Evaluation of Topical Anti-Inflammatory Potential of Mentha piperita L. Extract by Formulation of Microemulgel

A topical drug delivery system circumvents first-pass metabolism, gastrointestinal degradation and potential irritation linked with oral intake. This method of drug delivery is optimal for targeting skin-related ailments with localized action. The skin serves as a vital barrier for the human body, distinguishing internal biology from the external environment. Nonetheless, it is susceptible to inflammation. Microemulgel represents a dual drug delivery approach achieved by transforming liquid microemulsion into a semi-solid gel.¹ It’s regarded as a highly promising novel drug delivery system owing to its dual functionality through both emulsion and gel phases. Furthermore, research has demonstrated that combining emulsion with gel enhances its stability. The selection of the microemulsion system was based on its exceptional ability to solubilize and penetrate the skin effectively, while the gel component ensures sustained drug release, leading to prolonged drug residence time. The microemulgel drug delivery system emerges as the optimal choice for treating skin-related ailments, offering enhanced efficacy with reduced drug dosage.²

Treating inflammation often involves the use of plant-based remediesandextracts.Traditionallyrecognizedanti-inflammatory plants include Curcuma longa Linn., Zingiber officinalis Roscoe, Borago officinalis Linn., Oenothera biennis Linn. (Evening primrose), Harpagophytum procumbens (Devil’s claw) and Mentha piperita L. for instance, is indigenous to North India, Pakistan, Afghanistan, Tibet and Nepal. Its primary constituents include menthol (46.32%), menthofuran (13.18%), menthyl acetate (12.10%), menthone (7.42%) and 1,8-cineole (6.06%). It is used in the treatment of inflammation, it is used in the treatment of inflammation, M. piperita is utilized for a multitude of purposes such as addressing Irritable Bowel Syndrome (IBS), indigestion, bed sores, tension headaches, anxiety, insomnia, memory enhancement and various other applications, the research aims to formulate and evaluate a herbal microemulgel infused with M. piperita extract, aiming to scrutinize its in vitro anti-inflammatory attributes.³

While the oral route is commonly preferred for drug administration, both oral and intramuscular injections can lead to severe toxicity and adverse effects in patients. These effects may include gastrointestinal toxicity, renal failure and elevated liver enzymes at high doses. To address the challenges associated with these invasive delivery methods, a novel transdermal drug delivery system has been introduced. This system offers several advantages over oral and intramuscular injections, such as high absorption rates, ease of preparation, thermodynamic stability, avoidance of first-pass metabolism and ease of application. Additionally, its non-invasive nature and local effectiveness make it more patient-compliant compared to conventional delivery systems. The transdermal microemulgel, an enhanced form of emulsion, improves the solubility of poorly water-soluble drugs.⁴

Numerous research endeavors have shed light on the multifaceted biochemical impacts of M. piperita through both in vivo and in vitro investigations. Studies have showcased that various constituents of M. piperita exhibit a spectrum of beneficial effects, including anti-inflammatory, analgesic, immunomodulatory, antispasmodic, antihyperglycemic, anticancer, molluscicide, insecticidal, antiapoptotic, antibacterial, anti-sarcoptic, anxiolytic, anticonvulsant, antiulcer and antigastric properties.^5,6

We can improve drug solubility and skin penetration by creating a microemulgel containing M. piperita. This approach facilitates the development of an optimized formulation. Transforming a basic M. piperita microemulsion into a microemulgel can address challenges encountered with conventional topical drug delivery systems, offering a larger interfacial area for efficient drug absorption.

The M. piperita herb’s raw material was sourced from a nearby local nursery in Nagpur. Authentication of the plant material was carried out with the guidance of Dr. N. M. Dongarwar, associated with the Department of Botany at Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, utilizing a botanical specimen sheet. The authentication number for the M. piperita specimen sheet is 10429.

Following authentication, the M. piperita was dried, powdered and subsequently utilized to extract its essence.

The M. piperita powder underwent maceration with 70% alcohol for 48 hr. Afterward, the residue was subjected to extraction with 70% ethanol using a Soxhlet apparatus. The extracts obtained from both processes were then combined, followed by further concentration of the combined extract.

Different batches of microemulgel were formulated by utilizing diverse gelling agents and adjusting their concentrations. 2% M. piperita extract was incorporated into the emulsion.⁷ The gelling agents were hydrated in water and pH adjustment was carried out by adding triethanolamine. Afterward, the emulsion was incorporated into the gel and thoroughly stirred to form the microemulgel.⁸ Isopropyl myristate, Kolliphor PS 80: Transcutol P was gifted by Gattefosse India Pvt. Ltd., Mumbai Tween 80, Isopropyl Myristate (IPM), n-butanol, Carbopol 940 and triethanolamine were also purchased from Sigma-chemicals Nagpur. All solvents and materials employed were of analytical grade. Throughout the formulation processes, distilled water was consistently utilized.⁹

Pseudo-ternary phase diagrams were generated employing the aqueous titration method for four weight ratios (1:0, 1:1, 1:2 and 2:1) of Labrafac CC Myristate (S_mix). Within each phase diagram, M. piperita oil and the designated S_mix ratio were meticulously combined in varying weight ratios spanning from 1:9 to 9:1, each within separate glass vials. A total of 9 combinations of oil and S_mix (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1) were prepared to accurately delineate the boundaries of phases formed in the phase diagrams. A gradual titration with the aqueous phase was conducted for every combination of oil and S_mix, succeeded by visual scrutiny to distinguish transparent and effortlessly flowable Oil-in-Water (O/W) microemulsions.¹⁰

Phase diagram with S_mix 1:1 showed maximum microemulgel region, so from that, concentrations of oil (3-5%w/w) and S_mix (85-87%w/w) were pooled and several combinations of ME were formed using Box Behnken Design. To each combination of oil and S_mix (1:1), water was added dropwise with stirring at ambient temperature.¹¹

Heating-cooling cycles: The formulations underwent a series of six heating-cooling cycles, alternating between 4°C and 45°C. Each temperature was maintained for a minimum of 48 hr during storage. The formulations were thoroughly examined at both temperatures for signs of significant instability, such as phase separation, precipitation, cracking and similar issues.¹²

Centrifugation test: A centrifugation test was conducted on the formulations at 3,500 rpm for 30 min, with careful observation for any indications of phase separation.

Freeze-thaw cycle: The formulations were subjected to three freeze-thaw cycles, oscillating between temperatures of -21°C and +25°C, with each temperature held constant for at least 48 hr during storage.¹³

MEs were characterized for globule size and zeta potential using Zeta Sizer Litesizer DLS 500 Dadasaheb Balpande College of Pharmacy, Besa Nagpur.

0.17 g of monobasic potassium phosphate were accurately weighed and dissolved in 625 mL of distilled water. Similarly, 0.22 g of sodium hydroxide were weighed and dissolved in 2.8 mL of distilled water. The solutions were combined and diluted to a final volume of 25 mL to prepare a buffer solution with a pH of 6.8. An empty test tube was weighed before the experiment. The drug M. piperita microemulgel was dissolved in 10 mL of pH 6.8 phosphate buffer until precipitation occurred. The solution was then filtered and the filtrate was subjected to evaporation. After heating, the test tube was weighed again. The difference in weights before and after heating was calculated to determine the drug’s solubility in this buffer.^14,15

The pH 6.8 buffer solution was prepared and 10 mL of it were transferred to a separating funnel. To this, 10 mL of methanol were added. Then, 100 mg of M. piperita were accurately weighed and added to the separating funnel. The mixture was vigorously shaken for 30 min and then allowed to stand for 15 min to allow the layers to separate. Both layers were carefully extracted and filtered into separate beakers. The buffer extract was diluted with distilled water, while the methanol extract was diluted with methanol. Subsequently, both extracts were analyzed using a spectrophotometer.¹⁶

The physical appearance evaluation included analyzing color, homogeneity, consistency and overall appearance. Color assessment was conducted visually, while homogeneity was determined by rubbing the microemulgel between fingers. The appearance of the microemulgel was evaluated visually. Furthermore, the consistency of the microemulgel was tested by applying it to the skin.¹⁷

To assess spreadability, the microemulgel was placed between two petri plates and the diameter of the spread microemulgel ring was measured. A gram of microemulgel was weighed and deposited onto a petri plate, with another petri plate positioned on top. A weight of 50 g was applied to the upper petri plate for 60 sec. After this duration, the diameters of the circles formed by the spread microemulgel were measured three times and the average reading was calculated. This average was then utilized in the following formula.¹⁸

Where, S=Spreadability, M=Mass, L=Diameter and T=Time.

The pH of the formulated microemulgel batches was assessed using a digital pH meter. A quantity of 0.5 g from each batch was dissolved in 10 mL of distilled water. The electrode was then immersed in the solution to measure the pH.¹⁹

The stability studies of the microemulgel included storing samples under different conditions: 5°C, 25°C/60% RH, 30°C/65% RH and 40°C/75% RH, for 3 months. Assessments were carried out at 15-day intervals, examining the appearance, pH, viscosity and spreadability of the samples.²⁰

The droplet size of the microemulgel was examined using a scanning electron microscope. A calibrated micrometer slide with a droplet of microemulsion was observed under the eyepiece and the droplet size was measured and determined.

The viscosity of the microemulgel preparation was measured using the Brookfield Viscometer (DV-E Brookfield Viscometer Model-LVDVE). This viscometer consists of a stationary cup and a rotating spindle. The rotating spindle was immersed in the sample of microemulsion gel. Due to the higher viscosity of the gel preparation, a smaller spindle with a reduced diameter and surface area was employed. The spindle was rotated within the gel preparation until a consistent reading was achieved on the viscometer dial. This procedure was repeated three times to ensure the reproducibility of the results.²¹

The electrical conductivity test was conducted to ascertain whether the microemulgel was an oil-in-water or water-in-oil microemulsion. The conductivity (σ) of the formulated sample was determined using a conductivity meter, which involved using a probe and meter. Voltage was applied across two electrodes within a probe immersed in the microemulsion. The decrease in voltage, attributed to the microemulsion’s resistance, was utilized to calculate the conductivity.

Utilizing a DSC-60 calorimeter by SHIMADZU, samples underwent heating under liquid nitrogen from room temperature to 400°C at a rate of 10°C/min. Approximately 5 mg of the sample in a sealed aluminium pan was subjected to a DSC scan. Pure M. piperita, the vehicle and the microemulgel were individually tested and the resulting thermograms were compared.

To ascertain the type of emulsion, whether it was Oil-in-Water (O/W) or Water-in-Oil (W/O), 10 μL of methylene blue, a water-soluble dye, was introduced into the emulsion. This addition aimed to observe whether the dye dispersed uniformly throughout the emulsion or formed clusters.

The functional groups present in the preparation were identified through FT-IR scanning microscopy. As IR rays passed through the sample, the absorbed radiations were transformed into vibrations or stretching, revealing spectrum signals ranging from 4000 cm^-1 to 400 cm^-1. These signals provided insights into the functional groups and the types of bonds present, forming a molecular fingerprint of the sample

In testing the release rate of M. piperita from various microemulgel formulations, a Franz diffusion cell with a rabbit membrane was employed. The abdominal skin of the rabbit was shaved and excised and the integrity of the subcutaneous fat was assessed. The membrane was positioned with the mucous membrane facing upward and the epidermis downward, then secured the Franz diffusion cell facilitated the transfer of substances The transfer took place between the donor and receiver compartments of the cell. The cell was loaded with 9 mL of phosphate buffer at a pH of 6.8 and throughout the experiment, the receptor fluid was continuously stirred using externally driven magnetic bars at a speed of 300 rpm. M. piperita microemulgel was introduced into the donor chamber at intervals ranging from 0.5 to 48 hr, 0.5 mL samples were extracted from the receiver chamber for spectrophotometric analysis. These samples were promptly replaced with an equivalent volume of buffer solution. UV-visible spectrophotometry at 354 nm was employed for the analysis of the samples.²²

A 1 mL aliquot of the emulsion was mixed with 9 mL of buffer solution at pH 6.8 and stirred for 30 min. The mixture was left at room temperature for 24 hr before undergoing an additional 30 min of stirring. Subsequently, the solution underwent centrifugation at 4000 rpm for 30 min to yield a clear supernatant. From this supernatant, 1 mL was extracted and combined with 9 mL of buffer solution. This resulting solution was then analyzed using a UV spectrophotometer at 354 nm and the absorbance was recorded. The concentration of M. piperita was determined by referencing a standard calibrated curve of the drug.

To evaluate the anti-inflammatory potential of the M. piperita extract microemulgel, the HRBC membrane stabilization method was utilized. Around 2-3 mL of blood was obtained from a healthy donor and combined in equal proportions with Alsever’s solution. Iso-saline was then added, followed by centrifugation for 5 min to isolate the HRBC suspension. Equal amounts of the sample were added to the HRBC suspension, with concentrations of 100, 200 and 300 μg/mL prepared. The mixtures were then incubated at 37°C for 30 min and subsequently centrifuged for 5 min. Negative controls included Alsever’s solution and blood, while aspirin served as the standard. UV spectroscopy at a wavelength of 560 nm was utilized to analyze the supernatant for estimation.²³

A volume of approximately 0.02 mL of the sample was measured and combined with 0.2 mL of 1% Bovine Albumin. To this mixture, 4.78 mL of PBS (Phosphate Buffer Saline) was added and thoroughly mixed, followed by an incubation period of 15 min. Afterward, the mixture was heated in a water bath at 70°C for 5 min and then allowed to cool down. UV spectrophotometry was employed to measure the absorbance at a wavelength of 660 nm. Phosphate buffer served as the control, while A was utilized as the standard. Next, the percentage of inhibition of protein denaturation was determined through calculation.²⁴

M. piperita was harvested and air-dried before being crushed into coarse powder. One kilogram of this powder was then used for extraction with petroleum ether, employing a Soxhlet apparatus at temperatures ranging from 50°C to 60°C for 72 hr. Following the petroleum ether extraction, the remaining material underwent a second extraction with 95% ethanol in a Soxhlet apparatus, at temperatures between 60°C and 70°C for up to 72 hr. The concentrated alcoholic extract obtained was stored in a desiccator, while the residue was retained for further analysis.

Surfactants and co-surfactants were evaluated based on their emulsification ability and percentage transmittance. For the surfactant screening, 300 mg each of oil and surfactant were heated at 40-45°C for 30 sec. Subsequently, a 50 mg mixture was diluted to 50 mL with distilled water and left to stand for 2 hr. The percentage transmittance was then measured at 688 nm. Labrafac CC was chosen as the surfactant as it gave higher transmittance.

For the screening of Cosurfactant, a mixture of co-surfactant (100 mg), selected surfactant (200 mg) and oil (300 mg) was heated at 40-45°C for 30 sec and proceeded similarly as for the screening of surfactant. Isopropyl myristate was found to be better (Table 1).

Pseudo-ternary phase diagrams were generated utilizing the aqueous titration technique for four different weight ratios (1:0, 1:1, 1:2 and 2:1) of Kolliphor PS 80:Transcutol P (S_mix) In every phase diagram M. piperita oil and a specific S_mix ratio were meticulously combined in various weight ratios ranging from 1:9 to 9:1. A total of 9 different combinations of oil and S mix were prepared, including 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 were made to Precisely outlining To delineate the boundaries of the phases formed in the phase diagrams, a gradual titration with the aqueous phase was performed for each oil and S_mix combination. Visual inspection was subsequently employed to identify transparent and effortlessly flowable Oil-in-Water (O/W) microemulsions (Figure 1).

M. piperita oil-3%-5%, Water- 52%-55%, S_mix-45%-40% (Kolliphor PS 80: Transcutol P (1:1) (Table 2).

After subjecting the formulations to a series of stability tests, including heating-cooling cycles, centrifugation tests and freeze-thaw cycles, no cracking or phase separation was observed in the microemulgel, indicating its favourable thermodynamic stability.

This optimum size of M. piperita microemulgel showed that surfactant, co-surfactant and oil phases were highly compatible with each other as the surface tension increases the globule size decreases (Figure 2).

Figure 1:
Ternary phase diagram of concentration for S_mix 1:1.

The solubility of M. piperita microemulgel was assessed across various oils (IPM, Propylene glycol, benzyl alcohol and oleic acid), surfactants (Tween 60, Tween 80 and Span 20) and cosurfactants (n-butanol, polyethylene glycol and diethylene glycol). Among these, the highest solubility was observed in IPM oil, Tween 80 surfactant and n-butanol co-surfactant compared to the other oils and surfactants. Microemulsions formed with IPM, Tween 80 and n-butanol was noted to be clear and stable due to the superior compatibility of these surfactant-co-surfactant pairs with the oil.

The microemulsion of M. piperita exhibited a transparent appearance with a light yellow and emitted an aroma reminiscent of alcohol. Notably, the microemulsion remained stable without undergoing phase separation upon the addition of gel to the formulation, confirming its stability.

Using a drag and slip device, spreadability was assessed. The device was made up of a wooden block with a glass slide installed over it and a pulley fastened to one edge of the block. 2 g of microemulgel was placed over the glass slide over the wooden block and another glass slide with the same measurements was placed on top of it. The weight of around 80 g was suspended by attaching a thread to the upper glass slide with the aid of a hook and passed above the pulley. The time in sec taken by the slide to cover 7.5 cm on the fixed glass slide was recorded. The spreadability was then calculated by using the following formula:

Figure 2:
Measurement of globule size and zeta potential.

Figure 3:
Particle Size of M. piperita microemulgel (117 nm).

Where, M=weight tied to upper glass slide, L=length of glass slides and T=time taken to separate the glass slides from each other.

One-gram microemulgel was placed between the plate and cone (no.3) of the viscometer (Brookfield viscometer Cap 2000+) and viscosity was measured at 10 rpm for 30 sec. Viscosity was also measured at increasing shear rate. Measurement was done for 30 sec at each rate of shear.

The viscosity of the M. piperita microemulgel measured at 86.7±0.01 cps. The formulation displayed Newtonian flow behaviour, signified by its viscosity showing minimal variation when subjected to external forces such as stirring. This suggests that the microemulgel could endure slight stress and maintain consistent viscosity during both handling and storage (Table 3).

The pH of the microemulgel was determined to be 6.2, indicating that the elevated S/CO ratio contributed to the increase in pH of the microemulgel.

The stability study suggested that the formulation was physically and chemically stable when stored at 5°C, 25°C with 60% Relative Humidity (RH), 30°C with 65% RH and 40°C with 75% RH over a period of 3 months.

The size of the droplets was 117 nm. This optimum size of M. piperita microemulgel showed that surfactant, co-surfactant and oil phases were highly compatible with each other (Figure 3).

The conductivity of the M. piperita microemulgel registered at 1.4 μS/cm, confirming its classification as Oil in Water (O/W) microemulgel. Notably, pure water typically exhibits a conductivity. The elevated conductivity observed in the microemulsion was attributed to the presence of dissolved salts and oils. As the amount of dissolved salts and oils in the microemulsion increased, so did its conductivity. The higher conductivity of the M. piperita microemulgel stemmed from the enhanced solubility of oil, surfactant, co-surfactant and water within the formulation.

M. piperita exhibited a sharp endothermic peak at around 204.73°C indicating its pure crystalline cubic form.

Since the dye dispersed evenly within the microemulgel, it was inferred that the continuous phase consisted of water. Consequently, the M. piperita microemulgel was classified as Oil in Water (o/w) type of microemulsion.

The distinctive peaks or FT-IR bands of pure M. piperita were identified at 2955.18 cm^-1 (C-H) and 1165.99 cm^-1 (C=O), as depicted in (Figure 4). These quality peaks of M. piperita were also evident in the M. piperita microemulgel, without any additional auxiliary peaks or significant peak shifts noted. This absence of chemical deterioration or incompatibilities was further confirmed through DSC analysis, which revealed no incompatibilities between M. piperita and the various excipients used. Both M. piperita and the excipients exhibited peaks in the microemulgel thermograms, affirming their compatibility.

The maximum release of M. piperita microemulgel was 94% at 48 hr which indicated that M. piperita microemulgel has good topical release properties (Table 4).

The distinct peaks or FT-IR bands characteristic of pure M. piperita were detected at 2955.18 cm^-1 (C-H) and 1165.99 cm^-1 (C=O). These key peaks of M. piperita were similarly observed in the M. piperita microemulgel, with no additional auxiliary peaks or significant shifts noted. This absence of chemical deterioration or incompatibilities was further confirmed by DSC analysis, which demonstrated no incompatibilities between M. piperita and the various excipients utilized. Both M. piperita and the excipients displayed peaks in the microemulgel thermograms, confirming their compatibility.

The M. piperita microemulgel exhibited a drug content of 94.35%. This significant drug content indicates that the drug is highly soluble and exhibits compatibility with the excipients used in the formulation.

The anti-inflammatory activity of the extract and the optimized formulation was evaluated using two methods: the HRBC membrane stabilization method and the protein denaturation method (Table 5). Both the extract and the formulated microemulgel demonstrated significant anti-inflammatory activity. The protein denaturation caused by the extract was also examined and presented (Table 6).

Figure 4:
FT-IR Measurement of Percentage Transmittance (%T), Corresponding to M. piperita Peak at 1509.18 cm^-1.

In this study, a microemulgel was developed using an herbal extract of M. piperita to target inflammation. Through experimentation with various surfactants and co-surfactants in preformulation studies revels that M. piperita extract exhibited superior solubility in IPM oil and Tween 80 surfactant, with n-butanol acting as the cosurfactant, surpassing other alternatives. The combination of IPM, Tween 80 and n-butanol yielded a microemulgel that was transparent and remained stable due to their excellent compatibility, which outperformed other surfactant-cosurfactant-oil combinations tested. The formulation demonstrated both physical and thermodynamic stability even under accelerated conditions. The study of Pseudo ternary phase diagram demonstrates that S_mix ratio 1:1 provide more stable microemulgel formulation as compared with other S_mix ratios. In thermodynamic stability studies, it was observed that there no any separation as well as no cracking of microemulgel. Top of FormBottom of FormThe microemulsion containing M. piperita appeared clear and light yellow, emitting a fragrance reminiscent of alcohol. The spreadability of the formulation varied between 27.21±0.98 and 37.51±0.28. Droplet size averaged approximately 117 nm, while viscosity measured at 86.7±0.01 cps. The dye dispersed uniformly throughout the microemulgel.

The microemulgel containing M. piperita had a pH of 6.2, indicating that the higher ratio of surfactant to cosurfactant contributed to its elevated pH compared to pure water, which typically exhibits a conductivity of 1.4 μS/cm. In the FT-IR spectra, peaks were observed at 2955.18 cm^-1 (C-H) and 1165.99 cm^-1 (C=O). The maximum release of M. piperita from the microemulgel reached 94% within 48 hr, demonstrating excellent topical release properties. The M. piperita microemulgel showed a drug content of 94.35%. Both the extract and the formulated microemulgel displayed promising anti-inflammatory activity by preventing cell lysis and inhibiting protein denaturation.

In this study, advanced methods is adapted to formulate the microemulgel, this technique can be employed to enhance the solubility and skin permeability. An effective microemulgel was developed using 1% Kolliphor PS 80 as a gelling agent, resulting in sustained release properties and prolonged residence time. The M. piperita microemulgel exhibited a remarkable 94.35% drug content, indicating high solubility and compatibility of the drug with the excipients. Permeability studies revealed that the M. piperita microemulgel achieved 94% permeability within 48 hr, showcasing enhanced drug permeability facilitated by the microemulsion-based gel system. These findings suggest that this formulation holds promise as a topical delivery vehicle for M. piperita. Moreover, the formulated microemulgel demonstrated significant anti-inflammatory activity.

The author expresses deep gratitude to the management and the Department of Pharmaceutical Sciences at Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, for their invaluable support in facilitating the successful completion of the research work.