Therapeutic Potential of Black Seed Oil to Nuclear Factor Kappa B Levels of Patients with Metabolic Syndrome Risk

 

Rahmat A Hi Wahid1*, Endang Darmawan2

1Department of Pharmacy, Faculty of Science and Technology,

Universitas PGRI Yogyakarta, Yogyakarta, Indonesia.

2Department of Clinical Pharmacy, Faculty of Pharmacy, Universitas Ahmad Dahlan, Yogyakarta, Indonesia.

*Corresponding Author E-mail: rahmat@upy.ac.id

 

ABSTRACT:

Herbal medicine is one of the most used adjuvant and alternative therapies by people with metabolic syndrome (MetS) risk. This is alongside conventional MetS risk treatments. Black Seed Oil(BSO) is a medicinal plant most widely used worldwide as the most excellent form of healing medicine. This study aimed to evaluate the effect of BSO as adjuvant therapy on levels of NF-κB in patients with MetS risk. This prospective was conducted at Jetis I Public Health Center (PHC), Yogyakarta, Indonesia. After confirmation of diagnosis, sixty-two patients who fulfilled the inclusion and exclusion criteria were enrolled in this study. Approval from the institutional ethical committee was also obtained. The patients with MetS risk were divided into two groups (n=31). In group I (the BSO group), the patients have advised BSO 3mL/day for 20 days. In group II (the Control group), the patients have advised a placebo for 20days. The level of NF-κB was estimated using the enzyme-linked immunosorbent assay (ELISA) method. The median values between groups were tested with Mann-Whitney with a significance of P=<0.05 (95%). The favorable impact of BSO was seen in almost all variables, but the results were not statistically significant (P>0.05). Adjuvant therapy of BSO doses of 3 mL/day could not increase levels of NF-κB in the patients with MetS risk at Jetis I PHC, Yogyakarta, Indonesia. A larger study with adequate sample size and long-term supplementation with BSO is recommended.

 

KEYWORDS: Black seed oil (BSO), ELISA, Indonesia Public Health Center, Metabolic syndrome, NF-κB.

 

 


INTRODUCTION: 

Metabolic syndrome (MetS) is a group of metabolic disorders both lipid and non-lipid, which are risk factors for coronary heart disease consisting of central obesity and atherogenic dyslipidemia (consisting of elevated triglycerides and LDL-cholesterol and reduced HDL-cholesterol), hypertension, and abnormal glucose levels plasma1-5.

 

MetS prevalence increased in Indonesia6, associated with increased mortality and morbidity related to cardiovascular diseases. Based on epidemiological data, the prevalence and incidence of MetS in the world and Indonesia were found to be 20-40% or more people in different countries7,8.

 

The Framingham Offspring Study found that MetS perfections in participants aged 26-82 years were 29.4% in men and 23.1% in women8. The results of Basic Health Research (Riskesdas) in 2018 show the national prevalence of MetS risk is among the population aged >15 years, with criteria of diabetics at 8.5%, central obesity at 31.0%, and hypertension at 34.1%9.

 

The pathogenesis of MetS is related to many factors10-12. Rani et al. (2016) postulated that people with metabolic syndrome-related diseases generally experience oxidative stress caused by free radicals, which aggravate the disease and lead to complications. In conditions such as inflammation, characteristic of metabolic syndrome, free radicals are overproduced, so the body's defense system cannot cope with oxidative stress13. Oxidative stress (OS), a state of lost balance between the oxidative and antioxidative systems of the cells and tissues, results in the overproduction of oxidative free radicals and reactive oxygen species (ROS)14. Excessive ROS generated could attack the cellular proteins, lipids, and nucleic acids leading to cellular dysfunction, including loss of energy metabolism, altered cell signaling and cell cycle control, genetic mutations, altered cellular transport mechanisms, and overall decreased biological activity, immune activation and inflammation15-17.

 

Different models of oxidative stress on NF-κB related activities. ROS can activate and repress NF-κB signaling in a phase and context-dependent manner. The NF-κB pathway can have both anti- and pro-oxidant roles in the setting of oxidative stress18.

 

The transcription factor nuclear factor-κB (NF-κB) is a family of transcription factors and is of central importance in inflammation and immunity19,20. NF-κB in activated B cells is a protein complex that controls DNA transcription21. The NF-κB transcription factor gained interest as it is related to many human diseases. An NF-κB activation is a key event in the early diagnosis of diabetic pathology22. Oxidative stress and production of free radicals (FOR) are increased in diabetes23 and lead to the release of ROS and the activation of NF-κB24. NF-κB inhibitors that decrease the effects of ROS and antioxidants have been used in the treatment25,26.

 

Oxidative stress plays a significant role in the pathogenesis of MetS and damage to the body. Nakhostin-Roohi et al. (2013) proved that supplementation with antioxidant (methylsulfonylmethane) could help to attenuate the damage to the body induced by oxidative stress27. Antioxidant therapy as adjuvant therapy may be expected to improve the efficacy of standard treatment in MetS patients. An antioxidant agent should be developed as an adjuvant therapy candidate that can synergize with the standard therapy for MetS patients25.

 

Black seed oil (BSO) has been empirically used to treat various diseases in Indonesia and Asia28. Pharmacological effects of BSO have been reported in preclinical and clinical studies, such as anti-inflammatory, antioxidant, immunomodulatory, and anticancer29,30, antidiabetics-immunostimulants31,21, hepatoprotective and antidislipidemia32,33, chemopreventive effects induced by DMBA (7.12-dimethylbenz (a) antracene) in Sprague Dawley rats34, and effective antihypertensive agents35. In phase I, clinical trials demonstrated that oral administration of BSO for 20 days in healthy volunteers is safe and tolerable36. Its use has also been suggested as adjuvant therapy for patients with metabolic syndrome risk. However, 3mL/day of oil did not prove effective in the management of patients with metabolic syndrome risk27.

 

BSO functional activity can be derived from the main bioactive component, thymoquinone. Thymoquinone (TQ), the most prominent constituent of Nigella sativa seeds. It has antioxidant and anti-inflammatory properties that can significantly increase the amount of the repressive NF-κB p50 homodimer, and bound to the TNF-α promoter as revealed by electrophoretic mobility shift37.

 

Therefore, the present study will focus on the potential effect of BSO as adjuvant therapy on NF-κB levels in patients with MetS, so to find out the role of NF-κB as a target candidate for antioxidants and immunoregulators.

 

MATERIALS AND METHODS:

Materials:

BSO was standardized with a thymoquinone content of 2.72% (28) and then was formed in soft capsules immunax®. BSO soft capsules were prepared by the pharmaceutical industry with Good Manufacturing Practices (GMP) certificate.

 
The type of this research was a prospective interventional study. Ethical clearance for this study was reviewed and approved by The Research Ethics Committee, Universitas Ahmad Dahlan (No: 011801018). Subjects for the study were selected based on the inclusion and exclusion criteria to ensure the accuracy associated factors at MetS risk.
 
The inclusion criteria in this study are 1) having at least 3 items of 5 metabolic symptoms based on the following criteria: abdominal obesity, hyperglycemic, hypertension and dyslipidemia, 2) both sexes with the age group of >18 years 3) with the without coexisting disease (having MetS signs, especially diabetes).

 

The exclusion criteria were 1) pregnancy or lactating, 2) patients not routinely control for 3 months 3) senile patients 4) taking corticosteroid medicines 5) patients allergic to BSO, 6) Taking medication for chronic diseases and tuberculosis, herbal or supplementation, 7) samples of damaged blood specimens.

 

Design of the Experiments:

The subjects were randomly divided into two groups. A co-investigator was selected to create subject identification numbers to assign subjects into the group. A total of 62 patients with MetS risk divided into two groups. Group I(n=31) get BSO with a dose of 3mL/day (every morning and night) and compared to controls/placebo group II(n=31) during the intervention for 20days. Controls were administered twice daily after meals for 20days, with each capsule containing 3mL mineral oil. The capsules are similar to those containing BSO extract so that people cannot guess the contents of the capsules by looking at the medicine packaging. The rationale for using mineral oil as a placebo is based on its inert quality concerning research results, as well as easy availability. The NF-κB level examination was conducted at Phytochemical Laboratory of Universitas Ahmad Dahlan, Yogyakarta in November 2017.

ELISA analysis for NF-κB levels:

NF-κB levels were quantified using immunoassay by the ELISA method. The measurement of the NF-κB control procedure is performed by Wuhan Fine Biotech Co., Ltd reagent (China). An antigen coating is performed using 100μL calibrator diluent. In a standard 100μL (human NF-κB) and a sample pipetted on each wells which have been centrifuge. Furthermore, incubated for 90 minutes at 370C. Next washed with washing buffer 4 times @100 μL then added 100μL SABC on each wells and incubated at 370C for 30 minutes. Washed with washing buffer 4 times @100μL then added 90μL TMB substrate and incubated for 15-30 minutes at 370C. 50μL of stop solution added into each well and mix thoroughly. The results are read immediately with the ELISA reader microplate at a wavelength of 450nm and expressed in units of ng/mL (nanogram/millilitre).

 

Statistical analysis:

The subject demographic characteristics are presented descriptively (percentage). All of the data were analyzed using the Statistical Package of Social Science (SPSS) version 24. The Mann-Whitney test was used for the analysis of data. A probability of p<0.05 was considered significant.

 

RESULT:

Demographic characteristic of subjects:

The subjects used in this study were 62patients with MetS risk consisting of 15(24.2%) men and 47(75.8%) women with the highest number in the range of age group 40-60 years (59.7%).

 

Based on the level of education, patients who had less than senior high school were 35(56.5%) patients and 22 (35.5%) patients have finished high school. Based on work, there were 47(75.8%) patients have work and 15 (24.2%) patients not have work. Characteristics of the subjects are presented in Table 1. Table 1 showed that the prevalence of patients with metabolic syndrome risk affects as; sex, age, education and work factors.

 

Table 1: The characteristics data of patients with metabolic syndrome risk at Jetis I Public Health Center, Yogyakarta, Indonesia

Status

BSO 3 mL group

Controls group

Total

N

%

N

%

N

%

Sex:

 

 

 

 

 

 

Men

7

46.7

8

53.3

15

24.2

Women

24

51.1

23

48.9

47

75.8

Age, years:

 

 

 

 

 

 

20-40

0

0.0

2

100.0

2

3.2

>40-60 tahun

22

59.5

15

40.5

37

59.7

>60 tahun

9

39.1

14

60.9

23

37.1

Education:

 

 

 

 

 

 

Uneducated

3

60.0

2

40.0

5

8.1

<SHS

18

51.4

17

48.6

35

56.5

>SHS

10

45.5

12

54.5

22

35.5

Job status:

 

 

 

 

 

 

Work

26

55.3

21

44.7

47

75.8

Not work

5

33.3

10

66.7

15

24.2

<SHS: <Senior High School (Primary to Junior High School), >SHS: >Senior High School (Tertiary)

 

Analysis of clinical characteristic patients with metabolic syndrome risk between BSO and control group at Jetis I Public Health Center, Yogyakarta, Indonesia:

In the current study, a monitoring clinical conditions of the patients with MetS risk included body mass index (BMI), blood pressure (systolic and diastolic), blood glucose, triglycerides (TG), total cholesterol (TC) and high-density lipoprotein (HDL). The results of the clinical conditions of patients with MetS risk in both groups were analyzed using an independent t-test as shown in Table 2 and Figure. Table 2 and Figure 1 showed that there was no significant difference in both groups test in the mean body mass index (BMI), blood serum glucose, blood pressure (systolic and diastolic), and cholesterol levels (p>0.05).

 

Table 2: Results of analysis clinical condition of patients with metabolic syndrome risk based on doses group BSO 3mL/day and control/placebo group for 20 days at Jetis I Public Health Center, Yogyakarta, Indonesia (independent t-test)

Parameter

Mean ± SD

P-value

BSO 3 mL/day group N=31

Control group N=31

BMI±SD (kg/m2)

25.25±3.81

24.5±4.43

0.64

BPS±SD (mmHg)

140.23±17.07

139.00±13.24

0.07

BPD±SD (mmHg)

80.81±9.91

75.39±8.60

0.44

BG±SD (mg/dL)

202.71±91.02

265.29±76.37

0.64

TG±SD (mg/dL)

149.48±75.12

186.68±117.49

0.35

TC±SD (mg/dL)

186.35±51.79

180.12±47.78

0.22

HDL±SD (mg/dL)

45.61±6.32

44.32±5.49

0.19

Note: BMI, body mass index; BPS, blood pressure systolic; BPD, blood pressure diastolic; BG, blood glucose; TG, triglyceride; TC, total cholesterol; HDL, high-density lipoprotein

 

Analysis of NF-κB levels in patients with MetS risk between BSO and control group at Jetis I Public Health Centerl, Yogyakarta, Indonesia

In the results of NF-κB levels in patients with MetS risk, statistically there was no significant difference between the two groups (P>0.05) (Table 3).

 

Table 3: Results of analysis NF-κB levels of patients with metabolic syndrome risk based on doses group BSO 3 mL/day and control/placebo group for 20 days at Jetis I Public Health Center, Yogyakarta, Indonesia

Variabel

Status

Median Minimum-Maximum

P value

NF-κB

BSO 3mL/day group

0.085 (0.083-0. 104)

1.00 (Mann-Whitney)

 

Controls group

0.085 (0,083-0.102)

 

 

DISCUSSION:

Our results indicate that TQ does not affect nuclear expression and level of NF-κB in patients with MetS risk. The same results were also reported by El Gazzar et al. (2007) through their research that TQ had no effect on expression and NF-κB levels but modulated transactivation by inducing a p50 homodimer bond while blocking p65 binding to this site. According to him, TQ increased the number of repressive NF-κB heptimers and simultaneously decreases the amount of transactivation of NF-κB p65:p50 heterodimer bound to TNF-α promoters37.

 

The findings in this study contrary to the previous finding. Usta and Dede (2017) proved by in vivo models that giving TQ for 21 days was perorally capable of repairing oxidative DNA damage and decreasing NF-κB levels post-induction of streptozotocin (STZ)21. The research by Sethi et al. (2008) proves that TQ, the active compound in the Nigella sativa L. plant capable of suppressing TNF-α that induces NF-κB activation and capable of inhibiting NF-κB activation caused by various carcinogenic effects and inflammatory responses39. Agbaria et al. (2015) also proved that BSO produces anticancer activity and oxidative stress through inhibition of the NF-κB signalling pathway40.

 

Nuclear Factor Kappa B (NF-κB) is a family of transcription factors that include Rel-A (p65), Rel-B, c-Rel, NF-κB1(p50/p105), and NF-κB2 (p52/pyh100)41. Wardyn et al. (2015) explaining that NF-κB can be released from its inhibitor by the phosphorylation and ubiquitination process of I-κB by an enzyme IκB kinase (IKK). NF-κB released from its inhibitor translocates to the nucleus, binds to the κB motif on the promoter of many genes, and regulates transcription41. In addition, it has been shown that, in macrophages and other cells, NF-κB p50 homodimers are detected in the nucleus bound to a limited number of promoters 42,43. Also, NF-κB dimers have been shown to shuttle between the cytoplasm and the nucleus in resting cells and both NF-κB p65 and 50 accumulate in the nucleus in a variety of cell types in response to LPS and inflammation. NF-κB entering the nucleus is associated with increased transcription of several genes such as coding for chemokines, adhesion molecules (VCAM, ICAM), proinflammatory cytokines and inflammatory enzymes (iNOS) cytokines44.

 

Findings of differences in levels of NF-κB in this study with other studies may be due to length of therapy, the dose of therapy with BSO, and small sample size. A prospective study by Najmi et al. (2008) at a tertiary service center in North India with a total of 60 patients proving that 2.5 mL of BSO twice daily for 6 weeks was able to improve and lower total cholesterol, low-density lipoprotein (LDL) cholesterol, and plasma blood sugar (p = <0.05)45. In contrast with Hosseini et al. (2013) research in 70 patients at Baqiyatallah Hospital, Iran who were given 2.5mL BSO twice daily for 3 months were able to control blood glucose without any adverse side effects46.

 

CONCLUSION:

In conclusion, adjuvant therapy of BSO doses of 3 mL/day could not increase levels of NF-κB in the patients with MetS risk at Jetis I PHC, Yogyakarta, Indonesia. A larger study with adequate sample size and long-term supplementation with BSO are recommended.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors forever indebted to the intellectual Dr.dr.Akrom for him relentless support in the preparation of this research and manuscript. The authors wish to thank the Government Republic of Indonesia. This study is funded by State Financial Management Institutions, Ministry of Finance Republic of Indonesia Number: PRJ-1668/LPDP.3/2017.

 

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Received on 03.09.2022            Modified on 31.12.2022

Accepted on 16.05.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4597-4601.

DOI: 10.52711/0974-360X.2023.00748