Flavonoid and phenolic acid profile by LC-MS/MS and biological activity of crude extracts from Chenopodium hybridum aerial parts

Abstract Extracts from leaves and stems of Chenopodium hybridum were characterised for the presence and quantity of flavonoids and phenolic acids by LC-ESI-MS/MS. Five flavonoids and eight phenolic acids were detected for the first time in aerial parts of this plant species, the most abundant compounds being rutin (2.80 μg/g DW), 3-kaempferol rutinoside (2.91 μg/g DW), 4-OH-benzoic (1.86 μg/g DW) and syringic acids (2.31 μg/g DW). Extracts were tested for anti-inflammatory/antiarthritic, antihyaluronidase and cytotoxic activities against human prostate cancer (Du145, PC3) and melanoma cell lines (A375, HTB140 and WM793) of different malignancy. None of the extracts protected bovine serum albumin from heat-induced denaturation. Antihyaluronidase effect at the tested concentration was higher than standard naringenin. Cytotoxic activity was generally low with an exception of the extract from the leaves, which was found most effective against prostate Du145 cell line with 98.28 ± 1.13% of dead cells at 100 μg/mL.

The genus Chenopodium is included into the Amaranthaceae family and encompasses over 250 species, many of which are considered a rich source of flavonoids (Vysochina 2010). Apart from these, it accumulates diverse groups of compounds, including saponins, alkaloids, betalains, terpenes, minerals and primary metabolites (Dellagreca et al. 2005;Kokanova-Nedialkova et al. 2009;Mroczek 2015). Several Chenopodium species, like Ch.quinoa and Ch. ambrosioides, have important economic value due to their nutritional and medicinal properties (Javaid & Amin 2009;Vega-Gálvez et al. 2010;Harraz et al. 2015).
Chenopodium hybridum L. -maple-leaved goosefoot, is widely distributed in temperate regions, being a common species in Poland. Aerial parts of the plant were used in folk medicine as an analgesic agent (Kokanova-Nedialkova et al. 2009). To the best of our knowledge, there are no reports on Ch. hybridum phytochemical composition except for a reference to the presence of quercetin in this plant species (Kokanova-Nedialkova et al. 2009). In a previous study (Nowak et al. 2016), extracts from different plant parts of four Chenopodium species, in this Ch. hybridum, were evaluated for total phenolic and total flavonoid contents together with the assessment of their antioxidative properties. Moreover, cytotoxic activity was tested on human lung carcinoma A-549 and ovarian carcinoma TOV-112D cell lines, showing antiproliferative effect of Ch. hybridum herb extract against the latter cell line.
The aim of this work was identification and quantification of individual flavonoids and phenolic acids in extracts from the aerial parts of this plant species by LC-ESI-MS/MS. In addition, further biological activity testing was performed, which included assessment of protective effect on hyaluronic acid and denaturation of bovine serum albumin (BSA) in vitro to establish anti-inflammatory and antiarthritic potential of this plant species. Moreover, cytotoxic properties were examined against three melanoma and two prostate cancer cells lines of different metastatic potential.

Results and discussion
LC-ESI-MS/MS analysis allowed to identify in methanolic extracts from Ch. hybridum five flavonoids and eight phenolic acids. This was done by comparison of retention times and mass fragmentation pattern with data obtained for commercial standards. The concentrations of individual compounds, which were quantified by comparison of peak areas with the calibration curves obtained for the corresponding standards, are reported in Tables S1 and S2. Extracts from leaves and stems did not differ greatly in the composition of phenolics except for the lack of isoquercetin, gentisic acid and caffeic acid in the latter. Among phenolic acids, the most abundant compounds in both analysed plant parts, were 4-OH-benzoic and syringic acids. The levels of flavonoids were generally lower, with an exception of rutin and 3-kaempferol rutinoside, which appear to be the predominant compounds.
Published data from investigations on Chenopodium flavonoids refer to almost 30 species of world flora and indicate that 3-O-glycosides of kaempferol, quercetin and isoramnetin seem to be characteristic of this genus (Vysochina 2010). Our data on flavonoid profile in Ch. hybridum are in accordance with these results. There is, however, relatively little information concerning the quantity of phenolics in aerial parts of species of Chenopodium. Most reports refer to quantification of these compounds in the products of economic value, like Ch. quinoa seeds (Paśko et al. 2008;Repo-Carrasco-Valencia et al. 2010;Tang et al. 2015) and quinoa flour Many of reported pharmacological properties of Chenopodium species, such as healing, hypotensive, immunomodulatory, antimicrobial, analgesic activities, are attributed to the presence of flavonoids (Ibrahim et al. 2007;Vysochina 2010;Arora et al. 2014). Notes on ethnopharmacological use of Ch. hybridum in pain conditions urged us to test its antiinflammatory/antiarthritic potential in vitro by BSA antidenaturation assay (Mizushima & Kobayashi 1968;Williams et al. 2008). However, extracts from leaves and stems showed no protective effect as compared to diclofenac sodium in the tested concentration range of 5-500 μg/mL (Table S5).
Numerous reports indicate that together with phenolic acids, flavonoids contribute to the overall antioxidant activity of plant extracts (Li et al. 2009;Li et al. 2015;Nowak et al. 2016). Antioxidants play an important role in protection of our organisms against many disorders caused by oxidant damage, such as cancer, or neurodegenerative and cardiovascular disorders. Recently, also hyaluronidase inhibitors have been extensively studied as components that may hinder progress of these diseases. A positive correlation between the expression of hyaluronidase and tumour invasiveness and metastatic potential was reported (Załuski et al. 2015). Thus, in our study, we tested the extracts from Ch. hybridum as potential Hyal inhibitors. Samples of leaf and stem extracts (1 mg/mL) showed 8.96 and 9.70 % inhibition for 30 I.U./mg protein/mL, respectively, as compared to standard naringenin (3.49 % at 1 mg/mL). The results obtained in this assay suggest a significant inhibitory potential of extracts. The higher inhibition by extracts may result from the nature of chemical constituents of the extracts as a mixture. It is known that the activity of hyaluronidase is dependent on the presence of calcium ions, in turn, the extracts contain polyphenols acting as chelators.
Cytotoxic activity studies were performed on two panels of human cancer cell lines with metastatic potential. Skin panel included three malignant melanoma cell lines, namely A375, HTB140 and WM793. Prostate cancer cells tested were of low (Du145) and high (PC-3) metastatic potential. The cytotoxic effect was evaluated in the concentration range of 10-100 μg/ mL (Table S6). Extract from the leaves was found most effective against prostate Du145 cell line and WM793 melanoma cells, with 98.28 ± 1.13 % and 78.11 ± 2.05 % of dead cells at 100 μg/mL, respectively. Stem extract presented moderate cytotoxic activity only against melanoma A375 cell line, with 17.01 ± 0.08 % of dead cells at 100 μg/mL. It is noteworthy that the activity on prostate and melanoma cells of the extract from Ch. hybridum leaves was generally higher than that reported for ovarian and lung carcinoma cell lines (Nowak et al. 2016). Our results were also comparable to data obtained for the extract from the leaves of Ch. quinoa on rat prostate cancer cell lines and also on human melanoma HTB 140 cells (Gawlik-Dziki et al. 2013).

Conclusion
In this study, we reported for the first time the qualitative and quantitative data regarding flavonoids and phenolic acids in the aerial parts of Chenopdium hybridum as well as results of in vitro anti-inflammatory, antihyaluronidase and cytotoxic activity against melanoma and prostate cell lines assays. Despite records of the use of this plant species in folk medicine it has not been previously characterised chemically or pharmacologically. The type of flavonoid compounds identified in our study supports the notion that 3-O-glycosides of kaempferol and quercetin seem to be typical of the Chenopodium genus and may have chemotaxonomic significance. The results of biological activity assays demonstrate that extracts from Ch. hybridum show cytotoxic and antihyaluronidase activities at different magnitudes of potency. The leaf extract might be a potential supporting agent in prostate cancer therapy, but further studies are certainly needed.

Supplementary material
Includes experimental section and Tables S1-S6.