Development of microemulsion containing thai herbal remedy extract for treatment of urticaria

Abstract

Herbal medicine can be used as an alternative treatment to alleviate allergy symptoms in individuals with urticaria. The herbal remedy comprised four plant components, including Allium ascalonicum, Acanthus ilicifolius, Bambusa blumeana, and Rhinacanthus nasutus, in equal amounts. It was administered to impacted areas of the skin affected by allergies. Nevertheless, the disadvantages of herbal products include their limited ability to penetrate the skin. Microemulsion (ME) is a topical medication delivery device that enhances drug absorption into the skin. This work aims to develop and optimize a ME containing an herbal remedy extract. The construction methods utilized were pseudo-ternary phase diagrams and mixture design using system engineering software. Data were calculated and reported as means ± standard. Statistical significance was indicated when P < 0.05. The optimal herbal ME consisted of 4%–5% isopropyl myristate, 35%–45% Smix, and 46%–58% water. The stability studies demonstrated consistent physical properties and a low level of viscosity. The pH, particle size, polydispersity index, and zeta potential readings have remained stable after storage under nine heating and cooling cycles. The ME exhibited sustained release of rhinacanthin-C, with a steady release rate of 10.34% ± 0.03% from 0.5 h to 20.21% ± 0.11% at 8 h. The Kae-Lom-Pid proves to be suited for MEs and can serve as a model for pharmaceutical development.

INTRODUCTION

Hives, also known as urticaria, are dermatological conditions resulting from an allergic response. Allergens stimulate histamine and allergic cytokines, leading to rash, itching, redness, swelling, and lesions.[1] Topical medications, such as antihistamines and steroid creams, are commonly used to treat urticaria.[2] Nevertheless, these medications can cause adverse reactions, especially steroids.[3] Herbal and alternative medicine are additional options for relieving allergy symptoms in persons with urticaria. Furthermore, it exhibits a significant potential for effectiveness and few adverse effects.[4] The Thai traditional scripture has references to herbal formulations for urticaria treatment. The formula comprised four plans: Allium ascalonicum L., Acanthus ilicifolius L., Bambusa blumeana Schult. f., and Rhinacanthus nasutus (L.) Kurz, in equal proportions. This formulation has been called Kae-Lom-Pid (KLP) in Thai. The materials were boiled and applied to wash the skin affected by allergies.[5]

Earlier research demonstrated that this preparation’s plant components had anti-allergic properties. The acetonitrile extract of A. ascalonicum has demonstrated the ability to block mast cell granule release and alleviate allergic rhinitis symptoms.[6] The alcoholic solution obtained from A. ilicifolius suppressed allergy responses in an animal model.[7] It has been observed that the ethylacetate of R. nasutus has inhibitory effects on ß-hexosaminidase, TNF-α, and IL-4 in mast cells, with active chemicals rhinacanthin-C, rhinacanthin-D, and rhinacanthin-N.[8] Previous studies have indicated that using organic solvents for extraction produces extracts with strong anti-allergy properties. However, it was recommended to use water to produce this herbal remedy.[5] The solvent choice affects active compound concentration, with rhinacanthin showing reduced solubility in water-based extractions.[9] Our research primarily focuses on applying ethanolic extraction for a Thai herbal formulation extract. We also macerate the extract to decrease heat sensitivity of active chemicals. Moreover, the product development process can enhance the product’s effectiveness. Initially, this herbal composition was used as a cleaning solution but had limited contact duration and low skin penetration. Topical drug delivery systems, such as microemulsion (ME) and nanoemulsion, are utilized to improve skin absorption and prolong drug half-life.[10] The ME is a drug delivery technique characterized by particle sizes ranging from 20 to 200 nm, which allows for effective penetration into the dermis layer of the skin. Nevertheless, the formulation of ME must consider the chemical composition and the proportions of its constituents, such as the oil phase, surfactant, and cosurfactant.[11] This study aimed to develop a ME product using a herbal formulation extract, test its physical properties, and assess skin permeability using ex vivo methods. Our research produced a stable ME that effectively permeates the skin.

MATERIALS AND METHODS

Materials

The plant materials were collected from the central and eastern regions of Thailand. All plants were identified by comparing them with authenticated voucher specimens: A. ascalonicum (BK No.084791), A. ilicifolius (BK No.084901), R. nasutus (BK No.084900), and B. blumeana (TTM No.0005495) Several chemical reagents such as ethanol, acetonitrile, methanol (Bangkok, Thailand), and phosphoric acid (Tokyo, Japan). Isopropyl myristate (IPM), medium chain triglycerides (MCT), isododecane, tween-20, polyglyceryl-4 caprate, PEG-6 caprylic/capric glycerides (PEG-6), 1,3-propanediol, isopentyldiol, and 1,3-butylene glycol (Bangkok, Thailand). Column Phenomenex Luna 5 µm C18 100 Å, 250 mm × 4.6 mm.

Methods

Extraction of Kae-Lom-Pid formulation

The plant materials were cleansed and aired. Subsequently, the ingredients were broken down and combined in equal proportions to produce a KLP formulation. Then, maceration was performed in 95% ethanol for 3 days. The residue underwent maceration twice more. The filtrate was concentrated using an evaporator at 45°C until a consistent weight was achieved.

Analysis of chemical content in Kae-Lom-Pid formulation extract

High-performance liquid chromatography (HPLC) analyzed the chemical composition of the KLP formulation extract. A 10 μl of each sample was injected into a column. The mobile phases were water with acetonitrile (A) and 0.1% phosphoric acid (B). The gradient profiles of A: B were as follows: 75:25 for 35 min, 75:25–71.4:28.6 in 3 min, 71.4:28.6–95:5 in 2 min, and 95:5–75:25 in 3 min. Finally, maintain the 75:25 (A/B) ratio for 3 min. The flow rate was 1.0 ml/min at room temperature and 254 nm. Rhinacanthin-C was the marker.

Screening of microemulsion formulation components

A study was performed to determine the solubility of a KLP formulation extract (200 mg) in various substances (500 mg). The oil vehicles were IPM, isododecane, and MCT. The surfactant vehicles were Tween-20, polyglyceryl-4 caprate, and PEG-6. The co-surfactant were isopentyldiol, 1,3-propanediol, and 1,3-butylereglycol. The mixtures were stirred at 30°C ± 2°C for 48 h and then centrifuged at 10,000 rpm for 15 min. The concentration of active compounds was measured using HPLC.

Construction of pseudo-ternary phase diagrams

Pseudo-ternary phase diagrams were constructed using aqueous phase titration to identify the appropriate components and their concentration for a wide ME area. Initially, a mixture comprising of Smix was prepared with weight ratios of 2:1, 1:1, and 1:2. Each Smix was mixed with oil in different weight proportions. Deionized water was added by titration and vigorously stirred until equilibrium for the self-emulsifying ME. The transition from transparency to cloudiness indicated the ME region. The proportions of each constituent were quantified using Origin Pro 2021 (OriginLab Corporation, Northampton, MA).

Optimization of microemulsion using D-optimal mixture design

The D-optimal mixture design optimized the combination of water, oil, and Smix for a ME with optimal droplet sizes and characteristics. The experimental design was created using a limited ME region obtained from pseudo-ternary phase diagrams using Design-Expert® software (Stat-Ease Inc., Minneapolis, USA). The design built a batch matrix with three independent variables, measuring responses of droplet size and transmittance. Data were analyzed by fitting it with suitable polynomial equations. The significance of the models was using the ANOVA test. Response surface, contour, and overlay plots visually represented the variables. Formulations desired criteria into an attractiveness index ranging from 0 to 1, with values close to 1 being acceptable.

Physical and chemical properties of microemulsion of Kae-Lom-Pid formulation extract

The ME was visually examined for phase separation, and its pH level was assessed. In addition, droplet size, zeta potential, and polydispersity index (PDI) were analyzed using photon correlation spectroscopy and laser Doppler electrophoresis using the Zetasizer® Pro (Malvern Panalytical, Malvern, UK). Viscosity was determined using a Brookfield viscometer (Brookfield Engineering Laboratories, Inc. located in Middleboro, MA). All experiments were conducted three times.

Stability study of microemulsion using heating-cooling method

The ME with the herbal extract was stored in a glass vial at 4°C and 45°C for 24 h and each, repeating for 9 cycles.[12] Physical characteristics including visual appearance, droplet size, zeta potential, viscosity and pH, were evaluated at cycles 0, 3, 6, and 9.

In vitro drug permeation study using Franz diffusion cell

An in vitro skin permeation study was performed using a Franz diffusion cell with Strat-M® membrane.[13] The receptor chamber contained 12 ml of 40% ethanol in PBS, maintained at 32°C ± 1°C and 600 rpm. The ME 1 g was placed into a donor compartment at intervals of 15, 30, 45, 60, 120, 180, 240, 300, 360, 420, and 480 min. A 500 μl sample was taken from the receptor chamber and replaced with a fresh medium. The concentration of rhinacanthin-C in the samples was measured using HPLC.

RESULTS AND DISCUSSION

High-performance liquid chromatography analysis of rhinacanthin of Kae-Lom-Pid formulation extract

The peaks for rhinacanthin-C and rhinacanthin-N had retention times of 19.741 min and 20.551 min, respectively. The KLP extract had a concentration of 1.65% ± 0.05% w/w of rhinacanthin-C and 0.20% ± 0.00% w/w of rhinacanthin-N. Thus, rhinacanthin-C was selected as an active component and an analytical marker for the extract.

Selection of the oil phase, surfactant, and co-surfactant

The highest solubility of rhinacanthin-C was found in IPM (86.86% ± 1.40%), followed by MCT (76.62% ± 0.85%), and isododecane (39.05% ± 1.04%). Among surfactants, tween-20 demonstrated a higher solubility (74.63% ± 2.17%) compared to polyglyceryl-4 caprate (35.75% ± 0.98%) and PEG-6 (12.78% ± 0.33%). The co-surfactants showed the highest solubility in 1,3-propanediol (35.91% ± 0.78%), followed by isopentyldiol (34.50% ± 0.70%) and 1,3-butylene glycol (30.12% ± 0.63%).

This study examined the process of optimizing the components of a KLP ME. The oil phase used was IPM, which exhibited high solubility for rhinacanthin-C, was nontoxic, and easily permeated through a low-polarity structure.[14] In addition, ME was used to decrease the particle size using IPM as a nonaqueous medium for the formulation of reverse micelle.[15] Thus, IPM is appropriate for extracting active compounds with low polarity.[16] Tween-20 and polyglyceryl-4 caprate are surfactants with elevated hydrophilic–lipophilic balance values. They are proficient in dispersing low quantities of oils in aqueous solutions.[17] Isopentyldiol and 1,3-propanediol were the most effective co-surfactants, safe for skin, and commonly used in cosmetics and topical products.[18]

Pseudoternary phase diagram construction

The pseudo-ternary phase diagram indicates that using IPM, tween-20, and a co-surfactant mixture of isopentyldiol and 1,3-propanediol with water creates a small water-in-oil ME region. In contrast, using polyglyceryl-4 caprate instead of Tween-20 results in a larger oil-in-water ME [Figure 1a-f]. Tween-20 has a higher HBL value, requires less water for dissolution, resulting in a smaller ME area.[17,19] Polyglyceryl-4 caprate was chosen as a surfactant for this study because of its extensive ME area and suitability for sensitive skin.[20]

Figure 1.

Pseudo-ternary phase diagrams. The Smix ratios used in the diagrams are as follows: (a and d) 2:1, (b and e) 1:1, and (c and f) 1:2

The diagrams of IPM and Smix (polyglyceryl-4 caprate mixture with isopentyldiol:1,3-propanediol) ratio of 1:2 and water showed the largest oil-in-water emulsion area, while the surfactant ratio was at its lowest [Figure 1f]. Thus, these specific ingredients were selected to maximize ME efficiency using a D-optimal mixture design. Isopentyldiol and 1,3-propanediol are co-surfactants enhance the water phase volume stability and fluidity of MEs.[21,22]

Optimization of microemulsion formulation

The D-optimal mixture design was used to optimize the KLP ME formulation. IPM varied from 3% to 5%, Smix from 35% to 45%, and the water from 46% to 58%, with KLP extract fixed at 1%, ethoxydiglycol, disodium EDTA, phenoxyethanol, and 2-Amino-2-methyl-1-propanol at 3% of the total volume. The results indicate droplet diameters of the 19 formulations ranged from 118.8 to 245.7 nm, while the transmittance ranged from 96.38% to 99.31%. All droplet sizes are within the criteria for ME.[10]

A high-quality ME has to possess a large water phase ratio and minimal viscosity to enhance permeability and decrease the surfactant ratio.[23,24] The responses from nineteen runs were analyzed using response surface methodology with Design Expert software, revealing that the most suitable model was linear. To determine the optimal regression model, we analyzed all run results for high adjusted R² and predicted R² values, indicating excellent predictive capability. The regression model in the sequential model should have a significant P < 0.05. Similarly, the lack of fit should have an insignificant P > 0.05.[25] Our findings indicate that the fitted model to the response variable, droplet size, and transmittance as shown in Table 1.

Table 1.

The optimization of the regression model

Response		Fitted model		Sequential P		Lack of fit P		R 2		Adjusted R2		Predicted R2		CV (%)
Droplet size		Special cubic		0.0002		0.1481		0.9986		0.9978		0.9962		1.0600
Transmittance		Special quartic		0.0029		0.0730		0.9856		0.9741		0.9472		0.1367

The selection was based on the high adjusted R² and predicted R² values. In addition, the insignificance of lack of fit and the low coefficient of variation indicate high precision for repetition.[26] The polynomial regression equation for fitting experimental data is provided below.

Droplet size = 567.69A + 167.97B − 244.67C − 647.33AB − 1288.86AC − 67.45BC + 420.27ABC

Tramsmittance = 62.87A + 99.24B + 96.50C + 37.12AB + 50.82AC + 2.48BC − 29.35A²BC + 52.73AB²C − 54.86ABC²

The results displayed the interaction between the formulation and dependent variables through the regression model generated a contour plot and a 3D surface plot illustrating the relationship between droplet size and transmittance [Figure 2]. The findings indicated that increased oil and water phases significantly reduced droplet size [Figure 2a and b]. The higher oil phase ratio inhibits ME particle fusion and fission, while the water phase inhibits the hydrolysis reaction of ME, leading to smaller particles.[25,27] Although surfactants and co-surfactants did not affect droplet size, they reduced the surface tension of the oil and water phases.[28] Conversely, low transmittance was due to a low Smix ratio and a high water phase ratio [Figure 2c and d]. Surfactant and co-surfactant enhanced micelles formation and reduced the size of droplets.[29] Lower proportions of these result in less blending of water and oil phases, causing larger particles and increased turbidity.

Figure 2.

A contour plot and a 3D surface plot illustrating the impact of different ratios of microemulsion components on (a and b) the size of droplets and (c and d) the transmittance

An overlay plot analysis identified the optimal formulation with the lowest Smix ratio, smallest droplet size (119.5 ± 1.8 nm), and highest transmittance (97.95% ± 0.13%). The ideal composition was 5% IPM, 35% Smix (1:2), and 56% water, with a desirability of 81.1% [Figure 3]. Testing of the predicted composition showed a droplet size of 119.9 ± 2.1 nm and a transmittance of 97.82% ± 0.19%, with errors of 0.33% and 0.13%, respectively.

Figure 3.

The information is shown by (a) an overlay plot and (b) desirability plot

Stability testing of Kae-Lom-Pid microemulsion

Following nine heating-cooling cycles, the KLP MEs remained translucent and isotropic. No separation occurred in this investigation, which typically results from the combination of oil and water forming large particles and separations.[30] The pH of the KLP ME ranged from 5.17 ± 0.06 to 6.12 ± 0.06 within the acceptable pH range for topical treatments.[31] No significant changes were observed in droplet size, PDI, or zeta potential. Moreover, they exceeded the established parameters for ME. The droplet size values are smaller than the accepted standard of 200 nm.[32] Temperature variations affected both the pH level and the droplet size.[33] Following the heating-cooling cycles, parameters were altered. The PDI was below 0.3, indicating stability, uniformity, and appropriate droplet size.[34] The zeta potential ranged within the conventional threshold of –30 and 30 mV, due to the anionic surfactant causing a negative charge.[35,36] Moreover, the surfactant was dissolved in water and subjected to a low pH, increasing PDI.[37,38] The viscosity remained low and within the standard criterion for ME.[39] The fusion of hydrophilic, surfactant, and co-surfactant can increase particle size and viscosity.[40,41] Nevertheless, the KLP ME exhibited droplet size and viscosity that met the conventional standards, enhancing skin permeability. The value of stability as shown in Table 2.

Table 2.

Stability testing of the Kae-Lom-Pid microemulsion and base microemulsion under heating-cooling cycle (mean±standard error of mean)

Sample		Cycle		pH		Droplet size (nm)		PDI		Zeta potential (mV)		Viscosity (cP)
KLP ME		0		6.12±0.06		135.40±3.48		0.21±0.02		−44.53±1.28		11.55±0.09
		3		5.57±0.08*		136.50±6.21		0.20±0.02		−45.52±2.15		10.84±0.36
		6		5.34±0.04*		143.00±8.21		0.20±0.01		−42.93±1.01		10.68±0.29
		9		5.17±0.06*		142.40±7.83		0.20±0.02		−47.77±1.40		11.52±0.16
BaseME		0		6.35±0.27		107.30±6.67		0.20±0.01		−30.32±2.07		9.95±0.22
		3		6.12±0.26*		116.60±4.55*		0.20±0.01		−31.96±2.02		9.88±0.73
		6		5.73±0.31*		129.70±1.65*		0.20±0.01		−35.10±0.99		9.83±0.22
		9		5.46±0.36*		128.30±1.76*		0.20±0.01		−33.82±4.71		10.16±0.54

*P<0.05 compared to 0 cycle. PDI: Polydispersity index, KLP: Kae-Lom-Pid, SEM: Standard error of mean

Skin permeation of active compound in the Kae-Lom-Pid microemulsion

The findings indicated that the rhinacanthin-C was released after 0.5 h with a cumulative rate of 10.34% ± 0.03%. The KLP ME enhanced rhinacanthin release consistently over 8 h, reaching a cumulative rate of 20.21% ± 0.11% [Figure 4]. The ME’s diffusion rate was higher than that of cream or gel, which took 1 h to diffuse.[42,43,44] It is small and well-suited for permeating through the skin barrier without causing any injury to the skin.[22]

Figure 4.

Percentage cumulative of rhinacanthin-C releasing from Kae-Lom-Pid microemulsion

Our results can be concluded that the optimal KLP ME constituents were derived from a pseudo-ternary phase diagram and software analysis. Polyglyceryl-4 caprate was the most effective surfactant. The optimal composition included 5% IPM, 56% water, 35% Smix, 1% KLP extract, and 3% mixture of ethoxydiglycol, EDTA, phenoxyethanol, and 2-Amino-2-methyl-1-propanol. This formulation had a particle size smaller than 150 nm, 97.95% transmittance, and met ME standards. The KLP ME effectively released rhinacanthin-C, which is the active component, reaching 20.21% ± 0.06% after 8 h. It also remained stable during the heating-cooling experiment, indicating its potential longevity. However, the biological activity of the KLP emulsion should be assessed to determine its efficacy in future applications.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.