GC-FID Studies on the levels of Allyl Isothiocyanate in Mustard oil combined with Menthol/thymol and its Antimicrobial Investigation

 

Alyaziya Mohammed Al Aisaee¹, Hamida Ali Lawatia¹, Arwa Said Al Wardi¹,

Jamal Al Sabahi², Shah Alam Khan¹, Amal³, Mohammad Jawaid Akhtar¹*

¹Department of Pharmaceutical Chemistry, College of Pharmacy, National University of Science and Technology, PO 620, PC 130, Azaiba, Bousher, Muscat, Sultanate of Oman.

²Central Instrument Laboratory, College of Agriculture and Marine Sciences, Sultan Qaboos University, Oman.

³Department of Pharmacology and Biological Sciences, College of Pharmacy, National University of Science and Technology, PO 620, PC 130, Azaiba, Bousher, Muscat, Sultanate of Oman.

 

ABSTRACT:

Objectives: The excessive use of antimicrobials and rapid resistance to antibiotics is a major concern for health care providers. Hence there is a need to develop effective antimicrobials. Natural sources are considered as the potential source of antimicrobial agents. The study aimed to decrease the pungency or irritancy effects of the allyl isothiocyanate (AITC) contents present in the mustard oil after mixing it with natural phytochemicals such as menthol/thymol and to evaluate its effects as potential antimicrobials. Methods: The mustard oil was mixed with menthol, stirred for 12h and with thymol stirred for 7 days in the presence of triethylamine. The components were analysed for decrease in the allyl isothiocyanate contents by gas chromatography-flame ionisation detector (GC-FID). Results and Conclusions: The results showed 40% and 60% decrease in the allyl isothiocyanate content in mustard oil when mixed with menthol and thymol, respectively. In vitro antimicrobial experiments demonstrated that the tested bacterial species were highly susceptible to the mustard oil mixed with thymol or menthol as compared to the neat mustard oil except Klebsiella aerogenes. Mustard oil mixed with menthol (4.5 mg menthol/ 0.3 mL of mustard oil) exhibited the most potent antimicrobial activity as shown by zone of inhibition (in diameter). The most susceptible species was Bacillus subtilis (2 cm zone of inhibition as compared to neat mustard oil 1.2 cm). The study highlights the potential of mustard oil mixed with thymol or menthol as potential antimicrobials.

 

 

 

1. INTRODUCTION: 

The excessive and inappropriate consumption of antimicrobials is a major concern in post antibiotics-era in which infections are very difficult to treat¹. The effectiveness of these antimicrobials declines worldwide due to over or indiscriminate use and rapid development of resistance in microbes^2,3. Therefore, there is a need to develop new effective antimicrobials preferably from the natural products with a different mechanism of action^4-7. These natural agents are generally recognised as safe substances (GRAS) by Food and Drug Administration (FDA) and are easily approved without much complicated analyses. These provides food safety and food wastage against food borne pathogens (E. coli, Salmonella etc.) which may severely affect millions of people with serious results⁸.  Mustard oil seeds from the Brassicaceae family contains significant amounts of allyl isothiocyanate (AITC). Mustard seeds of Brassica juncea  contains >7g/kg and Brassica nigra contains  >10g/kg of AITC⁹. In plants AITC is formed by the hydrolysis of widely occurring inactive prodrug glucosinolate sinigrin by the action of endogenous plant enzymes myrosinase^10,5,11. It is used as a source of edible oil, traditional medicine and is also an excellent source of nutritional quality proteins. It is grown as condiment for the spice trade due to their specialized pungency taste^9,12,13. It possesses wide range of biological activities; anti-carcinogenic properties that prevent cancer development in the body¹², antifungal and antibacterial properties that cure skin disorders, antidiabetic, lipid lowering agent and relief from knee joint pain^14,15. It has potent antimicrobial action against bacteria, yeast, moulds, and foodborne pathogens¹⁶.  Additionally, it lowers the adhesive impulses in blood platelets, which helps to lessen the risk of heart failure¹⁷. The AITC is an exceptionally pungent toxic compound, a strong lachrymator and skin vesicant^18,19. The AITC (as antimicrobials) in its pure form is restricted in the food packaging because of extreme pungency and volatility and may reacts with the food products to give unpleasant off flavour¹¹. it also activates capsaicin-sensitive nerves that induces neurogenic inflammation and TRPA1 channels (noxious cold ion channel) expressed by peptidergic sensory fibres and indicating a possible role in pain processing^20,21.  The inflammatory irritant AITC present in the mustard oil to the dental pulp causes central sensitization (hyperexcitability of nociceptive neurons in CNS via peripheral tissue damage and inflammation)²².

 

Intracolonic instillation of mustard oil is a valid mouse model of acute visceral pain i.e., spontaneous pain-related and sensation of visceral hyperalgesia²³. Topical application of the mustard oil causes inflammation, pain, and activation of the thermal and mechanical stimuli¹⁸. Mustard oil can also cause IgE-mediated allergic reaction in some sensitive individuals²⁴. The presence of electrophilic nature of the isothiocyanate functional groups is assumed to be associated with the toxicity after reacting with biological thiols, amines, and alcohols in vivo⁹.

The present work is directed towards reducing the content of allyl isothiocyanate present in the mustard oil by mixing with thymol and menthol with an aim to diminish its pungency and retain or enhance its antimicrobial activity. The mustard oil mixed with menthol and thymol were evaluated against four strains of microbes i.e., Escherichia coli (E. coli), Bacillus subtilis, Staphylococcus aureus and Klebsiella aerogenes.

 

2. MATERIALS AND METHODS:

2.1. Chemicals and reagents:

All the chemicals including menthol and thymol used were of laboratory grades and procured from the S.D. Fine chemicals (Mumbai India) and Loba Chemie Pvt. Ltd (Mumbai, India). The drug standard (gentamicin, cephotaxime and trimethoprim) was purchased from HI MEDIA (Mumbai India).  The mustard oil was collected from a supermarket in Muscat and was from the seeds of Brassica nigra varieties. The lyophilized KWIK-STIK for the different bacterial strain was purchased from Bio Oman.

 

2.2. GC-FID conditions:

Analysis of the chemical composition in particular AITC level in the mustard oil mixed with thymol or menthol was performed on a gas chromatography coupled with flame ionization detector (GC-FID). The PerkinElmer 600GC system fitted with Rtx-5MS capillary column (30 mm x 0.25 mm I.D. 0.25 µm film thickness; maximum temperature of 350^oC) and coupled to a PerkinElmer flame ionization detector. Helium as carrier gas (99.9999%) ultra-high purity was applied with constant flow of 1.0 mL/min. The injection and transfer line temperature were adjusted to 250, 260^oC. The oven temperature program was 60^oC (held for 1 min) at a rate of 4^oC/min-260^oC (held for 4 min). The identification of the unknown compounds was performed by matching the spectra with GC spectrum libraries (NIST 2011 v.2.3 and Wiley, 9^th edition)^25-27.

 

2.3. Procedure for the mixing of mustard oil with menthol:

A mixture of mustard oil (10 mL; 0.001 mole of AITC) and the menthol (0.156 g; 0.001 mol) was stirred at room temperature for 12 h.

2.4. Procedure for the mixing of mustard oil with thymol:

To the mixture of 10 mL of the mustard oil (10 mL; 0.001 mole of AITC) anhydrous triethylamine 0.1mL and thymol (0.15 g; 0.001 mol) was added. The mixture was left stirred at room temperature for 7 days²⁸.

 

2.5. Antimicrobial activity:

2.5.1 Culturing of bacterial strains:

The bacterial strain was purchased from Bio Omanas lyophilized KWIK-STIK. It was cultured in the nutrient broth medium, mixed well, and was incubated for 24 h at 36 ±1^oC. Then subculturing was done in nutrient broth agar plate using a sterile cotton swab²⁹.

 

2.5.2. Agar well diffusion method:

Agar well diffusion method was used to evaluate the antimicrobial activity. The agar plate surface was inoculated by spreading uniformly the volumes of the microbial load in the entire agar surface using sterile cotton swab. The mustard oil mixed with thymol or menthol were tested against four bacterial strains viz., E. coli (ATCC 25922), B. subtilis (ATCC 11774), S. aureus (ATCC 29213) and K. aerogenes (ATCC 13048). A hole of 6-8 mm was punched aseptically with a sterile cork borer or a tip and a volume (300 µL) of the tested substance was introduced in to the well. The agar plate was incubated under suitable aerobic conditions (24 h at 36 ± 1 ^oC) during which the antimicrobial agents spread in the agar plate and inhibits the growth of the microbial strain tested. The diameter of zone of inhibition of the bacterial growth was measured in cm. Test were performed in duplicate^30-32.

 

3. RESULTS AND DISCUSSION:

 

3.1. Chemistry:

AITC present in mustard oil was stirred with menthol for 12 h to form conjugates, whereas it was stirred with thymol for 7 days in the presence of triethylamine. This was performed to reduce the contents of allyl isothiocyanate present in the mustard oil responsible for the pungency, irritancy and to increase the antimicrobial activity.

 

3.2. Chemical composition by GC-FID:

The compounds were identified by comparing the compounds libraries in the GC-FID data with the available literature (NIST 2011 v.2.3 and Wiley, 9^th edition)^{25, 33}. The compounds with their retention times (min), retention indices, area and % area obtained from the GC chromatogram are presented in the Table 1. The percentage area represents the amount of each compound present and from the results showed that the contents of allyl isothiocyanate was decreased to 40% when mustard oil was mixed with menthol and 60% decrease was noted after mustard oil mixed with thymol as compared to neat mustard oil. There is also a marginal decrease in the contents of 4-isothiocyanato-1-butene in the mustard oil mixed with menthol/thymol as compared to the neat mustard oil. The structure of the chemicals compounds used and identified through GC-FID are shown in Figure 1.

 

Table 1: GC-FID analysis of the mustard oil, mustard oil mixed with thymol and mustard oil mixed with menthol

Sample	Compounds	RT (min)	RI	Area	% Area
mustard oil	Allylisothiocyanate	6.767	846	276249	0.1
	4-isothiocyanato-1-butene	9.765	982	699838	0.24
mustard oil + menthol	Allylisothiocyanate	5.446	846	78865	0.06
	4-isothiocyanato-1-butene	8.051	982	275704	0.21
	Isopulegol	13.559	1159	112922	0.08
	Menthol	14.604	1164	7200000	54.13
	2-Ethylhexyl-2-propenoate	16.608	1215	246858	0.19
mustard oil + thymol	Allylisothiocyanate	5.506	60875	846	0.04
	4-isothiocyanato-1-butene	8.253	318114	982	0.23
	Thymol	17.856	1266	5200000	37.17
	Phenol,2-(1,1-dimethylethyl)-4-methyl	19.876	293467	1345	0.21

RT = retention time; RI = retention indices; % = percentage

 

Figure 1: The chemical structures of compounds with the help of GC-FID

 

 

3.3. Antimicrobial activity:

The zone of inhibition of mustard oil (neat), mustard oil mixed with menthol/thymol (menthol and thymol 4.5 mg per 0.3 mL of mustard oil) was determined against E. coli, B. subtilis, S. aureus and K. aerogenes. The results were compared with standard antibiotics which are shown in Table 2. Except K. aerogenes all the tested species were susceptible to the tested compounds. The highest susceptibility was shown by mustard oil mixed with menthol against Bacillus subtilis with a zone of inhibition 2 cm whereas mustard oil mixed with thymol showed 1.6 cm as compared to neat mustard oil (1.2 cm). The next susceptible organism was S. aureus with zone of inhibition of 2 cm for both the compounds as compared to the neat mustard oil (zone of inhibition 1.6 cm). The zone of inhibition extended marginally to 1.3 cm for both the tested compounds as compared to neat mustard of 1.2 cm. Growth of the K. aerogenes was not influenced by any of the tested compounds. The zone of inhibition against different strains of bacteria cultures are shown in Figure 2.

 

Table 2: Zone of inhibition (in diameter) against different strain of bacteria

Sample 15 mg thymol or menthol/mL of mustard oil	Escherichia coli	Bacillus subtilis	Staphylococcus aureus	Klebsiella aerogenes
Mustard oil	1.2 cm	1.2 cm	1.6 cm	1.2 cm
Mustard oil + Thymol	1.3 cm	1.6 cm	2.0 cm	1.2 cm
Mustard oil + Menthol	1.3 cm	2.0 cm	2.0 cm	1.2 cm
Antibiotics	2.8 cm (Cefotaxime 30 mcg/disc)	3.8 cm (Gentamicin 10 mcg/disc)	2.8 cm Trimethoprim (5 mcg/disc)	2.4 cm Gentamicin (10 mcg/disc)

 

Figure 2. Zone of inhibition against four strains of bacteria culture A) zone of inhibition against Escherichia coli B) zone of inhibition against Bacillus subtilis C) zone of inhibition against Staphylococcus aureus D) zone of inhibition against Klebsiella aerogenes.

 

4. CONCLUSION:

The current study showed that combining mustard oil with natural phytoconstituents such as menthol and thymol enhances their antibacterial activity spectrum against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The GC-FID results further confirmed a reduction in the amount of the allyl isothiocyanate responsible for the pungency and irritancy of the oil upon mixing with natural volatile phytoconstituents thymol and menthol. This may be due to a conjugate formation which will be further probed in the future studies.

 

5. FUNDING:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors

 

6. CONFLICT OF INTEREST:

The authors declare no competing or financial conflict of interests.

 

7. ETHICAL APPROVAL:

The research and Ethics and Biosafety Committee of the College of Pharmacy at National University approves the study (EBS/13/2021-2022) on 16^th May 2022.

8. CONSENT TO PARTICIPATE/CONSENT TO PUBLISH:

Not applicable

 

9. ACKNOWLEDGEMENTS:

We are grateful to the College of Pharmacy, National University of Science and Technology, Azaiba, Bousher, Muscat Sultanate of Oman for providing laboratory facilities. Support rendered by the Staff at Central Instrument Laboratory, Sultan Qaboos University for carrying out GC-FID is highly acknowledged.

 

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Accepted on 24.01.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(7):3326-3330.