HPLC Analysis and In Vitro and In Silico Evaluation of the Biological Activity of Polyphenolic Components Separated with Solvents of Various Polarities from Helichrysum italicum.

Helichrysum italicum has piqued the interest of many researchers in recent years, mostly for its essential oil, but increasingly for its polyphenolic content as well. In the current study, we examine the polyphenolic composition of H. italicum grown in Bulgaria. The polyphenolic complex was fractionated with solvents of various polarities, including hexane, chloroform, ethyl acetate, and butanol, in order to assess the biological impact of the components. HPLC-PDA and UHPLC-MS/MS were used to examine all fractions. The green coffee fingerprint profile was employed as a "surrogate standard" in the polyphenolic components detection approach. From the UHPLC-MS/MS analysis, we identified 60 components of the polyphenolic complex such as quercetin 3-O-glucuronide, quercetin acetyl-glycoside, isorhamnetin acetyl-glycoside, isorhamnetin caffeoyl-glycoside, quercetin caffeoyl-malonyl-glycoside, isorhamnetin coumaroyl-glycoside, coumaroyl-caffeoylquinic acid, and diCQA-acetyl-derivative were first reported in the composition of H. italicum. The biological activity of the fractions was evaluated in vitro and in silico, which included the fight against oxidative stress (hydrogen peroxide scavenging activity (HPSA), hydroxyl radical scavenging activity (HRSA), metal-chelating activity (MChA)) and nitrosative (nitric oxide scavenging activity) (NOSA)), in vitro anti-inflammatory, and anti-arthritic activity. Results are presented as IC50 ± SD µg/mL. The analysis showed that the EtOAc fraction was characterized by highest HPSA (57.12 ± 1.14 µg/mL), HRSA (92.23 ± 1.10 µg/mL), MChA (5.60 ± 0.17 µg/mL), and NOSA (89.81 ± 2.09 µg/mL), while the hexane and chloroform fractions showed significantly higher in vitro anti-inflammatory activity (30.48 ± 2.33 µg/mL, 62.50 ± 1.69 µg/mL) compared to the standard ibuprofen. All three fractions showed potential anti-arthritic activity (102.93 ± 8.62 µg/mL, 108.92 ± 4.42 µg/mL, 84.19 ± 3.89 µg/mL).

Characterization of Polyphenols from Chenopodium botrys after Fractionation with Different Solvents and Study of Their In Vitro Biological Activity.

In the present work, we have investigated the polyphenolic composition of Chenopodium botrys from Bulgaria. The polyphenols were fractionated with solvents of varying polarity (n-hexane, chloroform, ethyl acetate, and n-butanol). The fractions were analyzed by HPLC-PDA and UHPLC-MS. The ethyl acetate fraction contained mono- and di-glycosides of quercetin, di-glycosides of kaempferol, and isorhamnetin and monoglycosides of hispidulin and jaceosidine. We found quercetin triglycosides in the butanol fraction. The ethyl acetate and butanol fractions contained 168.82 mg/g Extr and 67.21 mg/g Extr of quercetin glycosides, respectively. The main components of the polyphenolic complex in C. botrys were 6-methoxyflavones (355.47 mg/g Extr), which were found in the chloroform fraction. The flavonoids pectolinarigenin, demethylnobiletin, and isosinensetin, and the glycosides of quercetin (triglycosides, acylglycosides), kaempferol, isorhamnetin, hispidiulin, and jaceosidine, were discovered and reported in Chenopodium botrys for the first time. We used in vitro methods to assess the biological activity against oxidative stress (hydrogen peroxide scavenging activity (HPSA) and hydroxyl radical scavenging activity (HRSA)), nitrosative stress (nitric oxide scavenging activity (NOSA)), anti-inflammatory activity (IAD inhibition), and anti-tryptic activity (ATA). Quercetin mono- and di-glycosides exhibited greater HPSA and HRSA (IC50 = 39.18, 105.03 µg/mL), while 6-methoxyflavones had a greater NOSA (IC50 = 146.59 µg/mL). The same components showed the highest ATA (IC50 ranging from 116.23 to 202.44 µg/mL).

The role of antioxidant defense in freezing tolerance of resurrection plant Haberlea rhodopensis.

Haberlea rhodopensis Friv. is unique with its ability to survive two extreme environmental stresses-desiccation to air-dry state and subzero temperatures. In contrast to desiccation tolerance, the mechanisms of freezing tolerance of resurrection plants are scarcely investigated. In the present study, the role of antioxidant defense in the acquisition of cold acclimation and freezing tolerance in this resurrection plant was investigated comparing the results of two sets of experiments-short term freezing stress after cold acclimation in controlled conditions and long term freezing stress as a part of seasonal temperature fluctuations in an outdoor ex situ experiment. Significant enhancement in flavonoids and anthocyanin content was observed only as a result of freezing-induced desiccation. The total amount of polyphenols increased upon cold acclimation and it was similar to the control in post freezing stress and freezing-induced desiccation. The main role of phenylethanoid glucoside, myconoside and hispidulin 8-C-(2-O-syringoyl-b-glucopyranoside) in cold acclimation and freezing tolerance was elucidated. The treatments under controlled conditions in a growth chamber showed enhancement in antioxidant enzymes activity upon cold acclimation but it declined after subsequent exposure to -10 °C. Although it varied under ex situ conditions, the activity of antioxidant enzymes was high, indicating their important role in overcoming oxidative stress under all treatments. In addition, the activity of specific isoenzymes was upregulated as compared to the control plants, which could be more useful for stress counteraction compared to changes in the total enzyme activity, due to the action of these isoforms in the specific cellular compartments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00998-0.

Antioxidant defense during desiccation of the resurrection plant Haberlea rhodopensis.

Maintaining a strong antioxidant system is essential for preventing drought-induced oxidative stress. Thus, in the present study we investigated the role of some non-enzymic and enzymic antioxidants in desiccation tolerance of Haberlea rhodopensis. The effects of high light upon desiccation on antioxidant capacity was estimated by comparing the response of shade and sun plants. The significant enhancement of the antioxidant capacity at 8% RWC corresponded to an enormous increase in flavonoid content. The important role of ascorbate-glutathione cycle in overcoming oxidative stress during drying of H. rhodopensis was established. The antioxidant capacity increased upon dehydration of both shade and sun plants but some differences in non-enzymatic and enzymatic antioxidants were observed. Investigations on the role of polyphenols in desiccation tolerance are scarce. In the present study the polyphenol profiles (fingerprints) of the resurrection plant Haberlea rhodopensis, including all components of the complex are obtained for the first time. It was clarified that the polyphenol complex of H. rhodopensis includes only two types of glycosides - phenylethanoid glucosides and hispidulin 8-C-glucosides. Upon desiccation the polyphenol content increase and the main role of phenylethanoid glucosides in the protection of H. rhodopensis was revealed.

Flavonoid content and radical scavenging potential of extracts prepared from tobacco cultivars and waste.

The flavonoid-rich natural products exert a wide range of pharmacological properties. This investigation aimed at obtaining extracts from tobacco cultivars and waste enriched with flavonoids and purified from nicotine and phenolic acids, and evaluating their radical scavenging potential. Extraction with a mixture of ethyl acetate-methanol (1:1, v/v) was employed resulting in 100% yield for flavonoids and 36% yield for phenolic acids. The crude extracts were purified using preparative column chromatography on silica gel. The content of flavonoids in the purified extracts varied from 8.8 ± 1.1% to 14.3 ± 1.8%. Nicotine was not detected in amounts higher than 0.3 µg mL(-1). The content of phenolic acids was lower than 1.0%. The radical scavenging potential of extracts from tobacco cultivars exhibited values from IC50 = 35.0 ± 3.1 to 64.6 ± 7.5 µg mL(-1). The extracts obtained by the proposed procedure are enriched with flavonoids with high radical scavenging potential and are purified from nicotine and phenolic acids. They can be regarded as potential biopharmaceuticals.