Formulation and Evaluation of Eucalyptus Oil-Based Emulsion for Enhanced Stability and Topical Application

Material

The fresh Eucalyptus oil was collected from Waghdole Aushadhalaya (Center for Quality Ayurvedic Medicines), Satara (Maharashtra), India.

 

Characterization of Eucalyptus oil

Organoleptic Properties

Eucalyptus oil was evaluated for its organoleptic properties such as color, odor, and taste. Determination of acid value

 

The acid value is a quantity that represents the mg of potassium hydroxide required to neutralize the free acids present in 1 g of a substance.

 

The sample was carefully weighed and dissolved in 50 ml of a mixture of equal quantities of ethanol (95 percent) and ether, which had been previously neutralized with 0.1 M potassium hydroxide to phenolphthalein solution. Because the sample did not dissolve in the cold solvent, the flask was attached to a reflux condenser and slowly warmed with regular shaking until the sample dissolved. 1 ml phenolphthalein solution was added, and the solution was titrated with 0.1 M potassium hydroxide until it was faintly pink after 30 seconds of shaking. [12]

 

From the expression, calculate the acid value.

Acid value = 5.61 n/w

Where,

n = the number of ml of 0.1 M potassium hydroxide required;

w = the weight in g of the substance.

 

Determination of saponification value

The saponification value is the amount milligrams of potassium hydroxide required to neutralize the free acids and saponify the esters in 1 g of the substance.

In this approach, 2 g of the sample was weighed and poured into a 200 ml borosilicate glass flask with a reflux condenser. About 25.0 ml 0.5 M ethanolic potassium hydroxide, plus a pinch of pumice powder, heated for 30 minutes on a water bath under reflux. Titrated with 0.5 M hydrochloric acid (‘a'ml) after adding 1 ml of phenolphthalein solution as an indicator. A blank determination was performed without the chemical under investigation (‘b' ml). [12]

From the expression, calculate the saponification value.

 

Saponification value = 28.05 (b - a)/w

Where, w = weight, in g of the substance.

 

Determination of ester value

The ester value is the amount of potassium hydroxide needed to saponify the esters in 1 g of the material. The acid value and saponification value of the material under investigation were determined. [12]

Calculated the ester value from the expression

Ester value = Saponification value - Acid value

 

Determination of iodine value

The iodine value is a number that expresses in grams the amount of halogen, computed as iodine that is absorbed by 100 g of a substance under the conditions given. In this method, weighed the accurate quantity of the substance was examined, placed in a dry 300 ml iodine flask or which has been rinsed with glacial acetic acid. Added 15 ml of chloroform and dissolved. Added slowly from a burette 25.0 ml of iodine monobromide solution, inserted the stopper, allow standing in the dark for 30 minutes, shaking frequently. 0.1 M sodium thiosulphate was titrated using 10 ml potassium iodide solution and 100 ml water, with a starch solution added at the end of the titration as an indicator. Take note of the needed ml (a). Without the substance under evaluation, repeat the procedure and record the number of ml required (b). [12]

 

Calculated the iodine value from the expression

Iodine value = 1.269 (b - a)/w

Where, w = weight, in g of the substance.

 

Determination of peroxide value

The peroxide value is the number of milliequivalents of active oxygen contained in 1000 g of the substance. In this procedure, precisely weighed 5 g of the substance transferred to a 250 ml glass stoppered conical flask, 30 ml of a mixture of 3 volumes glacial acetic acid and 2 volumes chloroform was added, swirled until completely dissolved, and 0.5 ml of saturated potassium iodide solution was added. Allow for 1 minute of standing time with intermittent shaking before adding 30 ml of water and titrating with 0.01 M sodium thiosulphate until the yellow color almost vanishes with continuous and vigorous shaking. Continue the titration by adding 0.5 ml of starch solution and shaking firmly until the blue color fades completely (‘a’ml). Make a blank decision without the substance under investigation (‘b’ml). In the blank determination, the volume of 0.01 M sodium thiosulphate must not exceed 0.1 ml. [12]

From the expression, calculate the peroxide value.

Peroxide value = 10 (a - b)/w

Where, w = weight, in g of the substance.

 

Boiling point

The boiling point of Eucalyptus oil was determined by micro controlled based melting point apparatus (Chemi Line). The sample was placed in a fusion tube with one end sealed shut. The capillary was then put into a silicone oil bath that was heated in a controlled manner using an electric heating coil. The temperature at which bubble formation occurs was noted as boiling point temperature and compared with the literature value. [13]

 

Refractive index

An Abbe's type refractometer was used to assess the refractive index of Eucalyptus oil at room temperature. [14]

 

Viscosity

The viscosity of Eucalyptus oil was determined by using an Ostwald viscometer. Calculated the viscosity from the expression. [14]

 

Identification of Eucalyptus oil

Infrared spectroscopy

The purpose of the IR investigation was to determine the purity of the medication and to determine which functional groups were present in the oil. A Fourier Transform Infrared spectrophotometer was used to determine it (FTIR, Alpha, Bruker). By scattering the sample, it was scanned over a wavelength range of 4000 to 500 cm^-1 with a resolution of 500 cm^-1. The spectrum was acquired by placing the pellet in the path of light. [15]

 

Drug –excipient stability study

Visual observations

Any visual changes in the samples subjected to drug-excipient compatibility trials were examined. Changes in color and nature were detected in the samples.

Infrared spectroscopy

A Fourier Transform infrared spectrophotometer was used to determine the IR (FTIR -410, Jasco, Japan).

 

Formulation and evaluation of emulsion

Optimization of formulation parameters and process factors

For optimization, the preparation method, selection of stabilizer, type and volume of organic solvent, stirring time, and speeds were changed and the characteristics of the prepared emulsion were evaluated. [16]

Preparation of emulsion

The oil phase of the emulsion was prepared by combining Span 20 with light liquid paraffin and Peppermint oil, as well as Eucalyptus oil. Tween 20 was dissolved in purified water to make the aqueous phase. Because Methylparaben and Propylparaben are hydrophobic, they were dissolved in the oil phase after being dissolved in Propylene glycol and combined with an aqueous phase. Both the oily and aqueous phases were heated to 70 to 80°C separately, then the oily phase was added to the aqueous phase and stirred continuously until the mixture reached room temperature. The emulsion was created and is kept in a tightly sealed airtight container. [17-18]

 

 

Table 1: Composition of emulsion

Batch/Ingredients	F1	F2	F3	F4	F5	F6	F7	F8
Eucalyptus oil (a)	2.1	2.1	2.1	2.1	2.1	2.1	2.1	2.1
Light liquid paraffin (a)	4.37	4.37	4.37	4.37	4.37	4.37	4.37	4.37
Peppermint oil (a)	1.05	1.05	1.05	1.05	2.1	2.1	2.1	2.1
Span 20 (a)	1.4	2.8	1.4	2.8	1.4	2.8	1.4	2.8
Tween 20 (a)	0.7	1.4	1.4	0.7	1.4	0.7	0.7	1.4
Methyl paraben (b)	0.003	0.003	0.003	0.003	0.003	0.003	0.003	0.003
Propyl paraben (b)	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
Propylene glycol (a)	5	5	5	5	5	5	5	5
Water (a)	q.s.
Where (a) is volume taken in ml and (b) is the weight taken in g.

Characterization of eucalyptus oil

Organoleptic properties

The Eucalyptus oil was found to be a pale yellow, slightly viscous liquid at room temperature. It possessed an unpleasant odor and a distinctly bitter taste. These organoleptic characteristics are consistent with the typical sensory properties reported for pure Eucalyptus oil.

 

Table 2: Characterization of eucalyptus oil

 

Boiling point

The boiling point of eucalyptus oil was found to be 178 to180°C.

Viscosity

The viscosity of eucalyptus oil was found to be 54.40 cPs at 29°C. While as per the literature standard it is to be 55.5 ± 0.37 cPs at 25°C. All experimental values were in good agreement with official values, it could be concluded that Eucalyptus oil was in a pure state.

Identification of Eucalyptus oil

Infrared spectroscopy

Figure 1: FTIR spectrum of Eucalyptus oil

 

Table 3: IR interpretation of eucalyptus oil

 

The IR spectrum of Eucalyptus oil (Figure 1 & Table 3) showed the presence of functional groups which are present in 1,8-cineole so, it indicated that 1,8-cineole is present in Eucalyptus oil.

 

Drug- excipient stability study

Visual observation

Visual inspection revealed no significant changes in the sample. There was no visible change in color. It suggested that eucalyptus oil might be compatible with the chosen excipients.

Infrared spectroscopy

FTIR spectrum of the Eucalyptus oil and another excipient like Carbopol 934 were compared with spectra of the formulation. An FTIR spectrum of formulation shows significant peaks of Eucalyptus oil indicating no interaction between Eucalyptus oil and excipient.

 

A

B

C

Figure 2: IR spectra of (A) Eucalyptus oil, (B) Carbopol 934, (C) Emulgel respectively

 

Table 4: IR interpretation emulgel

 

Emulsion Formulation and evaluation

Selection of emulsifier

The selection of emulsifiers was carried out depending upon their HLB value and their miscibility with the oil. The different combinations of emulsifiers were screened for the formulation of microemulsions such as Span 20 & Tween 60, Span 20 & Tween 80, and Span 20 & Tween 20. The optimization of emulsifier combination was fixed upon visual inspection such as clarity, phase separation, coalescence, and centrifugation. The combinations of Span 20 with Tween 60 and Span 20 with Tween 80 were show instability of emulsion. The combination of Span 20 and Tween 20 was not showing any instability of microemulsion. Hence, this combination was optimized to prepare stable microemulsion.

Optimization of the emulsion formulation

Optimization of the formulation was done based on stability studies. Throughout the trial, all other formulation and processing factors were held constant. Formulation of microemulsion was optimized by observing phase separation, creaming at room temperature. Batch F2 was not shown phase separation, coalescence in the formulation, also it was stable after the centrifugation test therefore batch F2 was selected as optimized formulation as it is stable microemulsion.

 

 

 

Table 5: Composition of optimized Batch F2

 

Evaluation of Microemulsion

Microscopic observation

Using an optical microscope, the emulsion was studied microscopically and photographs were made. The type of emulsion seen was oil in water (Figure 3).

Figure 3: Microscopic observation of emulsion

pH

At 30±1°C, the pH of the emulsion was measured with a digital pH meter (Systronics pH system 362). At 30°C, the pH was observed to be 5.28. The study revealed that concentrations of oils and Tween 20 plays role in changing the pH of a formulation.

 

Electrical conductivity

The emulsion's electrical conductivity was measured to be 188.2 µs. The existence of strong electrical conductivity in an emulsion indicates that water was the emulsion's continuous phase. As a result, the created emulsion was oil in water emulsion.

 

Viscosity

The viscosity of emulsion was measured by using Brookfield viscometer (Fungilab) at 100 rpm, at 29°C, it was found to be 41 cPs. The viscosity of emulsion was increased due to the presence of viscosity modifier agents or emulsifiers used. By retarding the inevitable rise of the oil droplets to the top, increased viscosity considerably improved emulsion stability.

 

Centrifugation

After the centrifugation of emulsion at 5000 rpm for 20 minutes, there was no phase separation or creaming observed. Hence it was concluded that emulsion formulation was stable.

Zeta potential and globule size analysis

Figure 4: Zeta potential of emulsion.

 

A highly negative or highly positive zeta potential value indicates good physical stability of the formulation. The zeta potential of the emulsion formulation (F2) was measured by using Zetasizer at 25 ℃, it was found to be +12.70 mV. High zeta potential in turns maximum stability of the emulsion. Ideally zeta potential for stable dispersion should be ±30 mV.

Figure 5: Particle size analysis of emulsion.

Particle size analysis of emulsion was shown in Figure 10.7 Emulsion showed average particle size 203nm.The polydispersity index of the emulsion was found to be 0. 221.The particle size was below 1000 nm; hence it was confirmed that the formed emulsion was microemulsion. The polydispersity index of the emulsion was less than 0.5, this indicated uniformity in particle size of droplets and directly it will affect stability of emulsion.