Chemical compositions and sleep-promoting activities of hop (Humulus lupulus L.) varieties

Byungjick Min, Yejin Ahn, Hyeok Jun Cho, Woong Kwon Kwak, Kyungae Jo, Hyung Joo Suh

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Among natural products with sleep-promoting activity, hops have been used since ancient times as a tranquilizer and hypnotic agent. This study investigated the sleep-promoting effects of extracts of various hop (Humulus lupulus L.) varieties in invertebrate and vertebrate models. The content of α-acids, β-acids, and xanthohumol was higher in hop 70% alcohol extracts than in hop hot water extracts. Among the alcohol extracts, Citra contained a high α-acid content (229.32 µg/mg), while Saphir showed high β-acid and xanthohumol content (66.37 and 4.23 µg/mg, respectively). In Drosophila melanogaster, Simcoe and Mosaic water extracts and Saphir and Simcoe alcohol extracts significantly increased total nighttime sleep. Total sleep time of mice with pentobarbital-induced sleep was significantly increased by Simcoe and Mosaic water extracts and Saphir and Simcoe ethanol extracts compared to the normal group. Oral administration of Simcoe water extract and Saphir alcohol extract improved sleep in the caffeine-induced insomnia model and upregulated the mRNA expression of GABAA (gamma 2 subunit) and GABAB receptors in mouse brains. Additionally, Saphir alcohol extract significantly increased the GABA content in mouse brains. Simcoe water extract and Saphir ethanol extract modulated GABAergic signaling to improve sleep-related behaviors, including sleep duration.

Original languageEnglish
Pages (from-to)2217-2228
Number of pages12
JournalJournal of Food Science
Volume88
Issue number5
DOIs
Publication statusPublished - 2023 May

Bibliographical note

Funding Information:
Ten hop varieties (Amarillo, Cascade, Centennial, Chinook, Citra, Herkules, Mosaic, Saaz, Saphir, and Simcoe) were obtained from Lotte Co., Ltd. (Seoul, Korea). Hot water or ethanol extracts of hops were prepared by mixing distilled water or 70% ethanol (500 mL), respectively, with hops (20 g) and refluxing twice for 1 h at 95°C. The extracts were filtered (No. 1, Whatman, Maidstone, UK), concentrated under reduced pressure, and freeze-dried to make a powder. Ten hop varieties (Amarillo, Cascade, Centennial, Chinook, Citra, Herkules, Mosaic, Saaz, Saphir, and Simcoe) were obtained from Lotte Co., Ltd. (Seoul, Korea). Hot water or ethanol extracts of hops were prepared by mixing distilled water or 70% ethanol (500 mL), respectively, with hops (20 g) and refluxing twice for 1 h at 95°C. The extracts were filtered (No. 1, Whatman, Maidstone, UK), concentrated under reduced pressure, and freeze-dried to make a powder. The yields of hot water and 70% ethanol extracts are expressed as a percentage of the dry matter of the raw material used to prepare the extract. The total polyphenol content was measured by the Folin–Ciocalteu method using gallic acid as a standard (Maksimovic et al., 2005), the total flavonoid concentration by the p-(dimethylamino) cinnamaldehyde method using catechin as a standard (Arnous et al., 2002), and the reducing sugar content by the 3,5-dinitrosalicylic acid method using glucose as a standard (Hu et al., 2008). The protein concentration was analyzed using a micro BCA protein kit (Thermo Fisher Scientific, Sunnyvale, CA, USA; Cat. #23225) according to the manufacturer's protocol. α-Acid (cohumulone and adhumulone) and β-acid (colupulone and adlupulone) contents were analyzed by HPLC as described previously with some modifications (Buckee & Baker, 1987) using a YMC-Triart C18 column (4.6 mm × 150 mm, 5 µm) and at 275 nm wavelength. The mobile phase was methanol:water:phosphoric acid (775:210:9 [v/v/v]) under isocratic conditions. Xanthohumol, quercetin-3-glucuronide, and kempferol-3-glucoside contents in the extracts were also analyzed using the YMC-Triart C18 column and at 372 nm wavelength. Mobile phases A and B were 0.025% trifluoroacetic acid in water and 0.025% trifluoroacetic acid in acetonitrile, respectively, and the flow rate was 1 mL/min. The gradient of the mobile phase was 65% A for 0 min, 25% A for 25 min, 5% A for 30 min, and 65% A for 35–40 min (Choi et al., 2017). Quercetin-3-glucuronide and kempferol-3-glucoside contents were analyzed using a YMC Triart C18 column (4.6 mm × 250 mm, 5 µm) and at 350 nm wavelength with 0.5% formic acid in water and 0.5% formic acid in acetonitrile as mobile phases A and B, respectively. The flow rate was 0.8 mL/min. The gradient of the mobile phase was 80% A for 0 min, 77% A for 5 min, 73% A for 20 min, and 80% A for 25–30 min (Lee et al., 2020). The wild-type D. melanogaster Canton-S strain (3 days old, male; Bloomington Drosophila Stock Center, Bloomington, IN, USA) was used. The locomotor activity of Drosophila administered with a mixture of hop extracts (2%), 5% sucrose, and 2% agar medium for 4 days was evaluated. The total activity of Drosophila and the sleep-promoting effects of hop extracts were evaluated using the Drosophila Activity Monitoring system (TriKinetics, Waltham, MA, USA). One fly was placed in each vial, allowed to acclimate in constant darkness for 3 days, and then behavioral patterns were analyzed for 4 days (n = 50). Data were analyzed using Actogram J (NIH, Bethesda, MD, USA). Sleep parameters were calculated based on the total number of activities recorded each day (Jo, Choi, et al., 2018). Six-week-old male ICR mice (Orient Bio Inc., Seongnam, Korea) were housed at 23−25°C and 55% relative humidity with a 12 h light–dark cycle and acclimatized for at least 1 week. Food and water were freely available. The mice were randomly divided into groups of seven mice. All animal experiments were approved by the Korea University Animal Experimental Animal Control Committee (KUIACUC-2021-0020, Seoul, Korea). Hop water extracts (Centennial, Mosaic, and Simcoe) and 70% ethanol extracts (Saphir and Simcoe) were dissolved in 0.9% physiological saline and orally administered (100 or 200 mg/kg). The positive control group was orally administered with the benzodiazepine alprazolam (Xanax; Pfizer, New York, NY, USA) at a concentration of 0.2 mg/kg. After 45 min, sleep was induced by intraperitoneal pentobarbital injection (42 mg/kg). Sleep latency and sleep duration were measured as previously described (Kim, Suh, et al., 2019). In the caffeine-induced insomnia mouse model, caffeine (40 mg/kg) was orally administered along with the hop extracts (200 mg/kg) to all groups except the normal group. After 45 min, pentobarbital (42 mg/kg) was administered intraperitoneally to evaluate the sleep-improving effect. Mice were orally administered with Simcoe water extract or Saphir ethanol extract (200 mg/kg) for 21 days (n = 7). Total RNA was isolated from mouse brains using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). mRNA expression was analyzed by quantitative real-time RT-PCR using the StepOne Plus Real-time PCR system (Applied Biosystems, Foster City, CA, USA) and Power TaqMan PCR Master Mix Kit (Applied Biosystems) as previously described (Kim, Jo, et al., 2019). The following genes were analyzed: GABAA receptor gamma 2 subunit (NM_0,08073.3), forward primer (FP) 5′-GCTTTGGTGGAGTATGGCAC-3′, reverse primer (RP) 5′-GGCCTTGAAGGAAAACATCCG-3′; GABAB receptor 1 (NM_01,9439.3), FP 5′-TCCGGAACGGGGAAAGAATG-3′, RP 5′-TTGTACTCGCCGACCTTCAC-3′; GABAB receptor 2 (NM_0,010,81141.1), FP 5′-GGAGCTAGCCATCGAGCAGA-3′, RP 5′-CGGTGTCGTAGAGTCGCAG-3′; 5-HT1A (NM_0,08308.4), FP 5′-CCGATCTCATGGTGTCAGTG-3′, RP 5′-ACATCCAGGGCGATAAACAG-3′; and GAPDH (NM_0,08084.3), FP 5′-CATCACTGCCACCCAGAAGACTG-3′, RP 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′. The GABA content in mouse brains was analyzed by HPLC. Mobile phases A, B, and C were water AccQ-tag eluent A (acetate–phosphate buffer), acetonitrile, and Milli-Q water, respectively. A Waters AccQ-Tag column (150 mm × 3.9 mm) and a fluorescence detector (250 nm excitation and 395 nm emission wavelengths) were used (Jo, Kim, Hong, et al., 2021). Experimental results are expressed as mean ± standard error of the mean. Statistically significant differences among the groups were determined by one-way analysis of variance followed by Tukey's multiple range test at a significance level of 5%, using the Statistical Package for Social Sciences version 12.0 (SPSS Inc., Chicago, IL, USA).

Publisher Copyright:
© 2023 Institute of Food Technologists.

Keywords

  • flavonoids
  • GABA receptor
  • Humulus lupulus
  • sleep
  • xanthohumol

ASJC Scopus subject areas

  • Food Science

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