The Effect of Black Cumin Seed Oil on Active Smokers Based on Interleukin-1β Activity and Neutrophil

Health problems due to exposure to cigarettes cause the body to fight against the occurrence of immune responses, one of which is leukocytes. The most common types of leukocytes are neutrophils, which are about 50-70% with a function as the body's line of defense against foreign substances. One of the toxic components in cigarettes is nicotine. Nicotine in cigarettes can induce the production of IL-1β. Black Cumin Seed Oil (BCSO) in various studies has been widely used as a supplement, especially as an immunomodulator. This study used a single-blind Randomized Controlled Trial (RCT) method, with a total of 39 test subjects divided into four groups for 30 days. Group 1 received placebo 3x1 capsules/day, group 2 received BCSO 3x1 capsules/day, group 3 received BCSO 3x2 capsules/day, and group 4 received BCSO 3x3 capsules/day. Data analysis used a one-way ANOVA test to see the average neutrophils between treatment groups. While the average levels of IL-1β using the method of Kruskall Wallis. The test results are said to be significant if p 0.05. The p value of the ANOVA test was 0.16 neutrophil levels. In Kruskal Wallis, the –p value for IL-1β is 0.43. BCSO administration does not affect IL-1β levels in smokers and BCSO doses 3x1 capsules/day can reduce neutrophil levels.


Introduction
The impact of smoking is one of the biggest health problems in the world. The World Health Organization (WHO) states that smoking can cause health problems that are the cause of death of approximately 6 million people per year. The risk of death from smoking in active smokers is higher than passive smokers (Hidayati et al., 2019). Cigarette smoke is high in free radicals and triggers a decrease in antioxidant capacity. The increase in free radicals in smokers and a decrease in the body's immune system will trigger various systemic diseases such as rheumatoid arthritis, cancer, diabetes mellitus, cardiovascular disease, neurodegenerative and asthma (Suryadinata et al., 2017). The three main toxic components found in cigarettes are carbon monoxide, tar and nicotine (Janah & Martini, 2017). High levels of tar and nicotine in the body cause immunological changes by suppressing the phagocytic activity of neutrophils (Yamaguchi, 2019).
Neutrophils are a type of leukocyte that plays a role in the body's defense mechanism (Utami et al., 2013). Neutrophils play a role in the immune response due to smoking by increasing free radicals in the body. Exposure to tobacco smoke can also cause oxidative stress and trigger an inflammatory response in the lungs (Suryadinata et al., 2017). During inflammation, neutrophils in the blood can increase four times to five times the normal number (Nusa et al., 2016).
Interleukin 1 beta (IL-1β) plays an important role in inflammation and immunity. IL-1β is produced by monocytes, macrophages, neutrophils and also by other cell types such as fibroblasts and epithelial cells (Moeintaghavi et al., 2017). Exposure to cigarette smoke causes changes in the epithelial structure which results in the release of proinflammatory cytokines such as TNFα and IL-1β by alveolar macrophages (Wahyuni, 2017).
Increased levels of cytokines and/or chemokines released by inflammatory cells after exposure to cigarette smoke function as chemoattractants that will exacerbate inflammation (Zuo et al., 2014).
Traditional medicine is widely used in improving the health of Indonesian people. Traditional medicine refers to efforts to strengthen natural healing. One of the traditional medicinal plants that is starting to get attention for its benefits is black cumin (Marlinda, 2015). Black cumin seeds are believed to maintain human health and are used to treat various diseases such as hypertension, rheumatism and treat various bacterial infections. In addition, it is important to maintain kidney, bile, liver function, and is used to improve the immune system (Sulistiawati, 2014). Black cumin is useful as an immunomodulator by increasing antibody titers and increasing monocytes and decreasing neutrophils (Amanulloh et al., 2019). The important ingredients of black cumin are thymoquinone, dithymoquinone, thymohydroquinone and thymol which can activate and awaken the immune system with its ability to increase levels of helper T cells, suppressor T cells and natural killer cells to maintain homeostasis (Ningtyas, 2012). To date, the benefits of taking BCSO on neutrophil counts and IL-1β levels have not been studied. Based on this description, this study was conducted to

Research Method Materials
Tools and materials used in this study included primary and secondary data collection sheets using a case report form (CRF), blood collection equipment, and a set of tools for blood analysis. The CRF was used to collect demographic and lifestyle data. Neutrophil levels were analyzed using a Hematology Analyzer. Measurement of vital signs, filling out CRF, and nutritional status and blood collection were carried out at residents' homes. Complete blood count was carried out at Nur Hidayah Hospital.

Method
This research is an experimental study with a Randomized Controlled Trial (RCT) design. The sampling technique used is purposive sampling, where the researcher determines the sample based on predetermined criteria. Eligible subjects were divided into four groups by simple random sampling. Subjects received either three-dose BCSO capsules or a placebo 3x1 for 30 days. Reviewing health conditions and side effects is carried out daily through smartphones.
Every three days, researchers made visits to test subjects to ensure compliance with BCSO administration. The compliance of the test subjects was measured by calculating the previously given BCSO difference.

Data Analysis
Analysis of the average levels of neutrophils used a one-way ANOVA test which previously had met the normality and homogeneity tests to see whether there was a significant difference between the treatment groups. The analysis compares the average levels of IL-1β between treatment groups using Kruskal Wallis

General Description of Respondents
The subjects of this study consisted of 39 active smokers volunteers who were divided into 4 treatment groups located in the working area of the Jetis 1 Public Health Center in Bantul. Demographic conditions of research subjects according to gender, education, status, occupation, smoking status, age, length of smoking (years) and number of cigarette consumption (sticks/day).
As shown in Table 1. the characteristics of respondents as subjects. The subject is male and smokers (100%), this is in accordance with research by Chinwong et al. (2018) where smoking behavior is higher among men than women and the average number of daily cigarette consumption is higher in men than women. The level of education is mostly senior high school (SHS) (61.5%). Level of education affects smoking behavior. Education can affect a person's way of thinking and behavior  Vol.18 No. 02 Desember 2021: 476-484 related to the risks that arise for health due to smoking (Juliansyah & Rizal, 2018). Most of them are married (66.7%) and their work is dominated as laborers (46.2%). The mean age was 40 years, the average length of smoking was 18 years and the average number of cigarettes consumed was 10 cigarettes/day. Examination of vital signs, namely the average blood pressure showed a result of 139/87 mmHg. There is a relationship between increased blood pressure and smoking behavior. Factors related to increased blood pressure include the number of cigarettes smoked, how to smoke and the frequency of smoking duration (Tisa, 2012).
Based on the Table 1, analysis was carried out One Way Anova, where the p-value in the age group was 0.80, blood pressure was 0.52, length of smoking was 0.63 and the number of cigarettes consumed per day was 0.66. From the results of the overall analysis, it was found that -p value > 0.05, which means there was no significant difference between in the placebo group, BCSO 3x1 capsules/day, BCSO 3x2 capsules/day and BCSO 3x3 capsules/day. This is possible because the length of exposure to cigarette smoke experienced by each individual is different.

Analysis of IL-1β Levels Between Treatment Groups
Nicotine in cigarettes can induce the production of IL-1β significantly in in vitro studies (Wu et al., 2014). This study is expected to show that BCSO can reduce levels of IL-1β. As shown in table 2, the average IL-1 after administration of BCSO in the placebo group was 16.61 pg/mL, the BCSO 3x1 group was 24.67 pg/mL, the BCSO 3x2 group was 20.83 pg/mL and the 3x3 group was 17.61 pg/mL. In normal healthy adults, serum IL-1β concentrations range from 0.5 to 12 pg/mL (O'Neill et al., 2013). When compared to normal values between IL-1β and placebo, it was not in the normal range, so BCSO administration did not affect IL-1β levels. The difference in IL-1β expression which is higher than in the placebo group is a sign of a more active inflammatory process (Astawa et al., 2017). Research by (Zikriah, 2014) stated that a dose of 250mg/kg black cumin ethanol extract was able to suppress lipopolysaccharide-induced interleukin 1β levels. Another study stated that black cumin extract was able to inhibit IL-1β (Amartey et al., 2019).
The average level of IL-1β levels obtained was analyzed by the Kruskal Wallis test. As shown in table 2, the pvalue is 0.43 > 0.05, which means that there is no significant difference between the average level of IL-1β in the placebo group, BCSO 3x1 capsules/day, BCSO 3x2 capsules/day and BCSO 3x3 capsules/day.

Analysis of the Level of Neutrophils between Treatment Groups
Neutrophils were the most common type of leukocyte, about 50-70% among other leukocyte cells.   (Kiswari, 2014). According to previous research, smoking can increase neutrophil levels in the blood (Prihandari, 2016).

PHARMACY: Jurnal Farmasi Indonesia
The immune system of smokers decreases in fighting bacterial infections compared to non-smokers so that it will increase the number of neutrophils (Zuo et al., 2014). In this study, it is hoped that the administration of BCSO can reduce neutrophil levels. As shown in Table 3, the mean neutrophils after being given BCSO for 30 days in the placebo group was 54, the BCSO 3x1 group was 52.87, the BCSO 3x2 group was 56.82 and the BCSO 3x3 group was 61.87.   Vol.18 No. 02 Desember 2021: 476-484 Analysis of the average level of neutrophil levels using one-way ANOVA test. Based on the results of the one-way ANOVA test in the table, the p-value is 0.16 > 0.05, which means that there is no significant difference between the mean neutrophil levels in the placebo group, BCSO 3x1 capsules/day, BCSO 3x2 capsules/day and BCSO 3x3 capsules/day.

Conclusion
The number of BCSO capsules consumed every day affects respondent compliance in consuming BCSO. The administration of BCSO did not affect IL-1β levels in smokers and the administration of BCSO 3x1 capsules/day could reduce neutrophil levels.