Immunomodulatory Effect of Nigella sativa Extract through the Improvement of IL-1β Level in Balb-c Mice Infected by M S ethicillin-resistant taphylococcus aureus

Nigella sativa (NS) has been used for many years as an anti-bacterial herbal treatment, such as M S , but the mechanism in bacterial elimination is still unknown IL-1β is a proinflammatory cytokine that helps the immune system eliminate microbes when it enters the body. The purpose of this study was to prove the immunomodulatory effects of Nigella sativa extract through increased IL-1β in dealing with MRSA infections. The design of this study was post-test only control group design using 25 male Balb-c mice infected with MRSA and randomly divided into five groups. Group K was infected with MRSA without being treated. All treatment groups were given NS extracts in different doses for 7 days before being infected with MRSA. P1 was given N. sativa extract 0.05ml, P2 (was given NS extract 0.25ml, P3 was given 0.5ml NS extract, and P4 was given 0.75ml NS. On the 8th day, blood was taken from the retroorbital plexus for IL-1 level examination using ELISA. Statistical tests were done using the One Way ANOVA test. The results showed the mean of IL-1β levels in group K was 11.65ng/L, P1 was 14.07ng/L, P2 was 16.66ng/L, P3 was 18.54ng/L, and P4 was 19.49ng/L ethicillin-resistant taphylococcus aureus .

Black cumin seeds were obtained from the Ting Bao herbal shop in Purwokerto, Banyumas, Central Java. Extracts were made at the Laboratory of Pharmaceutical Biology, Department of Pharmacy, Faculty of Health Sciences, . Black cumin seeds were extracted using maceration and vacuum evaporator methods with 96% ethanol solvent, and the results were diluted with Carboxyl Methyl Cellulose ( until a concentration of 20 mg/ml was reached.

RESULTS
The research data were processed and analyzed using the SPSS 22.0 for Windows Data were presented in the form of mean and standard deviation. Normality test with the Shapiro-Wilk on the measurement results of IL-1β levels taken 24 hours after MRSA infection showed normal data distribution, so the differences in IL-1β levels were analyzed using the One Way ANOVA.
The invasion of bacteria to the body will stimulate phagocytosis by macrophages. Macrophages, dendritic cells, and other cells recognize microbes and give respond by producing cytokines. One of these cytokines was IL-1β which work on the endothelial venules at the site of infection and initiate the process of migrating leukocytes into the tissues for bacterial elimination (1,7,11).

INTRODUCTION
This research was conducted to identify the potential of black cumin extract as an immunomodulator through increased IL-1β in Balb-c mice infected by MRSA. The reason researchers chose IL-1 to be evaluated in this study was because IL-1 is one of the early pro inflammatory cytokines that is expected to stimulate the immune system in eliminating extracellular bacteria such as MRSA β -Black cumin ( ) has long been used in several countries, especially in the Middle East and in several other Asian countries, including Indonesia. The active compound of the black cumin is monoterpen molecule that is extracted from the residue of black cumin oil containing thymoquinone (TQ) and thymohydroquinone (THQ).

Nigella sativa
, anti-bacterial, and antiinflammatory. Both sativa oil is rich in unsaturated fatty acids, linolenic acid, and stearidonic acid. Various studies have shown that has anti-inflammatory and immunomodulatory properties (9,10). tud on the immunomodulatory effects of been conducted, and it is proven that can improve the immune response in humans, increasing IgM production in murine lymph cell culture at concentrations can reduce IgM levels concentrations (11).

S y N. sativa N. sativa
The substance has properties to enhance the body's immune system TQ and THQ allegedly can form irreversible complexes with nucleophilic amino acids in proteins of bacteria causing the inactivation of the protein (8 The results showed that increasing dosage of N. sativa

METHOD
The independent variable in this study was the dose of extract while the dependent variable was IL-1β levels. Blood of the mice was taken 24 hours after MRSA injection and followed by IL-1β level examination. Mice were then terminated. IL-1β examination was carried out in the Research Laboratory, Faculty of Medicine, by using ELISA Kit.

Unsoed Bioassay Technology Laboratory Mouse IL-1β
This .This research applied animal ethics in handling the experimental animals. Ethical clearance number 3968/KEPK/VIII/2019 was obtained from the Health Research Ethics Commission before the study was begun. The study population was male Balb-c strain mice aged eight weeks and weighed 20-30 grams. Mice were obtained from the Pharmacology Laboratory of the Faculty of Medicine, Yogyakarta. Experimental animals were placed in cages and given food and drink ad libitum. Before treatment, mice underwent an adaptation for one week, and no mice were found sick or died. Mice were randomly divided into five groups. Control group (K) was mice infected with MRSA treatment, treatment 1 (P1) was mice treated with or 0.05ml extract for seven days and infected with MRSA 0.2ml ( cfu/ml), treatment 2 (P2) was mice treated with 0.25ml 2 or extract for seven days and infected with MRSA 0.2 ml ( cfu/ml), treatment 3 (P3) was mice treated with or 0.5ml extract for seven days then infected with MRSA, and treatment 4 (P4) was mice treated with 0.75ml extract for 7 days then infected with MRSA. The results showed an increase in the average IL-1β level along with the increasing dose of extract, N. sativa Previous also identified that black cumin extract could act as immunomodulator. administration of black cumin ethanol extract dose of 50mg/kg BW, 100mg/kg BW, and 200mg/kg BW in mice given antigen (Goat Eritrosit 5%) could increase the number of lymphocytes, monocytes, and reduce the number of segment neutrophils significantly (12). also found that extract dose of 5.2gr/day could infected by Salmonella thyphimurium (13). In contrast to research by Zainiyah that black cumin extract could reduce pro-inflammatory cytokines IL-6 in endothelial dysfunction of preeclampsiainduced Balb-c mice (14). Research conducted by Muhyi found no significant differences in the number of Th-17 cells and neutrophils in asthmatics who only received immunotherapy and placebo compared to those that received immunotherapy and extract and those who received immunotherapy and probiotics (15).

N. sativa
however difference test using ANOVA showed no significant difference among groups. studies Aldi and Suharti showed that Sari and Purnawati N. sativa increase macrophages nitric oxide (NO) production of Balb-c mice The difference in the nature of in this study to other studies may be caused by several factors including the way of extraction, the number of samples, the composition of the active ingredients of black cumin plants, the dose of black cumin extract, age of the significantly affects plant components and the results (16). Other studies found that has anti-bacterial activity against MRSA in vitro (17). Sulistiawati stated that N. sativa could an immunomodulator and also anti-inflammatory depending on the accompanying disease conditions because of the number of substances contained in black cumin plants (9).
N. sativa extract in related to the immunoregulatory effects and the plant that N. sativa serve as

DISCUSSION
One of the active of the black cumin is Thymoquinone (TQ).

compounds
Mohany and colleagues investigated Thymoquinone effects on pesticide induced immunotoxicity in male albino rats. Among several biochemical and histopathological changes, Imidacloprid insecticide treatment caused a On humoral immunity, significant decline in total Ig levels (especially IgGs) and a significant inhibition of antibody hemagglutination. Intra peritoneal injection of TQ reversed the immunological effects, leading to significantly increased total Ig levels and antibody hemagglutination. The findings of this study provided hints that TQ may potentially modulate the outcome of humoral immune responses (19)