Phytochemical analysis of aerial part of Ikonnikovia kaufmanniana and their protection of DNA damage

Abstract Ikonnikovia kaufmanniana is an endemic plant of Kazakhstan of which phytochemical analysis has not been reported. The present study found out that this species enriched with antioxidant chemicals. Isolation and structural identification processes reveal twelve phenolic compounds (1–12) having dihydroflavanonol, flavonol, isoflavone and flavanol skeletons. The annotation of individual components in the extract was carried out by LC-ESI-MS/MS to represent a chemotaxonomic marker of the target plant. The antioxidant activities of all compounds were screened using three different radical sources (DPPH, ORAC, and hydroxyl radicals). Most compounds (1–11) had significant antioxidant activity against three radical sources, and their efficacies were found to differ by their functionality and skeleton. The potential of the isolated compounds in preventing oxidative damage of DNA was evaluated with pBR322 plasmid DNA. Compounds (1, 5, 7, and 8) had protective effects on DNA damaged with 80% efficacy at 60 μM concentration. Graphical Abstract


Introduction
Reactive oxygen species (ROS) are unstable molecules that can damage cell structures, mainly by attacking double bonds of biomolecules. The ROS in a living organism can be quenched naturally, but the excess of ROS can lead to mutation, cell death, ageing and human degenerative disease. Antioxidative potential is commonly screened with several kinds of radical sources, which have application in understanding of the antioxidant characteristics (Schaich et al. 2015). In particular, the hydroxyl radical leads to the single-stranded cleavage of supercoiled plasmid DNA into a relaxed open circular form, reaction of which can be screened in gel electrophoresis . Plasmid DNA damage protection potential of antioxidants may avert carcinogenesis, mutagenesis and a lot of genetic disorders (Srinivas et al. 2018).
Ikonnikovia kaufmanniana (regel) lincz. belongs to Plumbaginaceae family and only species of Ikonnikovia genus (Wu et al. 1996;Komarov 2004). This species is an endemic plant which grows naturally in the South-eastern part of Kazakhstan (Akhmetova et al. 2014). The phytochemical study of I. kaufmanniana has not been reported so far.
Our study is focused on the isolation and identification of phytochemicals in I. kaufmanniana. The isolated compounds were further screened for their antioxidant capacities, including a DNA damage protection ability.

Results and discussion
The compounds isolated from ethyl acetate extract of I. kaufmanniana were identified by analyzing spectroscopic data and comparing them with literature data (Nonaka et al. 1983;Pereira et al. 2012). The compounds are dihydromyricetin potassium sulfate (1), gallocatechin (2), epigallocatechin (3), isoluteolin (4), myricetin (5), myricitrin (6), hyperoside (7), quercitrin (8), kaempferin (9), quercetin (10), dihydromyricetin (11), and rodiolinozide (12), as shown in Figure 1. This is the first phytochemical  investigation of the target plant. Therein, the annotation of individual components in the extract was carried out by LC-ESI-MS/MS analysis to represent a chemotaxonomic marker of I. kaufmanniana ( Figure S41). In the current study, three different assays were used for evaluation of antioxidant capacity: DPPH, hydroxyl radical scavenging activities and ORAC. Firstly, the isolated compounds were screened by DPPH radical scavenging activity assay. All phytochemicals apart from 12 had potent antioxidant actions against DPPH radical with IC 50 of 6.7 114.7 lM (Table S1). The most active DPPH scavengers were determined as compounds 1, 4, 5, and 11, with IC 50 s 24.0, 23.0, 16.9, and 6.7 lM, respectively. All compounds were also examined by ORAC assay, which follows the hydrogen atom transfer mechanism and is a better model of antioxidant reactions in food stuffs. This assay is based on the oxidation of fluorescein by the peroxyl radical (Tijerina S aenz et al. 2009). The most active compounds 3, 8, and 10 had significant antioxidant potentials with IC 50 s 18.9, 21.9, and 18.3 lmol TE/g, respectively (Table S1).
The hydroxyl radicals are capable of modifying biomolecules in the living cell, which causes damage in DNA strand and lipid peroxidation (Phull et al. 2018). Flavonoids (1-11) showed a significant hydroxyl radical scavenging effect, with IC 50 s ranging from 2.4 to 38.9 lM. The most potent antioxidants were found to be compounds 1, 5, 7, 8, and 11 with 9.7, 8.3, 6.8, 4.9, and 2.4 lM, respectively (Table S1).
Finally, the compounds (1-9) were evaluated for antioxidant potential concerning preventing oxidative damage of plasmid DNA. The supercoiled circular DNA (scDNA) was converted into an open circular form (ocDNA), and further a linear double stranded DNA molecule (lnDNA), by hydroxyl radicals, generated from Fenton's reaction. The evaluation of DNA damage was carried out following the procedure of Kim et al. (2018). Figure S49 shows the protective potentials of the compounds (1-9) in pBR322 plasmid DNA at a concentration of 60 lM. Inhibitory efficacies varied between 12.4 and 93.5% according to chemical structures (Table S1). Compounds 1, 5, 7 and 8 had over 80% protective effects from DNA damage and also showed dose-dependent manner ( Figure S47). The most active compound, quercitrin (8) protected DNA damage dose-dependently from 15 to 120 lM concentrations as shown in Figure S50.

Experimental
See Supplementary materials.

Conclusion
This work investigated for the first time the phytochemicals of I. kaufmanniana, which is an endemic plant of Kazakhstan. Phytochemicals pattern and specific annotation of individual components in the extract were carried out by LC-ESI-MS/MS to represent a chemotaxonomic marker of the target plant. The isolated compounds (1-11) showed potent antioxidant activity against three unique DPPH, ORAC, and hydroxyl radicals, as well as a significant protective effect on pBR322 plasmid DNA.