Plant extracts and essential oils are used in many fields, including the medical, chemical, pharmaceutical, and cosmetic industries [1]. Botanicals of plant origin, such as Trifolium pratense L. flowers, Glycyrrhiza glabra L. roots, and Myristica fragrans Houtt. essential oils are generally considered safe due to their natural source. It can prevent various degenerative diseases and possess many pharmacological activities such as antibacterial, anti-inflammatory, antiviral, antitumor, etc [2]-[4]. Antimicrobials of plant origin are promising options for treating infections phenolic compounds in plants can act synergistically and enhance valuable effects. These effects can be beneficiary in pharmaceutical forms.
The aim was to study the antimicrobial effects of two extracts (Trifolium pratense L. flowers, Glycyrrhiza glabra L. roots) and essential oil (Myristica fragrans Houtt.) and incorporate them together to create a stable emulsion.
Trifolium pratense L. flowers extract was prepared using ultrasound combined with thermal hydrolysis, and used solvent was 50 % ethanol solution. Glycyrrhiza glabra L. roots extract was done using ultrasound. The extraction solvent was water. Myristica fragrans Houtt. The essential oil was prepared by modified hydrodistillation [5]. The antimicrobial effects were investigated against standard cultures of non-spore bacteria (all bacteria were obtained from American Type Culture Collection (ATCC))—Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC 27853), Proteus vulgaris (ATCC8427), Bacillus cereus (ATCC 6633) and fungus Candida albicans (ATCC 10231). Emulsion with extracts and essential oil was prepared using excipients (maltodextrin, inulin, and/ or gum Arabic) and sodium alginate solution. The solution was homogenized for 15 min at 5000 rpm using homogeniser. Emulsion stability was tested using a centrifuge, particles' size and distribution were assessed using Mastersizer.
Trifolium pratense L. extract (33 mg/ml) inhibited the growth of Staphylococcus epidermidis, Staphylococcus aureus, and Bacillus cereus. Obtained results showed that this extract inhibits Gram-positive bacteria more than Gram-negative. The growth of other pathogens was not inhibited. Glycyrrhiza glabra L. extract (11 mg/ml) had expressed antimicrobial activity against Gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Bacillus cereus. Additionally, it suppressed the growth of the fungus Candida albicans. Essential oils are volatile liquids, so it is difficult to determine their effect due to evaporation. Nevertheless, essential oil (30 μg) suppressed the growth of Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae.
Six emulsion samples were prepared, and their stability was evaluated. Emulsion, which was prepared additionally using maltodextrin, inulin, gum arabic (ratio 62:43:15), alginate, all the extracts and essential oil, was the most stable, and its centrifugation index was 100%. Myristica fragrans Houtt. essential oil drop diameter in the emulsion was 0.571 μm. Evaluated percentiles were measured: D10 was 0.328 ± 0.051 μm, D50 was 0.46 ± 0.03 μm, and D90 was 0.89 ± 0.094 μm.
All of the extracts were more susceptible to Gram-negative bacteria than Gram-positive, but increasing extract concentration results can be more effective, so plant extracts should be concentrated in the final product in order to have a wide range antimicrobial agent. Created stable emulsion with both extracts and suspended essential oil can be used as the final pharmaceutical form or incorporated in other products for therapeutic benefits.