*3.3. Phytochemical Analysis*

The analysis of polyphenols in the green plum flesh and seed powders were carried out as a screening study of 39 different polyphenols chosen for their presence and prominence in fruit and in the mango, to which the green plum is related. The compounds identified in the various extracts are shown in Table 3. The compounds which had concentrations high enough to be quantified by UPLC-PDA are shown in Table 4.


**Table 3.** Compounds found in green plum flesh and seed extracts using ultra high pressure liquid chromatography—mass spectrometry (UHPLC-MS) QExactive in negative mode.

X, compounds present; [x], compound present in trace amounts; U, unbound; B, bound; M, methanol; E, ethanol; W, water. *n* = 1.

**Table 4.** Quantified phytochemicals in green plum flesh and seed unbound and bound extractions.


The green plum flesh and seed contain a number of compounds with antioxidant and antimicrobial activity as well as other beneficial properties. The high radical scavenging ability shown in the DPPH assay may be due to the presence of gallic acid, which was present in all extractions. Gallic acid is able to scavenge a variety of free radicals including singlet oxygen, hydroxyl, peroxyl and alkyl peroxyl and protects against UV cell damage [30]. *Trans*-ferulic acid is a low toxic component of plants and protects against pathogen invasion and has antioxidant, antimicrobial and anti-inflammatory activities [31]. It scavenges reactive oxygen species and reactive nitrogen species and nanoparticles containing it reduce lipid peroxidation [32].

Chlorogenic acid inhibits a range of bacterial pathogens by increasing outer membrane permeability, inducing the efflux of potassium from the cell, and causing rupture of the membrane and leakage of the cytoplasmic contents, including nucleotides [33].

Some flavonoid combinations can work synergistically in their antioxidant capacity, including kaempferol and quercetin (+19.9%) and catechin and kaempferol (+2.4%) [34], all found in the green plum flesh. Catechin and quercetin may mitigate adipose inflammation, oxidative stress and insulin resistance and have protective effects on human health [35]. They are antioxidants with radical scavenging activity and reducing properties [36] and quercetin is active against oxidation deterioration in emulsions [15].

Naringenin was also found and it has antioxidant, anti-carcinogenic and anti-inflammatory effects [37]. Ellagic acid has antioxidant functions and has an inhibitory effect on the growth of micro-organisms [38]. There is an abundance of ellagic acid in the Kakadu plum that contributes to its use as a natural food preservative [7], and it was found in both the flesh and seed of the green plum.

The unbound seed extractions were pink in colour and was analysed for anthocyanin. The spectrum was characteristic for anthocyanin compounds and the associated fragments in the LC-MS showed an *m*/*z* of 303.0482 and a structure of C15H11O7, which is indicative of a delphinidin aglycone. The quantification was done against a cyanidin 3-glucoside calibration curve and the concentration of the anthocyanin was 423 ± 8.04 μg cyanidin 3-glucoside equivalents/g DW in the seed. Anthocyanins have been found previously in the seed coats of common beans using a similar extraction method [39], and in the seed coats of black soybeans, including delphinidin-3-glucoside [40].

Antioxidants are important in food to prevent the oxidation that occurs naturally and causes off-flavours and rancidity, loss of fat-soluble vitamins, fatty acids and bioactives, and can sometimes form toxic compounds [41]. There is an increased interest from consumers and the food industry in replacing synthetic preservatives with natural ones, such as plant material that inherently has these properties [15]. Other Australian native plant foods are being incorporated into food products for their natural preservative effects [9]. Plant extracts can be used in the food industry to increase the shelf-life of food products, using their natural antioxidant and antimicrobial properties to control the growth of food-borne pathogens [42]. The green plum could potentially be used as a natural alternative to chemical additives to increase the shelf life and quality of food. The antioxidant and antimicrobial potential of the green plum indicate it may be able to be used as a functional ingredient.
