Bulletin of Entomological Research, Page 1 of 6 © Cambridge University Press 2015.
doi:10.1017/S0007485315000255
Antifeedant activity of xanthohumol and supercritical carbon dioxide extract of spent hops against stored product pests J. Jackowski1*, M. Hurej1, E. Rój2, J. Popłoński3, L. Kośny1 and E. Huszcza3 1
Department of Plant Protection, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 24a, 50-363 Wrocław, Poland: 2Supercritical Extraction Department, New Chemical Syntheses Institute, al. Tysiąclecia Państwa Polskiego 13a, 24-110 Puławy, Poland: 3Department of Chemistry, Wrocław University of Environmental and Life Sciences, ul. Norwida 25, 50-375 Wrocław, Poland Abstract
Xanthohumol, a prenylated flavonoid from hops, and a supercritical carbon dioxide extract of spent hops were studied for their antifeedant activity against stored product insect pests: Sitophilus granarius L., Tribolium confusum Duv. and Trogoderma granarium Everts. Xanthohumol exhibited medium deterrent activity against the adults of S. granarius L. and larvae of T. confusum Duv. The spent hops extract was more active than xanthohumol towards the adults of T. confusum Duv. The potential application of the crude spent hops extract as a feeding deterrent against the stored product pests is proposed. Keywords: antifeedant activity, xanthohumol, spent hops, stored product pests (Accepted 9 March 2015)
Introduction The flowers of the female hop plant (Humulus lupulus L.) are used mainly in the brewing industry to add bitterness and flavor to beer. They are a rich source of prenylated flavonoids. Xanthohumol (fig. 1) is the main prenylated flavonoid found in hops at a concentration of 0.1–1.0% of the cone dry mass (Stevens et al., 1999), and it displays a wide range of biological activities, e.g.: antioxidant, antimicrobial, antiviral, antiplasmodial, antiinflammatory, antiangiogenic and anticancer activity (Stevens & Page, 2004; Chadwick et al., 2006; Zanoli & Zavatti, 2008; Bartmańska et al., 2013). Several recent studies showed that xanthohumol has chemo-preventive properties relevant to the suppression of cancer development at the initiation, promotion and progression phases (Strathmann & Gerhäuser, 2012). Xanthohumol is converted into the corresponding isomeric prenylflavanone isoxanthohumol (fig. 1) during the brewing process of beer. Although
*Author for correspondence Phone: +48713201693 Fax: +48713201748 E-mail: [email protected]
isoxanthohumol has a significantly better solubility than xanthohumol, its biological properties are not so promising (Bartmańska et al., 2013). The high concentration and the external accumulation of xanthohumol in lupulin glands significantly facilitate the extraction of this lipophilic compound with chloroform or acetone (Stevens et al., 1997). However, the most convenient source of xanthohumol is spent hops – waste product of the large scale hop-processing industry, generated during the extraction of hop cones with supercritical carbon dioxide, which is currently the most accepted solvent for the manufacture of hops extracts for the brewing industry. Carbon dioxide extracts (50°C, 280 bar) contain almost all essential oils present in hops, a high ratio of bitter acids, a small amount of other hops components and moreover, they lack traces of unpleasant solvents (Guo-qing et al., 2005). The resulting spent hops can serve as suitable starting materials to obtain xanthohumol, because the extraction of hops with carbon dioxide under 300 bar causes polyphenols, including prenylflavonoids, to be retained almost quantitatively in the waste material. Flavonoids play an important role in plant development and physiology, especially during plant interactions with other organisms (Iwashina, 2003). They have been considered as factors of resistance to insects in several studies. They are
J. Jackowski et al.
2
Fig. 1. Chemical structures of xanthohumol (1) and isoxanthohumol (2).
involved in the constitutive as well as in the induced resistance phenomena. Some flavonoids act through direct toxicity while others affect insects as feeding deterrents or digestion inhibitors (Harborne & Grayer, 1993; Simmonds, 2001, 2003; War et al., 2012). Various hops extracts containing mainly hops bitter acids, β-acids and also spent hops extract were tested for their antifeedant activity against insects. These preparations were shown to be deterrent to mites (Jones et al., 2003), moths (Gökçe et al., 2009), aphids (Powell et al., 1997; Dancewicz et al., 2011) and beetles (Gökçe et al., 2012). Taking into account these data, the authors decided to examine whether the main prenylated flavonoid from hops – xanthohumol, and a supercritical carbon dioxide extract of spent hops – a waste material containing xanthohumol and isoxanthohumol as main flavonoid components, will exhibit deterrent activity against three selected species of insect stored product pests. Such results may have, in the foreseeable future, some applicative value for stored food and feed grain protection.
Materials and methods Tested compounds and materials Plant material
of SC CO2 of 220 kg h−1. The plant has two separators; one for high pressure separations up to 300 bar and the second up to 100 bar, usually 50–60 bar. Commercial carbon dioxide (99% purity, produced by Zakłady Azotowe, Puławy, Poland) was used for the extractions. Supercritical carbon dioxide extract of spent hops The spent hops were used as a raw material for extraction of xanthohumol. The following process parameters were used for the extraction: pressure – 850 bar, temperature − 80°C. The two-separator system has been used to collect the extract. The extract analysis The extract was dissolved in methanol and analysed by HPLC on Waters 2695 Alliance instrument with a photodiode array detector Waters 2996 (detection at 280 and 370 nm wavelength) using the analytical HPLC column Cosmosil Cholester 5 μm (4.6 × 250 mm) at the flow rate of 1 ml min−1. A linear solvent gradient from 45 to 95% aq MeOH containing 0.05% HCOOH over 39 min was used. Standards of xanthohumol and isoxanthohumol were purchased from Alexis Biochemicals (Switzerland).
Cones (hops) of H. lupulus cv. ‘Magnum’ were collected in 2013 in Lublin region (SE Poland).
Test insects and feeding deterrent activity tests Insects
Chemical compounds studied in the experiment reported in this article Xanthohumol (PubChem CID: 639665); Isoxanthohumol (PubChem CID: 513197) Xanthohumol (3′-[3,3-dimethylallyl]-2′,4′,4-trihydroxy-6′methoxychalcone) was isolated according to Stevens et al. (1997) from supercritical carbon dioxide extracted hops, obtained in the Production of Hop Extracts of New Chemical Syntheses Institute, Puławy, Poland. Xanthohumol obtained for this study was identical with the standard (Alexis Biochemicals, Switzerland). Spent hops Extraction with supercritical carbon dioxide (SC CO2) was carried out using the high pressure pilot plant comprising two extractors of 40 l each, designed by Natex, Austria. The plant was designed to operate at a maximum pressure of 1000 bar and temperature 100°C, and at a maximum mass flow rate
The tests of feeding deterrent activity were carried out using three species of stored product pests: Granary Weevil (Sitophilus granarius L.), Confused Flour Beetle (Tribolium confusum Duv.) and Khapra Beetle (Trogoderma granarium Everts). The species had been selected originally by Nawrot et al. (1986), for their stored product pest status and are recognized since then as a kind of standard set of model organisms used for screening the deterrent activity of chemical compounds (see Nawrot et al., 2009). Granary Weevil and Confused Flour Beetle are cosmopolitan, synanthropic species that occur only indoor in a temperate climate. S. granarius L. is feeding on stored grain of wheat, barley, rye, oats, millet and maize, as well as on buckwheat. T. confusum Duv. also feeds on flour, cereal bran and on dried bread. The reason for including T. granarium Everts is its tropical origin, as this fact broadens the possible scope of information about the activity of tested compounds and may render the results of the experiments important also for the regions to which this species is native. Nonetheless, T. granarium Everts is repeatedly reported
Antifeedant activity of xanthohumol and carbon dioxide spent hops extract from storage facilities in Poland as an introduced species. Although it does not complete its development outdoor for its high thermal requirements, it is an important pest of stored grain. (Coleoptera Poloniae. Information System about Beetles of Poland – KFP (Catalogue of Fauna of Poland). Database browser 2011, accessed in July 2014). The insects were reared in permanent darkness in climatic chambers. S. granarius L. was reared at 26 ± 1°C, T. confusum Duv. – at 30 ± 1°C and T. granarium Everts – at 31 ± 1°C. The relative humidity in all the chambers was maintained at 60 ± 5%. S. granarius L. was reared on wheat grain cv. NATULA, whereas T. confusum Duv. and T. granarium Everts – on commercially available oat flakes of the brand Górskie (Stoisław Mills, Poland), mixed at equal proportion with oat flour (VITACORN, Poznań, Poland). The temperature and humidity during the tests were always identical to those of the rearing conditions and the tests were also carried out in darkness. Wheat wafer test Wheat wafer disk bioassay, as described by Nawrot et al. (1986, 2009), was used to test the feeding deterrent activity of the purified xanthohumol and that of spent hops extract. The wheat wafer used in this method is produced of wheat flour and water in the process which does not involve extensive heating and baking as in the majority of bakery products. It also does not contain any additional materials such as fats or monosaccharides and in this sense it may distantly ‘resemble’ the main constituents of wheat grain, yet without a number of features typical for living biological material, to mention only respiration. The authors of the manuscript had made an assumption that the wafer is an inert, homogenous substrate, suitable for testing the biological activity of different target compounds in the most fundamental context, i. e., unaffected by any possible interactions with the treated material, as might be expected in the case of tests carried out on true grain. The compound or the extract was tested as 1.0% ethanol solutions. The wheat wafer disks of 1 cm diameter were dipped in the ethanol solution of the compound, in the extract or in the absolute ethanol (reference) using pincers, and placed on large glass Petri dishes, treatment and reference separately, in order to air-dry. After 30 min of drying, which allowed for the ethanol evaporation, the disks were weighed and placed on polystyrene Petri dishes of 90 mm diameter. After the weighing of the disks in all the treatments have been completed, the test insects, beetles or larvae, depending on the species, were placed on the Petri dishes which were then covered and placed in climate chamber conditions appropriate for the test species of insect used. All the insects necessary for one test had been always collected from their cultures 1–2 h before the beginning of the test and kept until that time in polyethylene tubes of 35 mm diameter and 60 mm height, loosely closed with a polyethylene screw-cap. Therefore transferring them all on the test dishes with wafers took
doi:10.1017/S0007485315000255
Antifeedant activity of xanthohumol and supercritical carbon dioxide extract of spent hops against stored product pests J. Jackowski1*, M. Hurej1, E. Rój2, J. Popłoński3, L. Kośny1 and E. Huszcza3 1
Department of Plant Protection, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 24a, 50-363 Wrocław, Poland: 2Supercritical Extraction Department, New Chemical Syntheses Institute, al. Tysiąclecia Państwa Polskiego 13a, 24-110 Puławy, Poland: 3Department of Chemistry, Wrocław University of Environmental and Life Sciences, ul. Norwida 25, 50-375 Wrocław, Poland Abstract
Xanthohumol, a prenylated flavonoid from hops, and a supercritical carbon dioxide extract of spent hops were studied for their antifeedant activity against stored product insect pests: Sitophilus granarius L., Tribolium confusum Duv. and Trogoderma granarium Everts. Xanthohumol exhibited medium deterrent activity against the adults of S. granarius L. and larvae of T. confusum Duv. The spent hops extract was more active than xanthohumol towards the adults of T. confusum Duv. The potential application of the crude spent hops extract as a feeding deterrent against the stored product pests is proposed. Keywords: antifeedant activity, xanthohumol, spent hops, stored product pests (Accepted 9 March 2015)
Introduction The flowers of the female hop plant (Humulus lupulus L.) are used mainly in the brewing industry to add bitterness and flavor to beer. They are a rich source of prenylated flavonoids. Xanthohumol (fig. 1) is the main prenylated flavonoid found in hops at a concentration of 0.1–1.0% of the cone dry mass (Stevens et al., 1999), and it displays a wide range of biological activities, e.g.: antioxidant, antimicrobial, antiviral, antiplasmodial, antiinflammatory, antiangiogenic and anticancer activity (Stevens & Page, 2004; Chadwick et al., 2006; Zanoli & Zavatti, 2008; Bartmańska et al., 2013). Several recent studies showed that xanthohumol has chemo-preventive properties relevant to the suppression of cancer development at the initiation, promotion and progression phases (Strathmann & Gerhäuser, 2012). Xanthohumol is converted into the corresponding isomeric prenylflavanone isoxanthohumol (fig. 1) during the brewing process of beer. Although
*Author for correspondence Phone: +48713201693 Fax: +48713201748 E-mail: [email protected]
isoxanthohumol has a significantly better solubility than xanthohumol, its biological properties are not so promising (Bartmańska et al., 2013). The high concentration and the external accumulation of xanthohumol in lupulin glands significantly facilitate the extraction of this lipophilic compound with chloroform or acetone (Stevens et al., 1997). However, the most convenient source of xanthohumol is spent hops – waste product of the large scale hop-processing industry, generated during the extraction of hop cones with supercritical carbon dioxide, which is currently the most accepted solvent for the manufacture of hops extracts for the brewing industry. Carbon dioxide extracts (50°C, 280 bar) contain almost all essential oils present in hops, a high ratio of bitter acids, a small amount of other hops components and moreover, they lack traces of unpleasant solvents (Guo-qing et al., 2005). The resulting spent hops can serve as suitable starting materials to obtain xanthohumol, because the extraction of hops with carbon dioxide under 300 bar causes polyphenols, including prenylflavonoids, to be retained almost quantitatively in the waste material. Flavonoids play an important role in plant development and physiology, especially during plant interactions with other organisms (Iwashina, 2003). They have been considered as factors of resistance to insects in several studies. They are
J. Jackowski et al.
2
Fig. 1. Chemical structures of xanthohumol (1) and isoxanthohumol (2).
involved in the constitutive as well as in the induced resistance phenomena. Some flavonoids act through direct toxicity while others affect insects as feeding deterrents or digestion inhibitors (Harborne & Grayer, 1993; Simmonds, 2001, 2003; War et al., 2012). Various hops extracts containing mainly hops bitter acids, β-acids and also spent hops extract were tested for their antifeedant activity against insects. These preparations were shown to be deterrent to mites (Jones et al., 2003), moths (Gökçe et al., 2009), aphids (Powell et al., 1997; Dancewicz et al., 2011) and beetles (Gökçe et al., 2012). Taking into account these data, the authors decided to examine whether the main prenylated flavonoid from hops – xanthohumol, and a supercritical carbon dioxide extract of spent hops – a waste material containing xanthohumol and isoxanthohumol as main flavonoid components, will exhibit deterrent activity against three selected species of insect stored product pests. Such results may have, in the foreseeable future, some applicative value for stored food and feed grain protection.
Materials and methods Tested compounds and materials Plant material
of SC CO2 of 220 kg h−1. The plant has two separators; one for high pressure separations up to 300 bar and the second up to 100 bar, usually 50–60 bar. Commercial carbon dioxide (99% purity, produced by Zakłady Azotowe, Puławy, Poland) was used for the extractions. Supercritical carbon dioxide extract of spent hops The spent hops were used as a raw material for extraction of xanthohumol. The following process parameters were used for the extraction: pressure – 850 bar, temperature − 80°C. The two-separator system has been used to collect the extract. The extract analysis The extract was dissolved in methanol and analysed by HPLC on Waters 2695 Alliance instrument with a photodiode array detector Waters 2996 (detection at 280 and 370 nm wavelength) using the analytical HPLC column Cosmosil Cholester 5 μm (4.6 × 250 mm) at the flow rate of 1 ml min−1. A linear solvent gradient from 45 to 95% aq MeOH containing 0.05% HCOOH over 39 min was used. Standards of xanthohumol and isoxanthohumol were purchased from Alexis Biochemicals (Switzerland).
Cones (hops) of H. lupulus cv. ‘Magnum’ were collected in 2013 in Lublin region (SE Poland).
Test insects and feeding deterrent activity tests Insects
Chemical compounds studied in the experiment reported in this article Xanthohumol (PubChem CID: 639665); Isoxanthohumol (PubChem CID: 513197) Xanthohumol (3′-[3,3-dimethylallyl]-2′,4′,4-trihydroxy-6′methoxychalcone) was isolated according to Stevens et al. (1997) from supercritical carbon dioxide extracted hops, obtained in the Production of Hop Extracts of New Chemical Syntheses Institute, Puławy, Poland. Xanthohumol obtained for this study was identical with the standard (Alexis Biochemicals, Switzerland). Spent hops Extraction with supercritical carbon dioxide (SC CO2) was carried out using the high pressure pilot plant comprising two extractors of 40 l each, designed by Natex, Austria. The plant was designed to operate at a maximum pressure of 1000 bar and temperature 100°C, and at a maximum mass flow rate
The tests of feeding deterrent activity were carried out using three species of stored product pests: Granary Weevil (Sitophilus granarius L.), Confused Flour Beetle (Tribolium confusum Duv.) and Khapra Beetle (Trogoderma granarium Everts). The species had been selected originally by Nawrot et al. (1986), for their stored product pest status and are recognized since then as a kind of standard set of model organisms used for screening the deterrent activity of chemical compounds (see Nawrot et al., 2009). Granary Weevil and Confused Flour Beetle are cosmopolitan, synanthropic species that occur only indoor in a temperate climate. S. granarius L. is feeding on stored grain of wheat, barley, rye, oats, millet and maize, as well as on buckwheat. T. confusum Duv. also feeds on flour, cereal bran and on dried bread. The reason for including T. granarium Everts is its tropical origin, as this fact broadens the possible scope of information about the activity of tested compounds and may render the results of the experiments important also for the regions to which this species is native. Nonetheless, T. granarium Everts is repeatedly reported
Antifeedant activity of xanthohumol and carbon dioxide spent hops extract from storage facilities in Poland as an introduced species. Although it does not complete its development outdoor for its high thermal requirements, it is an important pest of stored grain. (Coleoptera Poloniae. Information System about Beetles of Poland – KFP (Catalogue of Fauna of Poland). Database browser 2011, accessed in July 2014). The insects were reared in permanent darkness in climatic chambers. S. granarius L. was reared at 26 ± 1°C, T. confusum Duv. – at 30 ± 1°C and T. granarium Everts – at 31 ± 1°C. The relative humidity in all the chambers was maintained at 60 ± 5%. S. granarius L. was reared on wheat grain cv. NATULA, whereas T. confusum Duv. and T. granarium Everts – on commercially available oat flakes of the brand Górskie (Stoisław Mills, Poland), mixed at equal proportion with oat flour (VITACORN, Poznań, Poland). The temperature and humidity during the tests were always identical to those of the rearing conditions and the tests were also carried out in darkness. Wheat wafer test Wheat wafer disk bioassay, as described by Nawrot et al. (1986, 2009), was used to test the feeding deterrent activity of the purified xanthohumol and that of spent hops extract. The wheat wafer used in this method is produced of wheat flour and water in the process which does not involve extensive heating and baking as in the majority of bakery products. It also does not contain any additional materials such as fats or monosaccharides and in this sense it may distantly ‘resemble’ the main constituents of wheat grain, yet without a number of features typical for living biological material, to mention only respiration. The authors of the manuscript had made an assumption that the wafer is an inert, homogenous substrate, suitable for testing the biological activity of different target compounds in the most fundamental context, i. e., unaffected by any possible interactions with the treated material, as might be expected in the case of tests carried out on true grain. The compound or the extract was tested as 1.0% ethanol solutions. The wheat wafer disks of 1 cm diameter were dipped in the ethanol solution of the compound, in the extract or in the absolute ethanol (reference) using pincers, and placed on large glass Petri dishes, treatment and reference separately, in order to air-dry. After 30 min of drying, which allowed for the ethanol evaporation, the disks were weighed and placed on polystyrene Petri dishes of 90 mm diameter. After the weighing of the disks in all the treatments have been completed, the test insects, beetles or larvae, depending on the species, were placed on the Petri dishes which were then covered and placed in climate chamber conditions appropriate for the test species of insect used. All the insects necessary for one test had been always collected from their cultures 1–2 h before the beginning of the test and kept until that time in polyethylene tubes of 35 mm diameter and 60 mm height, loosely closed with a polyethylene screw-cap. Therefore transferring them all on the test dishes with wafers took
