Development and Evaluation of Essential Oil-based Lozenges using Menthol and Eucalyptus and in vitro Evaluation of their Antimicrobial activity in S.aureus and E.coli

 

Manas Ranjan Sahoo¹, Marakanam Srinivasan Umashankar¹*, Ramesh Raghava Varier²

¹Department of Pharmaceutics, SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur, Tamilnadu, India, 603203.

²AVN Ayurveda Formulation Pvt Ltd, Madurai, Tamilnadu, India, 600034.

 

ABSTRACT:

The throat lozenegs are most commonly used first line of treatment for relief of various throat infections. The most common ingredient used in the lozenegs are various herbal extracts and essential oil having local action of throat area. in the present studies a essential oil based lozenges were prepared using eucalyptus oil and menthol as active ingredient. Antibacterial activity was evaluated for menthol and eucalyptus oil against human pathogens E.coli and S.aureus. both the sugar based and sugar free base was used to develop two types of lozenges formulation. Quality parameters were evaluated for lozenges formulations such as hardness, friability, dissolving time, and moisture content. Compatibility study was carried out using thin layer chromatography and gas chromatography analysis. The eucalyptus oil and menthol displayed moderate antibacterial activity against E.coli and S.aureus. The lozenges were found to be having satisfactory quality parameters. GC and TLC analysis was found to be a suitable method for identification of menthol and eucalyptus oil in the lozenges. TLC was found to be a simple, quick and simple technique for rapid identification of the essential oil and to confirm the batch-to-batch consistency. ¹H-NMR and ¹³C-NMR was found to be useful tool for rapid identification of essential oils on basis of diagonostic peaks. Overall this study can be used for standard methods for quality control of the essential lozeneges formulation for the new formulation and product available in the market as well.    

 

 

INTRODUCTION:

Sore throat, throat infection, rhinitis and acute pharyngitis are most common symptoms of respiratory tract infections. These infections are caused majorly by virus and by bacteria upto a lesser extent. Throat lozenegs are commonly used for relief throat infection due to their convenience of administration and local actions. The lozenges are the unit solid and flavored dosage for that are designed to hold in oral cavity and slowly dissolve to release the actives for local action. Lozenges help in delivery of the actives in the site of action so reducing the dose^1-3.

 

 

Various research works has been undertaken on developing lozenges for some pharmaceutical drugs and supplements like nimesulide, theophylline, and ascorbic acid for better elegance, improved release, and better bioavailability and to improve the taste of bitter drug with flavours and sweeteners. This also improves patient compliance in paediatrics as well in the patients with swallowing difficulties^4-6. Lozenges containing combination menthol and eucalyptus oil are most commonly used in various cough drops for relief of the sore throat, inflammation and throat pain ⁷.  Menthol is an anti-tussive compound and also decreases the cough reflex through its TRPV1 mediated actions and cooling sensation at throat area⁸. Both menthol and eucalyptus oils are reported for antitussive, mucolytic, antimicrobial, antiviral, bronchodilatory and menthol reported for analgesic, coolent and mucociliary function ^9-10. Cineole one of the major compounds of eucalyptus oil has been found to be having mucolytic, bronchodilating and anti-inflammatory properties and found to be beneficial in bronchitis^11-12. In the present method we have prepared a lozenegs formulation using menthol and eucalyptus oil as active essential oil. The sugar based lozenegs was prepared from sucrose and liquid glucose and sugar free lozenges prepared using isomalt. The resulting formulation was further evaluated for various physical quality parameters. The preformulation characterization was done through ¹H-NMR and ¹³C-NMR analysis of menthol and eucalyptus oil and evaluation of their anti-bacterial activity.  

 

MATERIALS AND METHODS:

The essential oil eucalyptus oil and menthol was purchased from AOS product private limited-India. For sugar free lozenges, Isomalt was received from Beneo-Palatinit GmbH. Liquid glucose was received from Roquette and Pharmaceutical grade Sugar was used for sugar based hard-boiled lozenges. The chemical and solvents used were from Rankem and Merck. Silica gel 60-F 254 TLC plates were from sigma-aldrich. All the media used in the microbiology study were purchased from HiMedia.

 

Preformulation characterization:

Before using in the formulation the menthol and eucalyptus oil was analyzed by ¹H-NMR and ¹³C-NMR for confirming authenticity of menthol and eucalyptus oil. The NMR was recorded using the Varian-400 MHz model. For recording the NMR 20mg of menthol and 100µl of eucalyptus oil was dissolved in deuterated solvent CDCl₃ and the ¹H and ¹³C spectrum was recorded.

 

Preparation of hard boiled lozenges from menthol and eucalyptus oil:

The hard-boiled lozenges were prepared by using heating, melting and congealing method¹³. The preparation process for both sugar based and sugar free lozenegs are mentioned below. The lozenges formulations were designed to contain approximately 4mg menthol and 1mg of eucalyptus oils per one gm lozenges. After several trials of different compositions the lozenges were decided to provide ideal physical properties such hardness, texture and taste. The composition of both sugar and isomalt based lozeneges formulation are mentioned in below table-1.

 

Sugar based lozenges:

The lozenges were prepared from mixture of sugar and liquid glucose (table-1) by boiling with sufficient quantity of water to dissolve sugar entirely. The boiling was performed at temperature of 110ºC to 120°C to evaporate most of water. The fluid plastic mass is then cooled to temperature range of 90 to 100°C and at this stage essential oil menthol and eucalyptus oil is added into the molten matrix followed by vigorous mixing and immediate cooling to stabilize the essential oils in the lozenegs structure. The sugar free lozenges was prepared using similar heating congealing procedure using isomalt 70 % w/w instead of the sugar and liquid glucose. The lozenegs were than packed in air tight met-pet pouch to protect from moisture and temperature.  

 

Table 1 : Composition of both sugar and sugar free lozenges formulation

 

Evaluation of Lozenges Formulation:

Both the sugar based and sugar free lozenges were evaluated for quality parameters like hardness, weight variation, thickness, friability, and dissolving time. Thickness and diameter of the lozenges were measured by vernier caliper. Hardness of lozenges was measured using Monsanto-hardness tester. Friability was studied in Roche friabilator. For friability test 20 lozenges of both formulations were carefully weighed before testing and the lozenges were placed in the rotary-drum of friability tester. The drum was rotated for 100 revolutions and the lozenges were dusted weighed again and the weight loss was calculated. The loss of weight should be less than 1% to comply the quality. Weight variation was measured by weighing twenty lozenges and the average weight was calculated. Dissolving time was measure in USP disintegration apparatus at 37±2°C using phosphate buffer of buccal physiology pH=6.8 as medium. 

 

Compatibility study Excipient: Essential Oils:

Thin Layer Chromatography analysis of lozenges formulation:

The lozenges formulation was analyzed for presence menthol and eucalyptus oil to identify the presence of menthol and eucalyptus oil in lozenges. The TLC was developed in twin-trough twin-trough CAMAG TLC chamber with a mobile phase consist of hexane: ethyl acetate:formic acid (7.5:2.5:0.25; v/v/v). After development the TLC pate was derivatized with anisaldehyde sulphuric acid till the color zones were appeared and visualized under white light.

 

GC analysis of essential oil:

The essential oil eucalyptus oil was identified as cineole and menthol was identified by gas chromatography analysis. The GC analysis was accomplished with a Chemito GC instrument; GC 1000 model, using DB-5 capillary column (30mm x 0.32mm id, 1 µm), oven temperature program; 130ºC-230ºC. The Injector and detector temperatures 240°C and 250°C; carrier gas nitrogen (2.0mL/min) inj. volume-2µl. the identification of the components were performed, for both  columns, by comparison of their retention time with that of the authentic standards. The quantity of eucalyptus oil in terms of cineole equivalent was found to be 0.71 mg and 3.15 mg per 1 gm of sugar-based lozenges formulation and 0.72 mg and 3.69 mg per 1gm of sugar free lozenges formulation. 

 

Antibacterial activity of essential oil eucalyptus oil and menthol:

The antibacterial activity eucalyptus oil and menthol were determined using the well diffusion method. The antibacterial activity was performed on Mueller Hinton Agar plate. The bacterial pathogens of gram-negative bacteria E. coli- (MTCC 739) and gram-positive bacteria S. aureus (MTCC 7443) were swabbed on sterile agar plate. The samples of eucalyptus oil and menthol were added into the well and the plates were incubated at 37°C for 24h. The zones of inhibition were measured in millimeters. Each antibacterial assay was performed in triplicate. 

 

¹H-NMR of Eucalyptus Oil

 

¹³C-NMR Eucalyptus Oil

 

RESULTS AND DISCUSSION:

Preformulation characterization of Eucalyptus oil and Menthol:

¹H-NMR (CDCl₃-400MHz) d (ppm) :

0.79, 0.92, 0.94, 1.00, 1.09, 1.12, 1.14, 1.17, 1.22, 1.36, 1.40, 1.43, 1.46, 1.49, 1.56, 1.60, 1.61, 1.62, 1.64, 1.68, 1.88, 1.95, 1.97, 1.99, 2.00, 2.13, 2.26, 4.65,5.34, 5.14.

 

¹³C-NMR (CDCl₃-100MHz) d (ppm):

20.17, 20.75, 20.89, 22.36, 22.75, 22.93, 22.29, 23.42, 23.90, 24.04, 26.13, 26.29, 26.83, 27.37, 27.48,27.83, 28.80, 30.52, 30.74, 30.95, 31.19, 31.39, 31.43, 32.87, 33.63, 37.90,40.66, 41.02, 44.93, 46.96, 69.83, 73.82,108.32, 115.96, 120.50, 120.58, 126.19, 128.90, 135.04, 144.42, 145.78

NMR analysis of Menthol:

¹H-NMR (CDCl₃-400MHz) Chemical shift d (ppm):

3.40, 2.17,2.15, 2.13, 1.96, 1.67, 1.65, 1.63, 1.60, 1.57, 1.41, 1.30, 1.29, 1.09, 1.06, 0.97, 0.96, 0.95, 0.90, 0.87, 0.84, 0.83, 0.78.

 

¹³C-NMR (CDCl₃-100MHz) Chemical shift-d (ppm):

16.07, 20.99, 22.20, 23.10, 25.82, 31.61, 34.51, 45.02, 50.12, 71.55.

On the basis of the spectral diagnostic peaks and previous reported data 1,8-cineol was identified major constituents in the eucalyptus oil ^14-15.

 

 

¹H-NMR of Menthol

 

¹³C-NMR of Menthol

 

 

 

Physical properties of lozenges:

The prepared lozenges exhibited in good shape and hardness, the weight loss was less than 1% in the friability experiment without any cracked and broken lozenegs. Each of the lozenges was within the limits of weight variation. The quality control parameters of the lozenges are mentioned in below table-2.

 

Table 2:Evaluation of the lozenges

 

 

Compatibility study lozenges formulation:

TLC analysis:

The TLC of both the lozenges with menthol and eucalyptus oil was carried out. The lozenges showed spots of eucalyptus oil and menthol as blue and grey spots after derivatization with anisaldehyde sulphuric acid indicating presence of the active ingredients in the lozenges. The chromplate after derivatization with anisaldehyde H₂SO₄ is presented below fig-1. 

 

Menthol-lozenegs.formulation-eucalyptus oil 

Fig-1: TLC chromplate of lozenegs formulation with active ingredients menthol and eucalyptus oil

 

 

GC chromatogram of standards menthol and cineole

 

GC chromatogram of sugar free lozenges

 

GC chromatogram of sugar based lozenges

Fig-2: GC chromplate of lozenges formulation menthol and cineole

GC Analysis:

Eucalyptus oil and menthol were observed at around Rt of 3.80 and 4.00 respectively for the standards of menthol and cineole and the both lozenges formulation of sugar base and sugar free base. The identification of both the actives in the lozenges formulation substantiates stability of the eucalyptus oil and menthol in the lozenges formulation during the manufacturing process including the heating and congealing process irrespective of their volatile nature.  

 

In Vitro Evaluation of antibacterial activity:

Both the eucalyptus oil and menthol were evaluated for the antibacterial activity. The menthol displayed a zone of inhibition 03 mm in S.aureus and no activity on E.coli while eucalyptus oil displayed zone of inhibition of 4mm in both S.aureus and E.coli pathogens. The observed antibacterial activity of eucalyptus oil and menthol supports its traditional uses in various cough and cold drops. 

 

CONCLUSION:

The present study confirmed sugar base and sugar free excipients are ideal for preparation of lozenges formulation. The TLC was found to be an easier and simpler method for identification of the actives menthol and eucalyptus oil in the formulation coupled. Further GC analysis confirms presence of the essential oils in the lozenges formulation. The ¹H-NMR and ¹³C-NMR were found to be rapid and advance techniques for identification of essentials oils. Both menthol and eucalyptus oil was found to be stable in the formulation on basis of identification by gas chromatography and thin layer chromatography fingerprint. The lozenges prepared were having satisfactory quality parameters. These methods can be used for routine evaluation of the lozenges formulation available in the market.

 

ACKNOWLEDGEMENT:

The authors are thankful to AOS-India for providing the gift sample of menthol and eucalyptus oil and Benque-Germany for the gift sample of Isomalt and Roquette for providing sample of Liquid glucose. We are thankful to Amrutanjan Health Care Ltd for helping in the NMR experiment.

 

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.     Morokutti-Kurz M. Graf C. Prieschl-Grassauer E. Amylmetacresol/2,4-dichlorobenzyl alcohol, hexylresorcinol, or carrageenan lozenges as active treatments for sore throat. International Journal of General Medicine 2017; 10: 53–60. doi: 10.2147/IJGM.S120665

2.     Sousa R. Lakha DR. Brette S. Hitier S. A Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy and Safety of Ambroxol Hard-Boiled Lozenges in Patients with Acute Pharyngitis. Pulm Ther, 2019; 5:201–211. doi: 10.1007/s41030-019-00100-w   

3.     Sahoo MR. Umashankar MS, Varier RR. The research updates and prospects of herbal hard-boiled lozenges: a classical platform with promising drug delivery potential. International journal of applied pharmaceutics 2021; 13(2): 1-13. doi.org/10.22159/ijap.2021v13i2.40165

4.     Yousuf Ali M. Shamim Qureshi M. Hamed M. Das B, Rao KP. Design and Evaluation of Nimesulide Lozenges for Pediatrics. Research Journal of Pharmacy and Technology 2010; 3 (3):818-20. doi:10.5958/0974-360X   

5.     Bhandarkar A, Alexander A, Bhatt A, Sahu P. Agrawal P. Tripti B et al. Evaluation of Ascorbic acid Lozenges for the treatment of Oral Ulcer. Research Journal of Pharmacy and Technology 2018; 11(4):1307-12. doi:10.5958/0974-360X.2018.00243.3  

6.     Pravalika L. Shravan Kumar Y. Formulation and Evaluation of Theophylline Lozenges. Research Journal of Pharmacy and Technology 2021; 14(3):1601-6. doi:10.5958/0974-360X.2021.00284.5

7.     Wise PM, Paul AS. Dalton P. Sweet Taste and Menthol Increase Cough Reflex Thresholds. Pulm Pharmacol Ther 2012; 25(3): 236-41. doi:10.1016/j.pupt.2012.03.005 

8.     Addey D. Shephard A. Incidence, causes, severity and treatment of throat discomfort: a four-region online questionnaire survey. BMC Ear Nose Throat Disorder 2012; 12: doi:10.1186/1472-6815-12-9

9.     Valussi M. Antonelli M. Donelli D. Firenzuoli F. Appropriate use of essential oils and their components in the management of upper respiratory tract symptoms in patients with COVID-19. J Herb Med. 2021; 28: 100451. doi: 10.1016/j.hermed.2021.100451.

10.  Da Silva JK. Figueiredo PL. Byler KG. Setzer WN. Essential Oils as Antiviral Agents, Potential of Essential Oils to Treat SARS-CoV-2 Infection: An In-Silico Investigation. International Journal of Molecular Sciences 2020; 21(10): 3426. doi: 10.3390/ijms21103426.  

11.  Kamilla A. Bencsik T. Boszormenyi A. Kocsis B. Horvath G. Essential Oils and their vapours as potential antibacterial agents against respiratory tract pathogens. Natural Product Communication 2016; 11(11):1709-12. PMID: 30475513

12.  Fischer J. Dethlefsen U. Efficacy of cineole in patients suffering from acute bronchitis: a placebo-controlled double-blind trial Cough. 2013; 9: 25. doi: 10.1186/1745-9974-9-25

13.  Sahoo MR. Umashankar MS. Varrier RR. Formulation Development of Curcumin Loaded Lozenges: Extensive Characterization and In Vitro Evaluation of Antioxidant Activity, Anti-Inflammation and Antimicrobial Activity. International Journal of Pharmaceutical Research 2020; 12(2):2706-16. doi: https://doi.org/10.31838/ijpr/2020.SP2.310    

14.  Ghaffar A. Yameen M. Kiran S. Kamal S. Jalal F. Munir B et al. Chemical Composition and in-Vitro evaluation of the antimicrobial and antioxidant activities of essential oils extracted from seven eucalyptus species. Molecules 2015; 20: 20487-98. doi: 10.3390/molecules201119706  

15.  Gusain P. Ohki S. Hoshino K. Tsujino Y. Shimokawa N. Takagi M. Chirality-Dependent Interaction of d- and l-Menthol with Biomembrane Models. Membranes 2017; 7(4): 69. doi: 10.3390/membranes7040069.

 

Accepted on 22.03.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5283-5288.