Therapeutic potential of Nigella sativa extract against inflammatory markers interleukin-1&szlig; (IL-1&szlig;) and tumor necrosis factor-α (TNF-α) in rat models with high fat diet

Muhammad Ridwan; Nurkhalis Nurkhalis; Fauzul Husna; Dhanang Ali Yafi; Abdurrohman Izzuddin

doi:10.12688/f1000research.159199.1

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Research Article

Therapeutic potential of Nigella sativa extract against inflammatory markers interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in rat models with high fat diet

[version 1; peer review: 2 approved with reservations]

Muhammad Ridwan ^1,2, Nurkhalis Nurkhalis^1,2, Fauzul Husna³, Dhanang Ali Yafi^1,2, Abdurrohman Izzuddin^1,2

Muhammad Ridwan ^1,2, Nurkhalis Nurkhalis^1,2, [...] Fauzul Husna³, Dhanang Ali Yafi^1,2, Abdurrohman Izzuddin^1,2

PUBLISHED 06 Feb 2025

Author details Author details

¹ Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia
² Department of Cardiology and Vascular Medicine, Regional General Hospital Dr Zainoel Abidin, Banda Aceh, Aceh, 23126, Indonesia
³ Department of Pharmacology, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia

Muhammad Ridwan
Roles: Conceptualization, Investigation, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Nurkhalis Nurkhalis
Roles: Methodology, Project Administration

Fauzul Husna
Roles: Methodology, Project Administration

Dhanang Ali Yafi
Roles: Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Abdurrohman Izzuddin
Roles: Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

Nigella sativa L., known as black cumin, is thought to possess anti-inflammatory properties that may help alleviate related conditions. This study examined the effects of black cumin extract on levels of Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α), both inflammatory markers.

Methods

The experimental design included a control group used solely for post-testing. Five groups of Wistar rats were studied: a negative control group (N), a dyslipidemia group as a positive control (P), a dyslipidemia group given black cumin (P1), a dyslipidemia group treated with atorvastatin (P2), and a dyslipidemia group receiving both atorvastatin and black cumin (P3).IL-1β and TNF-α levels were measured using ELISA, and statistical analysis was conducted using ANOVA followed by the Duncan test.

Results

After treatment, the average IL-1β levels were 38.26 pg/mL (N), 102.16 pg/mL (P), 57.05 pg/mL (P1), 29.16 pg/mL (P2), and 54.06 pg/mL (P3). The Duncan test indicated no significant differences in IL-1β levels among groups N, P2, and P3 (p>0.05), while group P exhibited the highest IL-1β levels, significantly different from the others. For TNF-α, average levels post-treatment were 30.42 pg/mL (N), 22.02 pg/mL (P), 27.25 pg/mL (P1), 16.33 pg/mL (P2), and 13.29 pg/mL (P3). The Duncan test showed that group P3 had the lowest TNF-α levels, which were not significantly different from P2 (p>0.05) but significantly different from groups P, P1, and N (p<0.05).

Conclusions

In conclusion, black cumin extract effectively reduces IL-1β levels in high-fat diet rat models, while the combination of atorvastatin and black cumin extract yields the most significant reduction in TNF-α levels.

Keywords

Nigella sativa, Atorvastatin, Interleukin-1ß, Tumor Necrosis Factor-α, High-Fat Diet, Atherosclerosis

Corresponding author: Muhammad Ridwan

Competing interests: No competing interests were disclosed.

Grant information: This research was partly funded by Associate Professor research scheme from Syiah Kuala University, based on Agreement letter no 199/UN11.2.1/PT.01.03/PNBP/2023 (3rd of May 2023).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2025 Ridwan M et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Ridwan M, Nurkhalis N, Husna F et al. Therapeutic potential of Nigella sativa extract against inflammatory markers interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in rat models with high fat diet [version 1; peer review: 2 approved with reservations]. F1000Research 2025, 14:172 (https://doi.org/10.12688/f1000research.159199.1) First published: 06 Feb 2025, 14:172 (https://doi.org/10.12688/f1000research.159199.1) Latest published: 06 Feb 2025, 14:172 (https://doi.org/10.12688/f1000research.159199.1)

Introduction

Obesity is characterized by an abnormal or excessive accumulation of fat that can hinder the maintenance of optimal health. Overweight and obesity have increased substantially in recent decades, reaching epidemic proportions. Globally, at least 2.8 million deaths are linked to obesity or being overweight. While obesity was once primarily associated with high-income countries, it has now become common in low- and middle-income nations as well. According to 2022 statistics, prevalence rates vary significantly, from 12% in the South East Asian Region to 82.8% in the Western Pacific Region.¹ Nearly 40% of the world’s population is currently classified as overweight or obese, with projections indicating a steady increase by 2030.² This trend is also reflected in rising healthcare costs associated with obesity, which vary between countries, accounting for 3% to 21% of medical expenditures. Additionally, obesity is linked to noncommunicable diseases such as diabetes, hypertension, and cardiovascular conditions.¹

Excess macronutrients in adipose tissues can increase inflammatory mediators including interleukin-1b and tumor necrosis factor-α, and decrease adiponectin levels, leading to oxidative stress and pro-inflammatory state.³ The finding that pro-inflammatory cytokines are expressed in obesity was made about thirty years ago. This preliminary research served as a trigger for the currently recognised paradigm, which holds that inflammation can be harmful and that food overload increases inflammation by connecting the immune and metabolic systems.⁴

Inflammation is primarily mediated by the NF-κB and mitogen-activated protein kinase (MAPK) pathways. When these pathways are activated, proinflammatory cytokines including IL-6, IL-8, IL-1, and TNF-α are released, potentially leading to an inflammatory response.^5,6 Tumor necrosis factor (TNF) is one of the traditional, multifunctional pro-inflammatory cytokines; it was the first identified “adipokine” produced by adipose tissue, regulated by obesity, and is believed to contribute to metabolic diseases associated with obesity.⁷ Conversely, the IL-1 family of ligands and receptors has been extensively linked to the pathophysiology of both acute and chronic inflammatory conditions.⁴ Given the significant role of inflammation, recent studies have focused on identifying effective therapeutic strategies that leverage the anti-inflammatory properties of various compounds.⁸

Black cumin (Nigella sativa L.) is recognized for its ability to inhibit inflammation.⁹ Its anti-inflammatory properties, particularly those of Thymoquinone (TQ), are significant pharmacological benefits.¹⁰ Daily supplementation with 2000 mg of black cumin oil over two 8-week periods led to notable reductions in the blood mRNA expression levels of obesity-related pro-inflammatory genes, such as IL-6, IL-1β, and leptin, in overweight or obese women.¹¹ Additionally, long-term use of black cumin for 6 to 12 weeks can significantly decrease body weight and other anthropometric measures.¹² This study aimed to explore these effects by measuring Interleukin-1β (IL-1β) and Tumor Necrosis Factor-alpha (TNF-α) in rat models fed a high-fat diet after administering black cumin. The results were then compared with those from statins, which have shown a beneficial effect on reducing inflammatory markers in patients with metabolic syndrome and related conditions.¹³ The inflammatory markers chosen for this research were intended to evaluate both inflammatory pathways: TNF-α indicates the MAPK pathway, while IL-1β corresponds to the NF-κB pathway.

Methods

Study design

This study is fundamentally experimental in nature and utilized a post-test control group design. Conducted over eight weeks, from 1st August to the end of September 30, 2023. The research took place in the Experimental Animal Laboratory at the Faculty of Veterinary Medicine, Syiah Kuala University. The protocol for this preclinical investigation adhered to the Animal Research: Reporting in vivo Experiments (ARRIVE) Guidelines as set forth by the Institutional Animal Care and Use Committee.

Every rat was given a standard diet and given a week to acclimatise. 6 rats were fed a regular diet during the first stage (2nd–5th week), while 24 rats were provided a high-fat diet. In the second stage (6th–9th week), 24 rats on a high-fat diet were split into 4 groups [positive control (P); dyslipidemia received black cumin (P1); dyslipidemia received atorvastatin (P2); and dyslipidemia received black cumin and atorvastatin (P3)], whereas 6 rats on a standard diet were kept on the diet as a negative control (N). Each week, measurements of each rat’s body weight were made to track their weight gain. After collecting blood samples at the end of week 9 to measure IL-1β and TNF-α levels, the animals were put to sleep by intraperitoneal injection of ketamine at a dose of 15-20 mg/kg, followed by cervical dislocation.

Animals

Male Rattus novergicus strain Wistar white rats (n = 30) at 4 weeks of age, in good health, and weighing between 50 and 100 grammes were utilised in this investigation. The rats were procured from the Universitas Syiah Kuala Faculty of Veterinary Medicine. Every rat was given proper care, kept at a constant temperature of +24^o C, and given unlimited access to food and drink.

Black cumin and high fat diet

The seed of Black cumin came from Surakarta, Central Java, Indonesia, and was recognised at Javaplant Factory by its product number, 2065J91N. The maceration method was used to carry out the extraction, using three 24-hour cycles of 95% ethanol solvent and one 24-hour cycle of water solvent. Crude oil with residual ethanol solvent NMT 5000 ppm was the final extract form. Phytochemical analysis was carried out at the Chemistry Education Laboratory, Faculty of Teacher Training and Education, Syiah Kuala University, Banda Aceh, using the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2’-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) methods. The purpose of this study was to detect alkaloids, flavonoids, polyphenols, tannins, quinones, saponins, triterpenoids, and steroids.

Vitamin D3, 0.2% cholic acid, 2% egg yolk, 5% goat fat, and 92.8% conventional diet are all present in a high-fat diet. To make this feed, 1000 g of rat food in the form of corn rice is combined with 50 g of egg yolk and 100 g of goat fat. To get the yolk from cooked eggs, first heat goat fat until it melts. Next, 1000 g of corn rice is combined with egg yolk and goat fat. Every rat received 20 mg of diet every day.¹⁴

Data analysis

The data’s normality was determined using the Shapiro-Wilk test. The data analysis used a one-way ANOVA test (α=0.05) to compare IL-1β and TNF-α levels between groups. SPSS Statistics version 26 for Windows was applied to conduct this statistical test. To identify significant changes across groups, Duncan’s Test post hoc tests (α=0.05) were used.

Results

General descriptions

During the research, several rats died due to unknown cause, and leaving 5 mice from each treatment group. The rat’s body weight was calculated at the start of the study, week 4 (after giving the high-fat diet and before treatment), weeks 5, 6, 7, and 8. At the end of week 4, there was a significant difference in body weight between the groups given normal feed (N) with groups given atherogenic feed (P, P1, P2, P3). At the end of the 8th week, the average body weight of the rats starting from the highest, was the positive control group/P1 (286.6 gr), negative control/N (264.8 gr), atorvastatin group/P2 (233.6 gr), Nigella Sativa group/P1 (229.6 gr), and the Nigella Sativa and Atorvastatin groups/P3 (188.8 gr). Table 1 shows the rats’ average body weights throughout the research.

Table 1. Average body weight of rats during the study.

Groups	Body weight (gr) on week
Groups	0	4	5	6	7	8
N	65.0	198.8	214.6	226.2	248.6	264.8
P	63.6	245.4	262.6	261.0	276.4	286.6
P1	64.6	231.8	219.2	216.0	218.2	229.6
P2	66.2	236.2	233.6	224.4	229.8	233.6
P3	64.2	238.4	203.8	196.2	195.2	188.8
N	65.0	198.8	214.6	226.2	248.6	264.8

Effect of giving black cumin extract on IL-1β and TNF-α levels

The average levels of IL-1β after treatment were 38.26 pg/mL (N), 102.16 pg/mL (P), 57.05 pg/mL (P1), 29.16 pg/mL (P2), and 54.06 pg/mL (P3). The levels of IL-1β varied significantly between the groups (p=0.000). The results of further tests (post hoc) using the Duncan Test showed that there were no significant differences in IL-1β levels between N, P2, and P3 groups (p>0.05). The P group was the group with the highest IL-1β levels, and was significantly different from the IL-1β levels in the other groups ( Table 2 and Figure 1).

Table 2. Mean distribution of IL-1β according to treatments (pg/mL).

Groups	Mean	SD	95% CI	P value
N ^ab	38.26	5.47	32.53 – 44.00	0.000
P ^c	102.16	25.13	75.79 – 128.54
P1 ^b	57.05	17.51	38.68 – 75.42
P2 ^a	29.16	8.76	19.97 – 38.35
P3 ^ab	54.06	21.43	21.19 – 66.17

Figure 1. Mean distribution of IL 1 according to treatments.

Different superscripts indicate significant differences.

The average levels of TNF-α after treatment were 30.42 pg/mL (N), 22.02 pg/mL (P), 27.25 pg/mL (P1), 16.33 pg/mL (P2), and 13.29 pg/mL (P3). TNF-α levels vary significantly between groups. The results of further tests (post hoc) using the Duncan Test showed that the lowest levels of TNF-α was in P3 group, and was not significantly different from the P2 group (p>0.05), but were significantly different from P, P1, and N group (p<0.05). The P group did not differ significantly from the P1 group (p>0.05), the P2 group did not differ significantly from the P group (p>0.05), and the P1 group did not differ significantly from the N group (p>0.05) ( Table 3 and Figure 2).

Table 3. Mean distribution of TNF-α according to treatments.

Groups	Mean	SD	95% CI	P value
N ^a	30.42	5.45	24.69 – 36.15	0.000
P ^bc	22.02	5.45	16.30 – 27.74
P1 ^ab	27.25	5.21	21.78 – 32.73
P2 ^cd	16.33	3.27	7.93 – 24.74
P3 ^d	13.29	5.23	7.80 – 18.78

Figure 2. Mean distribution of TNF according to treatments.

Different superscripts indicate significant differences.

Discussion

Nigella sativa L. is rich in bioactive compounds such as thymoquinone (TQ), dithymoquinone, thymol, and thymohydroquinone. It is widely recognized in traditional medical systems, including Unani, Tibb, Ayurveda, and Siddha. The oil and seeds have long been used in a variety of culinary and medical uses. Nigella sativa L. seeds have been widely used to treat a variety of diseases and disorders, and in Islamic literature, they are recognized as one of the most efficient therapeutic agents. Regular use of these seeds is recommended in Tibb-e-Nabwi (Prophetic Medicine).¹⁵ Among the identified compounds, TQ is the most prominent, and its therapeutic benefits are largely attributed to this component.¹⁶ Previous research has demonstrated that TQ possesses several pharmacological properties, including antioxidant, lipid-lowering, and anti-inflammatory effects.^17–20

Severe obesity is defined not just by an excessive buildup of body fat but also by the infiltration of immune cells into adipose tissue and associated inflammation.^1,2 Numerous clinical consequences of obesity, especially type 2 diabetes and atherosclerotic cardiovascular disease, are associated with this low-grade systemic inflammation.^3,4 Substantial evidence highlights the significant role of IL-1β in atherosclerosis.²¹ In patients with atherosclerosis, both IL-1β protein and mRNA levels are markedly elevated compared to those in healthy individuals, and these levels are positively correlated with disease severity.²² Additionally, genetic variations in IL-1 have been associated with coronary heart disease (CHD).²³ Furthermore, increased susceptibility to atherosclerosis, related to clonal hematopoiesis in peripheral blood cells, is partially regulated by the NLRP3/IL-1β pathway.^24,25

The findings of this study are consistent with previous research. TQ treatment decreased the expression of NLRP3, caspase-1, IL-1β, and IL-18 genes, which were increased by a high-cholesterol diet. Following a high-cholesterol diet, TQ supplementation in LDL-R-/- mice lowered NF-κB protein expression.²⁶ Pyroptosis, a form of programmed cell death driven by NLRP3 activation, is linked to the exacerbation of hyperlipidemia. When NLRP3 is activated, it triggers caspase-1, which modifies IL-1 and cleaves the precursors of the inflammatory cytokines IL-1β and IL-18. This mechanism stimulates the production of pro-inflammatory cytokines, which promotes pyroptosis.²⁷

In this study, the administration of Nigella sativa extract was found to effectively reduce TNF-α levels. While significant differences were observed between the P1 and P2 groups, the combination of Nigella sativa and atorvastatin in the P3 group resulted in the lowest TNF-α levels among all samples. The P3 group also showed significant differences from the other treatment groups. The reduced TNF-α levels in the P group may be linked to the obesity present in the treatment samples, as this group had the highest body weight among all treatment groups. Several studies indicate that, in cases of obesity, lower TNF-α levels may arise due to the increased production of TNF-α receptors in adipose tissue. This phenomenon is one of the foundational concepts of the Obesity Paradox.^28,29

In a study involving pediatric patients, Boeck et al. reported that no detectable TNF-α was found in the serum of obese individuals.³⁰ However, another study indicated that obese individuals produce higher amounts of TNF-α protein per mass or per unit of DNA from their fat tissue compared to lean control subjects.³¹ Myeloid cells release TNF-α, a powerful pro-inflammatory cytokine that stimulates MAPK and NF-κB signaling pathways. This leads to the production of additional inflammatory cytokines, including IL-1β and IL-6.³² In rodents, TNF-α is overexpressed in the adipose tissue of obese animals.³³ In humans, TNF-α levels are elevated in the plasma and adipose tissue of those who are obese, with levels decreasing following weight loss.³⁴

By suppressing adipogenic genes such CCAAT/enhancer-binding protein (C/EBP) and peroxisome proliferator-activated receptor gamma (PPARγ), TNF-α is known to prevent preadipocytes from maturing into mature adipocytes. The recruitment of more cells is made possible by this inhibition, which promotes the growth of adipose tissue mass.³⁵ Furthermore, TNF-α inhibits genes linked to lipid absorption and storage after being activated by NF-κB.³⁶ Adipocytes create the hormone adiponectin, which is essential for preserving the peripheral glucose and lipid balance. It also lowers the mRNA levels of this hormone. Additionally, because TNF-α promotes the synthesis of other cytokines, including IL-6, its effect on the immune response is mostly indirect.³⁷

Several TNF-related factors are associated with atherosclerosis and elevated circulating IL-6,³⁸ acute phase proteins like C-reactive protein (CRP),³⁹ fibrinogen,⁴⁰ and triglycerides, total cholesterol (TC), and low-density lipoprotein (LDL) are all increased in this lipid profile, but HDL and the HDL/TC ratio are low. Consequently, TNF-α is an early mediator of the acute phase response, aiding in the recruitment of leukocytes during inflammatory responses and promoting the synthesis of chemokines, IL-6, and CRP.⁴¹

In numerous clinical trials, the effects of Nigella sativa ingestion on biomarkers for oxidative stress and inflammation have been investigated, though the results remain mixed. For instance, a study found that taking 3 g/day of Nigella sativa oil for 8 weeks led to significant reductions in inflammatory markers, specifically hs-CRP and TNF-α, in obese women.⁴² Conversely, research by Hadi et al. indicated that a dosage of 1 g/day of Nigella sativa oil for 8 weeks did not alter TNF-α and SOD levels in patients with type 2 diabetes mellitus.⁴³ Additionally, there is some evidence suggesting that Nigella sativa oil may be more therapeutically effective than the seed form of the plant.¹⁵

Ethical considerations

This study has been ethically assessed by Syiah Kuala University’s Faculty of Veterinary Medicine’s Medical Research Ethics Commission and it was registered on July 31, 2023, under the number 243/KEPH/VII/2023. Rats were put to sleep at the end of the ninth week by cervical dislocation after intraperitoneal injections of xylazine and ketamine. The reporting for this animal study follows the ARRIVE guidelines.

Data availability

Zenodo: ARRIVE checklist for ‘Therapeutic Potential of Nigella sativa Extract against Inflammatory Markers Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) in Rat Models with High Fat Diet’, https://doi.org/10.5281/zenodo.13958154.⁴⁴

Zenodo: Master Data, https://doi.org/10.5281/zenodo.14207758.⁴⁵

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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39. Mendall MA, Patel P, Ballam L, et al.: C Reactive protein and its relation to cardiovascular risk factors: A population based cross sectional study. Br. Med. J. 1996; 312(7038): 1061–1065. PubMed Abstract | Publisher Full Text | Free Full Text
40. Fowkes FGR, Housley E, Rattray A, et al.: Cross-linked fibrin degradation products, progression of peripheral arterial disease, and risk of coronary heart disease. Lancet. 1993; 342(8863): 84–86. PubMed Abstract | Publisher Full Text
41. Krishnaswamy G, Kelley J, Yerra L, et al.: Human endothelium as a source of multifunctional cytokines: Molecular regulation and possible role in human disease. J. Interf. Cytokine Res. 1999; 19(2): 91–104. PubMed Abstract | Publisher Full Text
42. Mahdavi R, Namazi N, Alizadeh M, et al.: Nigella sativa oil with a calorie-restricted diet can improve biomarkers of systemic inflammation in obese women: A randomized double-blind, placebo-controlled clinical trial. J. Clin. Lipidol. 2016; 10(5): 1203–1211. PubMed Abstract | Publisher Full Text
43. Hadi S, Mirmiran P, Daryabeygi-Khotbesara R, et al.: Effect of Nigella sativa oil extract on inflammatory cytokine response and oxidative stress among people with type 2 diabetes mellitus: A randomized, double-blind, placebo controlled trial. Prog. Nutr. 2018; 20: 127–133.
44. Muhammad Ridwan R, Nurkhalis N, Husna F, et al.: Therapeutic Potential of Nigella sativa Extract against Inflammatory Markers Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) in Rat Models with High Fat Diet. Zenodo. 2024. Publisher Full Text
45. Muhammad R, Nurkhalis N, Husna F, et al.: Therapeutic Potential of Nigella sativa Extract against Inflammatory Markers Interleukin-1β (IL-1β) and Tumor Necrosis Factor-α (TNF-α) in Rat Models with High Fat Diet. Zenodo. 2024. Publisher Full Text

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Author details Author details

¹ Department of Cardiology and Vascular Medicine, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia
² Department of Cardiology and Vascular Medicine, Regional General Hospital Dr Zainoel Abidin, Banda Aceh, Aceh, 23126, Indonesia
³ Department of Pharmacology, Faculty of Medicine, Universitas Syiah Kuala, Banda Aceh, Aceh, 23111, Indonesia

Muhammad Ridwan
Roles: Conceptualization, Investigation, Methodology, Project Administration, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Nurkhalis Nurkhalis
Roles: Methodology, Project Administration

Fauzul Husna
Roles: Methodology, Project Administration

Dhanang Ali Yafi
Roles: Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Abdurrohman Izzuddin
Roles: Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research was partly funded by Associate Professor research scheme from Syiah Kuala University, based on Agreement letter no 199/UN11.2.1/PT.01.03/PNBP/2023 (3rd of May 2023).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Ridwan M, Nurkhalis N, Husna F et al. Therapeutic potential of Nigella sativa extract against inflammatory markers interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in rat models with high fat diet [version 1; peer review: 2 approved with reservations]. F1000Research 2025, 14:172 (https://doi.org/10.12688/f1000research.159199.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Reviewer Report 30 Jul 2025

Aisyah Hasyila Jahidin, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, selangor, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.174891.r398634

The manuscript is generally well-prepared; however, the reference list could be updated to more effectively reflect recent developments in this field. At present, only around 30% of the cited works are from the past five years. Given the growing research ... Continue reading

The manuscript is generally well-prepared; however, the reference list could be updated to more effectively reflect recent developments in this field. At present, only around 30% of the cited works are from the past five years. Given the growing research interest in Nigella sativa, inflammation, and high-fat diet models, incorporating more recent literature would strengthen the scientific relevance and contextual grounding of the study.

The overall study design is appropriate and aligns with the research objectives. The inclusion of both negative and positive controls, along with well-structured treatment groups involving Nigella sativa, atorvastatin, and their combination, is commendable. Nonetheless, several methodological aspects require clarification to enhance transparency, reproducibility, and scientific rigour.

Firstly, the study duration is described as eight weeks, yet the timeline detailed in the manuscript, comprising a one-week acclimatisation period followed by phases of diet induction and treatment, appears to span nine weeks in total. The authors are encouraged to clearly delineate the duration and purpose of each experimental phase (acclimatisation, induction, treatment).

Secondly, the daily feed amount is reported as “20 mg/day,” which seems implausibly low for rats and may reflect a unit error. Clarification on this point is necessary.

Thirdly, the dosing information for Nigella sativa extract and atorvastatin is incomplete. The methods section should clearly describe the dose, frequency, route of administration (e.g., via feed or oral gavage), and duration of treatment. This information is particularly important in the combination treatment group (P3), where it should be clarified whether the atorvastatin dose was the same as in the single-treatment group or adjusted accordingly.

It is also unclear whether randomisation was applied during animal group allocation, or whether outcome assessors were blinded. If such practices were not followed, this should be explicitly stated, along with a brief discussion of potential sources of bias.

The statistical analysis is generally acceptable. While Duncan’s post hoc test can be used, it is considered less conservative than alternatives such as Tukey’s HSD or Bonferroni, which are more commonly recommended in recent biomedical research. The authors may wish to justify their choice of post hoc test or acknowledge this as a limitation.

The underlying source data appear to have been provided, which supports reproducibility. However, the manuscript states that some animals died during the experiment, leaving five rats in each treatment group. Yet, the dataset shows six animals per group, with no indication of attrition or exclusions. This discrepancy should be addressed. If animals were excluded, the final group sizes should be updated in both the methods and results sections, and the statistical analysis revised accordingly. If replacements were made or if the earlier statement was inaccurate, this should be corrected to maintain transparency.

With regard to results presentation, several improvements are suggested. First, since body weight was measured across multiple timepoints, a line graph would be more appropriate than a summary table. This would allow readers to more clearly visualise weight trends over time. Additionally, there is a likely error in the text, where the positive control group is incorrectly referred to as P1 instead of P. The data for the negative control group (N) also appear twice in the body weight table. Furthermore, I recommend using “g” for grams, rather than “gr,” which is not standard in scientific writing.

Second, the graphs provided do not include any indicators of statistical significance (e.g., asterisks or superscript letters). Including these on the graphs is important for clarity, enabling readers to interpret key differences without needing to cross-reference tables.

Third, while superscript letters are used in tables to indicate significant differences, there is no legend or footnote explaining their meaning. A simple note stating that groups sharing the same letter are not significantly different (p > 0.05) would improve clarity.

Fourth, all figures should include complete legends specifying the number of animals per group (n), the statistical test applied, and what the error bars represent (e.g., mean ± SD). This would enhance the scientific transparency and clarity of the data.

Finally, in the section reporting TNF-α results, the description includes numerous non-significant pairwise comparisons, which may obscure key findings. It would be clearer and more impactful to highlight only the statistically significant differences, particularly the finding that TNF-α levels were significantly lower in the P3 group compared to N, P, and P1.

The discussion provides useful background on Nigella sativa and its active constituents, particularly thymoquinone, and is well supported by literature. However, the section could be strengthened by more directly integrating and interpreting the study’s own findings, especially regarding IL-1β and TNF-α reductions in the combination treatment group, and comparing them with related studies.

To improve focus and clarity, especially if constrained by word count, the authors may consider condensing some of the general background and detailed mechanistic explanations (e.g., pyroptosis, adipogenesis) that are not directly supported by the data. This would provide more space to highlight novel findings, potential mechanisms, and the significance of combining Nigella sativa with atorvastatin. A more structured discussion separating key findings, biological rationale, comparison with prior studies, and limitations would enhance coherence.

The manuscript currently lacks a dedicated conclusion section. I recommend including a brief concluding paragraph to summarise the primary findings, reinforce their implications, and acknowledge key limitations, such as the lack of blinding, uncertainty in dosing methods, and issues with sample attrition. Including such a section would enhance the completeness and scientific integrity of the manuscript.

Lastly, please ensure consistency in the use of either British or American English throughout the manuscript, in accordance with the journal’s language guidelines.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Pharmacology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 09 Apr 2025

Maha Mahmoud Mohamed, Ain Shams University, Cairo, Cairo Governorate, Egypt

Approved with Reservations

https://doi.org/10.5256/f1000research.174891.r369316

Your Report

The authors describe the use of a 'standard diet' and 'high-fat diet (HFD)' in the methods section. However, the exact composition is not provided. Please clarify the detailed composition of both diets to ensure

Your Report

The authors describe the use of a 'standard diet' and 'high-fat diet (HFD)' in the methods section. However, the exact composition is not provided. Please clarify the detailed composition of both diets to ensure the reproducibility of the experiments. If commercially sourced, provide the manufacturer and diet reference. If custom-made, specify the formulation."
The manuscript mentions the use of black cumin (Nigella sativa), but the form (e.g., whole powder, defatted powder, oil, or extract) and the dose (e.g., mg/kg body weight, percentage in diet) are not specified. Please clarify the form of black cumin
The manuscript describes the following experimental groups: 1-Control, 2- Dyslipidemia (P), Dyslipidemia + Black Cumin (P1), and so on. Please clarify by what method(s) was dyslipidemia confirmed.
While the Methods section describes phytochemical analyses (DPPH, ABTS, and metabolite screening), the Results section lacks all corresponding data Please provide the missed data
The manuscript inconsistently describes the animal model: Page 3, Methods states "rats (Rattus norvegicus)" and Page 4, Results mentions "mice " Please: Clarify which species was actually used (rats or mice)
Please revise all instances of mass unit abbreviations from "gr" to the standard SI unit "g" for grams. This aligns with: International System of Units (SI) conventions
Table 1 presents comparative data between experimental groups, but lacks indications of statistical significance (e.g., asterisks, letters, or p-values). Please:

a. Add appropriate statistical annotations to highlight significant differences between groups (control vs. treated, etc.)
b. Specify in the table footnote:

- The statistical test used
- The significance levels

8. The titles of Tables 2-3 and the figures lack sufficient detail to stand alone as informative descriptors. Please revise all titles to clearly state the key variables/markers measured and the experimental groups compared
9. There are many typographical and grammatical errors throughout the manuscript. Therefore, I recommend a professional round of language editing before the paper is indexing.
10. Could you check abbreviations for consistency? Some were not fully spelled out. It might be beneficial to include a list of acronyms.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

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Comments on this article Comments (0)

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VERSION 1 PUBLISHED 06 Feb 2025

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Maha Mahmoud Mohamed, Ain Shams University, Cairo, Egypt
Aisyah Hasyila Jahidin, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, selangor, Malaysia

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Reviewer Report

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30 Jul 2025 | for Version 1

Aisyah Hasyila Jahidin, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, selangor, Malaysia

5 Views Cite this report Responses(0)

Approved With Reservations

The manuscript is generally well-prepared; however, the reference list could be updated to more effectively reflect recent developments in this field. At present, only around 30% of the cited works are from the past five years. Given the growing research interest in Nigella sativa, inflammation, and high-fat diet models, incorporating more recent literature would strengthen the scientific relevance and contextual grounding of the study.

The overall study design is appropriate and aligns with the research objectives. The inclusion of both negative and positive controls, along with well-structured treatment groups involving Nigella sativa, atorvastatin, and their combination, is commendable. Nonetheless, several methodological aspects require clarification to enhance transparency, reproducibility, and scientific rigour.

Firstly, the study duration is described as eight weeks, yet the timeline detailed in the manuscript, comprising a one-week acclimatisation period followed by phases of diet induction and treatment, appears to span nine weeks in total. The authors are encouraged to clearly delineate the duration and purpose of each experimental phase (acclimatisation, induction, treatment).

Secondly, the daily feed amount is reported as “20 mg/day,” which seems implausibly low for rats and may reflect a unit error. Clarification on this point is necessary.

Thirdly, the dosing information for Nigella sativa extract and atorvastatin is incomplete. The methods section should clearly describe the dose, frequency, route of administration (e.g., via feed or oral gavage), and duration of treatment. This information is particularly important in the combination treatment group (P3), where it should be clarified whether the atorvastatin dose was the same as in the single-treatment group or adjusted accordingly.

It is also unclear whether randomisation was applied during animal group allocation, or whether outcome assessors were blinded. If such practices were not followed, this should be explicitly stated, along with a brief discussion of potential sources of bias.

The statistical analysis is generally acceptable. While Duncan’s post hoc test can be used, it is considered less conservative than alternatives such as Tukey’s HSD or Bonferroni, which are more commonly recommended in recent biomedical research. The authors may wish to justify their choice of post hoc test or acknowledge this as a limitation.

The underlying source data appear to have been provided, which supports reproducibility. However, the manuscript states that some animals died during the experiment, leaving five rats in each treatment group. Yet, the dataset shows six animals per group, with no indication of attrition or exclusions. This discrepancy should be addressed. If animals were excluded, the final group sizes should be updated in both the methods and results sections, and the statistical analysis revised accordingly. If replacements were made or if the earlier statement was inaccurate, this should be corrected to maintain transparency.

With regard to results presentation, several improvements are suggested. First, since body weight was measured across multiple timepoints, a line graph would be more appropriate than a summary table. This would allow readers to more clearly visualise weight trends over time. Additionally, there is a likely error in the text, where the positive control group is incorrectly referred to as P1 instead of P. The data for the negative control group (N) also appear twice in the body weight table. Furthermore, I recommend using “g” for grams, rather than “gr,” which is not standard in scientific writing.

Second, the graphs provided do not include any indicators of statistical significance (e.g., asterisks or superscript letters). Including these on the graphs is important for clarity, enabling readers to interpret key differences without needing to cross-reference tables.

Third, while superscript letters are used in tables to indicate significant differences, there is no legend or footnote explaining their meaning. A simple note stating that groups sharing the same letter are not significantly different (p > 0.05) would improve clarity.

Fourth, all figures should include complete legends specifying the number of animals per group (n), the statistical test applied, and what the error bars represent (e.g., mean ± SD). This would enhance the scientific transparency and clarity of the data.

Finally, in the section reporting TNF-α results, the description includes numerous non-significant pairwise comparisons, which may obscure key findings. It would be clearer and more impactful to highlight only the statistically significant differences, particularly the finding that TNF-α levels were significantly lower in the P3 group compared to N, P, and P1.

The discussion provides useful background on Nigella sativa and its active constituents, particularly thymoquinone, and is well supported by literature. However, the section could be strengthened by more directly integrating and interpreting the study’s own findings, especially regarding IL-1β and TNF-α reductions in the combination treatment group, and comparing them with related studies.

To improve focus and clarity, especially if constrained by word count, the authors may consider condensing some of the general background and detailed mechanistic explanations (e.g., pyroptosis, adipogenesis) that are not directly supported by the data. This would provide more space to highlight novel findings, potential mechanisms, and the significance of combining Nigella sativa with atorvastatin. A more structured discussion separating key findings, biological rationale, comparison with prior studies, and limitations would enhance coherence.

The manuscript currently lacks a dedicated conclusion section. I recommend including a brief concluding paragraph to summarise the primary findings, reinforce their implications, and acknowledge key limitations, such as the lack of blinding, uncertainty in dosing methods, and issues with sample attrition. Including such a section would enhance the completeness and scientific integrity of the manuscript.

Lastly, please ensure consistency in the use of either British or American English throughout the manuscript, in accordance with the journal’s language guidelines.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Pharmacology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

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Reviewer Report

18 Views

09 Apr 2025 | for Version 1

Maha Mahmoud Mohamed, Ain Shams University, Cairo, Cairo Governorate, Egypt

18 Views Cite this report Responses(0)

Approved With Reservations

Your Report

The authors describe the use of a 'standard diet' and 'high-fat diet (HFD)' in the methods section. However, the exact composition is not provided. Please clarify the detailed composition of both diets to ensure the reproducibility of the experiments. If commercially sourced, provide the manufacturer and diet reference. If custom-made, specify the formulation."
The manuscript mentions the use of black cumin (Nigella sativa), but the form (e.g., whole powder, defatted powder, oil, or extract) and the dose (e.g., mg/kg body weight, percentage in diet) are not specified. Please clarify the form of black cumin
The manuscript describes the following experimental groups: 1-Control, 2- Dyslipidemia (P), Dyslipidemia + Black Cumin (P1), and so on. Please clarify by what method(s) was dyslipidemia confirmed.
While the Methods section describes phytochemical analyses (DPPH, ABTS, and metabolite screening), the Results section lacks all corresponding data Please provide the missed data
The manuscript inconsistently describes the animal model: Page 3, Methods states "rats (Rattus norvegicus)" and Page 4, Results mentions "mice " Please: Clarify which species was actually used (rats or mice)
Please revise all instances of mass unit abbreviations from "gr" to the standard SI unit "g" for grams. This aligns with: International System of Units (SI) conventions
Table 1 presents comparative data between experimental groups, but lacks indications of statistical significance (e.g., asterisks, letters, or p-values). Please:

a. Add appropriate statistical annotations to highlight significant differences between groups (control vs. treated, etc.)
b. Specify in the table footnote:

- The statistical test used
- The significance levels

8. The titles of Tables 2-3 and the figures lack sufficient detail to stand alone as informative descriptors. Please revise all titles to clearly state the key variables/markers measured and the experimental groups compared
9. There are many typographical and grammatical errors throughout the manuscript. Therefore, I recommend a professional round of language editing before the paper is indexing.
10. Could you check abbreviations for consistency? Some were not fully spelled out. It might be beneficial to include a list of acronyms.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Respond to this report

Responses (0)

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Therapeutic potential of Nigella sativa extract against inflammatory markers interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in rat models with high fat diet

Abstract

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Methods

Study design

Animals

Black cumin and high fat diet

Data analysis

Results

General descriptions

Table 1. Average body weight of rats during the study.

Effect of giving black cumin extract on IL-1β and TNF-α levels

Table 2. Mean distribution of IL-1β according to treatments (pg/mL).

Figure 1. Mean distribution of IL 1 according to treatments.

Table 3. Mean distribution of TNF-α according to treatments.

Figure 2. Mean distribution of TNF according to treatments.

Discussion

Ethical considerations

Data availability

References

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