Trifolium pratense

Raw materials (with abbreviations) selected for the preparation of extracts used in this study included:

Alv L–aloe leaves, Aloe vera (L.) Burm. f.;

Am Fr–black chokeberry fruits, Aronia melanocarpa (Michx.) Elliott;

Arv H–common mugwort herb, Artemisia vulgaris L.;

Bv R–beetroot roots, Beta vulgaris L.;

Co F–common marigold flowers, Calendula officinalis L.;

Ea H–field horsetail herb, Equisetum arvense L.;

Ep F–purple coneflower flowers, Echinacea purpurea (L.) Moench;

Ep L–purple coneflower leaves, Echinacea purpurea (L.) Moench;

Hp H–St. John’s wort herb, Hypericum perforatum L.;

Hr Fr–sea-buckthorn fruits, Hippophae rhamnoides L.;

Lc S–red lentil seeds, Lens culinaris Medik.;

Mc F–chamomile flowers, Matricaria chamomilla L.;

Ob H–basil herb, Ocimum basilicum L.;

Pm H–broadleaf plantain herb, Plantago major L.;

Poa H–common knotgrass herb, Polygonum aviculare L.;

Ps S–pea seeds, Pisum sativum L.;

Pta L–common bracken leaves, Pteridium aquilinum (L.) Kuhn;

Sg L–giant goldenrod leaves, Solidago gigantea Ait.;

So R–comfrey roots, Symphytum officinale L.;

To F–common dandelion flowers, Taraxacum officinale (L.) Weber ex F.H. Wigg.;

To L–common dandelion leaves, Taraxacum officinale (L.) Weber ex F.H. Wigg.;

To R–common dandelion roots, Taraxacum officinale (L.) Weber ex F.H. Wigg.;

Tp F–red clover flowers, Trifolium pratense L.;

Ur L–nettle leaves, Urtica dioica L.;

Ur R–nettle roots, Urtica dioica L.;

Vo R–valerian roots, Valeriana officinalis L.

First, a 4% sodium alginate solution was prepared from distilled water and alginic acid sodium salt. It was used throughout the experiment for emulsion preparation as the shell material. Emulsion with Trifolium pratense L. and Glycyrrhiza glabra L. extracts, and Myristica fragrans Houtt. essential oil were prepared as follows: solution with excipients (maltodextrin, inulin, and/ or gum Arabic) was mixed with sodium alginate solution (stirred for 15 min with a magnetic stirrer MSH-20A (Witeg, Wertheim, Germany)) and then extracts with essential oil were added. The solution was homogenised for 15 min at 5000 rpm using an IKA T18 homogeniser (IKA-Werke GmbH & Co., KG, Staufen, Germany).
The emulsion’s stability was tested using a centrifuge Sigma 3-18KS (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The test was repeated three times using 23 °C temperature, 3000 rpm, and the duration was 5 min. The centrifugation index (CI) was calculated to evaluate emulsion stability.
$$\mathrm{CI}(\%)=\frac{{\mathrm{V}}_{\mathrm{e}}}{{\mathrm{V}}_{\mathrm{i}}}·100$$
where V_e is the volume of the remaining emulsion after centrifugation and V_i is the volume of the initial emulsion.

Since the AOX gene sequences of P. sativum were not publicly available, prior to the expression analysis, the sequences and gene structure of the AOX gene family members needed to be identified in this plant species. For this, all pea sequences homologous to AOX Glycine max sequences were retrieved, and a BLAST search was performed at the pea genome database (available at https://urgi.versailles.inra.fr/Species/Pisum/Pea-Genome-project, accessed on 3 June 2021). AOX sequences retrieved from G. max were used as queries because it belongs to the same family Fabaceae. Sequences identified in the P. sativum database were further used as secondary queries. To verify the homology with AOX of the sequences identified at the pea genome database, a Blastn analysis at the NCBI (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/, accessed on 3 June 2021) was conducted.
For PsAOX gene structure analysis, the software Splign (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi?textpage=online&level=form, accessed on 5 July 2023) was used. To compare PsAOX gene structure with other AOX genes from other plant species, genomic and transcript sequences of five additional species belonging to the Fabaceae family (Glycine max, Medicago trucatula, Phaseolus vulgaris, Trifolium pratense, and Vigna unguiculata) were retrieved from EnsemblPlants databases (https://plants.ensembl.org/index.html, accessed on 3 June 2021). The GSDS 2.0 software (available at http://gsds.gao-lab.org/, accessed on 21 July 2023) was used to draw the scheme with the exon/intron composition.
To get the correct classification of P. sativum AOX genes (PsAOX), protein sequences were aligned with Glycine max AOX sequences, and the classification adopted was based on [33 ]. CLC Genomics Workbench 11.0.1 software (ClCbio, Aarhus N, Denmark) was used to edit AOX sequences and perform alignment.
Protein subcellular localization and position of the cleavage sites of mitochondrial targeting signals were predicted by using the translated peptide corresponding to exon 1 in the TargetP software [34 (link)] (freely available at http://www.cbs.dtu.dk/services/TargetP/, accessed on 4 July 2023). The prediction of putative isoelectric point (pI) and the molecular weight was conducted by using the PeptideMass tool, freely available at Expasy software (http://web.expasy.org/peptide_mass/, accessed on 4 July 2023).
To better evaluate the relation between the identified sequences, a phylogenetic relationship study was conducted using the deduced peptide sequences of the pea genes and genes from Liliopsid (14 species) and Magnoliopsid (38 species) species retrieved from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 3 June 2021) (details of sequences are in Supplementary Materials Tables S1 and S2). Retrieved sequences were aligned in MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/, accessed on 15 June 2021), following the default settings to generate an output Pearson/FASTA file. MEGA 7 [35 (link)] was used to construct a phylogenetic tree using the neighbor-joining (NJ) method [36 (link)] with bootstrap analysis using 1000 replicates, “number of differences” as the substitution model and “pairwise deletion” for gaps/missing data treatment. For a graphical view, the tree was edited in Fig Tree v14.0 software ([37 ], Edinburgh, UK) (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 16 June 2023).