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Tolerability and safety of black cumin seed oil (Bcso) administration for 20 days in healthy subjects

Akrom Akrom1,2*, Endang Darmawan1

1Faculty of Pharmacy, Ahmad Dahlan University, Yogyakarta, DIY, Indonesia

2Ahmad Dahlan Drug information and Crisis Center, Yogyakarta, DIY, Indonesia

*Corresponding Author:
Akrom Akrom
Head of Ahmad Dahlan Drug Information and Crisis Center
Universitas Ahmad Dahlan, Yogyakarta, Indonesia

Accepted date: February 28, 2017

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Abstract

The objective of this research was to determine the effect of black cumin seed oil (BCSO) on hematology, level of aspartat aminotransferase (AST)/alanin aminotransferase (ALT), level of ureum and creatinine, and also immune response in healthy subjects. This study applied a phase 1 of clinical trial. The 36 healthy subjects were divided into three groups, 12 subjects each group. Group I, subjects received 1 × 3 soft capsules of BCSO. Group II, subject received 2 × 3 soft capsules of BCSO and in groups III subject received 3 × 3 soft capsules of BCSO. Each soft capsule contained of 0.5 ml standardized BCSO. On the 21st day, the blood cell, level of ALT and AST and level of ureum and creatinine were measured. The average number of blood cells, levels of ALT/AST and level of ureum and creatinine then were been analyzed with one way ANOVA test. Statistical analysis was performed on 95% confidence level. The results showed that the administration of three doses of BCSO soft capsule did not increase number of blood cells, level of ALT/AST and level of ureum and creatinine (p>0.05). BCSO also did not affect number of CD4+ Th and CD4+CD25+ Treg cells and also expression of IFN-γ (p>0.05). The consumption of BCSO at three doses 1.5, 3, and 4.5 ml/d for 21 days in healthy subjects were tolerable and safety.

Keywords

Black cumin seed oil, Phase 1 clinical trial, Safety, Soft capsule immunax.

Introduction

Black cumin seed oil (BCSO) has often been used by people as an alternative and complementary medicine. BCSO contains a lot of unsaturated fatty acids such as linoleic acid and linolenic acid. It also contains of essential oil with active substances such as thymoquinone, and nigellone (dithymoquinone) [1-3]. Unsaturated fatty acids and thymoquinone are powerful antioxidants and immunomodulatory agents [4-7]. The mechanism actions of antioxidant and anti-inflammatory effects of BCSO have been investigated both in preclinical [8-10] and clinical studies [11-13].

Unfortunately, the administration of a combination of BCSO and other herbal preparations has been reported cause hepatic dysfunction in Indonesia. Furthermore, the administration of a combination between BCS and Phyllantus niruri increases the activity of ALT and AST in hospitalized-patients. But, other clinical trials showed that 300-500 mg/d of BCS powders supplied with standard drugs in patients improve the profile blood cholesterol, lowering the percentage of HbA1C and blood pressure in patients menopause and non-menopause and is not accompanied by increase in SGPT and SGOT or their harmful side effects [14-16].

This study was aimed to determine the effect of BCSO at dose of 1.5, 3 and 4.5 ml/d for 20 days on peripheral blood, liver and kidney function in healthy subjects to study the safety of BCSO. The influence of BCSO on immune responses was observed by measuring the expression of IFN-γ in T lymphocytes and the number of CD4+ Th and CD4+CD25+ Treg cells. In this study, BCSO was prepared in soft capsules to be more acceptable by the subjects.

Methods

Materials

BCSO was standardized for the concentration of thymoquinone and then was formed in soft capsules immunax (SCI). Specification of BCSO is presented in Table 1. The capsules were given to the subjects orally daily at 1.5 ml (1 × 3 SCI), 3 ml (2 × 3 SCI) and 4.5 ml (3 × 3 SCI).

Content of BCSO Relative concentration (%)
Thymoquinone 2.72
Caprilic acid 0.21
Capric acid 0.15
Lauric acid 0.1
Myristic acid 0.18
Palmitic acid 12.27
Palmitoleic acid 0.28
Heptadecanoic acid 0.1
Oleic acid 0.07
Linoleic acid 2.85
Linolenic acid 0.1
Eicosanoic acid 3.15
Eicosenic acid 0.15
Eicosadienoic acid 0.25
Arachidonic acid 0.03
Eicopentanoic acid 0.03
Behenic acid 0.06
Docoheksanoic acid 0.04
Teracosanoic acid 0.02

Table 1: The content of BCSO in the soft capsules immunax (SCI).

Subjects

The inform consent and the ethical clearance for this study was reviewed and approved by Research Ethics Committee, Ahmad Dahlan University (No: 011503029). Respondents for the study were selected based on the inclusion and exclusion criteria. The inclusion criteria were men/women aged 18-60, be healthy evidenced by a health certificate, and willing to be the respondent (by filling the informed consent). The exclusion criteria were woman pregnancy and hypersensitive history to ingredient of Nigella sativa oil. Physical examination and clinical examination of prospective subjects are conducted to ascertain the health status.

Recruitment of subjects

The brochures/leaflets, the recruitment procedure, the informed consent, the research procedures as well as case report form have been reviewed and approved by the Ethics Committee of Ahmad Dahlan University. Researchers distributed the brochures and the announcement about the recruitment of volunteers of BCSO safety testing in the campus of Ahmad Dahlan University and Gadjah Mada University as well as in some public facilities in Yogyakarta. Interested people should call on the phone number listed in the brochure or leaflet. For prospective volunteers who have signed up are then invited for an explanation of the purpose, benefits, and procedures as well as the consequences and the rights of research volunteers. After signing the informed consent, the subjects performed a physical and clinical examination to ascertain their health status. A total of 36 volunteers received health certificates and signed the informed consents.

Experimental design

A total of 36 healthy subjects were randomly divided into three experimental groups. Group 1 was given 1 × 3 BCSO soft capsules (SC) (1.5 ml/d), group 2 was given 2 × 3 BCSO SC (3 ml/d) and group 3 was given 3 × 3 BCSO SC (4.5 ml/d). BCSO SC were orally administered every day for 20 days. In day 21, the peripheral blood, the levels of AST/ALT, BUN and creatinine level, number of CD4+ Th and CD4+ CD25+ Treg cells and expression of IFN-γ were determined.

Blood pressure and heart rate measurement

Measurement of blood pressure and heart rate is performed three times during the study by professionals. Blood pressure was measured in the left arm using a mercury sphygmomanometer, supine sleeping position, wide cuff adapted to the size of the arm, as recommended by WHO. Each blood pressure measurement was repeated 2 times with 30 minutes interval time between measurements. The heart rate was calculated on the radial artery and repeated 3 times. The value of blood pressure and heart rate were presented as average value.

Peripheral blood, renal and liver function analysis

Peripheral blood was taken and collected from the cubital vein by authorized analyst. The blood was then examined for the peripheral blood and kidney function by measuring level of urea and creatinine using hematoanalyzer as performed in a previous study [17].

Expression of IFN-γ and number of CD3+CD4+ Th and CD3+CD4+CD25+ treg cells analysis

Immune response was observed by measuring the expression of IFN-γin T lymphocytes and the number of CD4+ Th and CD4+CD25+ Treg cells. IFN-γ expression and the number of CD4+ Th and CD4+CD25+ Treg cells been measured using flow cytometry.

Data analysis

The significant difference between groups was tested by one way Anova analysis. The difference intern group before and after treatment were tested by two-way ANOVA. The statistical test was performed at 95% confidence level. The average difference in blood pressure and heart rate between measurements in one group was analyzed with repeated ANOVA.

Results

Demographic characteristic of respondents

Respondents who participated in this study are 36 subjects consisting of 10 (22.92%) men and 26 (77.08%) women. Characteristics of the respondents are presented in Table 2. Respondents in this study aged 18-60 years. Based on body mass index (BMI), there were 15 (40.83%) people having BMI <25 (non-obese) and 21 (59.17%) subjects having BMI ≥ 25 (obese). Based on a history of hypertension owned respondents, there were 2 (5.17%) people with history of hypertension and 34 (94.83%) had no history of hypertension. Based on the level of education, respondents who had finished high school were 9 (25.00%) people and 27 (75%) respondent have passed university. While based on marital status, 5 (14.58%) respondents are categorized as married and 31 (85.42%) people are unmarried. Statistically the sex, age, BMI, history of hypertension, education, occupation and marital status of respondents were not different (p>0.05).

Characteristic of the respondents n % p
Sex      
Men 10 27.8 0.35
Women 26 72.2
Age      
≤ 25 years 30 83.33 0.947
>25 years 6 16.67
Body mass index (BMI)      
<25 (kg/cm2) 15 41.7 0.918
≥ 25 (kg/cm2) 21 58.3
History of Hypertension      
Yes 2 4.17 0.837
No 34 95.83
Education      
High School 9 25.00 0.056
Graduate program 27 75
Occupation      
Student 28 79.17 0.734
Non-government 6 14.58
entrepreneur 2 6.25
Marital status      
Married 5 14.58 0.673
Not married 31 85.42
Health assurance      
Private health assurance 3 8.3 0.056
Public heath assurance 10 27.8  
Not health assurance 23 63.9  

Table 2: Characteristic of respondents consummated BCSO 1.5; 3; 4.5 ml/d.

Clinical characteristic of respondents

The clinical condition of the volunteers before treatment are presented in Table 3. As shown in Table 3, the clinical condition of the subject prior to the administration BSCO under similar conditions. Levels of hemoglobin, number of erythrocytes, leukocytes and platelets within the normal range and comparable between treatment groups (p>0.05). The results of the number of leukocytes to monocytes, neutrophils, eosinophils and basophils in all three groups were comparable (p>0.05).

`
Clinical characteristic Treatment Groups p
Group I (n=12) Group II (n=12) Group III (n=12)
Age (years) 25.83 ± 6.58 24.08 ± 4.48 23.83 ± 4.45 0.6
Body Weight (Kg) 57.42 ± 11.33 58.00 ± 16.18 61.50 ± 17.66 0.8
Pulse (frex/menutes) 73.00 ± 4.39 76.16 ± 7.79 75.50 ± 5.60 0.6
Systolic blood pressure (mmHg) 116.25 ± 16.80 117.00 ± 14.19 114.35 ± 17.89 0.8
Diastolic Blood Pressure (mmHg) 73.75 ± 10.02 73.33 ± 13.70 73.75 ± 12.45 0.7
Hb (pg) 13.28 ± 13.28 13.04 ± 1.17 14.19 ± 0.74 0.6
Erithrocytes (×106/mL) 4.885 ± 0.37 5.02 ± 0.43 5.15 ± 0.51 0.2
Leucocytes (×103/mL) 8.22 ± 2.05 7.42 ± 1.68 9.26 ± 4.45 0.5
Thrombocytes (×103/mL) 311.17 ± 73.73 302.17 ± 85.96 299.90 ± 56.35 0.6
Limphocytes (%) 30.58 ± 4.85 36.41 ± 8.48 31.67 ± 9.61 0.6
Monocytes (%) 7.67 ± 1.30 7.00 ± 1.28 6.17 ± 1.11 0.3
Neutrophil (%) 57.92 ± 6.68 53.83 ± 9.67 59.42 ± 10.48 0.8
Eosinophil (%) 3.83 ± 2.62 2.75 ± 1.76 2.75 ± 1.54 0.9
ESR hour 1 (mm/h) 14.92 ± 11.06 16.33 ± 12.87 15.67 ± 12.18 0.8
ESR hour 2 (mm/h) 34.67 ± 24.42 32.75 ± 22.17 32.25 ± 20.33 0.9
MCV (fl) 81.94 ± 7.02 81.49 ± 7.82 83.02 ± 5.67 0.5
MCH (pg) 27.41 ± 2.63 26.81 ± 3.48 27.75 ± 1.90 0.5
MCHC (%) 33.44 ± 0.72 32.82 ± 1.41 33.45 ± 0.66 0.4
RDW (%) 13.77 ± 1.86 13.66 ± 1.75 13.05 ± 0.78 0.6
Glucose (mg/dl) 89.17 ± 13.23 94.33 ± 14.67 93.33 ± 16.52 0.7
Total cholesterole (mg/dl) 177.00 ± 23.33 197.25 ± 24.38 183.67 ± 31.26 0.2
Triglyceride (mg/dl) 97.08 ± 34.91 149.33 ± 95.34 99.42 ± 53.89 0.1
ALT (mg/dl) 23.58 ± 29.26 18.03 ± 16.80 20.06 ± 17.69 0.9
AST (mg/dl) 22.47 ± 21.51 20.76 ± 11.62 19.19 ± 9.18 0.9
BUN (mg/dl) 7.96 ± 1.80 8.43 ± 2.31 7.55 ± 2.31 0.4
Creatinine (mg/dl) 0.71 ± 0.11 077 ± 0.16 0.76 ± 0.16 0.5
IFN-γ (%) 3.06 ± 1.95 2.19 ± 1.51 3.44 ± 2.86 0.6
CD4+ Th Cells (%) 46.19 ± 7.55 41.49 ± 8.61 39.30 ± 9.17 0.6
CD4+CD25+ Treg Cells (%) 24.30 ± 6.71 28.30 ± 9.50 25.20 ± 6.17 0.5

Table 3: Clinical characteristics of the healthy subject before BSCO administration.

Analysis of blood pressure and heart rate

In this research, monitoring clinical conditions of the respondents included blood pressure and heart rate. Clinical examination includes blood pressure, temperature; rhythm and heart rate were performed on day 0, 10 and 20. The average of blood pressure of the respondents is presented in Table 4.

Groups Parameters Blood pressure (mmHg) and pulse (frex/men) (mean ± SD) p
Control Day 10  Day 20
Group I (n=12) Systolic BP (mmHg) 116.25 ± 16.80 109.50 ± 11.29 110.83 ± 11.64 >0.05
Diastolic BP (mmHg) 73.75 ± 10.02 70.08 ± 7.26 69.58 ± 9.40 >0.05
Pulse (frex/menut) 73.00 ± 4.39 75.00 ± 5.42 76.33 ± 8.97 >0.05
Group II
(n=12)
Systolic BP (mmHg) 117.00 ± 14.19 116.67 ± 14.03 112.50 ± 12.88 >0.05
Diastolic BP (mmHg) 73.33 ± 13.70 70.00 ± 9.04 65.83 ± 11.83 >0.05
Pulse (frex/menit) 76.16 ± 7.79 78.33 ± 8.97 74.16 ± 7.00 >0.05
Group III
(n=12)
Systolic BP (mmHg) 114.35 ± 17.89 112.08 ± 14.37 107.92 ± 16.98 >0.05
Diastolic BP (mmHg) 73.75 ± 12.45 72.91 ± 9.40 70.41 ± 12.14 >0.05
Pulse (frex/menit) 75.50 ± 5.60 74.83 ± 5.74 77.33 ± 7.92 >0.05

Table 4: The average of blood pressure between control and treatment groups treated by BCSO.

Overall, the average of the systole and diastole blood pressure both on day 0 and day 10 was not significant (p>0.05). While there was a significant difference in the average of blood pressure on day 0 and day 10 compared to day 20 both systole and diastole (p<0.05). However, there are no significant differences in the average blood pressure in both systole and diastole between the groups treated with control (p>0.05). BCSO did not significantly affect to the blood pressure and heart rate than the control group. Thus, the consumption of BCSO for 20 days does not significantly affect blood pressure in healthy subjects.

Blood composition and liver & renal function analysis

The administration of 1 × 3, 2 × 3 and 3 × 3 SC BCSO for 20 days in healthy respondents did not affect the blood components. The number of blood cells such as leukocytes, erythrocytes and platelets within normal limits and there was no significant difference between treatment groups (p>0.05). The normal value of hemoglobin is >13 ml/dl in men or >12 mg/dl in women. Hb value of all groups was >13 mg/dl. The number of erythrocytes for normal condition is 3-7 × 103 cell/dl. The number of erythrocytes in all groups was >4 ×106 cells/dl. The results showed that administration of a 1 × 3, 2 × 3 and 3 × 3 BCSO SC in healthy respondents did not affect blood cell number.

Table 5 revealed that the administration of 1 × 3, 2 × 3 and 3 × 3 BCSO SC for 20 days did not affect the levels of AST, ALT, BUN, and creatinine. The levels of BUN, creatinine, and ALT/AST of subjects within the normal range. The levels of BUN, creatinine and AST/ALT after the treatment did not differ between the experimental groups (p>0.05). ALT and AST are useful biomarkers of liver function. ALT and AST values on all treatment groups were in the normal range. In recent years it was reported that the administration of BCSO combined with other herbal preparations caused hepatic dysfunction, but our study indicate that administration for 20 days BCSO does not affect liver function and kidney function.

Blood components Treatment groups p
Group1 treated 1.5 ml BCSO Group2 treated 3 ml BCSO Group3 treated 4.5 ml BCSO
Hemoglobin (pg) 13.33 ± 1.47 13.62 ± 1.60 14.12 ± 0.57 >0.05
Erythrocyte (× 106/ml) 4.87 ± 0.31 5.09 ± 0.41 5.06 ± 0.46 >0.05
Hematocrit (%) 39.53 ± 3.79 4.0 ± 3.87 41.69 ± 1.3 >0.05
Leucocyte (× 103/ml) 8.34 ± 1.64 8.44 ± 1.85 76.1 ± 1.63 >0.05
MCV (fl) 81.30 ± 7.3 80.98 ± 8.0 82.84 ± 5.57 >0.05
MCH (pg) 27.38 ± 2.75 26.9 ± 3.45 28.04 ± 1.98 >0.05
MCHC (%) 33.66 ± 0.81 33.13 ± 1.32 33.86 ± 0.56 >0.05
RDW (%) 13.97 ± 2.29 13.75 ± 1.80 13.14 ± 0.80 >0.05
Thrombocyte (× 103/ml) 302 ± 70.56 312 ± 80.71 305 ± 52.66 >0.05
Lymphocyte (%) 31.75 ± 4.98 33.33 ± 7.86 33.58 ± 8.24 >0.05
Monocyte (%) 7.00 ± 1.54 6.67 ± 1.23 8.42 ± 3.90 >0.05
Neutrophil (%) 57.83 ± 6.28 57.67 ± 8.82 55.42 ± 10.76 >0.05
Eosinophil (%) 3.42 ± 2.81 2.33 ± 1.44 2.82 ± 2.02 >0.05
ESR hour 1 (mm/h) 14.58 ± 11.0 14.00 ± 11.0 16.08 ± 11.07 >0.05
ESR hour 2 (mm/h) 34.25 ± 20.67 30.75 ± 22.12 32.75 ± 20.63 >0.05
Glucose (mg/dl) 89.17 ± 13.23 94.33 ± 14.67 93.33 ± 16.52 >0.05
Total cholesterol (mg/dl) 177.00 ± 23.33 197.25 ± 24.38 183.67 ± 31.26 >0.05
Triglyceride (mg/dl) 97.08 ± 34.91 149.33 ± 95.34 99.42 ± 53.89 >0.05
AST (mg/dl) 22.81 ± 21.70 19.18 ± 7.80 18.52 ± 3.38 >0.05
ALT (mg/dl) 19.57 ± 25.59 16.13 ± 14.53 16.82 ± 11.96 >0.05
BUN (mg/dl) 8.17 ± 1.68 9.13 ± 3.05 7.83 ± 2.27 >0.05
Creatinine (mg/dl) 0.72 ± 0.0.07 0.78 ± 0.15 0.77 ± 0.14 >0.05
IFN-γ expression (%) 4.52 ± 2.84 3.65 ± 1.54 5.02 ± 2.60 >0.05
CD4+Th (%) 53.49 ± 7.76 49.94 ± 6.94 46.24 ± 5.33 >0.05
CD4+CD25+Treg (%) 13.23 ± 5.99 12.11 ± 2.18 12.44 ± 3.19 >0.05

Table 5: The clinical characteristic of healthy subject after administration 1.5, 3 and 4.5 ml/day BCSO for 20 days (each group n=12 subject).

Expression of IFN-γ and Number of CD4+ Th and CD4+CD25+ Treg cells analysis

The administration of 1 × 3, 2 × 3 and 3 × 3 BCSO SC for 20 days in healthy subjects did not affect the number of CD4+ Th cells, CD4+CD25+ Treg cells and expression of IFN-γ in lymphocytes. Table 5 shows that the BCSO administration for 20 days in healthy subject did not affect the number of CD4+ Th and CD4+CD25+ Treg cells and also expression of IFN-γ (p> 0.05). BCSO may increase the immune response. Our preclinical study demonstrated that administration of BCSO increases macrophage activity and T lymphocytes in rats exposed by dimethylbenzantracene (DMBA) [17].

Acknowledgements

We acknowledge to all volunteers who are willing to become study subjects. Thanks are also expressed to the Ministry of Higher Education, Research and Technology, Indonesian Government for funding this research.

References