An optimal polyherbal formulation with antimicrobial and anti-oxidant properties

Abstract

Introduction: Since ancient times, spices and herbs have been traded, valued, and used for the enhancement of taste, flavour, and colour of food and beverages. Their role in preservation has been investigated in several in vitro and in vivo studies. Many herbs and spices, other than their culinary uses, have health-promoting properties and have been used by different cultures and in various traditions to prevent and manage illnesses and diseases. Such health benefits among others include antibacterial, antifungal, antiviral, anti-oxidant, anti-inflammatory, antidiabetic, and anticancer activities. A number of studies have demonstrated synergism as an effective strategy to enhance the bioactivity of plant extracts and essential oils. Thus, using this framework, this study was conducted to effectively determine the ideal combination of crude extracts or neat essential oils of some common culinary herbs and spices that would exhibit the most favourable anti-oxidant and antimicrobial activities, and incorporate these into an optimal polyherbal formulation. Materials and methods: A total of 51 crude extracts (methanol, dichloromethane, water) and 15 neat essential oils were chemically profiled using ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) and gas chromatography coupled with mass spectrometry (GC-MS), respectively. The major constituents in the extracts and essential oils were identified based on the available data from the literature. The anti-oxidant activities of the extracts and essential oils were evaluated independently, and in combination (1:1) using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ferric reducing anti-oxidant power (FRAP) assays. Whereas the minimum inhibitory concentration (MIC) assay was used to determine the antibacterial activities of the individual extracts and essential oils and their combinations (1:1) against three Gram-positive and three Gram-negative bacteria. The antimicrobial and anti-oxidant interactions were determined by calculating the sum of fractional inhibitory concentration index (ΣFICI). The Design of Experiments (DOE) (MODDE 9.1®) software analysis was used to optimize synergy potential of the herbal extracts by generating a statistically significant model that predicted the most effective ratio required for the polyherbal combination, as validated experimentally. A polyherbal capsule was formulated from blending various spice extracts based on DOE model predictions. The formulation was evaluated for toxicity using Brine shrimp lethality assay (BSLA), and the antioxidant and antibacterial activity of the formulation was also determined. Results: The major compounds that were identified in the analysed essential oils were limonene, linalool, 1,8-cineole, β-caryophyllene, α-pinene, β-pinene, eugenol, estragole, cinnamaldehyde, thymol, citral, terpinene-4-ol, trans-sabinene hydrate, γ- terpinene, thujone (α, β), menthone and menthol. A total of 45 bioactive compounds , mainly phenolic acids and flavonoids were identified in the analysed crude extracts by comparing mass spectra and retention time (Rt) with those of commercially available reference standards. Where standards were not available, the compounds were tentatively identified by comparing mass spectra in terms of m/z fragments with data reported in the literature. Compounds such as apigenin, caffeic acid, proanthocyanidins, catechin, and rosmarinic acid were common in numerous analysed extracts. Each extract and essential oil displayed variable anti-oxidant activity. A total of 27 combinations were assessed for anti-oxidant interactions using the DPPH, ABTS, and FRAP assays. From these combinations, 3.8% was synergistic in the DPPH assay, 7.7% in the FRAP and ABTS assays, whilst 50.0% of the combinations were additive using the DPPH and FRAP assays, and 19.2% with the ABTS assay. Antagonism was shown in 11.1% with the DPPH assay, and 3.7% with ABTS and FRAP assays. Many of these combinations had an indifferent effect (37.0% with DPPH and FRAP assays, and 66.7% with ABTS assay). Out of the 14 combinations of the extracts assessed for antibacterial interactions, 16.7% were synergistic, additive, and antagonistic, whilst 50.0% of the combinations had an indifferent effect. A total of 21 essential oil combinations were evaluated for antibacterial interactions. of these combinations, 66.7% were antagonistic, whereas 33.3% were indifferent. None of the essential oils combinations showed additive or synergy interactions. The optimum combination for anti-oxidant properties was predicted by DOE to be the mixture containing crude methanol extracts of Mentha piperita L. (55.0%), Thymus vulgaris L.(44.0%), and Zingiber officinale Roscoe (1.0%). The optimal antimicrobial mixture contained crude methanol extracts of Rosmarinus officinalis L.(59.5%), Salvia officinalis L.(40.0%), and 1.8 (0.5%). A polyherbal capsule containing a mixture of R. officinalis, S. officinalis, and S. aromaticum methanol extracts was formulated successfully. The formulation demonstrated a dosedependent mortality to the brine shrimps. The formulation exhibited less than 50% mortality at concentrations below 500 µg/mL. The formulation demonstrated notable anti-oxidant and antibacterial activity Conclusion: An optimal polyherbal capsule containing R. officinalis (59.5%), S. officinalis (40.0%), and S. aromaticum (0.5%) methanol extracts, with antimicrobial and anti-oxidant properties was formulated successfully by calculating ΣFICI and using Design of Experiment (DOE).