ANTIOXIDANT CHARACTERISTICS OF METHANOL AND ETHANOL EXTRACTS OF SELECTED VARIETIES OF HUMULUS LUPULUS

high content of biologically active substances. The results showed a connection between the content of α-bitter acids and the total content of polyphenols in hops. The highest concentration of polyphenols, corresponding to 2.52 g/dm 3 of gallic acid, was the ethanol extract of the Polaris variety, prepared at a temperature of 100 °C from cryo-pulverized hops, whose content of α-bitter acids is up to 20 %. With the use of the ABTS cationic radical higher antioxidant activity, on average, was determined in the methanolic extracts, on the other hand, with the use of the DPPH radical, the ethanolic extracts showed a higher antioxidant activity. Using the ABTS method, the highest total antioxidant capacity was determined in the methanol extract of the Polaris variety, prepared from pulverized hops at a temperature of 100 °C, namely 25.50 mmol/dm 3 Trolox equivalents. The DPPH method had the highest antioxidant efficiency, 94.9 %, determined in the methanol extract of the Polaris variety prepared from cryo-pulverized hops by extraction at a temperature of 100 °C. By comparing the results of analyzes of several hop varieties, the Polaris variety appears to be suitable for the preparation of functional beer.


INTRODUCTION
Common hop (Humulus lupulus) is a perennial herbaceous liana from the Cannabeaceae family cultivated by humans since time immemorial (Alonso-Esteban et al., 2019).In brewing, the flowers of the female plants are used, which are 2.5-5 cm long, cone-shaped cones that develop shortly after pollination.Cones contain a diverse amount of phytochemicals in the glandular trichomes (so-called lupulin glands) on the lower part of the bracts, which are responsible for the effects of hops on human health (Almaguer et al., 2014;Liberatore et al., 2018).Lupulin is secreted from the lupulin glands (Esslinger 2009), in which the primary metabolites are bitter resins and aromatic substances, responsible for the characteristic taste and aroma of hops (Stevens et al., 1998).Secondary metabolites are hop oils, resins, and polyphenols.The content of this metabolites in the dry matter of the hop cone ranges from 4-14 % (Yan et al., 2019).Hop resins are divided into soft and hard resin fraction.Bitter acids, known as -bitter acids mainly represented by humulone and -bitter acids, mainly represented by lupulone, are included in the soft resin fraction.These substances have the most significant effect on the bitterness of beer (Čermák et al., 2015;Ban et al., 2018).In addition to hop resins, lupulin contains polyphenols, which can be divided into flavan-3-ols (catechin, epicatechin, proanthocyanidin), phenolic acids (ferulic acid), flavonols (quercetin and kaempferol) and in smaller quantities prenylated flavonoids (xanthohumol, desmethylxanthohumol, isoxanthohumol, 6prenylnaringenin and 8-prenylnaringenin) (Gorinstein et al., 2007).The content of prenylated flavonoids, which are currently of the greatest scientific interest, depends on the variety, agroecological conditions of growing and storing hops.Their amount is generally reported to be around 5 g in 100 g of dry hop cone mass However, it follows from the results of the research so far that for the assessment of the antioxidant potential of hops, the sources of variability in the results are the variety and growing conditions of hops, the method of solubilization and extraction of biologically active substances, as well as the analytical methods used.In general, it is necessary to realize that many substances in hops act synergistically, thus combining antioxidant effects with anti-inflammatory or antiproliferative effects (Zugravu et al., 2022).

Plant materials
Three varieties of common hops (Humulus lupulus L.) which differences in the declared content of bitter acids were analysed (Html 1, Html 2, Html 3) (Tab.1).
Hops are an important ingredient in beer composition.In recent times, attention has been focused on the positive effects of hops as a plant with a high content of substances with antioxidant properties.Knowing the total concentration of polyphenols or the total antioxidant capacity of individual hop varieties could contribute to the creation of the so-called functional beer with a high content of biologically active substances.The results showed a connection between the content of α-bitter acids and the total content of polyphenols in hops.The highest concentration of polyphenols, corresponding to 2.52 g/dm 3 of gallic acid, was the ethanol extract of the Polaris variety, prepared at a temperature of 100 °C from cryo-pulverized hops, whose content of α-bitter acids is up to 20 %.With the use of the ABTS cationic radical higher antioxidant activity, on average, was determined in the methanolic extracts, on the other hand, with the use of the DPPH radical, the ethanolic extracts showed a higher antioxidant activity.Using the ABTS method, the highest total antioxidant capacity was determined in the methanol extract of the Polaris variety, prepared from pulverized hops at a temperature of 100 °C, namely 25.50 mmol/dm 3 Trolox equivalents.The DPPH method had the highest antioxidant efficiency, 94.9 %, determined in the methanol extract of the Polaris variety prepared from cryo-pulverized hops by extraction at a temperature of 100 °C.By comparing the results of analyzes of several hop varieties, the Polaris variety appears to be suitable for the preparation of functional beer.

Polaris
Germany 20 % 6,0 % hint of pine and mint special types of craft beers universal The homogenous preparation of hops was prepared by two procedures, namely mechanical homogenization, and cryo-pulverization after freezing with liquid nitrogen.During mechanical homogenization, 50 g of natural hop pellets were homogenized manually in a mortar (designation N).When preparing the cryopulverized sample, 200 ml of liquid nitrogen was added to 50 g of hop pellets.The frozen hops were then mechanically homogenized into a fine powder (designation C).

Extraction of prenylated flavonoids
The extraction system Dionex™ ASE™ 350 (Thermo Fisher Scientific, USA) was used for the extraction of hop flavonoids.The extraction was carried out with polar solvents 99.8 % (v) methanol and 99.9 % (v) ethanol in six repeated five-minute extraction cycles at a pressure of 10.5 MPa and a temperature of 50 °C, 100 °C, 150 °C or 200 °C.Determination of the total polyphenols content and the total antioxidant capacity was carried out in 6 repetitions.

Determination of the total polyphenols content
Folin-Ciocalteu reagent was used for the determination of total polyphenols content.25 µl of extract and 1 ml of Folin-Ciocalteu reagent (1:9, v/v) were added to 1 ml of demineralized water.The solution was vigorously vortexed.After 5 minutes, 1 ml of saturated Na2CO3 was added and let to incubation for 15 minutes at room temperature.The total polyphenols content was determined from the absorbance value at 515 nm using a calibration line (0.1-5.0 g/L gallic acid) in mg/L GAE.

Determination of the total antioxidant capacity by the ABTS method
The cationic radical ABTS + • was generated by the reaction of 8 mmol/L 2,2azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) with 3 mmol/L K2S2O8 in a ratio of 2:1 (v/v) in the dark at room temperature for 12 hours.Before determining the total antioxidant capacity, the ABTS + • solution was diluted with 96% ethanol (v) so that the absorbance of the solution at a wavelength of 734 nm was equal to the maximum absorbance of the calibration line, prepared by determining the antioxidant capacity of 0 -1 mmol/L Trolox.To determine the antioxidant capacity, 25 µl of extract was added to 2 ml of ABTS + • solution.The reaction took place at room temperature for 30 minutes in the dark.The antioxidant capacity of the extract in mmol TE/L was determined from the absorbance value at 734 nm using the calibration line.

RESULTS AND DISCUSSION
Three varieties of common hops (Humulus lupulus L.) were analysed, which differ significantly in the content of bitter acids.The Saaz Late variety has a low content of these substances (2.69 % α-bitter acids and 4 % β-bitter acids), the average content of bitter acids is typical for the Premiant variety (7.3 % α-bitter acids and 3.5 % β -bitter acids), on the other hand, the Polaris variety is characterized by a high content of bitter acids (20 % α-bitter acids and 6 % β-bitter acids).

The total polyphenols content
The method of determining the total polyphenols content in plant material by reducing the Folin-Ciocalteu reagent was used by several authors The highest total polyphenols content was determined in the ethanol extracts (Figure 1) of the Polaris variety, namely in the extract obtained at a temperature of 100 °C (2.52 g/L GAE in extract C, respectively 2.51 g/L in extract N).Cryopulverization had no significant effect on the extractability of total polyphenols from hops.The biggest differences were determined in the Saaz Late extract at an extraction temperature of 150 °C, when compared to the N homogenate due to cryo-pulverization, total polyphenols content in the extract increased by 16 %, and in the Premiant C extract, total polyphenols content increased by 10% at a temperature of 100 °C.The most suitable extraction temperature when using ethanol as an extraction agent was 100 °C.

Figure 1 The total polyphenols content in ethanol extracts of hops
In extracts prepared using methanol, the mass yield of polyphenols was lower than in ethanol extracts (Figure 2).Also in this case, the highest total polyphenols content was determined in the Polaris C extract (2.43 g/L GAE).The mass yield of polyphenols was lower by 3.6 % compared to the ethanol extract.The highest total polyphenols content was obtained at a temperature of 100 °C.When comparing N and C methanol extracts, the mass yield of polyphenols due to the cryopulverization of Saaz Late was higher by 20 %, Premiant by 3 % and Polaris by 6 %.Processing hop pellets into extracts can be an effective way to reduce the amount of hops used in the production of beers enriched with polyphenols (Bramforth, 2006) They analyzed the extracts by HPLC and found that polar phenolic compounds were extracted at low temperatures, while less polar phenolic compounds were extracted at higher temperatures.Water's polarity was reduced by higher temperatures, allowing it to solvate non-polar compounds and extract them.The study of total polyphenols content showed that an extraction temperature higher than 100 °C reduces total polyphenols content in hop extracts.Thermal degradation of these substances takes place, while this process also involves treatment, or material homogenization and type of solvent.Several authors (Lapornik et al., 2005;Turkmen et al., 2006;Turkmen et al., 2007;Xi et al., 2009;Inglett et al., 2010) state that the extraction of polyphenolic substances will increase by using dilute solvents, e.g.ethanol : water 1:1 (v/v).

Kowalczyk et al. (2013)
found that a 50 % aqueous ethanol solution was a better solvent for these substances from plant materials than pure ethanol.
The reactions of antioxidants with transition metals or direct reactions of quenching or scavenging of free radicals are most often used in them.They are methods of evaluating the ability of substances to eliminate radicals and methods that assess the redox properties of substances  3).For comparison, the difference between the same prepared Polaris C and Polaris N extract was 13 % against N.The lowest antioxidant capacity was determined in the Saaz Late N extract (11.27 mmol/L TE in the extract obtained at 50 °C), which corresponds to the declared lowest content of α-bitter acids from the analyzed hops (2.69 %).Cryopulverization of hops increased the ethanol extractability of substances with antioxidant action at the most suitable temperature of 100 °C in the analyzed hops by 11.8 %.Arsene et al. (2015) found a direct correlation of antioxidant activities and the total polyphenols content in the hop's ethanolic extract.A good antiradical activity, corresponding to 0.78 -0.99 mg/ml was determined in methanol extracts of hops, which had a higher polyphenolic content of 7.12 mg GAE/g (Keskin et al., 2019).
Figure 3 The total antioxidant capacity of ethanol extracts of hops Polaris C methanol extract obtained at 100 °C (25.50 mmol/L TE, Figure 4) had the highest antioxidant capacity, while Saaz Late N extracted at 150 °C (10.30mmol/L TE) had the lowest antioxidant capacity.Cryo-pulverization of hops increased the methanol extractability of substances with antioxidant action at the most suitable temperature of 100 °C in the analyzed hops by 15.2 %.

Figure 4 total antioxidant capacity of methanol extracts of hops
The ABTS method was used to determine the antioxidant activity in methanol extracts of Styrian Golding, Hersbrucker, Hallertau Blanc, Cascade Cryo Hops®, Pacifica Jade, Pacifica, Topaz hops.The Hallertau Blanc variety had the highest activity, 1.036 µmol TE/mg of dry hop, on the other hand, the extract from the Cascade variety showed the lowest antioxidant capacity, only 0.114 µmol TE/mg of dry hop (Iannone et al., 2022).

Antioxidant efficiency
The ability to scavenge the DPPH radical was widely used to determine the antioxidant activity of crude extracts or purified compounds from plants.The results are expressed as percentage inhibition of radical activity.The highest antioxidant efficiency of all analyzed extracts, up to 94.9 %, was shown by the Polaris C methanol extract prepared from cryo-pulverized hops by extraction at a temperature of 100 °C (Figure 5  456.08 µg/ml.In contrast, the extract prepared using 55 % ethanol had an activity of only 184.9 µl/ml (Wu et al., 2020).

CONCLUSION
Currently, in brewing practice, in addition to classic cold hopping, beer is enriched with biologically active substances with an antioxidant effect, as well as the addition of pure hop extracts.The results of the work showed a connection between the content of α-bitter acids and the total content of polyphenols in hops.Ethanol extraction under high pressure at 100 °C is an effective preparation in which an extract with an application-interesting content of antioxidants is obtained.Cryo-pulverization of hops increases the yield of these substances, which supports the use of Cryohops® in brewing practice.Of the hop varieties that were analyzed, the Polaris variety appears to be the most suitable for the preparation of beer with functional properties.

(
Ciriminna et al., 2018).More specifically, Karabin et al. (2012) report that the content of xanthohumol is 0.2 -1.1 g in 100 g dry matter of hop cone and according to Miligan (2009) 8-prenylnaringenin occurs only in very low concentrations.Xanthohumol and 8-prenylnaringenin, which is known as the most potent phytoestrogen of hops (Sun et al., 2022; Lecomte et al., 2023) are investigated as potential anticancer agents, with significant antiproliferative activity against cancer cells (Busch et al., 2015).Xanthohumol has the potential of a drug that could slow the malignant progression of breast cancer (Kim et al., 2013).Hops have long been known in the pharmacopoeia as a plant that alleviates the symptoms of many health problems.Its use was essential in the treatment of toothache, leprosy, fever, during treatment of stomach problems, anxiety and it was even considered a medicament for insomnia (Korpelainen et al., 2012).Significant use of hops is the action of its flavonoids in alleviating postmenopausal discomfort (Keiler et al., 2015).Nowadays, it is confirmed that the bioactive substances of hops have a wide range of therapeutic and anti-inflammatory properties, the antimicrobial properties of hops against several microorganisms have also been proven (Oliveira Neto et al., 2017).Biologically active substances of hops can act as scavengers of free radicals, metal chelating agents, modulators of enzymatic activity and inhibitors of cell proliferation (Karabin et al., 2015).Thanks to these positive properties, these hop components are constantly being tested in primary or secondary prevention against some chronic degenerative diseases.The main role in these pathological processes is played by oxidation mechanisms in the organism (Zugravu et al., 2022).Hops show a very high antioxidant activity, slowing down or preventing the oxidation of substrates, thereby reducing oxidative stress in cells and the organism (Lang et al., 2024).However, the disadvantage of several natural antioxidants is that, in their free form are sensitive to modifications caused by various physicochemical factors, thereby losing their antioxidant properties (Sharma et al., 2023).Based on these findings, there is an effort to prepare hop extracts with a high content of active polyphenols, which could find application in functional foods, nutraceuticals, pharmaceuticals, or cosmetics (Astray et al., 2020; Tronina et al., 2020; Sun et al., 2022).

Figure 5 Figure 6
Figure 5 Antioxidant efficiency of ethanol extracts of hops

Table 1
Overview of hop varieties

(Sánchez- Rangel et al., 2013; Blainski et al., 2013; Kamboj et al., 2015; Lamuela- Raventós, 2017).
Methanol and ethanol extracts, prepared from pellets of natural hops homogenized mechanically (N) and from hops cryo-pulverized using liquid nitrogen (C), were analyzed.This modification of the sample was carried out because there are currently several commercial hop preparations available in the form of Cryohops®, modified by this method.It is a procedure in which, before pelleting, fresh hop heads are first frozen with liquid nitrogen under a low oxygen content at a temperature of -28 o C and only after freezing are they pelleted (Tembo, 2020).Publications by several authors indicate that such processing of hops should preserve a higher content of biologically active components of hops.Their content should be up to twice that of hops processed by classic pelleting (

Iannone et al., 2022
The total polyphenols content in methanol extracts of hopsThere is a connection between the amount of α-bitter acids and the total content of polyphenols in hops.In the analyzed varieties, the total content of polyphenols in ethanol and methanol extracts obtained at 100 °C decreases in the order Polaris > Premiant > Saaz Late.In methanol extracts from seven varieties of hops (Styrian Golding, Hersbrucker, Hallertau Blanc, Cascade Cryo Hops®, Pacifica Jade, Pacifica, Topaz) total polyphenols ranged from 27.4 to 49.8 mg GAE/g dried hops ( (Kowalczyk et al., 2013)ethanol and dichloromethane were used as extraction agents, currently subcritical CO2 and ethanol are mainly used in terms of safety and environmental impact(Kowalczyk et al., 2013).Due to its non-polar nature, subcritical CO2 extraction is selective for aromatic components and hop resins, not for polyphenols (Mander et al., 2010).Substances with a polar character, including polyphenols, are dissolved by ethanol (Stevens et al., 2004), ).Different authors report very different contents of total polyphenols in the methanol extract, e.g. is 2.39 mg GAE/g (Fărcaş et al., 2017), 7.4 µg GAE/g (Maliar et al., 2017).Polyphenols are generally considered to be thermally labile compounds (Rajbhar et al., 2015).In traditional extraction procedures, the highest yields of these substances are obtained at temperatures of 60 -80 °C (Casazza et al., 2012).On the other hand, high pressure extraction studies prefer temperatures above 100 °C.For example, increasing the extraction temperature to 180 -200 °C meant an increase in both total polyphenols content and antioxidant capacity in the canola flour extract (Nandasiri et al., 2019).Ibañez et al. (2003) carried out a sequential extraction of medicinal rosemary at temperatures of 100, 150 and 200 °C, in which they used water as a solvent.