Sunday, March 3, 2024

Utilizing the sublingual form of squalene in COVID-19 patients: a randomized clinical trial – Scientific Reports


Potassium hydroxide (KOH), FeCl36H2O (97%), FeCl24H2O (99%), acetone, ethanol, hexane, ammonium hydroxide (27–30%), Tween 80, and Span 80 were purchased from Merck Company (Darmstadt Merck, Germany). Pumpkin seed oil was procured from Zarband Company, Iran. Standard squalene was purchased from Sigma Aldrich Company for Gas Chromatography (GC) analysis.


Squalene extraction

SQ is naturally widespread in animals, plants, fungi, and bacteria22. The primary source of SQ is shark liver oil. SQ extraction from this source is restricted by animal protection regulations23. SQ is also extracted from plant sources24.

Here, SQ extraction from pumpkin seed oil was performed according to our previous studies25,26,27. In summary, pumpkin seed oil was applied as the main source of SQ extraction with a novel method. At first, fatty acids (FAs) adsorption on the magnetic iron oxide nanoparticles was performed. Then, the solvent extraction method was applied to separate SQ from other components. Briefly, a coprecipitation reaction was used to prepare magnetic nanoparticles (MNPs) in the form of iron oxide (Fe3O4). Then, the saponification reaction was applied for pumpkin seed oil. Pumpkin seed oil and ethanolic KOH were poured in a container. The container was heated at 80 °C for 1 h. To evaporate all the solvent, the solution was heated in an oven for 4 h. Finally, a soap‐like dried sample was obtained. MNPs were added to this sample that was dissolved in water. This sample was placed in an oven at 130 °C for FAs adsorption on the MNPs surface. Simultaneously, water was removed from this sample. The dried sample was washed with acetone to separate extracted SQ, and MNPs were separated with a magnet. Afterward, hexane, distilled water, and ethanol were added to the remaining solvent and placed in a decanter to separate hexane containing SQ from other materials. Finally, hexane was removed under argon, and SQ was extracted.

Microemulsion preparation

For the preparation of the sublingual form of SQ, a microemulsion formulation with the SQ concentration of 10 mg/ml was applied. The titration approach was used to create a microemulsion. For this purpose, a mixture of hydrophilic and hydrophobic nonionic surfactants was used. Surfactant components were mixed in a mass ratio of 9:1 (Tween80:Span80), SQ was added to the surfactant mixture in a mass ratio of 1:5 (SQ: surfactants), and double distilled water was gently added under moderate agitation (magnetic stirring). The final formulation for sublingual usage includes 10 mg SQ, 45 mg Tween 80, 5 mg Span 80, and 1 ml water.


The purity of extracted squalene was measured using GC‐FID analysis (6890 Series GC system) was applied. A dynamic light scattering analyzer (DLS, CORDOUAN) was used to assess the hydrodynamic diameter of a microemulsion sample in deionized water. TEM (LEO-Germany) analysis was used to assess the morphology and size of the sample. The zeta potential of the microemulsion sample was calculated using a zeta meter (Zeta CAD).


Study population Patients were selected from adults (age > 18 years) admitted to the emergency and infectious department of Shohada Hospital, Ghaen (Iran) between November 2021 and January 2022. Cases with the signs of diagnostic and symptoms of COVID-19, including fever, acute loss of sense and smell, fatigue, dry cough, lymphocytopenia, elevated CRP with or without contact with verified cases of COVID-19, or patients with individual characteristics in HRCT, including ground-glass opacities, bilateral abnormalities, vascular enlargement, lower lobe involvement, and posterior predilection (1), or who confirmed with a real-time PCR test, eligible patients who had respiratory symptoms (including dyspnea, chest pain, or discomfort), oral temperature > 38 °C and SpO2 < 93%, have entered the study.

The sample size was calculated according to the findings of the previous study27, 300 for each study group, using the following formula (α = 0.01 and β = 0.95):

$${\text{N}} = \left( {{\text{Z}}_{{{1} – \alpha /{2}}} + {\text{Z}}_{{{1} – \beta }} } \right)^{{2}} *{\text{P}}_{{1}} \left( {{1} – {\text{P}}_{{1}} } \right) + {\text{P2}}\left( {{1} – {\text{P}}_{{2}} } \right)/\left( {{\text{P}}_{{1}} – {\text{P}}_{{2}} } \right)^{{2}}$$

Demographic and baseline characteristics, including age, sex, body mass index (BMI), history of diabetes mellitus (DM), hypertension (HTN), IHD (ischemic heart disease), chronic heart disease (CKD), immunosuppressive disease, chronic respiratory disease, the number of vaccination doses, and vaccine type, were recorded.

Exclusion criteria were a history of mental retardation, being directly admitted to the ICU, pregnancy, breastfeeding, a suspected or confirmed history of alcohol or substance use disorder, and having participated in other drug trials in the past month.

After obtaining written informed consent, eligible patients were divided into two groups of standard treatment (control group, N = 301) and SQ plus standard treatment (intervention group, N = 301) using a web-based randomization tool and an allocation concealment mechanism by staff whom were not involved in the study.

Treatment protocol Standard treatment included oxygen therapy, dexamethasone 8 mg daily or prednisolone 40 mg daily for 10 days, remdesivir 200 mg for the first day of admission and then 100 mg daily for 5 days, and heparin 5000IU subcutaneous TDS (for BMI ≥ 40, 7500 IU SC TDS) or enoxaparin 40 mg SC once daily (for BMI ≥ 40, 40 mg SC BID). In the case group, standard treatment plus SQ dosage was applied. 5 drops of sublingually SQ was applied every 4 h for up to 5 days.

Study variables Vital signs, clinical characteristics, and probable adverse effects of squalene were collected and recorded continuously during hospital admission. Symptom evaluators were blinded.

Follow-up Patients were followed up for 30 days after discharge from the hospital in terms of the need for re-hospitalization and mortality rate.

Figure 1 shows a schematic of patient screening, enrollment, randomization, and clinical protocol that is applied in this study.

Figure 1

Screening, enrollment, randomization, and clinical protocol.

Statistical analysis

SPSS 18 was used to analyze the data. The data analyst was unfamiliar with the study groups. Quantitative factors were expressed as median standard deviation and qualitative variables as number and percent. The Chi-square and T-test was employed to analyze the difference between study groups in terms of baseline characteristics, different types of treatment, and outcomes in the following period. A nonparametric analytical tool (Binary logistic regression) was employed to determine the critical influential factors related to death, re-hospitalization, and duration of hospitalization due to COVID-19. A P value of 0.05 was deemed significant.


The study protocol was approved by the MUMS ethics committee (IR.MUMS.REC.1400.143) and IRCT (IRCT20200927048848N3, date: 10/10/2021). We confirm that all methods were performed in accordance with the relevant guidelines and regulations according to the Helsinki Declaration. Informed consent was obtained from all participants and/or their legal guardians.

Problems anticipated

Following participants 1 month after admission was our major challenge in this study. For dealing with this problem, we recorded all the participant addresses and also their phone number as well as their cell phone. To confirm the death of participants, we also recorded their national number and checked them in the death registry database.

Limitations of the study

One of the limitations of this study was the presence of intervening factors such as age, co-morbidities of the patients and patients’ personal medications, which could influence the relationship between SQ use and death. Another limitation of this study was the impossibility of subgroup analysis for all outcomes due to time constraints and a lack of research manpower (this research was conducted during the peak of the Iran’s COVID-19 crisis), so only the outcome of the need for oxygen therapy (a clinical outcome) was subjected to subgroup analysis. As a result, subgroup analysis was not performed based on characteristics such as SQ treatment duration and prescription dose. Furthermore, conducting the study in one center and the possibility of recall error were other limitations of this study.

However, critically ill COVID-19 patients had lower chance of receiving SQ treatment during hospitalization, this bias could account for the link between not utilizing SQ and poor outcomes.

Duration of the project

Squalene extraction and characterization: September–October 2021 (2 months).

Clinical trial sampling and following-up: November 2021-January 2022 (3 months).

Data entry and statistical analysis: February–March 2022 (2 months).

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