Deodorant Activity of Black Cumin Seed Essential Oil against Garlic Organosulfur Compound

The deodorant activity of black cumin (Nigella sativa L.) seed, a spice used to flavor curry and vegetable foods in Southwest Asia, against garlic (Allium sativum L.) organosulfur compounds related to human malodor was evaluated. Black cumin seed essential oil showed remarkable deodorant activity against garlic essential oil. The mode of action of this deodorant activity was presumed to be that black cumin seed essential oil covalently reacted with the organosulfur compounds in garlic. Therefore, thymoquinone, which is a major constituent in black cumin seed essential oil, and allyl mercaptan, which is one of the organosulfur compounds produced by cutting garlic, were reacted in vitro, and the products were purified and elucidated using spectroscopic data. As a result, these substances were identified as different allyl mercaptan adducts to dihydrothymoquinone. This chemical reaction was presumed to play a key role in the deodorant activity of black cumin seed essential oil.


Introduction
Black cumin (Nigella sativa L.) seed is a species of the Ranunculaceae family, and thymoquinone is a major component of black cumin seed essential oil. Black cumin seed essential oil (BCO) exhibits various beneficial effects, including restoring the antioxidant defense system, increasing the activity of antioxidant-related enzymes, decreasing inflammatory biomarkers, suppressing pro-inflammatory mediator production, preventing endothelial dysfunction, and improving hepatic, kidney, heart, and immune system functions [1].
Garlic (Allium sativum L.) is a species of the Amaryllidaceae family and is used as a food and spice worldwide. This plant exhibits various functions, such as antibacterial, antiviral, antioxidant, and anti-inflammatory effects [2]. However, garlic food products contain various organosulfur compounds that lead to bad breath, and the odor lasts for a long time after ingestion. For this reason, many people often hesitate to eat garlic products [3].
Halitosis and a peculiar garlic odor are common after the ingestion of garlic products. In the oral cavity, garlic fragments result in methyl mercaptan (MM) and hydrogen sulfide, leading to bad breath from these food products. MM and hydrogen sulfide are common causes of malodorous breath exhaled from the normal human body. In addition, other organosulfur compounds, including allyl mercaptan (AM) and allyl methyl sulfide (AMS), result from digestion in the stomach, and these chemicals are released as exhaled breath and excreted through sweat glands and sebaceous glands by the bloodstream, leading to body odor [4].
Since MM is a primary factor for halitosis, it has been studied extensively [1]. There have been several reports concerning halitosis prevention using plant materials against MM. Thyme (Thymus vulgaris L.) and rosemary (Rosmarinus officinalis L.) have both shown

Botanical Essential Oils
Black cumin (N. sativa) seed essential oil (BCO, thymoquinone content of 41.5% (v/v)) was purchased from Arjuna Natural (Cochin, India). Garlic (A. sativum) essential oil (GEO) was purchased from Plant Lipids (Cochin, India). Both BCO and GEO were manufactured industrially by steam distillation.

Isolation of AMTS and DATS from GEO
GEO (100 g, including AMTS 10.0% and DATS 45.2%) was distilled under reduced pressure (temperature, 70 • C; vacuum, 0.5 kPa for 1 h), and garlic distillation oil (GDO, 24.1 g, including AMTS 15.7% and DATS 9.4%) and its residue of GEO after distillation (RGO, 68.4 g, including AMTS 9.4% and DATS 67.5%) were obtained. GDO (8 g) was then loaded into a preparative high-performance liquid chromatography (HPLC) system (Shimadzu), using the following parameters: column, L-column ODS (separation column 20 mm I.D. × 250 mm, particle size 5 µm, guard column 10 mm I.D. × 20 mm, particle size 5 µm, Chemicals Evaluation and Research Institute, Tokyo, Japan); column temperature, ambient; mobile phase, water:acetonitrile = 25:75; flow rate, 18.9 mL/min; injection volume, 400 µL; and a photodiode array detector. UV absorption was monitored at 190 and 214 nm. Under these conditions, the two fractions with peaks at 8 min and 10 min were collected and concentrated to obtain GDO Fraction 1 (yield 3.24 g) and GDO Fraction 2 (yield 0.52 g), respectively. GDO Fraction 1 was again purified in the same preparative HPLC system with a different method using a mobile phase of water:acetonitrile = 30:70 and an injection volume of 50 µL. The fraction with a peak at 10.5 min was collected and concentrated to obtain GDO Fraction 3 (yield 0.33 g) [17][18][19]. GDO Fraction 2 and GDO Fraction 3 were identified as DATS (purity 95.8%) and AMTS (purity 98.4%) by GC-MS analysis, respectively. Furthermore, these isolated compounds were identified using NMR spectra.

GC-MS Analysis
The following GC-MS analytical conditions were used: column, InertCap Pure-WAX (60 m × 0.25 mm I.D., 0.25 mm film thickness, GL Sciences, Inc., Tokyo, Japan); injection temperature, 250 • C; injection volume, 1 µL; split ratio, 80:1; carrier gas flow, 1.2 mL/min (helium, constant flow); temperature program, 50 • C (2 min hold), 50-240 • C at 2.5 • C/min and 240 • C (50 min hold); ion source temperature, 230 • C; and quadrupole temperature, 150 • C. Ionization was performed using the electron impact ionization method at 70 eV (scan range 25-350 m/z). The constituents of the essential oil were identified using the NIST MS library (NIST 08) (National Institute of Standards and Technology, Standard Reference Data, Gaithersburg, MD, USA) and the Wiley MS Library (Version 7) (Cerno Bioscience, Las Vegas, NV, USA) along with retention indexes. The content of each component was shown as the area percentage relative to the total area of detected peaks in the GC-MS analysis.

Evaluation of Deodorant Activity
The deodorant activity of BCO against GEO and the organosulfur compounds in garlic was evaluated using the headspace method. The disintegration test solution (pH 1.2) used was diluted, 1st fluid for disintegration test, pH 1.2/1st fluid for dissolution test, pH 1.2 (×10) (Wako 066-06441, Fujifilm Wako Pure Chemical, Tokyo, Japan), with ion-exchanged water (pH 1.2 test solution). The phosphate buffer (pH 7.0) used was an instant phosphate buffer (RM-102-3 L, LSI Medience Corporation, Tokyo, Japan). GEO and the eight major organosulfur compounds in garlic were prepared with ethanol to defined concentrations (GEO, 300 ng/µL; AM, AMS, DAS, AMDS, DMTS, and DADS, 10 ng/µL; AMTS, 500 ng/µL; and DATS, 1000 ng/µL). These concentrations of organosulfur compounds were adjusted under PFPD saturation. The BCO ethanolic solution (1 mL) and pH 1.2 test solution or pH 7.0 buffer (5 mL) were mixed in a 30 mL glass vial and sealed with a silicone cap. After adding 50 µL of GEO or organosulfur compound ethanolic solution, the glass vial was shaken strongly for 1 min. The mixture was then left standing at 37 • C for 60, 120, and 180 min with GEO; 10 min with AM; 120 min with AMS, DAS, and AMDS; and 180 min with DMTS, DADS, AMTS, and DATS. The organosulfur compounds released to the headspace of the glass vial were collected with a gastight syringe, and the sampled headspace gas (1 mL) was injected into a GC equipped with a PFPD to calculate the peak area of the target organosulfur component. The deodorant activities of the test chemicals were calculated using the following equation [21]: Deodorant activity (%) = (C − S) C × 100(C, peak area of control; S, peak area of sample). (1)

Reaction Monitoring between Thymoquinone and AM
The fixed amount of ethanolic solution (1 mL) of thymoquinone (400 µg) and the fixed amount of ethanolic solution of AM (0.05 mL) were added to the buffer solution (pH 1.2 or 7.0, 5 mL), sealed, and kept at 37 • C for 10, 30, or 60 min. The buffer solution was similar to that used in the evaluation of the deodorant activity. The amount of thymoquinone (400 µg) was equivalent to 1 mg of BCO. In this test, the applied concentrations of AM were 50, 5, and 0.5 µg/mL.

Statistical Analysis
All data were analyzed statistically by one-way analysis of variance followed by multiple comparison tests using the Tukey-Kramer test. The analyses were performed using Bell Curve for Excel (Version 3.21, Social Survey Research Information Co., Ltd., Tokyo, Japan) an add-in software for Excel.

Organosulfur Compound Composition of GEO
The major organosulfur compounds in GEO according to the GC-MS analysis were allyl mercaptan (AM), allyl methyl sulfide (AMS), diallyl sulfide (DAS), allyl methyl disulfide (AMDS), dimethyl trisulfide (DMTS), diallyl disulfide (DADS), allyl methyl trisulfide (AMTS), and diallyl trisulfide (DATS). The area percentages of each organosulfur compound toward the total area of the GC peaks in GEO were 0.14%, 2.00%, 6.24%, 3.90%, 7.17%, 13.2%, 16.6%, and 40.2%, respectively. The major constituent of GEO was DATS (Figure 1). The organosulfur compound compositions of GEO from air-dried and freeze-dried garlic have been found to be characterized by an amount of organosulfur compound (84.3-98.9%) consisting of DATS (37.3-45.9%), DADS (17.5-35.6%), and AMTS (7.7-10.4%) [22]. In essential oil analyses of garlic varieties, the chemical composition has been found to consist of AMS  [23]. Based on these reports, the GEO used in this study was considered to be a typical GEO.  [23]. Based on these reports, the GEO used in this study was considered to be a typical GEO.

Deodorant Activity of Black Cumin Seed Essential Oil (BCO) against GEO
In a 30 mL glass vial, BCO (10 mg) showed remarkable deodorant activity at pH 1.2 at 180 min after treatment. The deodorant activities of AMS, DAS, AMDS, DMTS, DADS, and AMTS were 56.3%, 72.4%, 59.0%, 69.5%, 28.4%, and 32.0%, respectively ( Figure 2). The deodorant activities of AMS, DAS, AMDS, and DMTS were time-dependent, but DADS and AMTS were time-independent between 60 min and 120 min (Figure 3). We presume that DADS and AMTS have bulky structures and highly lipophilic properties compared to other compounds, and DADS and AMTS were difficult to volatilize into the headspace (Figure 1).

Deodorant Activity of Black Cumin Seed Essential Oil (BCO) against GEO
In a 30 mL glass vial, BCO (10 mg) showed remarkable deodorant activity at pH 1.2 at 180 min after treatment. The deodorant activities of AMS, DAS, AMDS, DMTS, DADS, and AMTS were 56.3%, 72.4%, 59.0%, 69.5%, 28.4%, and 32.0%, respectively ( Figure 2). The deodorant activities of AMS, DAS, AMDS, and DMTS were time-dependent, but DADS and AMTS were time-independent between 60 min and 120 min (Figure 3). We presume that DADS and AMTS have bulky structures and highly lipophilic properties compared to other compounds, and DADS and AMTS were difficult to volatilize into the headspace (Figure 1).   Unfortunately, AM and DATS were not detectable under the selected analytical conditions. Presumably, the amount of AM in GEO was very low and DATS had a high boiling point, and they could not sufficiently vaporize into the headspace to be detected by GC with a PFPD (Figure 1).

Deodorant Activity of BCO against Organosulfur Compounds
In a 30 mL glass vial, the 0.1 mg treatment of BCO showed remarkable deodorant activity against 0.5 µg of AM (75.7% at pH 7.0, 18.7% at pH 1.2). Under the same conditions, the 10 mg treatment of BCO showed deodorant activity against 0.5 µg of AM (100% at pH 7.0, 70.2% at pH 1.2) ( Table 1). The deodorant activity of BCO was dose-dependent, but it was decreased at pH 1.2 compared to pH 7.0. Additionally, the deodorant activity of BCO was effective against all test organosulfur compounds at pH 1.2 ( Table 2). This result suggests that BCO may decrease the amount of organosulfur compound derived from garlic in the stomach, and thus reduce halitosis due to belching. As previously mentioned, common halitosis involves oral odors, and many reports have involved tests performed at pH 7.0. However, in this study, we focused on deodorant activity in the stomach rather than the oral cavity. Specifically, we evaluated deodorant activity in a gastric environment at pH 1.2, since the garlic halitosis almost always arises from the decomposition of garlic organosulfur compounds in the stomach. As shown in Table 1, our comparative studies of pH 1.2 and pH 7.0 showed that the deodorant activity against AM tended to be lower under the pH 1.2 condition. Although GEO has low AM content, a considerable amount of AM is detected in the breath of humans who consume garlic [24]. The odor of garlic is known to derive from alliin. When raw garlic is crushed or minced, alliin is converted to allicin by alliinase. However, allicin is unstable and easy to decompose into various organosulfur compounds, such as DADS, DATS, and ajoene. Furthermore, DADS and DATS are converted to AM, followed by conversion to AMS [14,25]. These results suggest that BCO may act as a deodorizing agent, thereby preventing halitosis after garlic meal ingestion.

Isolation and Identification of Reactants between Thymoquinone and AM
A few products of the reaction between thymoquinone and AM were isolated using silica gel flash chromatography with hexane and ethyl acetate as the mobile phase. The elucidation of the structures of these products was performed using 1 H NMR, 13 C NMR, and 2D NMR (HSQC, HMBC) spectral data. Three reaction products, compounds (1-3) were identified in this study. Compounds 1 and 2 were AM added to dihydrothymoquinone, a reduced form of the major natural monoterpene thymoquinone in BCO (Figure 4). Compound 3 had a dithio-moiety from one extra AM added to 2 (Figure 4). This result suggests that the deodorizing mechanism of BCO against AM may have occurred due to the conversion of AM to these chemicals.

Isolation and Identification of Reactants between Thymoquinone and AM
A few products of the reaction between thymoquinone and AM were isolated using silica gel flash chromatography with hexane and ethyl acetate as the mobile phase. The elucidation of the structures of these products was performed using 1 H NMR, 13 C NMR, and 2D NMR (HSQC, HMBC) spectral data. Three reaction products, compounds (1-3) were identified in this study. Compounds 1 and 2 were AM added to dihydrothymoquinone, a reduced form of the major natural monoterpene thymoquinone in BCO (Figure 4). Compound 3 had a dithio-moiety from one extra AM added to 2 (Figure 4). This result suggests that the deodorizing mechanism of BCO against AM may have occurred due to the conversion of AM to these chemicals. A previous report concerning the prevention of bad breath due to garlic used various botanical sources containing polyphenols, such as apple, tea, lemon, parsley, and mint [26]. In our present study, AM was trapped with thymoquinone covalently and converted to a non-volatile phenol compound. However, a similar reaction could not be observed in the other tested sulfides in this study (data not shown). This fact suggested that a thiol moiety was essential to this reaction. A previous report described that some organosulfur compounds are converted to AM [27]; therefore, decreasing the AM amount may prevent AMS generation in the body.
A comparison of the effects of non-fat milk and whole milk on the deodorant activity of garlic reported that whole milk was more effective. However, whole milk showed the highest efficacy in comparison with water, whole milk, and 10% casein sodium aq., and it was presumed that the fat contained in whole milk suppressed the volatility of the garlic-derived DAS and AMS [13]. Similarly, the deodorant activity of BCO presumed that A previous report concerning the prevention of bad breath due to garlic used various botanical sources containing polyphenols, such as apple, tea, lemon, parsley, and mint [26]. In our present study, AM was trapped with thymoquinone covalently and converted to a non-volatile phenol compound. However, a similar reaction could not be observed in the other tested sulfides in this study (data not shown). This fact suggested that a thiol moiety was essential to this reaction. A previous report described that some organosulfur compounds are converted to AM [27]; therefore, decreasing the AM amount may prevent AMS generation in the body.
A comparison of the effects of non-fat milk and whole milk on the deodorant activity of garlic reported that whole milk was more effective. However, whole milk showed the highest efficacy in comparison with water, whole milk, and 10% casein sodium aq., and it was presumed that the fat contained in whole milk suppressed the volatility of the garlic-derived DAS and AMS [13]. Similarly, the deodorant activity of BCO presumed that not only covalent trapping between thymoquinone and AM but also high hydrophobicity interactions occurred in this study.

Reaction Monitoring between Thymoquinone and AM
The time-dependent reactions between thymoquinone and AM at pH 7.0 were measured. Although the amounts of thymoquinone and AM did not decrease drastically, the amounts of their adducts, compounds 2 and 3, increased in a time-dependent manner (Figure 5a-c). Similarly, the amounts of AM and thymoquinone did not decrease drastically, but the amounts of their adducts, compounds 1-3, increased in a time-dependent manner at pH 1.2 (Figure 5d-f). The amounts of reactants 1-3 were dependent on the acidic conditions in this study ( Table 3).
The time-dependent reactions between thymoquinone and AM at pH 7.0 were measured. Although the amounts of thymoquinone and AM did not decrease drastically, the amounts of their adducts, compounds 2 and 3, increased in a time-dependent manner (Figure 5a-c). Similarly, the amounts of AM and thymoquinone did not decrease drastically, but the amounts of their adducts, compounds 1-3, increased in a time-dependent manner at pH 1.2 (Figure 5d-f). The amounts of reactants 1-3 were dependent on the acidic conditions in this study (Table 3).  When 50 µg of AM was added to 400 µg of thymoquinone at pH 1.2, compound 1 occurred in concentrations of 4.05, 8.12, and 10.08 ng/mL at 10, 30, and 60 min after mixture, respectively. At pH 7.0, the concentrations of compound 1 were all not detected (N.D.) at 10, 30, and 60 min after mixture, respectively. Similarly, the concentrations of compound 2 were 1.81, 2.96, and 5.66 ng/mL at pH 1.2 at 10, 30, and 60 min, respectively. At pH 7.0, the concentrations of compound 2 were 422.69, 605.32, and 732.26 ng/mL at 10, 30, and 60 min, respectively. The concentrations of compound 3 were 0.08, 0.22, and 0.68 ng/mL at pH 1.2 and 163.23, 231.97, and 261.43 ng/mL at pH 7.0 at 10, 30, and 60 min, respectively.
The reaction between AM and thymoquinone was promoted at pH 7.0 in comparison with pH 1.2. These results were similar to those of the deodorant evaluations using HS-GC.

Conclusions
The results obtained for the deodorant activity of BCO against garlic odor suggest that this botanical resource applies to the control of oral malodor after consuming garlic meals. Previous papers about deodorant activity had evaluated this under oral environmental conditions of pH 7.0, while this study also evaluated gastric environmental conditions of pH 1.2. In this study, we discovered that BCO was effective in suppressing bad breath after intaking a garlic meal. We presumed that a part of the mechanism that is a major constituent of BCO, thymoquinone, trapped AM, which is an organosulfur produced from garlic meals in the human body causative of garlic bad breath. However, this deodorant activity of BCO did not overwhelm the odor, and the other non-reacted garlic organosulfur compounds remained in the human body. These residual intact garlic organosulfur compounds, such as DADS and AMTS, may have various valuable bioavailability in the human body. Recently, we showed that garlic organosulfur compounds had the potential to slow the procession of Alzheimer's disease by the inhibition of related enzymes located in the brain [28]. Although the deodorant activity of BCO is effective in controlling oral malodor from garlic meals, it is better to adjust the intake time to interval eating for garlic bioavailability. It is a critical point from a good human health point of view that the bioavailability of garlic organosulfur compounds increases and the deodorant activity of BCO follows. Finally, research on the deodorizing mechanisms of BCO is likely to continue in the future. People who consume garlic products for health benefits, such as revitalizing cells and the bloodstream, could prevent bad breath due to the odor of garlic by taking BCO.