© N. Wangpradit et al., Published by EDP Sciences, 2022

Highlights

This study aims to determine the rHLB of Amesiodendron chinense (Merr.) Hu oil which is the natural oil that has not to reported before, therefore the exact rHLB of Hu oil lead to the development of further stable formulation or another grateful benefit of this oil that can apply in the future. In this study, the rHLB of Hu oil was found at 6.2. The rHLB value was applied to calculate the required quantity of the emulsifier blend for the successful development of stable cream containing Hu oil. The cream containing Hu oil was developed with 3% of an emulsifier; the combination of Tween80 and Span60. The rHLB of the oil phase used was between 9.33 and 10.33. The stable creams were successfully formulated by utilizing determined rHLB.

1 Introduction

Most of the pharmaceutical as well as cosmetic, semisolid formulations are biphasic systems (emulsion, cream, and lotion), where hydrophilic-lipophilic balance (HLB) plays a vital role by managing the tension between two immiscible liquids at their interface (Alam et al., 2020). The exact knowledge of the required HLB (rHLB) value of different ingredients in the emulsion guides the determination of rHLB of the system (oil phase of formulation), which ultimately helps in the development of a stable in the final formulation (Meher et al., 2013). Fortunately, rHLB of almost all of the ingredients including emulsifiers used in emulsions such as cetyl/stearyl alcohol, stearic acid, white beeswax, Tween series, Span series, glycerol stearates, and many more are already reported in the literature, whereas aromatic oils are still not properly investigated and reported. Amesiodendron chinense (Merr.) Hu oil is an aromatic oil from SAPINDACEAE. It is reported to be used for the treatment of skin disease by Thai folk medicine. Unfortunately, the simple cream preparation using Amesiodendron chinense (Merr.) Hu oil was instability (Tiptiwa, 2008).

In the present study, a pharmaceutically and cosmetically elegant cream formulation of Amesiodendron chinense (Merr.) Hu oil with different cream bases was attempted to develop. During the formulation process, difficulty was experienced in the step of selecting the suitable emulsifiers to produce a stable emulsion. These complications further guided to determine the rHLB value of Amesiodendron chinense (Merr.) Hu oil, which is not reported in the literature. Therefore, currently, there is an attempt to determine the rHLB value of Amesiodendron chinense (Merr.) Hu oil. To ensure the correctness of the applied method, the rHLB value of coconut oil was also determined simultaneously. Utilizing the determined rHLB of Amesiodendron chinense (Merr.) Hu oil, the formulation of cream containing Amesiodendron chinense (Merr.) Hu oil was developed and also the evaluation was assessed for different physical parameters such as consistency, color, pH, viscosity, homogeneity, and phase behavior during the accelerated stability test. These parameters possibly ensure the reproducible quality of a specific product.

2 Materials and methods

2.1 Materials

Amesiodendron chinense (Merr.) Hu oil was collected from a local healer in Phatthalung province, Thailand. Botanical identification was performed by Asst. Prof. Dr. Katesarin Maneenoon, an ethnobotanist at the Faculty of Traditional Thai Medicine, Prince of Songkla University. Chemical ingredients used in formula development include, coconut oil, Tween80, Span60, stearic acid, white beeswax, glycerol monostearate (GMS), isopropyl myristate, propylparaben were purchased from L.B. science, Thailand. All were cosmetic/pharmaceutical grade.

2.2 Preparation and evaluation of emulsion

To determine the rHLB of Amesiodendron chinense (Merr.) Hu oil/coconut oil, the formulation was developed by the formula composed of 3 main parts; 3% was an emulsifier, 5% was an oil phase, and 92% was the aqueous phase. In the 3% emulsifier, Tween80 (HLB = 15) and Span60 (HLB = 4.7) were chosen and blended in the different ratios. 5% of oil phase consist of Amesiodendron chinense (Merr.) Hu oil and coconut oil while 92% water was taken as the aqueous phase. The emulsion was prepared using the phase inversion temperature technique. An emulsion was prepared by mixed of the required amount of Tween80 was dissolved in the aqueous phase and Span60 was dissolved in the oil phase. Till the equal temperature of the two phases, the aqueous phase was poured into the oil phase with continuously gentle stirring for 10 min or until becomes homogenously. The blended emulsion of Amesiodendron chinense (Merr.) Hu oil and coconut oil by different ratios of a chosen emulsifier could produce the HLB value range from 5 to 15 shown in Table 1 (first series emulsion). The first series of emulsions were physically evaluated for any changes in appearance, color, and instabilities for 30 days, the most stable emulsion was identified by the determination of the creaming index (CI) of all formulas. The most stable emulsion from the first series emulsion (HLB5-8) was selected to generate the second series by configuring the HLB to be more detailed. The emulsions in the HLB range of 5.0–7.8 were formulated as a second series emulsion and subjected to other evaluation parameters.

2.3 Creaming index

CI was recorded on the 3rd, 7th, 15th, 30th days. CI was calculated by taking the ratio of the total height of the cream (H_C) layer and the total height of emulsion (H_E) as follows:

$$\%{\text{CI = (H}}_{\text{C}}{\text{ / H}}_{\text{E}}\text{) × 100}$$(1)

2.4 Formulation of cream containing Amesiodendron chinense (Merr.) Hu oil

The determined rHLB value of Amesiodendron chinense (Merr.) Hu oil from the previous investigation. The further utilization of the rHLB was to develop the cream formulation, as shown in Table 2. The cream was prepared as an oil-in-water disperse system by heating the oil phase and aqueous phase separately. Then, two phases of an emulsion were mixed and continuously stirred for 30 min to result in a cream. After the preparations, the physical parameters including physical appearance, pH, viscosity, homogeneity, and phase separation were characterized. In addition, the visual appearance of the formulation, such as consistency, color, and the odor was observed. The pH was measured by using ColorpHast^TM pH-indicator strips (Merck, Darmstadt, Germany). Rotational rheometer DV-III Ultra equipped with a TC spindle was used to measure the viscosity of an emulsion at a rotation rate of 20 rpm at 25 °C ± 2 °C.

2.5 Microscopic analysis of the cream structure

The cream containing Amesiodendron chinense (Merr.) Hu oil was stained with water-soluble dye (Amaranth), and their morphology was analyzed using a light microscope (AxioLab. A1, Carl Zeiss, Germany) connected to an EOS 80D camera (Canon, Japan). All images were captured using a Zeiss Axiovision LE software (Carl Zeiss, Germany).

2.6 Stability test

The stability test was performed with the heating and cooling method for 6 cycles at 4 °C and 45 °C. The physical parameters of the cream were carried out before and after 6 cycles of the accelerated stability test.

2.7 Statistical analysis

Data were presented as mean ± SD with n = 3. Data were analyzed using Student’s t-test on SPSS software and considered statistically different at p < 0.05.

3 Results and discussion

This study aims to determine the rHLB of Hu oil, which is the natural oil that has not to reported before, therefore the exact rHLB of Hu oil lead to the development of further stable formulation or another grateful benefit of this oil that can apply in the future. The method performed is the reliable method to determine the rHLB of newly discovered oil based on a previous study (Meher et al., 2013). In addition, this study uses coconut oil as a standard to confirm the correctness of the rHLB value. For the stability test was assessed by heating and cooling method (Petchsomrit et al., 2020), which is the reliable method for testing the stability of the formula. The picture of all stable formulations was shown in Figure 1.

Fig. 1 Stable cream containing Amesiodendron chinense (Merr.) Hu oil after 6 cycles of accelerated stability test under 4 and 45 °C.

3.1 Creaming index

An emulsion is simply defined as “a system comprised of two immiscible liquids, one of which is dispersed as droplets (the dispersed or internal phase) throughout the other (the continuous or external phase)” (Jafari et al., 2008). Thermodynamically unstable is one of the properties of emulsions; over time, an emulsion tends to break down into an oil phase and aqueous phase (Hosseini et al., 2015). Various physicochemical mechanisms can indicate the instability of an emulsion, such as gravitational separation or creaming, coalescence, flocculation (partial) coalescence, and Ostwald ripening (Birdi, 2008, Hosseini et al., 2015). In biphasic systems, creaming is a natural phenomenon to indicate the destabilization of an emulsion (Robins et al., 2002). The creaming index is a value used to define the stability of an emulsion, by the emulsion with a low creaming index displays good creaming behavior and represents the stability of an emulsion. The low creaming index value of 0–20% indicates the low serum separation and higher emulsion stability (Zungur et al., 2015). It provides an initial idea of the nearest stable emulsion range that may be useful for defining rHLB (Meher et al., 2013). For 30 days, all the emulsions contain Amesiodendron chinense (Merr.) Hu oil (AC) and coconut oil (CC) were studied for creaming behavior and their percent CI was determined. The percent CI of Amesiodendron chinense (Merr.) Hu oil (AC) and coconut oil (CC) emulsions, which are stored at room temperature, are shown as follows in Table 3. The phase separation of aqueous and oil was observed in formula AC5–AC11 and CC5–CC11 with the HLB value ranging from 9 to 15 within 30 days of study. While the emulsion with HLB 5–7 has no phase separation (AC1–AC3), as shown in Table 3. As in the previous report, the best emulsion formulated was prepared with a surfactant or a mixture of surfactant with the HLB values which is close to the desired HLB value (rHLB) oil phase used in the formula (Orafidiya and Oladimeji 2002, Schmidts et al., 2010). Therefore, in the second series emulsions of Amesiodendron chinense (Merr.) Hu oil and coconut oil with selected HLB ranges from 5–7 were re-formulated with a more precise HLB value to determine the rHLB of Amesiodendron chinense (Merr.) Hu oil. The creaming index can provide indirect information about the extent of aggregation of the droplet occurring in an emulsion; the higher the creaming index, the faster the droplets move, and therefore more droplet aggregation has occurred that ensures the instability of an emulsion (Zarena et al., 2012). Thus, to monitor the most stable formula, the lowest percent CI was found in the formulations AC15 and CC13 with HLB 6.2 and 5.4. Therefore, the rHLB value of the Amesiodendron chinense (Merr.) Hu oil was 6.2, While the rHLB value of coconut oil is 5.4 close to Nattinee and Tanita's study, which reported an rHLB value of coconut oil as 5.8 (Nattinee and Tanita, 2011).

3.2 rHLB and cream formulation

All selected ingredients for formulation development are acceptable grades for cosmeceutical/pharmaceuticals. The chosen emulsifier in the formulation included Tween80 and Span60, while the stiffening agent was also added into the formula composed of stearic acid and glyceryl monostearate. To preserve the best condition; propylparaben was used as a preservative because exhibits a broad spectrum of antimicrobial activity and could prevent the contamination of bacteria that possibly occurs from the natural oil in the formula. Even some studies indicated that paraben is possibly toxic but in a population with normal skin, the paraben is practically non-irritating and non-sensitizing (Soni et al., 2002). While the Amesiodendron chinense (Merr.) Hu oil was used as a skin-conditioning agent in the formulation development. According to the previous reports, the best stable emulsions are formulated with emulsifiers or the mixer of emulsifiers, which possess HLB values close to the required HLB of the oil phase in the formula. Therefore, to the complete emulsification process of two immiscible phases, the suitable emulsifier with an rHLB value is very important to formulate the emulsion system with different proportions of oil and water phases (Schmidts et al., 2010, Alam et al., 2020). Consequently, the HLB of the ingredient needs to be known before proceeding with the emulsification process. Based on the theory that emulsions with an optimum emulsifier concentration will result in less percent CI (Orafidiya and Oladimeji, 2002) thus, the required HLB of Amesiodendron chinense (Merr.) Hu oil was determined in the previous step. The HLB value of the oil and emulsifier was determined according to the reported method. Finally, the rHLB for the oil phase is the sum of the HLB in combination with an emulsifier, as shown in Table 4. To develop the stable system of cream containing Amesiodendron chinense (Merr.) Hu oil, the quantity of emulsifier (Tween80) required to blend with another emulsifier (Span60) to reach the rHLB for the oil phase were calculated and shown in Table 5.

The concept of rHLB was used to design emulsion-based stable formulations. After the homogenization of the oil and aqueous phase, all formulation F1-F5 containing the ingredients at different proportions of stearic acid and emulsifiers (shown in Tab. 1) were able to form the stable emulsion system. The formulations had a suitable combination of emulsifiers with rHLB values that equal the oil phase. The results showed the overall rHLB value contributed by the oil phase of formulations F1-F5 was 10.33, 10.04, 9.71, 9.33, and 9.73, respectively (Tab. 4), and the overall HLB value contributed by the emulsifier combination of formulations F1-F5 was 10.33, 10.06, 9.71, 9.33, and 9.71, respectively (Tab. 5). This suitable in the contributed HLB of the oil phase and rHLB achieved by the combination of an emulsifier is the reason for the stability observed in the formulations, F1-F5. Therefore, it can be inferred that the specific ratio and percentage of an emulsifier blend is unique in the formulation development. If using the emulsifiers with different HLB values, maybe vary in the concentration of emulsifiers to stabilize the same emulsion (Meher et al., 2013).

3.3 Physical stability

Cream containing Amesiodendron chinense (Merr.) Hu oil was stained with a water-soluble dye and analyzed by light microscopy to confirm that the cream was an oil-in-water emulsion because the aqueous color solution was stained outside an oil droplet as shown in Figure 2. The optimized formulation of the cream containing Amesiodendron chinense (Merr.) Hu oil based on the HLB system study was subjected to an accelerated stability test, including centrifugation and heating-cooling cycles. The optimized cream did not exhibit any sign of instability under the given stress conditions. The parameters observed for physical stability were the visual appearance (consistency, color, and odor), pH, viscosity, homogeneity, and phase behavior. These parameters remain unchanged after 6 cycles of the heating-cooling of stability test. The pH values of the formulations were ranged from 4–5 that good as skin pH (Mishra et al., 2014). All formulations of cream containing Amesiodendron chinense (Merr.) Hu oil showed a pH quite similar to the pH of human skin. The viscosity of the cream formulations was found to be between 20 847 ± 1778 and 32 536 ± 1400 cPs; The value of this viscosity is included in the range of quality requirements of cream preparations ranging from 2000–50 000 cPs (Ermawati et al., 2017). The results demonstrated that the formulations were able to retain their integrity without showing any signs of instability (Tab. 6).

Fig. 2 The morphology of cream containing Amesiodendron chinense (Merr.) Hu oil as stained as stained by water-soluble dye and imaged by light microscopy. The scale bar indicates 20 μm.

4 Conclusions

In this study, the rHLB of Amesiodendron chinense (Merr.) Hu oil was found at 6.2 while the rHLB of the coconut oil is 5.4 as in a standard report. The cream containing Amesiodendron chinense (Merr.) Hu oil was developed with 3% of an emulsifier; the combination of Tween80 and Span60. The rHLB of the oil phase used was between 9.33 and 10.33. The blended emulsifier used in the O/W formulations could achieve the rHLB and result in the stability of the formulation system. The rHLB concept was applied to calculate the required quantity of the emulsifier blend for the successful development of stable cream containing Amesiodendron chinense (Merr.) Hu oil. The developed cream represents the favorable physicochemical properties for dermal use. However, efficacy evaluation of the developed cream should be further conducted to elucidate in-vitro and in-vivo studies in further study.

Conflict of interest

The authors declare the absence of any conflicts of interest.

Acknowledgments

We gratefully acknowledge the funding from the Research Institute for Health Science, Individual Research Grants (Grant No. WU-IRG-65-007) and Biomass and Oil Palm Center of Excellence, Walailak University, Tha Sala, Nakhon Si Thammarat 80160, Thailand. Traditional Thai Medical Research and Innovation Center, Faculty of Traditional Thai Medicine, Prince of Songkla University, Thailand.

References

All Tables

All Figures

	Fig. 1 Stable cream containing Amesiodendron chinense (Merr.) Hu oil after 6 cycles of accelerated stability test under 4 and 45 °C.
In the text

	Fig. 2 The morphology of cream containing Amesiodendron chinense (Merr.) Hu oil as stained as stained by water-soluble dye and imaged by light microscopy. The scale bar indicates 20 μm.
In the text