INTRODUCTION:

Skin is the first and the largest defensive barrier against numerous pathogenic microorganisms, withstands the variations from external environment keeping the skin healthy, more resilient and less sensitive [1]. It comprises of various protective layers that performs vital putative functions including regulating body temperature, maintaining the balance of body fluids and creating a hostile environment for microbes. Additionally, skin possesses natural micro biome which ceases the advancement of pathogenic bacteria by combating for the nutrients and its binding sites ultimately inhibiting their growth by the production of metabolic products.

Various species like Brevibacterium, Staphylococci, Streptococci, Corynebacterium, and Candida constitutes the skin’s natural microbiome [2][3]. When the skin’s condition is compromised, then its capability to work as an effective barrier can be flawed. Commonly found skin infections are caused by Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, Brevibacterium, P. acnes, Trichophyton [4] etc., which have evolved over a course of time and now became antibiotic resistant strains due to their overuse, incomplete course of treatment or due to the incorrect prescription of currently applicable conventional antimicrobials and thus, leading to unresponsive prognosis against already available antibiotics. Though, the majority of currently available treatments for dermal infections are symptomatic and are less efficacious in restoring the quality of life and ameliorating the damage thus, search for new targeted therapeutic interventions are required to overcome, from these challenges [5]. The complementary alternative medicinal (CAM) system inspired by traditional knowledge and therapies are newly found great help to target and eliminate the skin infections and wounds. Almost 60-80 % of the world’s population relies on this and believing it to be the most prevalent form of medicinal approach worldwide. Consequently, essential oils (EOs) are also known to be one of the most efficacious therapeutic agents against dermal infections with immense therapeutic benefits, one such form of EO, extracted from the leaves of native Australian shrub Melaleuca alternifolia is Tea tree oil (TTO) [6][7]. It is known for its antimicrobial, antioxidant, antifungal and anti-inflammatory properties and has many terpenoid sub groups [8]. Among them, terpinen-4-ol is the main active component which has shown potential benefits in treating transdermal disorders such as eczema, psoriasis, acne and fungal infections as it is effective against various pathogenic microbe species especially Staphylococcus aureus [9] and Micrococcus luteus [10][3]. Though the mechanism of action for terpenes still remains to be completely understood, it does involve disintegration of permeability barriers of cell membrane structures with decreased chemiosmotic control and finally also causing cytoplasmic membrane disruptions [11]. They further progresses for mutagenesis in membrane proteins and interruption in membrane ion transport process, fluidity, permeability and cellular respiration process. Also, TTO is reported to exhibit anti quorum sensing activity and thus, restricts the bacteria from being virulent [12]. Therefore, inhibiting the microbes further for their swarming, biofilm formation and bacterial toxins secretion processes. Besides, being highly rich in hydrocarbons content such as terpenes including, monoterpenes, diterpenes, sesquiterpenes and oxygenated compounds such as carbonyls and alcohols, it does show higher volatility, and irritability, that restricts it from the direct dermal application [13]. They are known to be chemically sensitive; hence, can get decomposed easily by direct exposure to heat, light, humidity, and oxygen [14]. Thereby, in order to overcome such pharmaceutical concerns and to enhance the overall therapeutic efficacy of Melaleuca alternifolia, encapsulating it in an efficient and biodegradable nano carrier system is required, that would eventually support in overcoming the problems of volatility, bioavailability and would increase its effective resident time at the target site (skin) [15].

MATERIALS:

For the formulation, pure and standardized Melaleuca alternifolia oil was purchased from Forest Glen Organics, Sydney, Australia, Eucalyptus oil from Oriental Traders, Karnataka, India. Ascorbic acid, Dialysis membrane (D9650), DPPH (2, 2-Diphenyl-1-picrylhydrazyl), Gallic acid, Tween 80 were procured by Sigma Aldrich Ltd., USA. Chloroform, Ethanol, and methanol were purchased from Hi-Media, India. All the other chemicals used in the experiment were of analytical grade.

METHODOLOGY:

Preparation of TTO loaded microemulsion:

The oil in water (o/w) type microemulsion system was prepared by aqueous titration method, where TTO ME formulation was developed by selecting eucalyptus oil for the oil phase, tween 80 as surfactant and ethanol as co-surfactant along with water as aqueous phase [16]. The optimized formulations were subjected for thermodynamic stability testing and after approving their stability they were further characterized by particle size [17], poly dispersibility index [18], zeta potential [19], transmission electron microscopy, Fourier transform infrared [20] , release kinetics [21] and rheological studies [22] analysis.

Quantitative analysis of Melaleuca alternifolia:

Melaleuca alternifolia (TTO) is been reported to possess higher content of hydrocarbons, oxygenated compounds and alcoholic groups making TTO physiochemically highly volatile and sensitive [23]. Thus, quantitative analysis of TTO was done through highly sensitive gas chromatography-mass spectrometry (GC-MS) [24] method to mark the essential phytocompounds present in the compound and then later followed by terpenoid and phenolic estimation methods.

Gas chromatography-mass spectrometry (GC-MS) analysis of TTO:

Quantification and identification of major components of TTO were performed as per the reported Tranchida et al. GC – MS estimation method [24]. As reported in many studies the estimation of M. alternifolia exhibits a high percentage of monoterpene group, specifically by Terpinen-4-ol [16] (~48%), neo-dihydrocarveol (~7%) and 1, 8-cineole (~6%). The stock sample preparation was done by mixing 1% (v/v) of TTO in acetone, and injected (1µL) to GC –MS unit (GC-6890N with a mass selective detector 5973 (quadrupole mass analyzer) from Agilent Technologies (Thailand) Co., Ltd. Bangkok, Thailand). The run time of samples was estimated to be for 25 minutes at a temperature variation of the oven between 40 – 250ºC with helium as a carrier gas and then chromatogram was recorded.

Total Terpenoid Estimation:

Terpenoids are found in abundance and are structurally distinct too, from other classes of phytocompounds. They consist of other subgroups like terpenes, diterpenes, and sesquiterpenes, reported to be responsible for enormous pharmacological activities. The quantitative analysis of the total terpenoid contents [25] in the TTO, ME and TTO ME was analyzed spectrophotometrically, in triplicates, using Linalool as a standard. For estimation,100µl of each of the test samples (TTO, ME and TTO ME) or the standard at different concentrations (10 - 50µl/ml) were mixed with 1.5ml of chloroform and vortexed at 1792g for 5 minutes followed by addition of 100µl of Sulphuric acid with further incubation in dark for another 5 minutes at 37°C. Thereafter, the reddish-brown precipitate acquired was further dissolved in 1.5ml of 95% (V/V) methanol and estimated at 538 nm.

Total Phenolic Estimation:

The presence of the total phenolic content in the test samples (TTO, ME, TTO ME) was determined by the Folin-Ciocalteu method [26] and gallic acid was taken as standard. Then, 100 µl of the test sample was mixed with 0.5 ml of 0.2 N Folin-Ciocalteu reagent and 4ml of 7.5% of Sodium carbonate (Na₂CO₃) one after the other. The phenolic component in the test sample was oxidized by the reagent at an alkaline pH, resulting in a blue colored molybdenum-tungsten complex. The mixture was then incubated for 10 minutes at 37°C followed by another one minute at 65ºC and estimated at 765 nm.

Antioxidant estimation:

Radical Scavenging Assays i.e. DPPH (2, 2-diphenyl-1-picryl hydrazyl) and ABTS (2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays were performed to analyze the antioxidant properties of the test samples (TTO, ME, TTO ME). DPPH is a dark-colored crystalline powder composed of stable, free-radical molecules that accept hydrogen ions from an antioxidant. The antioxidant effect is proportional to the disappearance of DPPH radicals in test samples [27] and the monitoring of reduction in DPPH radicals count with UV spectrometer has become the most commonly used procedure due to its accuracy and simplicity. DPPH predicts strong absorption maxima at 517 nm (purple). The color changes from purple to yellow which is followed by the formation of DPPH radicals upon absorption of hydrogen from an antioxidant. This reaction is stoichiometric with respect to the number of hydrogen atoms being absorbed. Thereafter, the antioxidant effect can be easily evaluated by following the decreasing trend of UV absorption at 517 nm. Determination of DPPH radicals scavenging activity was estimated by taking a 1mM solution of DPPH in methanol and also a different concentration of the test samples ranging from 10-100µl/ml in methanol was prepared and 800µl of the total test samples were added to 200µl of DPPH. The absorbance was measured at 517 nm.

Similarly, ABTS assay, [16] is based on the ability of testing samples to scavenge the radical ions and prohibit the oxidation of ABTS. It is a greenish- blue stable radical chromophore, in which the radicals are originated in response to the reaction occurring between ABTS (7mM) solution and potassium persulfate (2.45mM). The reaction mixture comprising 500µl of ABTS in addition to the equal amount of potassium persulfate was incubated for 16hrs in dark at 37ºC and diluted further with water. Thereafter, 20µl of the test samples (10-100µl/ml) along with 980µl of ABTS reagent were mixed and stirred continuously for 2 minutes and incubated again for 30 minutes. The absorbance was taken at 734 nm to estimate the scavenging effect of the same. The reduction of free radicals was analyzed. Ascorbic acid (10-50µg/ml) was taken as a standard for both the assays and scavenging of the test samples (%) was determined by the following calculation:

(Ac-As)

Scavenging effect (%) = ----------- × 100

Where; Ac- the absorbance of the control; As - the of the test sample.

Hydrogen peroxide scavenging activity:

Hydrogen peroxide (H₂O₂) is the free radical reactive oxygen species (ROS) which are produced as the by-products of the normal aerobic metabolism and gets increased during a stressed condition, infection, exercise, radiation, etc. H₂O₂ is not toxic itself but eventually, it can be converted into even more toxic radicals such hydroxyl radicals and thus, its removal is important [31]. H₂O₂ scavenging activity is based on direct UV-Vis absorption measurement (A230). The basic principle involves the colorimetric determining of the presence of hydrogen peroxide in the optimized sample. The determination of H₂O₂ scavenging activity was estimated by taking the different concentrations of the test sample ranging from 10-100µl/ml in PBS (50mM solution phosphate buffer, pH 7.4) followed by addition of 500 µl of H₂O₂in each of the sample. Thereafter, the reaction mixture was incubated for 10 minutes at room temperature and then absorbance was measured at 230 nm. Percentage of scavenging activity of H₂O₂ was evaluated and compared with the reference compound (Ascorbic acid) which was taken as the standard by the below-mentioned equation:

control absorbance - sample absorbance

H₂O₂ = ------------------------------------------------×100

activity (%) control absorbance

Nitric oxide scavenging activity:

Nitric oxide scavenging activity is based on the diazotization reaction which was originally described by Griess in 1879 [28]. It involves the chemical reaction between sulphanilamide and naphthyl ethylenediamine dihydrochloride (NED) under acidic conditions. Both of them compete for the nitrite in the Griess reaction. Hence, this reaction detects the presence of NO2- in a variety of test samples. Determination of nitric oxide (NO) [29] scavenging activity was estimated by taking ascorbic acid as standard and adding 0.25 ml of sodium nitroprusside (10mM) in 1 ml of test samples which were taken in different concentrations (10-100µl/ml) and incubated for 3 hours at 25°C. Thereafter, 500 µl of Griess reagents were added in all the samples and absorbance was taken at 546 nm for the purple colored chromophore. Percentage of scavenging activity of NO [31] was evaluated by the below-mentioned equation:

Ic - Is

Inhibition (%) = -----------------× 100

Where;

Ic - the absorbance of the control;

Is- the of the test sample.

Antimicrobial activity:

Diffusion Method (Disk-Diffusion):

The bacterial strains, Micrococcus luteus, and Staphylococcus aureus, both being gram-positive bacteria and known to cause a variety of skin ailments were selected for this study [30]. The antimicrobial activity of Tea tree oil (TTO) was compared with Eucalyptus oil (EPO), ME (Microemulsions only), and TTO ME (TTO loaded microemulsion) by disk diffusion assay on the mentioned microbial strains. These microbial strains were cultured on an agar plate and kept in incubation for 24 hours at37ºC to attain growth. Thereafter, these agar plates were then inserted with sterile paper discs of 2mm, infused with 20 µl of the test samples (TTO, EPO, ME, TTO ME, aqueous and positive control) and incubated again at 37ºC for 24 and 48 hours respectively. The antimicrobial activity was determined by measuring the diameters of the zone of inhibitions of test samples against the diameters of the zone of inhibitions of positive control. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of all the samples were determined through the disk diffusion analysis, where the MBC was looked upon for the least concentration of TTO and TTO ME. Further, where no visible growth was observed, MIC was looked upon for the least concentration of TTO and TTO ME where the visible growth was observed.

Dilution Method:

The growth inhibition in the primary culture of Micrococcus luteus and Staphylococcus aureus was also analyzed in a medium by taking 20ml of Luria broth supplemented with 100 µl of bacterial strains respectively and incubating it at 37ºC for 24hours [5][30]. Subsequently, 50µl of each bacterium was added from the primary culture into the 25ml of Luria broth media and kept in the incubator again at pre mentioned conditions. After the attained growth, 100µl of test samples (TTO, EPO, ME, TTO ME, aqueous and positive control) were added in each of the culture media. The absorbance of the same was recorded at 600nm after 24 and 48hours respectively for all the samples.

RESULTS AND DISCUSSION:

Preparation of TTO ME:

Tea tree oil loaded microemulsion system (TTO ME) was fabricated and optimised earlier with particle size of 149.1 ± 1.34 nm, Poly dispersibility score of 0.297 ± 0.005 with zeta potential of −3.19 ± 0.04 mv) using eucalyptus oil, tween 80, and ethanol as, oil, surfactant, and cosurfactant phases, respectively. The particle size analysis and other characterizations done with the formulation suggests it to be a more stable and efficacious therapeutic system in comparison to only TTO and suitable to apply on various in vitro and in vivo skin disorders models.

Quantitative estimation of Melaleuca alternifolia by GC – MS method:

The gas chromatography-mass spectrometry estimation (GC – MS) (Figure 1) of TTO reflected the presence of essential putative therapeutic categories like - Terpinen-4-ol, α-pinene, α-terpinene, D-Limonene, 1,8-cineole, γ- terpinene and α-terpinolene in the TTO which are known to exhibit various antioxidant, antimicrobial and anti-inflammatory properties. (Table 1). This result scanning was further utilised for the in vitro phytocompound (Terpinen-4-ol) release and its analysis as it is the prime component of TTO.

Figure 1- Representing the gas chromatography-mass spectrometry (GC – MS) chromatogram of pure Melaleuca alternifolia oil showing peaks of all forms of terpene, limonene and cineole groups present in the same.

Total Terpenoid and Phenolic Estimation:

The presence of total terpenoid and phenolic content in Melaleuca alternifolia -Tea tree oil (TTO), Microemulsion and Tea tree oil encapsulated Microemulsion (TTO ME) determined by the UV-Vis spectroscopy method. Gallic acid and Linalool standard were prepared (10mg/ml in methanol) and (200 µl/ml) respectively. The absorbance of the total terpenoid estimation was taken at 538 nm and the presence of the total phenolic content was observed at 765 nm for the test samples. The total terpenoid concentration present in Melaleuca alternifolia (TTO) is 80.03±0.02 mg/ml, Microemulsion is 71.27 ± 0.10 mg/ml and TTO ME is 89.79 ± 0.05 mg/ml. Similarly, the total phenolic concentration present in Melaleucaalternifolia is 79.9 ± 0.02 mg/ml, Microemulsion is 77.82 ± 0.09 mg/ml and TTO ME is 87.21 ± 0.03 mg/ml.

The Terpenes are known to restore and improve skin and also prevents the skin from oxidative damage and reduced inflammation. Whereas, the Phenols or polyphenols act protectively and inhibits the progression of certain disorders and also eliminates minor skin problem like wrinkles, acne and etc. The Terpenoid and phenolic estimation is done clearly states that TTO and ME both contain an adequate quantity of terpenes and polyphenols and when combined synergistically TTO ME can do wonder in eliminating the skin disorders.

Antioxidant estimation:

The antioxidant activity of DPPH assay (Figure 2) and ABTS assay (Figure 3) were plotted against various concentrations (0 – 100 µl/ml) of test samples (Eucalyptus oil, TTO, ME and TTO ME). The AO activity in DPPH assay exhibited the highest antioxidant activity of 98 ± 1.02% by TTO ME in comparison to ascorbic acid (95 ± 1.23%), TTO (92.4 ± 1.6%), eucalyptus oil (88.9 ±1.72%) and ME (83.8 ± 0.56%). Similarly, measurement of AO activity through ABTS assay again displayed the highest antioxidant activity of TTOME (99 ± 0.12 %) with significant scavenging of free radicals from the extract as compared to ascorbic acid (96 ± 0.21 %), TTO (93.9 ± 1.22%), eucalyptus oil (90.8 ± 2.2%) and ME (89.3%.8 ± 1.38%). Therefore, it was well reflected from the results that TTO ME has a maximum antioxidant activity followed by standard (AA), TTO, Eucalyptus oil and Microemulsion without TTO (ME). It was clear that with an increase in the concentration of TTO ME, better activity was displayed as compared to the TTO in the original state. DPPH and ABTS assay are highly sensitive even towards the weakest AOs and can very well determine both hydrophilic and lipophilic compounds even at the minimum concentration and thereby TTO ME even at its lowest concentration reported high AOs of (DPPH= 79.99±0.31%) and (ABTS=89.1± 0.12%)in comparison with TTO (DPPH= 71±0.11%) and (ABTS=80±0.61%) as displayed in Figure 2 and Figure 3 respectively. The possible reasons for enhanced activity are the microemulsion system altogether with its excipients is reported to reflect its own antioxidant properties thus enhancing the overall efficacy of the formulation.

Figure 2 -The Graph depicting scavenging effect (%) of DPPH assay -Eucalyptus oil, ME, TTO and TTOME at different concentrations.

Figure 3-The Graph depicting scavenging effect (%) of ABTS assay -Eucalyptus oil, ME, TTO and TTOME at different concentrations.

Stability studies of test samples:

Stability of TTO-ME (ETE-C) and TTO in the original state were examined after storing them at room temperature (37ºC) for a year and testing the retention ability of their AO [30] content left after storing them for a mentioned time by the methods-ABTS and DPPH assay. The DPPH assay exhibited a significant loss of AO in TTO (DPPH = 69.4 ± 1.7%) in comparison with TTO ME (DPPH=89±1.03%). Similarly, degradation of the therapeutic compounds with the loss of AO activity in TTO (ABTS=62.1 ± 1.62%) which was compared with TTO ME (ABTS=85 ± 0.42 %).

The overall comparison of the stored samples with the freshly prepared samples of TTO and TTO ME depicted a prominent loss of the AO activity as the freshly prepared samples had a higher content of AO i.e. TTO ME -(DPPH=98 ± 1.02%) and (ABTS=99 ± 0.12%). Similarly, TTO (DPPH=92.4 ± 1.6%) and (ABTS=93.9 ± 1.22%). Therefore, from the comparative data, we can conclude that the Microemulsion system significantly enhances the stability and the shelf life of the TTO. The stability analysis studies prove that the therapeutic phytoconstituents particularly the terpenoids were retained that impart the antioxidant properties [20].

Hydrogen peroxide scavenging activity:

The scavenging behavior of both the test samples (TTO and TTO ME) was dose-dependent as it increases with the increase in concentration. TTO ME showed % inhibition of 95.5 ± 1.61 % at its highest concentration of 100 µl/ml whereas; the % inhibition for standard was found to be 94.7 ± 2.23 %, TTO = 89.9 ± 1.6 %, Eucalyptus oil= 85.2± 2.12%and ME =78.6 ± 0.23 % on the same concentration as depicted in Figure 4. Hence from the results obtained, we can sequence the scavenging activity of hydrogen peroxide of various test samples as TTO ME > AA > TTO >Eucalyptus oil>ME.

Figure 4-The Graph depicting H₂O₂ activity (%) of Eucalyptus oil, ME, TTO and TTOME at different concentrations.

Nitric oxide scavenging activity:

The percentage inhibition of free radical, either by scavenging it or inhibiting it from further formation was plotted against various concentration (0 – 100 µl/ml) of test samples (TTO and TTO ME) TTO ME showed % inhibition of 96.5±1.72 % at its highest concentration of 100 µl/ml whereas; the % inhibition for standard was found to be 94.5±2.23 %, TTO = 85.8±1.6, Eucalyptus oil= 82.1±0.08%and ME = 80.66± 0.2 % on the same concentration as shown in Figure 5. It was well reflected from the results that the data represented by NO studies indicates that the samples are concentration dependent and thereby exhibit enhanced scavenging potential of TTO ME followed by standard (AA), TTO, Eucalyptus oil and microemulsion without TTO (ME).

Figure 5 - The Graph depicting NO Inhibition activity (%) of Eucalyptus oil, ME, TTO and TTOME at different concentrations.

Anti-microbial activity of Melaleuca alternifolia loaded microemulsion:

Diffusion Method (Disk-Diffusion):

The antimicrobial activity of Eucalyptus Oil, TTO and TTO loaded Microemulsion was assayed by disc diffusion assay against Micrococcus luteus and Staphylococcus aureus. The TTO-ME was found to be effective against both Micrococcus luteus(Figure 6)and Staphylococcus aureus(Figure 7)with a maximum zone of inhibition exhibited with 29±1.32 mm and 25 ± 0.78 mm respectively (Table 2).TTO is reported to exhibit anti-quorum sensing activity and thus, restricts the bacteria from being virulent and further lead to mutagenesis in membrane proteins and interruption in membrane ion transport process, fluidity, permeability, and cellular respiration process. Ultimately causing cytoplasmic membrane disruptions and cell lysis.

Figure 6 -Disk diffusion assay treatment of Micrococcus luteus, where (a) Antibiotic, (b) control, (c) TTO, (d) Eucalyptus oil, (e)ME and (f) TTOME

Figure 7-Disk diffusion assay treatment of Staphylococcus aureus, where (a) Antibiotic, (b) TTO, (c) Eucalyptus oil, (d) control, (e)ME and (f) TTOME

Table 2-The antimicrobial effect of Tea Tree Oil, Microemulsion encapsulated Tea Tree Oil and Microemulsion against M. luteus and S. aureus strain (Zone of Inhibition in millimetres ± S.D).

S. No	Microbial Strain	Gentamycin (mm)	TTO (mm)	EPO (mm)	ME (mm)	TTOME (mm)
1.	Micrococcus luteus	23 ± 2	20 ± 1	19.5 ± 2	17.5±1	29±2
2.	Staphylococcus aureus	21±3	19±1	17±2	15.5±3	25±3

Dilution Method:

The antimicrobial effect of TTO ME was also determined by using the dilution analysis. The bacteria i.e. Micrococcus luteus and Staphylococcus aureus were chosen as targets. The six prepared flasks, were inoculated with 100µl of test samples including Eucalyptus oil, Tea Tree Oil (TTO), Tea Tree Oil loaded microemulsion (TTOME), Microemulsion without Tea Tree Oil (ME) and the antibiotic resistant to both the bacteria (Gentamycin) respectively leaving one conical flask as a control. The readings were observed at 600nm after every 6,24 and 48 hours in each flask in order to determine the effect of the TTO ME on the two strains i.e. Micrococcus luteus (Figure 8) and Staphylococcus aureus (Figure 9).

TTO ME was the most effective in comparison with TTO, indicating that TTO combined with ME enhances the therapeutic efficacy of TTO, which enhance its antimicrobial properties and thereby, leading to the lysis of the bacterial cell membrane.

Figure 8-The graph representing the results of treatment by Eucalyptus Oil, TTO, TTO-ME and ME against Micrococcus luteus.

Figure 9 -The graph representing the results of treatment by Eucalyptus Oil, TTO, TTO-ME and ME against Staphylococcus aureus.

CONCLUSION:

The present study was conducted to evaluate the increased therapeutic efficiency and stability of TTO ME along with its enhanced shelf life due to the faster degradation of terpenoids, as reported. TTO is already been recognized to possess various antimicrobial, antioxidant and anti-inflammatory properties but due certain limitation listed such as volatility, and irritability, that restricts it from the direct dermal application and can get decomposed easily by direct exposure to heat, light, humidity, and oxygen. Thereby, in order to overcome such concerns and to enhance the overall therapeutic efficacy of Melaleuca alternifolia, encapsulating it in an efficient and biodegradable Nanocarrier system is required, and thus we had developed a ME system earlier, and in this study, we have evaluated if we could achieve the aims as mentioned above. From this study, we can conclude that the AO and stability efficiency of TTO has enhanced after formulating it into the ME system (TTO ME). Collectively, the experimental results suggested that the TTO ME formulation can effectively scavenge ROS and could provide effective protection against oxidative damage. The said ME were found to be equally efficient (in fact slightly more) in exhibiting their AO activity which might have been enhanced due to the already existing intrinsic activity of the carrier system components (ME). Furthermore, the major constituents of the extract were also found to be almost constant after 12 months’ storage at room temperature. Besides this, the amount and ratios of excipients (oil, surfactants, and cosurfactants) used for the formulation of ME are listed and are under the GRAS limits and that may be the reason that formulation was found to be quite safe. The stability and shelf life of the formulation were also augmented in comparison to the pure extract. Thus, the developed ME of TTO serves as a potential and effective delivery system and further.

ACKNOWLEDGEMENT:

The researchers are thankful to the Department of Biotechnology and Bioinformatics, Jaypee Institute of Information Technology, Noida, Uttar Pradesh for providing all the necessary facilities to execute this work. Also, they are grateful to SMITHA lab (IIT Delhi), SAIF (Punjab University, Chandigarh) and Essential Oil Association of India, Noida, Uttar Pradesh for their specialised analytical facilities for the successful completion of this research work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 20.08.2019 Accepted on 30.10.2019

Research J. Topical and Cosmetic Sci. 10(2): July- Dec. 2019 page 48-56.

DOI: 10.5958/2321-5844.2019.00011.6

Components of M. Alternifolia	Chemical Structure	Retention Time (RT)	Composition (%)
α-pinene		5.374	2.4
α-terpinene		7.623	10.5
D-Limonene		8.018	0.9
1,8-cineole		8.087	4.9
γ- terpinene		9.151	22.6
α-terpinolene		10.250	2.9
Terpinen-4-ol		14.575	40.6
α-terpineol		15.010	2.6