Screening of the antioxidant activity of extracts from Hedera helix leaves using the HPLC/ABTS method

Hedera helix is widely used as a remedy to treat the respiratory infections and cold accompanied with cough due to its anti-inflammatory effect. In addition, the antioxidant activity of its extracts has been confirmed. It is explained by the high content of flavonoids and phenolic acids among all phytochemicals of H. helix leaves. However, it remains uncertain which exactly components are responsible for the antioxidant activity and what is the best way to perform extraction. Aim. To determine the antioxidant profile of different extracts from H. helix leaves using the in vitro HPLC method combined with the ABTS reagent. Materials and methods. Extraction of H. helix leaves was conducted with different solvents (from 20 % methanol to 100 % methanol using an ultrasound bath); the method described in the Pharmacopeia was also used. A Waters chromatograph was used to determine the antioxidant profile. Results. About 90 % of the components responsible for the antioxidant activity were determined using the HPLC method proposed. Among them, chlorogenic acid and 3,5-caffeoylquinic acid showed the highest activities. Other components, such as neochlorogenic acid, hyperoside and 3,4-caffeoylquinic acid were revealed as components with the antioxidant scavenging activities in H. helix lextracts. Conclusions. The results obtained indicate that extracts from H. helix leaves possess the high antioxidant scavenging capacity. In addition, the in vitro HPLC method proposed can be used for the primary screening of components in the plant raw material.

Guidelines for ethical conduct when using animals in research require the application of alternative methods for pharmacological studies of pharmaceutical products [5]. According to the guidelines, at the stage of the primary screening of drug pharmacological activities, it is preferable to replace the experimental animals with other types of models, such as in vitro analysis, cell cultures, computer modeling. Docking provides all required information about biological activities using no animal in the research [6]. Also, the implementation of alternative procedures for pharmacological activities remains in high demand. This research is aimed to perform the HPLC method for the in vitro antioxidant studies of H. helix samples.

Plant raw material
The leaves of H. helix for this experiment were sampled in different European countries, such as Lithuania (Naujoji Akmene), Ukraine (Kharkiv), Czech Republic (Prague), Austria (Vienna).
After collection, the samples were air-dried at the ambient temperature with protection from direct sunlight.

Sample preparation
The samples were prepared according to Bezruk et al. [7]. Briefly, 1.0 g of accurately weighted powder of H. helix leaves were extracted thrice with 15 ml of methanol (or 20 %, 50 %, 70 % methanol) on an ultrasound bath for 15 minutes at ambient temperature. All supernatants were mixed and diluted to 50.0 ml with the solvent used for extraction.
The samples of H. helix were prepared according to the European Pharmacopoeia [8]. Concisely, 1.0 g of accurately weighted powdered leaves were extracted with 50 mL of the mixture of 80 % methanol under reflux on a water bath at 80 °C for 1 hour, after that a cooled solution was filtered through cotton. The cotton used and the residue were extracted with 30 mL of the same solution for 30 minutes. The extracts were mixed and diluted to the volume 100.0 mL with the same solution.
Chromatographic conditions HPLC-PDA and HPLC-ABTS were performed as described in Bezruk et al. [8]. A Waters Alliance 2695 (Waters, Milford, USA) separation system coupled with Waters 2487 UV/Vis and Waters 996 PDA diode-array detector (DAD). The separation of components was performed with the ACE C18 column (250 mm × 4.6 mm, particle size -5 µm). Using the PDA detector the mobile phase containing phytochemicals was mixed with the ABTS solution in the reaction coil, as described in the previous papers [9][10][11]. The ABTS post-column chromatograms were registered at 650 nm. The calibration curve was constructed with a Trolox standard.

Results and discussion
The components were identified compared to standard retention times and their UV-spectra. Twenty substances were found in the studies (Fig.). However, only five of them showed the antioxidant scavenging activity. Chlorogenic acid and 3,5-caffeoylquinic acid were the dominant components and explained from 58 % up to 93 % of the total  (Table). On the other hand, leaves from Prague showed the lowest concentration among the samples studied; however, its values were relatively significant from 5.317 up to 9.257 µmol TE/g DM for all extracts.
Among all solvents, extracts of 100 % methanol showed the highest antioxidant activity. This data was shown in our previous research [8]. Mostly, components of methanol extracts were in the following decreasing order: 3,5-caffeoylquinic acid > chlorogenic acid > hyperoside > 3,4-caffeoylquinic  acid > neochlorogenic acid. The same order could be found almost in each sample. However, leaves collected in Vienna had a higher amount of neochlorogenic acid than 3,4-caffeoylquinic acid. Phenolic components depended on the structural characteristics and showed a correlation between their concentration in the raw material and antioxidant scavenging capacities. The HPLC/ABTS method proposed (post-column assay) possessed the possibility to determine components with substantial activities and fast initial reactions. Consequently, the procedure is suitable for the detection and quantification of fast-acting high-capacity antioxidants to determine the main markers. Hence, this method is worth considering for the primary screening of antioxidants instead of using animals. Another point to consider is that the antioxidant activity markers can be advantageous to provide the quality and effectiveness of the plant raw material with promising health effects.

CONCLUSIONS
The extracts from H. helix leaves have shown the high antioxidant scavenging capacity; the main biomarkers responsible for the activity have been found. The method proposed provides the acceptable determination of every component, as well as the measurement of their activity. Thus, the procedure mentioned can be applied for screening of antioxidant effects in complex mixtures of the plant raw material and herbal medicines.
Conflict of interests: authors have no conflict of interests to declare.