METHODOLOGY/PRINCIPAL FINDINGS: The cytotoxic effect of thymoquinone was assessed using an MTT assay, while the inhibitory effect of thymoquinone on murine WEHI-3 cell growth was due to the induction of apoptosis, as evidenced by chromatin condensation dye, Hoechst 33342 and acridine orange/propidium iodide fluorescent staining. In addition, Annexin V staining for early apoptosis was performed using flowcytometric analysis. Apoptosis was found to be associated with the cell cycle arrest at the S phase. Expression of Bax, Bcl2 and HSP 70 proteins were observed by western blotting. The effects of thymoquinone on BALB/c mice injected with WEHI-3 cells were indicated by the decrease in the body, spleen and liver weights of the animal, as compared to the control.
CONCLUSION: Thymoquinone promoted natural killer cell activities. This compound showed high toxicity against WEHI-3 cell line which was confirmed by an increase of the early apoptosis, followed by up-regulation of the anti-apoptotic protein, Bcl2, and down-regulation of the apoptotic protein, Bax. On the other hand, high reduction of the spleen and liver weight, and significant histopathology study of spleen and liver confirmed that thymoquinone inhibited WEHI-3 growth in the BALB/c mice. Results from this study highlight the potential of thymoquinone to be developed as an anti-leukemic agent.
METHODS AND RESULTS: TQRF was extracted from N. sativa seeds using supercritical fluid extraction. The regulatory effects of TQRF at 80 microg/ml and TQ at 2 microg/ml on LDLR and HMGCR gene expression were investigated in HepG2 cells using quantitative real-time PCR. The TQ content in TQRF was 2.77% (w/w) and was obtained at a temperature of 40 degrees C and a pressure of 600 bar. Treatment of cells with TQRF and TQ resulted in a 7- and 2-fold upregulation of LDLR mRNA level, respectively, compared with untreated cells. The mRNA level of HMGCR was downregulated by 71 and 12%, respectively, compared with untreated cells.
CONCLUSION: TQRF and TQ regulated genes involved in cholesterol metabolism by two mechanisms, the uptake of low-density lipoprotein cholesterol via the upregulation of the LDLR gene and inhibition of cholesterol synthesis via the suppression of the HMGCR gene.
TRIAL DESIGN: The study is a randomized, placebo-controlled, adaptive clinical trial with parallel group design, superiority framework with an allocation ratio of 1:1 among experimental (HNS) and placebo group. An interim analysis will be done when half of the patients have been recruited to evaluate the need to adapt sample size, efficacy, and futility of the trial.
PARTICIPANTS: All asymptomatic patients with hospital or community based COVID-19 exposure will be screened if they have had 4 days exposure to a confirmed case. Non-pregnant adults with significant exposure level will be enrolled in the study High-risk exposure (<6 feet distance for >10min without face protection) Moderate exposure (<6 feet distance for >10min with face protection) Subjects with acute or chronic infection, COVID-19 vaccinated, and allergy to HNS will be excluded from the study. Recruitment will be done at Shaikh Zayed Post-Graduate Medical Institute, Ali Clinic and Doctors Lounge in Lahore (Pakistan).
INTERVENTION AND COMPARATOR: In this clinical study, patients will receive either raw natural honey (0.5 g) and encapsulated organic Nigella sativa seeds (40 mg) per kg body weight per day or empty capsule with and 30 ml of 5% dextrose water as a placebo for 14 days. Both the natural products will be certified for standardization by Government College University (Botany department). Furthermore, each patient will be given standard care therapy according to version 3.0 of the COVID-19 clinical management guidelines by the Ministry of National Health Services of Pakistan.
MAIN OUTCOMES: Primary outcome will be Incidence of COVID-19 cases within 14 days of randomisation. Secondary endpoints include incidence of COVID-19-related symptoms, hospitalizations, and deaths along with the severity of COVID-19-related symptoms till 14th day of randomization.
RANDOMISATION: Participants will be randomized into experimental and control groups (1:1 allocation ratio) via the lottery method. There will be stratification based on high risk and moderate risk exposure.
BLINDING (MASKING): Quadruple blinding will be ensured for the participants, care providers and outcome accessors. Data analysts will also be blinded to avoid conflict of interest. Site principal investigator will be responsible for ensuring masking.
NUMBERS TO BE RANDOMISED (SAMPLE SIZE): 1000 participants will be enrolled in the study with 1:1 allocation.
TRIAL STATUS: The final protocol version 1.4 was approved by institutional review board of Shaikh Zayed Post-Graduate Medical Complex on February 15, 2021. The trial recruitment was started on March 05, 2021, with a trial completion date of February 15, 2022.
TRIAL REGISTRATION: Clinical trial was registered on February 23, 2021, www.clinicaltrials.gov with registration ID NCT04767087 .
FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). With the intention of expediting dissemination of this trial, the conventional formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines.