Preparative Method of Mahajak Oil Formula and Formulation Development using Ethosome Technology

Abstract

Mahajak is a polyherbal formula described in the official Thai medicinal textbook, “King Narai’s Medicinal Formulas,” since the Ayutthaya period of the Thai Kingdom. It consists of nine herbal ingredients, including sesame oil, kaffir lime (Citrus hystrix DC.), cumin (Cuminum cyminum L.), fennel (Foeniculum vulgare Miller subsp. var. vulgare), garden cress (Lepidium sativum L.), black cumin (Nigella sativa L.), dill (Anethum graveolens L.), long pepper (Piper retrofractum Vahl.), and camphor. Mahajak is topically applied for skin pruritus, muscle pain, and wounds. The conventional preparative method of Mahajak was a combination of deep-fried and infused aromatization techniques. However, these production process is time-consuming and considered a bottleneck in the supply chain.

In recent years, ultrasonically assisted extraction (UAE) is widely employed for the extraction of biologically active compounds in aromatized vegetable oil. Applying this technique reduces the processing time of herbal extraction and produces the extract with high yielding phytochemical contents. UAE also allows for the production of high-quality oil with fast and cost-effective technology.

The present study aimed to compare the biological activities of Mahajak samples which prepared by 3 methods, including conventional preparative method (CMOF), modified conventional preparative method (MMOF), and UAE method using sonication times of 10, 20, and 30 minutes (UMOF10, UMOF20, and UMOF30). All samples were evaluated for in vitro antioxidant activities using DPPH and ABTS methods, anti-inflammatory activities using nitric oxide assays, anti-bacterial activities for Staphylococcus aureus DMST 8440 and Streptococcus pyogens DMST 17020 using disc diffusion and micro broth dilution methods. The percentage yield and the chemical profiles of all samples were determined using GC-MS and principal component analysis.

The results showed that the antioxidant activities of the MMOF and UAE samples (UMOF10, UMOF20, and UMOF30) were not significantly different from those of the CMOF sample. The IC50 of Mahajak samples, determined using DPPH and ABTS tests, ranged from 90.38-99.42 µg/mL. Ascorbic acid exhibited stronger antioxidant properties than all Mahajak samples. However, the UAE technique was found to be more effective in terms of anti-inflammatory activities (p<0.05), as UMOF10 and UMOF20 showed the most potent anti-inflammatory activity (IC50 of 63.12 ± 4.12 and 62.37 ± 2.82 μg/mL, respectively) compared to CMOF (IC50 = 74.27 ± 3.54 μg/mL). For the antibacterial activity determination, all Mahajak samples exhibit the inhibition zone against S. pyogens DMST 17020 and S. aureus DMST 8440 ranging from 8.53–10.30 mm. The minimum inhibitory concentration (MIC) were observed at the concentration of 15.38 and 18.18 µL/mL, respectively. The minimum bactericidal concentration (MBC) were more than 15.38 and 18.18 µL/mL, respectively.

In addition, the UMOF20 and UMOF30 samples had higher percentages of oil recovery than that of CMOF, MMOF, and UMOF10 (p<0.05). The GC-MS analysis indicated all UAE samples presented similar fingerprints to that of CMOF. Ten major chemical components of Mahajak samples, including betapinene, grandlure III, D-camphor, palmitic acid, cis-9-hexadecenal, octadecanoic acid, oxypeucedanin, gamma-tocopherol, sesamin, and gamma-sitosterol were identified and their contents were higher in the UAE samples.

The study also developed an ethosome of Mahajak using the classical cold method. The ethosomal system used in the formulations contained 2-4% (w/w) phospholipids, 20-40% (w/w) ethanol, and aqueous phase to 100% (w/w). The optimized MOF sample (UMOF20) was formulated into nine formulas (F1-F9) as the ethosomal dosage form. The entrapment efficiency and physicochemical properties of each formulation were evaluated by varying the concentrations of phospholipids and ethanol. Among the nine formulations, UMOF20 ethosome F1 was found to be the optimal formulation with a robust system having a pH of 4.77±0.02, vesicle size of 225.00 ± 3.60 nm, polydispersity index (PDI) of 0.35±0.01, zeta potential of -11.87±0.55, and percent entrapment efficiency (EE) of sesamin and sesamolin at 94.13±0.21 and 99.14±0.07, respectively. .

Stability studies were conducted on the most satisfactory formulation of UMOF20 F1 ethosome at temperatures of 40 ± 2°C for 90 days. The stability of ethosomes can be determined by monitoring the vesicle size, size distribution, zeta potential, pH, and physical characteristics of ethosomes. The vesicle size, PDI, zeta potential, and pH were 239.12 ± 3.65 nm, 0.35 ± 0.01, -11.8 ± 0.32 mV, and 4.56 ± 0.10, respectively. Based on these results, it was concluded that UMOF20 ethosome F1 was stable. However, after the stability test at 60 and 90 days, the vesicle size, PDI, and zeta potential increased, indicating that the ethosome system tended to decrease in stability.The appropriate UMOF20 F1 ethosome provided significantly higher skin permeation parameters, such as Q24 (23.46±0.06 μg/cm2), Jss (1.92±0.08 μg/cm2/h), and Kp (0.05±0.002 X10-3 cm/h), compared to UMOF20 Q24 (0.55±0.02μg/cm2), Jss (undetected), and Kp (undetected). Overall, the study demonstrated the potential of using UMOF20 ethosome F1 as a stable and effective delivery system for the active ingredients in Mahajak formulas.

In conclusion, the results of this study revealed that UAE technique has an impact on Mahajak production. It was found to improve the percentages of oil recovery and demonstrate the anti-oxidant, anti-inflammatory, and anti-bacterial properties of the Mahajak preparation, although it had no significant effect on the chemical components. Additionally, the study developed an ethosomal formulation containing the MOF formula.

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