Artemisinin and Lignans Extracts: Innovative Adjuncts to Standard COVID-19 Treatment Protocols.

2.1 Patients

This Randomized Controlled Clinical Trial (RCT) consist of 180 patients diagnosed with COVID-19, both male & female, their age between 20-70 years old, were involved in this study from private clinics within the Al-Ilam, Al-Turath, and Al-Ma’alif areas, as well as near the Darweesh intersection, Al-Karkh district, Baghdad, Iraq, where the participating outpatient clinics are located. Pregnant women and patients with severe hepatic or renal impairment were excluded. Inclusion criteria included confirmed SARS-CoV-2 infection via RT-PCR, presence of mild-to-severe symptoms, and willingness to provide informed consent.

Patient classification was based on the World Health Organization (WHO) clinical severity criteria for COVID-19, which categorize patients into mild, moderate, and severe cases according to clinical presentation, oxygen saturation levels, and comorbidity status.¹³

Group 1 (Mild Symptoms): Consist of 60 patients showing mild symptoms, such as low-grade fever, a mild cough, and fatigue.

Group 2 (Moderate Symptoms): Consist of 60 patients exhibiting more severe symptoms, including persistent fever, significant fatigue, and dyspnea without hypoxia.

Group 3 (With Coexisting Diseases): Consist of 60 patients presenting with severe COVID-19 symptoms accompanied by underlying comorbidities such as diabetes mellitus, hypertension, or asthma. Each of the three main groups will be divided into two subgroups:

Written informed consent will be requested from each participant, and the study will be authorized by the appropriate ethics committee in accordance with the Declaration of Helsinki's tenets.

2.2 Ethical approval

The study was approved by the Institutional Review Board of [College of Pharmacy, Al-Bayan University], approval no. [ALCOP/IRB/2022/147], dated [10-Sep-2022]. All participants signed informed consent forms prior to enrolment.

2.3 Method of Artemisinin extraction

Artemisia annua aerial parts and Linum usitatissimum (flaxseed) were purchased from certified local herbal suppliers in Baghdad. Dried aerial parts of Artemisia annua were harvested and air-dried under shaded conditions to preserve bioactive compounds. The dried plant material was finely powdered using an electric grinder. A total of 10 kilograms of the powder were extracted using ethanol (95%) in a Soxhlet apparatus for 6 hours. The ethanol extracts were then evaporated under reduced pressure at 40°C using a rotary evaporator to yield a concentrated artemisinin extract. The crude extract was purified by recrystallization using a hexane and ethyl acetate mixture (1:1, v/v) resulting in purified artemisinin extracts having a concentration of 1.5% w/w. using a sterile amber glass container to store the purified extracts at 4°C until further use.^14,15

2.4 Method of Lignans Extraction

Mature Linum usitatissimum seeds were thoroughly cleaned and finely ground into powder. A total of 5 kilograms of the powdered seeds underwent extraction using 70% ethanol (v/v) at 60°C for 4 hours with continuous stirring. After passing through Whatman No. 1 filter paper, the resultant extract was concentrated at 50°C under reduced pressure using a rotary evaporator. Lignans were separated by liquid-liquid partitioning using hexane and ethyl acetate (1:1, v/v). A pure lignan extract with a three percent w/w lignans content was produced by purifying the ethyl acetate fraction using silica gel column chromatography. Finally, the extract was stored in a sterile, dark jar at 4°C to ensure stability.¹⁶

2.5 Preparation of mixed extract

The pure lignan and artemisinin extracts were combined in a 2:1 weight ratio. To achieve uniformity, 3.33 grams of the lignans extract (3% w/w lignans) and 6.67 grams of the artemisinin extract (1.5% w/w artemisinin) were carefully mixed for every ten grams of the final formulation using a vortex mixer. The resulting mixture was then stored in sterile, sealed containers at 4°C to preserve its stability until consumption. The final dosage form of the combined extract was a dry powder that was administered orally in hard gelatin capsules.

2.6 Intervention protocol

All patients in the intervention groups received 10 g of the mixed extracts for 14 days through the oral route, together with COVID-19 standard therapy. Patients in the control groups received only the standard clinical therapy, that involve supportive therapy, steroids, & antiviral medication.

2.7 Parameters measured

Primary outcomes: Recovery rate, symptom resolution time, viral clearance (RT-PCR).

Secondary outcomes: Hospitalization duration, adverse effects. Clinical evaluations included vital signs, chest X-ray, renal function tests, and blood glucose monitoring.

2.8 Statistical analysis

Data will be processed & analyzed utilizing SPSS version 22 for statistical significance. The following statistical methods will be applied; Chi-square test will be utilized to compare the recovery rates between the intervention and control groups across all severity levels. One-way Analysis of Variance (ANOVA) will be performed to assess differences in continuous variables, such as symptom duration and time to viral clearance, between the two groups. T-tests will be used to compare the mean recovery times between the intervention and control subgroups. Kaplan-Meier survival analysis will be conducted to evaluate the time to recovery and viral clearance for both groups.

3.1 Recovery rates

According to the results of this study, the intervention group showed significant improvements in recovery rates. The recovery rate was 95% in the intervention group compared to 80% in the control group for the first group (mild symptoms), 85% in the intervention group versus 65% in the control group for the second group (moderate symptoms), and 70% in the intervention group compared to 50% in control group for the third group (patients with coexisting diseases), as illustrated in Table 1. This study was conducted over a 12-month period, including patient recruitment, intervention, & the follow-up phase.

3.2 Symptom resolution time

The mean time to complete symptom resolution is shorter in the intervention group, with an average of 7 ± 2 days compared to 10 ± 3 days in the control group.

3.3 Viral clearance

Compared to 60% of patients in the control group, 80% of patients in the intervention group tested negative by RT-PCR within a period of ten days, indicating a quicker viral clearance in this group.

3.4 Hospitalization

When compared to the control groups, the average hospital stays for patients who needed to be hospitalized in the intervention groups decreased by 2.5 days, suggesting a substantial benefit from the intervention.

3.5 Safety profile

There were no serious side effects noted during this investigation. Headaches (10%) and nausea (8%) were among the mild side effects that were observed, but they went away on their own in 24 hours without the need for further treatment.

4.1 Recovery rates and symptom resolution

According to our research, group 1's recovery rate significantly improved (95% in the intervention group compared to 80% in the control), suggesting a substantial increase in disease resolution. This finding is consistent with the study of Nair et al., that reported the extracts of Artemisia annua decrease cytokine production & viral replication, thus improving the clinical outcomes.¹⁷ Other study done by Nie et al., & Jassim RA, et al., demonstrate artemisinin adjunct treatment improve the viral clearance & decrease the severity of the symptoms in COVID-19 infected cells.^18,19 The efficacy of the combined extracts is further reinforced by shorting the mean symptoms resolution time (7 ± 2 days in the intervention group vs. 10 ± 3 days in the control). This finding aligns with the study done by Shi et al., that had proven use of artemisinin extract would reduce the duration of COVID-19 symptoms through its antiviral & immunomodulatory characteristics.²⁰

4.2 Viral clearance

In this RCT, 80% of intervention patients were RT-PCR negative by day 10 days versus 60% in the control group with faster viral clearance. This result is in line with the findings of Cao et al., who demonstrated the strong inhibitory effect of artemisinin and its derivatives against SARS-CoV-2 in vitro.²¹ Additionally, Zhou et al., observed that artesunate had a strong blocking impact on viral entrance and reproduction, which resulted in the virus being cleared quickly.²²

4.3 Hospitalization duration

The potential of artemisinin and lignans in treating severe COVID-19 cases is demonstrated by the average 2.5-day decrease in hospital stays among patients in the intervention group. This result is in line with the findings of Dolivo et al., who found that by lessening the severity of cytokine storms and lowering lung inflammation, artesunate treatment considerably reduced hospital stays and improved clinical outcomes.²³

4.4 Safety profile

The combination extract therapy appears to be well tolerated based on the minor and temporary side effects (headaches in 10% of patients and nausea in 8%). This is in line with research by Farmanpour-Kalalagh K et al., who examined the safety profile of artemisinin-based therapies and discovered no serious side effects in COVID-19 patients.²⁴

4.5 Comparison with standard treatment protocols

Antiviral medications, corticosteroids, and supportive care were the main COVID-19 treatment regimens employed in the control group. These therapies have limits, especially in severe cases, even if they have demonstrated efficacy in treating symptoms. Artemisinin and lignans seemed to enhance recovery outcomes due to their combination of antiviral and immunomodulatory qualities. Hunt et al., 's study, which demonstrated that extracts from Artemisia annua not only reduce viral loads but also modify immune responses to prevent excessive inflammation, is supported by this.²⁵

4.6 Limitations

The fairly short monitoring time and the absence of people who were pregnant or had children are two of the limitations. The research study was conducted on a single site and had a moderate sample size, which would have limited the applicability of the findings. Statistical analysis of biochemical and immunological markers, such as cytokine profiles and inflammatory mediators, may have provided a deeper knowledge of the mechanisms of action. Additionally, the study did not evaluate the pharmacokinetic properties or extended bioavailability of the oral capsule formulation. Future multicenter studies with larger and more diverse populations are required to validate these findings and assess long-term safety and efficacy.