INTRODUCTION:

Black cumin honey is produced by bees that feed on the black cumin flower's nectar, darker in color, distinctive scent, and flavor. Honey was used among Indian, Arabic, and Egyptian gentility as a gift or drink, a preservative, and treatment for wounds, rashes, or sore throat¹. There are about 300 types of natural honey in which the main components of honey are carbohydrates (95–97%), proteins, amino acids, vitamins, minerals, flavonoids, and organic acid^2,3. Honey has therapeutic properties such as antibacterial, anti-inflammatory, antimutagenic, antiviral, antifungal, expedite wound healings, anti-diabetic and anti-tumor effects⁴. The Prophet Muhammed (PBUH) has described black cumin as a "remedy for every illness except death." Black cumin is also mentioned in the Holy Bible for its curative property^5,6.

The antibacterial activity of natural honey and black cumin was reported previously^7,9. Djakaria et. Al¹⁰ reported the antibacterial activity of several Indonesian honeys against P. acnes. However, there is no report on the antibacterial effects of black cumin honey on Propionibacterium acnes and Pseudomonas aeruginosa¹¹. Propionibacterium acnes is a gram-positive bacterium that presents on human skin, oral cavity, conjunctiva, large intestine, and ear canal¹². P. acnes is responsible for developing acne vulgaris and postoperative infections¹³. It has been reported that forest honeybee can decrease malondialdehyde levels in patients with mild acne vulgaris¹⁴. Pseudomonas aeruginosa is a gram-negative rod bacteria that thrives in soil, water, and animate surfaces. P. aeruginosa can cause infections that vary from skin infections to life-threatening infections such as sepsis and chronic airway infections¹⁵.

Here we reported the antibacterial activity of black cumin honey against P. acnes and P. aeruginosa as a topical cream formulation to develop a new antibacterial herbal medicine.

MATERIAL AND METHODS:

Chemicals and reagents:

Stearic acid, cetyl alcohol, sorbitol, propylene glycol, triethanolamine, glycerin, methylparaben, distilled water, nutrient broth, nutrient agar, and black cumin honey was purchased from Medan, North Sumatra, Indonesia.

Instrumentation:

The proposed work was carried out on a Shimadzu UV-visible spectrophotometer (model UV-1800 series), which possesses a double beam double detector configuration with a1 cm quartz matched cell. All weighing was done on electronic balance (Sansui-vibra DJ-150S-S). A Fast clean ultrasonicate cleaner (India) was used for degassing the mobile phase.

Phytochemical Analysis:

The chemical compounds in black cumin honey were evaluated with GC-MS (Shimadzu GC-2010 Plus)at Research Center for Geotechnology, LIPI – Bandung, Indonesia. The column's specification was 30 m in length, 0.25mm in diameter, and 0.11μm film thickness. The conditions were set with a helium carrier gas, column temperature 270°C, injection volume 5μL, split mode 10.

Analysis of flavonoid compounds was carried out using HPLC (HPLC Agilent Technologies 1260 Infinity II) at Research Center for Chemistry, LIPI – Serpong Indonesia. The column used was VDSpher PUR 100 C18-E with 150mm x 4.6mm, 3.5μm. Column temperature 40C, the flow rate of 0.8mL/minute, the time set 15 minutes, injection volume 5μL, detector UV with 260nm and 370nm.

Preparation of Cream:

The cream formulations used are; stearic acid 12 g, sorbitol 5g, propylene glycol 3g, cetyl alcohol 0,5 g, triethanolamine 1g, glycerin 1-5 drops, methylparaben 0,1g, and distilled water ad 100ml. All of the materials needed were weighted and separated into the oil phase and water phase. The oil phase was prepared by melting stearic acid and cetyl alcohol with a water bath. The water phase was prepared by dissolving sorbitol, propylene glycol, triethanolamine, glycerin, and methylparaben with weighted hot water. When the oil phase and water phase were at the same temperature, the water phase was added into the oil phase and was kept stirring until the cream base was formed. Then, black cumin honey was slowly added to the cream base and was kept stirred until homogenous cream was formed. Black cumin honey cream was formulated with the concentration of 1%, 5%, 10%, 15%, 20% and 30% (w/w).

Physical Stability Test:

The stability test included organoleptic (color, odor, and phase separation), homogeneity, pH measurement test, and emulsion type test^16,17. Creams were observed for four weeks at room temperature.

Disc Difussion Assay:

The creams were individually tested against test bacteria. The test bacteria cultures used were P. Acnes (ATCC 6919) and P. Aeruginosa (ATCC 27853) obtained from the Microbiology Laboratory, University of Sumatera Utara, North Sumatera, Indonesia.

Into a sterile petri dish, 0.1ml of suspension and 15 ml of nutrient agar medium was poured and homogenized, and keep at room temperature. Inoculated agar plates and filter paper discs containing black cumin honey were placed on the agar surface. The Petri-dishes were then incubated for 24 hours at 37°C. Then the diameters of inhibition growth zones were measured¹⁸.

Statistical Analysis:

The results obtained were expressed as Mean ± SD. The two-way analysis of variance (ANOVA) was used to test for statistical significance with p<0.05.SPSS software was used for statistical analysis of experimental data.

RESULT AND DISCUSSION:

GC-MS Analysis:

Fig.1: GC-MS chromatogram of black cumin honey

Black cumin honey was analyzed by GC-MS regarding the volatile compounds, which may contribute to the antibacterial effects. The NIST library search is used to identify the chemical compounds. The GC-MS procedure is based on the ethanol extraction 1:1 (v/v) with sonication. A total of 25 compounds from black cumin honey were identified (Figure 1, Table. 1). The compounds' profile showed that the main components were saccharides and organic fatty acids^19-21. Saccharide compounds such as maltol, sucrose, d-allose, and glucofuranose were identified in black cumin honey. Studies on saccharide compounds revealed that saccharide could damage bacterial cell walls and cell membranes, increase cell permeability, thus causing cell death^22,23. The amino acid, such as asparagine, was also identified. A study showed an increase in the antibacterial activity of oligosaccharides when conjugated with asparagin²⁴. Organic fatty acids, including furan-2-carboxylic acid, octanoic acid, butanedioic acid, hexanoic acid, dodecenoic acid, cyclohexane carboxylic acid, benzoic acid, and hexanedioic acid, were identified. Organic fatty acid components in natural honey have shown antibacterial effects^25-28. A study showed that furan-2-carboxylic acid had antibacterial activity against Bacillus sp.²⁹. Hexanoic acid (caproic acid) and octanoic acid (caprylic acid) had been reported to inhibit S. aureus and E. Coli³⁰. Benzoic acid had been reported to inhibit E. coli, K. pneumonia, B. cereus, A. flavus, and A. parasiticus³¹. Hexanedioic acid has been reported with the antibacterial activity against S aureus, MRSA, K. pneumonia, and S. dysenteriae³². Fatty acids can decrease pH value and destabilize the bacterial cell membrane's phospholipid layer, changing the membrane permeability, cellular functions leading to cell lysis^25,27.

Table 1: The chemical compounds of black cumin honey detected by GC-MS

Compound	Ret. Time	Compound	Ret. Time
Ethanol	1258	Butanedioic acid	16.325
2-isopropoxyethylamine	1.458	Butane	16.908
Acetamide	1.583	Hexanoic acid	17.133
dl-Glyceraldehyde	5.758, 7.775	Sucrose	18.017
Maltol	12.008	D-Allose	18.383
Furane-2-carboxylic acid	12.075	Beta-d-Xylofuranoside	19.167, 19.583
4H-Pyran-4-one	13.167, 15.508	Trans-2-Dodecenoic acid	19.283
Propanal	13.492	Cyclohexanecarboxylic acid	19.667
2-Furancarbocaldehyde	14.608	1,6-Anhydro-beta-D-glucofuranose	19.842
1,2,3-propanetriol	14.808	Benzoic acid	21.858
d-Glycerol-d-ido-heptose	15.267	Hexanedioic acid	27.867
Methylene asparagines	15.975	Tetrapentacontane	30.217
2-Octanoic acid	16.125

HPLC Analysis:

The formulated cream was oil-in-water The number of flavonoid compounds was analyzed by the HPLC method with a DAD detector. The result showed that flavonoid (quercetin) was present in black cumin honey with a concentration of 0.223mg/g (Fig 2).

Fig 2: HPLC-DAD chromatogram of black cumin honey

Quercetin is a flavonol class of flavonoids and one of the most found flavonoids in honey. The inhibitory activity of quercetin against S. typhi, S. typhimurium, E. coli, V. cholera, P. vulgaris, S. Aureus, and P. aeruginosa has been reported^33-35,43. The antibacterial activity of flavonoid is attributed to three mechanism pathways; (a) Inhibition of nucleic acid synthesis, (b) inhibition of cytoplasmic membrane function by influencing the porin and permeability, and (c) inhibition of energy metabolism^35,36,44.

Physical Stability Test:

An The physical stability test result indicated no odor and color change, homogeneous, and no phase separation. These results indicated that black cumin honey cream was physically stable (Table 2). The cream's pH was in the range of ideal pH of skin balance (pH 4.5–6.5). The formulated cream was oil-in-water emulsion.

Table 2: The physical stability test of formulated cream

Parameter	Weeks
Parameter	1	2	3	4
Odor	No	No	No	No
Color	No	No	No	No
Phase separation	Good	Good	Good	Good
Homogeneity	Good	Good	Good	Good
pH	6.0	6.0	5.9	5.9
Emulsion type	O/W

Disc Difussion Assay:

The antibacterial activity of black cumin honey cream from various concentrations showed varying results (Table 3). The highest antibacterial activity against P. acnes and P. aeruginosa was shown in 30% concentration with d= 11.2mm and 10.8mm, respectively. The lowest antibacterial activity against both P. acnes and P. aeruginosa was shown in 1% concentration with d = 6.3mm and 6.2mm, respectively.

Table 3: The inhibition zone (mm) of formulated cream

Concentration	*P. acnes*	*P. aeruginosa*
1% black cumin honey cream	6.3 ± 0.1	6.2 ± 0.2
5% black cumin honey cream	7.2 ± 0.3	7.0 ± 0.1
10% black cumin honey cream	8.8 ± 0.2	8.4 ± 0.3
15% black cumin honey cream	9.4 ± 0.4	9.1 ± 0.3
20% black cumin honey cream	10.4 ± 0.3	9.8 ± 0.4
30% black cumin honey cream	11.2 ± 0.2	10.8 ± 0.3
Black cumin honey	16.8 ± 0.5	16 ± 0.2

*Values are given as means of 3 replicates ± standard error.

These data showed that inhibition diameter increased as the cream concentration increased. Our findings were in concordance with a study conducted in 2011, which showed that 10% Manuka honey 10+ UMF cream and 30% Manuka honey 10+ UMF cream exhibited inhibitory ability against P. acnes³⁷.

Based on the literature, the antibacterial effect is related to osmolarity, water activity, pH, hydrogen peroxide, and phytochemical properties³⁸. Honey, like other saturated sugar syrups and sugar pastes, has high osmolarity that is sufficient to stop microbial growth. The high osmotic effect in honey is due to its high sugar content. Osmotic pressure is exerted on bacterial cells, and through osmosis, water is transported out of bacterial cells. This causes cells unable to grow and proliferate39. Honey has low water activity with ranges from 0.56 to 0.62. Most microbes cannot grow in low water activity because it will affect bacterial growth and enzyme activity. When the suspending medium's essential water activity is low, water will be withdrawn from microbial cells, and intracellular fluid will be concentrated until equilibrium is reached. Microbial growth and metabolism cease when intracellular fluid concentration is beyond a critical value. Although the cells are still alive, they cannot replicate in a low water activity environment^2,40,45.

The pH ranges from 3.2 to 4.5 are low enough to inhibit bacterial growth since most bacteria positively grow at a pH of 6.5 to 7.5. The acidic pH in honey is due to the organic fatty acids in honey^38,41. Hydrogen peroxide is well-known for its antimicrobial and antiseptic properties. Honey produces hydrogen peroxide through the activation of the enzyme glucose oxidase. However, the typical concentration of hydrogen peroxide produced in honey is around one mmol/l, but it is still effective as an antimicrobial agent^39,41. Phytochemical factors are non-peroxide antibacterial factors, consisting of flavonoids, organic acids, and phenols. These compounds have antibacterial activity and are stable under heat or light, either with dilution⁴². The dark color of honey reflects a higher amount of flavonoids³⁸.

Representative pictures of experimental condition and formulated cream shown in figure 3 suggest a stabilized cream could be used as a new potential therapy for antibacterial purposes.

Fig 3: A- Antibacterial activity against Propionibacterium acnes, B- Antibacterial activity against Pseudomonas aeruginosa, C- Formulated black cumin honey cream

CONCLUSION:

The present study showed that phytochemical compounds such as fatty acids, amino acids, saccharides, and flavonoids are detected in black cumin honey. The antibacterial assay showed great antibacterial activity of formulated cream. The bioactivity might be related to the high osmolarity, low water activity, low pH, hydrogen peroxide, and honey's phytochemical properties.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Universitas Prima Indonesia for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 01.12.2020 Modified on 09.02.2021

Research J. Pharm. and Tech 2021; 14(11):5691-5695.

DOI: 10.52711/0974-360X.2021.00989