Effect of essential oil of black cumin (Nigella sativa) on hematological profiles and total cholesterol levels of Wistar rats exposed by cigarette smoke

Cigarette smoke contains toxic substances such as carbon monoxide, lead, cadmium, tar and hydrogen cyanide which can triggeroxidative stress and causeerythrocyte membrane damage and hemoglobin oxidation. In addition, it also contains nicotine which can increase the total cholesterol levels. Black cumin containing thymoquinone has been known for its antioxidant and anticholesterol activities. This study aimed to investigate the effect of black cumin extract on hematological profiles and total cholesterol levels of Wistar rats exposed by cigarette smoke. It was an experimental study with randomized posttest only control group design. Twenty Wistar rats (Rattus norvegicus) that divided into four groups were used in this study. The normal control group (N) was provided with standard feed,the negative control group (C) was exposed to the cigarette smoke with two pieces of cigarettes/day for 14 days, the treated groups were given black cumin extract 200mg/kg (T1) and 400mg/ kg (T2) and exposed by cigarette smoke two pieces of cigarettes/day for 14 days. On day 15, blood samples from the rats were taken through the sinus orbitalis and then the erythrocyte, the hemoglobin and the total cholesterol levels were examined. Data were analyzed using the one-way ANOVA test and continued by the post-hoc test. The results showed the number of erythrocytes and hemoglobin levels of the T2 group was significantly higher than those of the C group (p<0.05). Although, total cholesterol levels of the T2 group was lower than that of other groups, however it was not significantly different (p>0.05). In conclusion, the administration of black cumin extract at 400mg/kg significantly increases the erythrocytes numbers and the hemoglobin levels in Wistar rats exposed by cigarette smoke.


INTRODUCTION
Cigaretteis being a problem throughout the world. In 2014, it was reported that as many as 650 million people consumed cigarettes. In Indonesia, cigarette consumption ranks third in the world. Cigarette not only affected the health of active smokers, butalso the passive smokers in Indonesia. 1 In 2020, World Health Organization (WHO) estimatedaround six million people die from diseases caused by cigarette consumption, anually. 2 Cigaretteis dangerous to health due toitcontainstoxic substances such as carbon monoxide (CO), tar, nicotine and lead (Pb) which harmful to human body. 3 These toxic substances can trigger oxidative stress due to free radicals produced. A radical molecule is a molecule that consisted by unpaired electrons and in an active conditions. Thesereactive molecules can damage cellular macromolecules such asproteins, lipids, carbohydrates and nucleic acids by giving rise to oxidative stress and increasing lipid peroxidase (LPO) activity.
Those free radicals content may changescell membranesstructure of the erythrocyte that lead to lysis which cause a decrease in the number of erythrocytes followed by a decrease in the hemoglobin levels. In addition, the tar content in cigarettes may damages the spinal cordthus disrupt the formation of red blood cells. 4 Carbon monoxide easily boundswith hemoglobin thus become carboxy-hemoglobin. Other chemicals in cigarette can interfere the hemebiosynthesis thus reduce the hemoglobin levels in blood. 5 Besides causing abnormalities in erythrocytes and hemoglobin, cigarette smoke may also causing an increased in the total cholesterol levels. The enterednicotine within the body would triggerthe release of catecholamine, cortisol, and growth hormones. This hormone activated the adenylcyclase inadipose tissue, thereby increasing lipolysis and releasing free fatty acids into the plasma. This, eventually, causes change in lipid profile such as increased the levels of triglycerides and the very lowdensity lipoproteins (VLDL). 6 High blood cholesterol levels can have a negative impact on health, including increases the risk of atherosclerosis, hypertension, and coronary heart disease. 7 Alternative medicine using natural producthas been practiced for a long time to cure various health problems. Various medicinal plants have been used traditionally to treat various illness. Nigella sativa L., well known as black cumin or black seed, is one of natural medicine used in various regions in the world for wide range of illness. Black cumin is a shrub originating from the Mediterranean region and spread to India and Pakistan. However, this plant spreads to Southeast Asia including in Indonesia. 8,9 Black cumin contains major active substances well known as thymoquinone, thymohydroquinone, and thymol which exhibited various biological activities.
In vitro study revealed that N. sativa epicotyl suspension cultures have a high antioxidant activity, 10 where as in in vivo study on mice showed that black cumin oil has anti immunotoxic activity caused by DMBA (dimethylbenzantracene) induction. 11 In addition, dietary supplementation of N. sativa powdered seeds was reported ameliorate the serum lipid profile in rats. 12 In this study, the effects ofblack cumin (N. sativa) extract on erythrocytes and hemoglobin and total cholesterol levels in Wistar rats exposed by cigarette smoke. Two weeks of cigarette smoke exposure was applied and 2 doses of the black cumin extract were used according to the previous studies. 13,14

Essential oil of N. sativa preparation
Black cumin seeds were purchased from a local grocery store CV Almanar Herbafit in Yogyakarta. Extraction of essential oil of the black cumin was carried out using asteam distillation method. Five hundred g of black cumin seed sample was taken in a distillation flask and 1000mL of water was added. The condenser and the collection vessel were installed. The distillation flask was heatedand distillate was collected in the conical flask. This distillate had two layers, one dense layer and other less dense layer. This was then separated using a separating funnel. The less dense upper layer was the black cumin oil. Then the black cumin seeds oil was separated from water by 96% ethanol.

Animal treatments
Is was an experimental study using posttest only control group design. The protocol of the study was approved by the Bioethics Commission of Faculty of Medicine, Universitas Sultan Agung Semarang. The study was conducted in The Biology Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Negeri Semarang. Twenty fourmale Wistar rats (Rattus norvegicus) aged 8-12 weeks with 150-200 g body weight were used in this study. The rats were maintained for seven daysunder standard laboratory condition at room temperature in a 12 h light-dark cycle with free access to rat feeds and water ad libitum. Following after adaptation, the rats were then randomly divided into four groups with six rats in each group. The normal control group (N) was provided with standard feed, the negative control group (C) was exposed to the cigarette smoke with two pieces of cigarettes/day for 14 days, the treated groups were given black cumin oils 200 mg/kg (T1) and 400 mg/kg (T2) and exposed by cigarette smoke two pieces of cigarettes/day for 14 days. The cigarette smoke exposure was conducted 30 min before administration of black cumin oils.

Erythrocytes and hemoglobin analysis
On the day15, rats blood samples were taken from sinus orbital cantus for erythrocyte counts, hemoglobin levels, and total cholesterol level sex aminations. The erythrocytes countand hemoglobin levels were analyzed by Sysmex pocH-100i haematology analyzer. Erythrocytes were reported in total numbers (10 6 cells/L) and hemoglobin levels were reported as proportion of mg/dL.

Total cholesterol analysis
The total cholesterol levels were determined using CHOD-PAP method. The CHOD-PAP reagent was added in three tubes marked as Blank, Standard and Test, each 1000µL. Then 10μL of distilled water was added in Blank, 10μL of standard (200mg/dL) in Standard and 10μL serum in Test. The solutions were mixed properly and incubated at 37°C for 10 min. The serum total cholesterol levels was measured using a spectrophotometer (Thermo-Genesys 10S UV) at 546 nm against reagent blank and reported in mg/dL.

Statistical analysis
Data was presented as mean ± standard deviation (SD). The Saphiro Wilk and Levene tests were employed for data distribution and homogeneity. Considering the data obtained were normal and homogenous, the One Way ANOVA was employed, followed bythe Post Hoc test. A p value <0.05 was considered as significant result.

RESULT
Normality test of the erythrocytes count, hemoglobin levels, and cholesterol levels data both using the Saphiro-Wilk test and the Lavene homogeneity test showed the p values> 0.05 (TABEL 1). It was indicated that data was normally distributed and had a homogeneous variance. Therefore, the One Way ANOVA followed by the Post Hoc test was employed. The erythrocytes count, hemoglobin levels, and total cholesterol levels of all groups are presented in TABLE 2.
The erythrocytes countof C group was lower than that of N group (TABLE 2  and 3), although it was not significantly different (p>0.05), whereas the Hb levels of C group was significantly higher that of N group (p<0.05). It was demonstrated, the cigarette exposure could decrease in the Hb levels but not in the erythrocytes count of rats. In addition, the total cholesterol levels of C group was higher than that of N group (TABLE 2 and 3), although it was not significantly different (p>0.05). It was demonstrated, the cigarette exposure could not increase of the cholesterol levels of rats.
The erythrocytes count and Hb levels of T2 group was significantly higher than those of N, C and T1 group (p<0.005) as presented in Although the total cholesterol levels of T2 groups tended to lower compared to the N, C and T1 groups (TABLE 2), however they were not significantly different (p>0.05). It was demonstrated that there was no effect of the black cumin oils on the total cholesterol levels of rats.

DISCUSSION
The effect of N. sativa oils on erythrocytes count and hemoglobin levels No significantly decrease in the erythrocytes counts of rats after the cigarette smoke exposure (C)could occur due to lack of time and length of the exposure. In this study, the cigarette smoke exposure was only carried out for 14 days. Wulandari et al. 13 reported that smoke exposure time significantly reduce the number of erythrocytes at 28 days. The study reported that 1 to 4 cigarettes/day can reduce the number of erythrocytes and hemoglobin levels in Wistar rats exposed to cigarette smoke. 13 Reduction in the number of erythrocytes and hemoglobin can be caused by the presence of oxidants which causes lipid peroxidase in red blood cell membranes, then lead to lysis in red blood cell membranes. 15,16 In this study, significantly different in the erythrocytes count of the rats was observed after administration of black cumin oils at dose of 400 mg/kg BW (p<0.05) but not at dose of 200 mg/kg BW. This result isin accordance with previous study which reported that black cumin oils at dose of 400 mg/kg has protective effect to free radicals from cigarette smoke, whereas not at dose 200 mg/kg BW. 17 Black cumin extract contains substances such as polyphenols, thymoquinone, dithymoquinone, thymohydroquinone and thymol which have high antioxidant sactivity against free radicals. Thymoquinone can protect the erythrocyte membrane from oxidants that enter the cell to prevent hemolysis in erythrocytes. 18 A study conducted by Kanter et al. 19 showed that black cumin extract dose of 2 mL/kg BW for 30 days can protect blood including red blood cells from the toxic effects of cadmium.
To decrease the hemoglobin levels, cigarette smoke has the same mechanism as to decrease the number of erythrocytes, because hemoglobin is one of the components forming erythrocytes. This study shown that black cumin oils had an effect on increasing hemoglobin levels and protection from cigarette smoke free radicals. The administration of 400 mg/kgBW black cumin oils for 14 days showed a significant increase in hemoglobin levels, when compared to the C group.
Thymoquinone in black cumin extract can also inhibits the formation of hydroxyl radicals which oxidize the hemoglobin. In addition, thymoquinone as an antioxidant can prevent synthesis inhibition of hem. 20,21 Baskoro et al. 22 stated that the dose of black cumin extract that can significantly increase Hb levels is at500 mg/kg body weight. Although it mentioned that the number of cigarettes and the exposure duration of cigarette smoke was 4 cigarettes/day and given for 21 days, which is greater dose and longer period than in this study. 22 The effect of N. sativa on total cholesterol levels This study used black cumin oils as an exogenous antioxidant. It has been known to reduce total cholesterol levels by inhibiting the enzyme HMG CoA reductase. 9,12 Meanwhile, smoking is known to increase the lipid profile of adult smokers over the age of 20 years. 6 However, according to this study it was indicated that the cigarette smoke is not able to increase the total cholesterol levels in Wistar rats after 14days smoke exposure period. The black cumin oils also could not significantly reduce the total cholesterol levels in Wistar rats. The inability of black cumin extract to reduce LDL cholesterol levels was also reported by Khairunnisa et al. 23 It was reported that given black cumin extract up to 2 g/kg BW still has not been able to reduce LDL cholesterol levels of rats that was exposed to cigarette smoke.
Although in this study black cumin oilscould not reduce the total cholesterol levels of rats exposed to cigarette smoke, but the effect of black cumin to reduce cholesterol levels was reported in previous studies. An in vivo study conducted by Kocyigit et al. 12 using a supplement of powder from black cumin at various doses of 100, 200, 400 and 600mg/kg BW can produce a significant decrease in cholesterol after the fourth week of treatment. While in this study, although the dose of black cumin oils used was the same as the Kocyigit's study, the length of time for administration was only 14 days.
Although the effects of black cumin on the cholesterol of animal study are not consistent. Studies conducted using human subjects showed the effectiveness of black cumin. For example, different preparations of black seed including seed powder (100 mg-20 g daily), seed oil (20-800 mg daily), thymoquinone (3.5-20 mg daily), and seed extract (methanolic extract especially), could reduce plasma levels of total cholesterol, low-density lipoprotein cholesterol (LDL-C) and triglycerides. But the effect on highdensity lipoprotein cholesterol (HDL-C) was not significant. Nigella sativa and thymoquinone have been reported to be safe and well tolerated with no severe adverse effect. 24 Other meta-analysis study conducted by Sahebkar et al. 25 also shown a significant association between N. sativa supplementation and a reduction in totalcholesterol. A greater effect of N. sativa seed oil versus seed powder was observed on serum total cholesterol and LDL-C levels. An increase in HDL-C levels was found only after N. sativa seed powder supplementation. Nigella sativa has a significant impact on plasma lipid concentrations, leading to lower total cholesterol, LDL-C, and TG levels. While the increased HDL-C was associated with N. sativa powder only. According to Sahebkar et al. 25 further RCTs are needed to explore the N. sativa benefits on cardiovascular outcomes.

CONCLUSION
In conclusion, the black cumin oils at dose of 400 mg/kg BW significantly increase the erythrocytes count and hemoglobin levels of rats after cigarette smoke exposure. Meanwhile, there is no effect of black cumin oils on total cholesterol levels of the rats.