordance with the results of the blood gas examination and the respiratory insu ciency was assessed by calculating the PaO 2 -to-FiO 2 ratio (PaO 2 /FiO 2 ).Patients with PaO 2 /FiO 2 less than 100 were placed into the prone position in accordance with a local protocol [31].A highresolution computed tomography (CT) technique with arti cial intelligence software (Thoracic VCAR software with Parenchymal Analysis, GE Healthcare, USA) was used to assess the severity and quantitatively measure lung injury.In all participants, CT was performed immediately before admission to the ICU.A control CT was performed 2-3 days after extubation or immediately after discharge from the COVID zone in the ICU.

Study protocol, measured variables and outcomes
Observations were performed at four time points: 1) immediately after admission to the ICU (baseline), 2) 3 days after admission to the ICU, 3) on the 5th day of treatment and 4) on the 7th day of treatment.The degree of the in ammatory response was measured by NLR, PLR, D-dimer and plasma IL-6 concentration.Primary outcomes were mortality rates, which was determined at 7 and 28 days after admission to the ICU.Secondary outcomes were the dynamics and evolution of the in ammatory parameters and the evolution of CT imaging.

Statistical analysis
Statistical analysis was performed using Statistica 13.1 software (StatSoft, USA).Means and standard deviations (SD) were calculated for normally distributed variables, whereas non-Gaussian distributed variables were presented as medians and inter-quartiles range.The Kolmogorov-Smirnov test was used to analyse the normality of the data distribution.Categorical variables were compared using the χ 2 and Fisher exact tests, and Yates correction was applied.The value at ICU admission was regarded as baseline.The unpaired student's ttest was used to analyse variables with normal distribution.Nonparametric data were statistically analysed using the Wilcoxon signed-rank test and the Kruskal-Wallis test.Additionally, the Pearson test was used for analysis of any correlation in normally distributed variables, whereas Spearman's rank test was used for interpoint and intergroup comparisons for variables with a non-Gaussian distribution.Kaplan-Meier estimation was performed for survival probability analysis.A value of p < 0.05 was considered signi cant.

Study population
Seventy-two adult critically ill patients treated for COVID-19 with severe respiratory failure were included in the present study.A total of 22 patients were excluded from the nal analysis: 11 were excluded because informed consent could not be obtained, and 4 died within 7 days with incomplete data.Additionally, seven patients were also excluded due to incomplete data or consent withdrawal after recovery.Finally, fty patients (18 female and 32 male) aged 22 to 83 years (mean 58 ± 17) were studied.Twenty-ve patients were randomly assigned to the Xn-group and were treated with Xn, and 25 received 0.9% NaCl and were allocated to the control group.The relevant demographic data and comorbidities are presented in Table 1.

Primary endpoints
Overall mortality was 34%.Seventeen patients died between days 7 and 28 of treatment: 5 (20%) in the Xn group and 12 (48%) in the control group (χ 2 = 5.56, p < 0.05 and χ 2 with Yates correction = 4.25 and p < 0.05, Fig. 2).All patients treated with Xn who survived were discharged from the ICU to the pulmonology or rehabilitation ward and then discharged home in good clinical condition.In the control group that did not receive Xn, none of the patients were discharged directly home.These patients were discharged to another pulmonology hospital followed by another hospital, and their outcome could not be determined.

Changes in in ammatory markers
The mean baseline value of NLR was 20.8 ± 16 in all participants, and was comparable in both groups (21.5 ± 14.2 vs. 20.2 ± 17.6 in the Xn-treated and control groups, respectively).Treatment with Xn resulted in a near 5fold signi cant reduction in NLR at day 7 compared with baseline.In contrast, no signi cant NLR-decrease was observed in the control group (Table 2).In patients who survived, the NLR decreased on the 3rd and 7th day of treatment with Xn and on the day 7 in the control group (Fig. 3).
The platelet-to-lymphocyte ratio decreased on the 3rd and 7th day of treatment in the Xn-group and on the 5th and 7th day in the control group (Table 2).Changes in both groups were comparable, and no signi cant differences were noted.
The mean baseline value of plasma IL-6 concentration was 279.1 ± 380.1 pg/mL in all patients.IL-6 levels were comparable in both groups (298.4 ± 453.5 pg/mL vs. 256.7 ± 337.5 pg/mL in Xn-treated and control groups, respectively).Treatment with Xn reduced plasma IL-6 concentrations on the 3rd and 7th days, whereas these values were reduced on the 5th and 7th days in the control group (Table 2).In patients who survived, plasma IL-6 concentration decreased on days 3, 5 and 7 in both groups.The decrease in plasma IL-6 concentration was more pronounced in the Xn group (Fig. 4).
In both groups, D-dimer levels decreased on the 3rd, 5th and 7th days; however, treatment with Xn resulted in a more pronounced reduction compared to the control group (Table 2).In patients who survived, D-dimers decreased in both studied groups, but their concentrations were signi cantly lower in patients treated with Xn on the 3rd and 7th days (p < 0.05).

Discussion
In the present study, we documented that treatment with Xn signi cantly reduced the severity of the in ammatory response, as re ected by the plasma IL-6 concentration and NLCR, improved outcomes and reduced the mortality rate.Additionally, Xn at a daily dose of 4.5 mg/kg body weight improved the oxygenation index and reduced the length of mechanical ventilation.The mechanism responsible for these phenomena seems to be complex and pleiotropic.

Presumed pathophysiologic mechanisms
Xanthohumol is the most abundant prenylated avonoid in hops.Beer is the most important dietary source of Xn and other related prenyl avonoids.Concededly, the brewing process induces thermal isomerization of Xn to isoxanthohumol (IXn), but Xn can be converted into IXn in the stomach [32,33].An in vitro study showed that both forms can be biotransformed by human liver microsomes to glucuronides, hydroxylated metabolites, and cyclic dehydrometabolites [30,33,34].The bioavailability of Xn is dose dependent and increases linearly with increasing oral dose [30].Xn has strong antioxidant and anti-in ammatory properties and protects cells from injury induced by upregulated angiotensin-2 activation [23-26, 35, 36].An experimental study showed that angiotensin-2 stimulates the production of reactive oxygen species (ROS) via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and Xn and its major bioactive metabolite IXn strongly inhibit this process, preventing oxidative-related endothelial injury [35,37].It has been shown previously, that Xn inhibits the viral-encoded cysteine protease (main protease of CoV-2) in a dose-dependent manner [29].Importantly, this protease is necessary for viral replication.Another reported pathomechanism is based on a reduction in the intensity of viral replication related to the inhibition of diacylglycerol acyltransferase (DGAT) [20].Massive viral replication is associated with metabolic cell damage, and the rapid upregulation of lipid biosynthesis, particularly triacyglycerol, plays a crucial role in this process.The last step in triacyglycerol synthesis is catalysed by DGAT, the inhibition of which reduces the availability of fuel for viral replication.Xanthohumol strongly inhibits DGAT activity in a dose-dependent manner, and its antiviral effect has been noted in cardiomyocytes and type II alveolar epithelial cells -the major portal of CoV-2 infection [20].
Interestingly, Xn also has the most potent activity, improving cell viability from all chalcones extracted from Humulus lupus [36].In the present study, we observed relatively quick improvement in blood oxygenation and CT-lung imaging after 7 days of treatment with Xn.Therefore, we can speculate that the use of Xn at a dose of 4.5 mg/kg body weight may be a safe and effective adjuvant therapy in severe COVID-19 patients.

Anti-in ammatory properties
The therapeutic effect of Xn can also be explained by its anti-in ammatory properties.We noted a signi cantly lower IL-6 concentration and NLR in patients treated with Xn.Rapid and massive production of proin ammatory cytokines, particularly IL-6, is associated with the severity of COVID-19.In the present study, the plasma IL-6 concentration upon admission was 100-fold higher than normal, and the addition of Xn to the treatment regimen resulted in a rapid and signi cant decrease in its concentration.Several studies documented the strong anti-in ammatory properties of Xn with reduction in proin ammatory cytokines and the number of macrophages in injured tissues [24,38,39].Administration of Xn reduced plasma IL-6 levels by approximately 80% [40].An animal study showed that Xn effectively reduced tumour necrosis factor alpha (TNF-α), IL-6 and IL-1β secretion and suppressed high-mobility group box 1 protein (HMGB1) and inducible nitric oxide synthase (iNOS) expression [38].A decrease in the production and release of proin ammatory cytokines is associated with the suppression of nuclear factor-kappa B (NF-κB), which inhibits T-cell proliferation [29,38,39].Interestingly, anatomopathological examination of animal lungs revealed a signi cantly lower neutrophil in ltration in injured lungs, and lung damage was markedly reduced in animals treated with Xn compared to those treated with remdesivir [20,39].In the present study, CT-scans also showed markedly less consolidations and bilateral diffuse mixed densities of the lung in patients treated with Xn compared to controls.All our patients tolerated Xn well and none of them had adverse effects.Therefore, we suggest adding Xn as an adjuvant to standard therapy in COVID-19 patients.
The neutrophil-lymphocyte count ratio is frequently used as a marker of the severity of in ammation and outcome [41][42][43][44][45]. Elevated values of NLR predict poor outcome in patients treated for traumatic brain injury [41], mesenteric ischaemia [42] or sepsis [43].Importantly, NLR has also been proposed as a sensitive marker of endothelial dysfunction following viral infection [44].Progressive endothelial damage following viral infection, including CoV-2, induces massive glycocalyx injury, leading to endothelial in ammation with uncontrolled neutrophil activation, vasoconstriction and coagulation disorders [3,4,45].Anatomopathological examination of lungs from patients with COVID-19 showed the presence of viral inclusion and massive in ammation in endothelial cells [46][47][48].The virus binds to the angiotensin-converting enzyme 2 (ACE-2) receptor, displaying profound tropism for the human vascular endothelium and the lungs [47,48].In amed endothelial cells induce proin ammatory cytokine production, leading to general hyperin ammation with subsequent in ux of activated monocytes, neutrophils, and other immune cells.An increase in blood neutrophils with low lymphocyte count may predict poor outcome.It has been shown that an increase in NLR above 10 is a strong predictor of fatal outcome in critically ill COVID-19 patients [48,49].

Endotheliopathy
Severe COVID-19 has been linked to endotheliopathy and vasculitis, which has been documented in several studies [1-4, 50, 51].Elevated plasma D-dimer concentrations, which are brin degradation fragments, is associated with an increased risk for morbidity and mortality in COVID-19 patients [52,53].The virus possesses a strong a nity for the vascular endothelium, leading to lymphocytic endotheliitis with in ltration of in ammatory cells around the vessels and endothelial apoptotic cell death [54].A rapid increase in the concentration of proin ammatory cytokines, such as IL-6 and TNF-α, reduces the physiological antithrombotic and anti-in ammatory functions of endothelial cells, and triggers the procoagulopathy cascade [55].Hence, extensive in ammation may disturb the crosstalk between the endothelium, platelets and the coagulation system, leading to the formation of clots in the microvascular circulation of several organs, especially the lungs.Xanthohumol inhibits in ammatory-induced endothelial dysregulation, exerting antiangiogenic and antiin ammatory effects via the reduction of NF-κB activity, a well-established angiogenic and in ammatory factor [37,38,56].Interestingly, an experimental study documented that Xn at a dose of 10 mg/kg body weight administered twice daily during 7 days improves blood velocity and reduces the risk of arterial thrombosis, decreasing the incidence of pulmonary embolism [57].Additionally, treatment with Xn does not affect other coagulation factors, prothrombin time (PT), activated partial thrombin time (APTT), or thrombin time (TT), but it insigni cantly inhibited platelet activation and adhesion on collagen-coated surfaces [57].In the present study, we noted a much more profound decrease in D-dimers in the Xn-group compared to the control group.
Additionally, changes in lung CT were also more pronounced in the Xn-group.Therefore, we can speculate that Xn reduces vascular damage and the formation of microarterial thrombosis; however, this hypothesis should be con rmed in further studies.

Limitations
Despite promising ndings, our study has several limitations.First, because of the small number of patients treated with Xn the power of our analysis was signi cantly reduced.Second, our analysis of Xn-related antiin ammatory effects was based on commonly assessed variables.Several experimental studies have documented that Xn reduces many circulating proin ammatory cytokines in different diseases [8, 23-26, 38, 58, 59].Third, we did not analyse blood Xn and its metabolites concentrations.Previous studies showed that Xn is a safe and nontoxic supplementary product; however, its interaction with other anti-in ammatory medications has not been well documented.Additionally, we did not analyse an effect of Xn on blood glucose levels.
Experimental studies have shown that Xn may be favourable for glucose metabolism, and treatment with Xn at a dose of 60 mg/kg body weight per day effectively reduced plasma glucose, total cholesterol and LDLcholesterol concentrations [40,60].A reduction in plasma glucose concentration following Xn administration was only noted in male mice, whereas higher liver concentrations of Xn and its metabolites were found in female mice [60].It has been well established that IL-6 affects glucose homeostasis.Increased IL-6 levels impair insulin action, whereas inhibiting IL-6 improves hepatic insulin sensitivity [61,62].In the present study, the blood glucose concentration was maintained with continuous insulin administration, and the dose of insulin was not analysed.Therefore, we hypothesise that Xn affected glucose metabolism via a decrease in IL-6 concentration; however, this hypothesis should be con rmed in further studies.
Fifth, oestrogen and others sex hormones activity were not monitored.

Figure 1 Chemical
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Figure 2 The
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Figure 3 Evolution
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Figure 4 Evolution
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Figure 5 Sample
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Table 1
Baseline demographic data and comorbidities.NS -not statistically signi cant.* p < 0.05 -differences with baseline in SOFA evaluation, S ** p < 0.01 -differences in SOFA score after the exclusion of patients, who died between days 7 and 28 (Student t-test)

Table 3
day in only 4 patients, and 8 of them required percutaneous tracheostomy due to the necessity of prolonged mechanical ventilation for up to 14 days.PaO 2 /FiO 2 decreased in both groups; however, the changes were more pronounced in the Xn-group (Table2).Evolution of lung injury measured with the high-resolution computed tomography (CT) technique combined with arti cial intelligence software with percentage of the pulmonary parenchyma and affected automatic detection of pathology (emphysema, normal parenchyma, ground glass opacity and consolidation).Percentages are expressed as mean with standard deviation (SD).Baseline -CT performed immediately before admission to the ICU.** p < 0.01, *** p < 0.001 -changes between baseline and control lung pathologies assessed by arti cial intelligence software, † p < 0.05, † † † p < 0.001 -differences between lung pathologies observed in the Xn and control groups.
Importantly, Xn presents oestrogen activity by increasing the levels of 8-prenylnarigenin, which strongly reduces the in ammatory response and proin ammatory cytokine release[60, 63, 64].Additionally, 8-prenylnarigenin also shows anti-in ammatory and vascular-protective properties, which could have had a signi cant impact on our patients[64].Sex steroids are potent immune modulators and suppress the production of proin ammatory cytokines, such as IL-6, IL-1β and TNF-α[65].An experimental study showed a reduction in IL-6 production following oestrogen administration, and clinical observation documented a negative correlation between plasma oestrogen concentration and lung functionality in COVID-19 patients [66, 67].Another clinical observation documented a signi cantly increased mortality rate and severe respiratory failure in males compared with females[68].Oestrogen supplementation was also associated with a decreased risk of death in postmenopausal women[69].In the present study, the numbers of males and females were comparable in the studied group.However, only one woman died in the Xn group, and two died in the control group.Therefore, we hypothesise that the Xn-related increase in the oestrogen concentration might play a role in outcome because the limited number of patients and lack of hormone control preclude drawing such a conclusion.In the present study, we con rmed the bene cial effects of adjuvant therapy with Xn in critically ill COVID-19 patients requiring mechanical ventilation.Based on our ndings, we hypothesise that Xn may also improve the clinical course of COVID-19 in patients with only slight symptoms and may reduce the risk for developing severe respiratory failure, needing mechanical ventilation, however this hypothesis must be con rmed in further studies.ConclusionsXanthohumol appears to be a promising adjuvant treatment for COVID-19 patients with severe respiratory failure who require mechanical ventilation.Treatment with Xn improved the clinical course and reduced the severity of the in ammatory response and mortality rate.Further studies in a large cohort of patients are needed to con rm these ndings.
Declarations Ethical Approval and Consent to participateThis study was conducted in accordance with the Declaration of Helsinki and applicable regulatory requirements, and was approved by the Institutional Review Board and the Bioethics Committee of Medical University at Lublin, Poland (KE-0254/201/2020).Informed consent was obtained from all patients.Additionally, legal representatives were informed about the main purpose of this study.Consent for publicationFigures Page 19/24