Potential Effects of Nigella Sativa on T Lymphocytes and Inflammatory Cytokine Profile in Pediatric Systemic Lupus Erythematosus: A Randomized Controlled Trial
Phase: NA
Conditions: SLE (Systemic Lupus)
Sponsor: University of Brawijaya

 
 
 
 
 
 
Title: 
Effects of Nigella Sativa on T Cells, Cytokine Profile, and 
SLEDAI Score in Pediatric SLE Patients 
NCT number: 
NCT ID not yet assigned 
Document date: 
31 July 2024 

Objective(s) of the Study: 
The aim of this study was to determine the immunomodulating effect of N. sativa on T 
lymphocyte cell activity, various inflammatory cytokines, and SLEDAI Score in paediatric 
SLE patients. 
Study design : 
This is a clinical trial study, pre and post test control group design, that has received 
permission from the ethics committee of Health Research Ethics Commision General 
Hospital Dr. Saiful Anwar Malang with number (400/089/K3/302/2021). The research design 
is single blind and uses a placebo group as the control.  
Methods: 
A total of 32 SLE paediatric patients were randomly divided into two groups, the N. sativa oil 
treatment group and the placebo group. Each patient consumed 1 gram of N. sativa oil 
capsule or 1 gram of starch capsule daily for 8 weeks. N. sativa oil capsules were purchased 
from CV Rizki Abadi, Tuban, East Java, Indonesia, (POM TR.183314171). Standard therapy 
in all patients was Prednisone, Hydroxychloroquine, and Mycophenolate acid. Every week, 
the research team monitored the patients by phone to determine the progress of the 
patients' condition and any possible side effects. 
Research Procedures 
1. Blood Collection 
Blood samples were taken twice, at the beginning of the study and at the end of the 
treatment at the RSSA Clinical Pathology Laboratory by laboratory staff. The volume 
of blood taken in each research sample was 8 ml. Furthermore, blood samples were 
made serum through a centrifugation process at 3000 rpm for 5 minutes, and stored 
at 4°C for measurement of the patient's immunological profile. 
 
2. Measurement of T lymphocyte cell profile  
From the peripheral blood samples obtained earlier, PBMC isolation was carried out. 
PBMCs were isolated using Ficoll gradient density centrifugation. The PBMC 
suspension was incubated overnight at 37°C in RPMI 1640 media with 100 U/mL 
penicillin and streptomycin, 2 mM glutamine, and 10% FBS. The PBMCs were 
collected, washed, and stained with fluorescein isothiocyanate-conjugated anti-
human CD4. After surface labeling, cells were stained with phycoerythrin (PE)-
conjugated anti-human IFN-γ to identify Th1. Th2 detection was carried out using PE-
conjugated anti-human IL-4. Treg detection was performed using CD25+Foxp3-PE 
antibodies. Th17 identification was carried out using CD4+IL-17A-PE antibodies. All 
antibodies were using brands from BD Bioscience (San Jose, US). 
 
Afterwards, the analysis was carried out using a BD FACS Calibur flow cytometer 
(Becton Dickinson, USA). The cell population percentages were organized and 
analyzed using the CELL Quest pro software application. 

3. Measurement of cytokine levels 
Serum levels of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-17, and IL-10 were measured at 
both baseline and endpoint of the trial. They were measured using commercially 
available cytokine CBA (Cytometric Bead Array) flow cytometry kits, which can 
measure multiple analytes at the same time. All antibodies were using brands from 
Becton Dickinson (US) and the software to analyse it was CELL Quest Pro Software 
from Becton Dickinson (New Jersey, USA). 
 
4. Scoring SLEDAI 
SLE disease activity was measured using SLEDAI 2K. The data required are clinical 
manifestations, including arthritis, fever, elevated dsDNA, and other conditions such 
as photosensitivity and arthralgia. Each clinical manifestation was given a score, and 
the scores were then summed to obtain the total SLEDAI 2K score. 
Ethical clearance: 
Please find attached 
Statistical analysis 
Data for each patient were collected in the form of a data collection sheet. Numerical data 
were analysed by Independent T Test if the data were normally distributed or by Mann-
Whitney test if the data were not normally distributed. For pre and post treatment, each 
group (N. sativa treatment and placebo), if normally distributed using Paired T Test, but if not 
normally distributed using Wilcoxone test. Statistical tests were considered meaningful if the 
p value was ≤ 0.05. All calculations were performed with the help of SPSS for Windows 25.0 
software.