What Are the Medical Benefits of Humulus Lupulus L?

Hops (Humulus lupulus L.), also known as hop, yeast flower, fragrant hop, snake hop, snake hop grass, and Tang grass flower, are climbing perennial herbs belonging to the genus Humulus in the family Cannabaceae [1]; they are primarily distributed in northern Xinjiang and are also cultivated in North China, Northeast China, Zhejiang, Shandong, and other regions [2]; they have a bitter taste and neutral in nature, and has the effects of strengthening the stomach, calming the mind, aiding digestion, promoting urination, and relieving coughs and phlegm. It is commonly used to treat loss of appetite, insomnia, edema, abdominal distension, hysteria, tuberculosis, cystitis, pleurisy, and other conditions [3].

Hops are a plant with both medicinal and edible uses, primarily used in the brewing process of beer, and is a basic raw material for beer production. It is classified into four types: Type A (aromatic flowers), Type B (hybrid type), Type C (with indistinct characteristics), and Type D (bitter flowers). Its extracts contain complex components, including resins, essential oils, flavonoids, polyphenols, tannins, and choline, among other chemical compounds [3]. The abundance of active components confers beer flower extracts and their compound formulations with pharmacological effects such as antibacterial, antitumor, antioxidant, hypoglycemic, hypotensive, and estrogen-like activities [3]. Therefore, beer flowers have gradually emerged as a new focus in medical research.

This paper reviews recent advances in the pharmacological effects and mechanisms of beer flowers, providing a theoretical foundation for their further development, utilization, and functional research.

1 Active ingredients

1.1 Resins

Based on their solubility in organic solvents, they are classified into soft resins and hard resins. Soft resins refer to the portion soluble in low-boiling-point hydrocarbons, which are the primary sources of hops' aroma and bitterness. The main components are α- acid [4] and β- acid [5], both derivatives of ketones, which are highly reactive and easily oxidized or reduced; hard resins refer to the portion insoluble in hexane within total resins, classified as unidentified resins. Although α-acid does not contain hydroxyl groups in its molecular structure, it exhibits weak acidity due to the presence of an enol group. It has very low solubility in cold water and is only slightly soluble in boiling water. Under conditions of heating, dilute alkali, or light exposure, it readily undergoes isomerization reactions to form iso-α-acid. Isomeric α-acids have a strong bitter taste and are much more soluble than α-acids. They are the primary source of bitterness and preservative properties in beer. β-acids are present in lower concentrations than α-acids and have weaker bitterness, preservative properties, and solubility. They are more prone to oxidation, forming β-resins, which impart a mild bitterness to beer and serve to complement and refine its flavor.

1.2 Flavonoids

Flavonoids in hops are primarily classified into three categories: chalcones, flavones, and flavonols [6]. Flavonoids are a major active component in hops. Humulone [7] is a unique flavonoid component of hops that has garnered significant attention. It is produced by the hop cone glands and is a natural isoprenoid flavonoid compound.

1.3 Essential Oils

The volatile components in hops are collectively referred to as hop essential oils [8], primarily composed of hydrocarbons and oxygenated compounds, with a portion containing sulfur compounds. The aromatic compounds have an unpleasant odor and have a negative effect on the aroma of hops; whereas the oxygen-containing compounds have a mild and pure aroma (e.g., linalool has a woody aroma, and geraniol has a rose-like aroma), which are the primary components of the elegant aroma in beer.

1.4 Other

It also contains tannins, choline, fructose, glucose, lipids, waxes, etc. [8].

2 Pharmacological Effects

As a raw material for brewing beer, hops also have medicinal value, with the medicinal part being the unripe green flower spikes. It is commonly used in folk medicine for making tea. Research has revealed the following main pharmacological effects.

2.1 Antimicrobial

Studies have shown that both α- and β-acids in hops exhibit good antibacterial activity [9-10], particularly against Gram-positive bacteria (including Staphylococcus, Bacillus, Streptococcus, Micrococcus, and Mycobacterium tuberculosis), with significant inhibitory effects. Among these, the antibacterial activity of humulone in β-acids is stronger than that of lupulone in α-acids. This is primarily due to the high lipophilicity and large distribution coefficient of humulone, which allows it to easily penetrate the wax layer of Mycobacterium tuberculosis, exerting a special affinity and thereby inhibiting its growth.

Bogdanova et al. [11] studied the biological activity of humulone and caryophyllene in hops and the effects of humulone on sensitive and drug-resistant Staphylococcus aureus. They found that these compounds reduced the number of bacteria released from biofilms, with humulone exhibiting the strongest effect, followed by humulone; humulone and humulone not only penetrate biofilms and reduce the number of bacteria within them, but also reduce the number of surviving bacteria to zero at high concentrations (humulone 60 μg/ml, humulone 125 μg/ml). They reported in [12] that these components (including α-acid, β-acid, and humic acid) not only effectively inhibit G+ bacteria reference strains but also resistant strains resistant to methicillin and vancomycin. However, no inhibitory effect was observed against G- bacteria. Among the substances detected in hops, humulone was identified as the hop component with the most effective antimicrobial properties; in enterococci, α-bitter acid exhibited the strongest inhibitory effect.

2.2 Antioxidant

The intrinsic antioxidant capacity of hops [13-14] is primarily associated with the polyphenolic compounds present in hops. Guo Miao et al. [15] isolated polyphenolic extracts from spent hops and administered them orally to hyperlipidemic mice at doses of 200–800 mg/(kg·d) for five weeks. resulting in significant reductions in total cholesterol and triglyceride levels in the liver, as well as low-density lipoprotein cholesterol levels and atherosclerosis indices in the serum, while high-density lipoprotein levels in the serum significantly increased. Additionally, the levels of lipid peroxidation products (malondialdehyde) in mouse livers were significantly reduced, and the activity of catalase was significantly increased. The activity of superoxide dismutase in red blood cells and livers, as well as the activity of glutathione peroxidase in whole blood and livers, were all significantly increased. The results indicate that hop polyphenols have significant lipid-lowering and antioxidant effects on hyperlipidemic mice.

Japanese researchers have found [16] that the isohumulone in hops can prevent Alzheimer's disease (AD) and cognitive dysfunction. They discovered that isohumulone activates the peroxisome proliferator-activated receptor gamma (PPARγ), leading to an increase in microglia and inhibition of inflammatory responses. In experiments, oral administration of isohumulone to normal mice resulted in a significant increase in double-positive anti-inflammatory microglia and CD11b and CD206 in microglia in the brain; in AM5×FAD mice, immunohistochemical analysis showed that compared with the control group, reducing amyloid beta in the cerebral cortex by 21%, and significantly reducing inflammatory cytokines (such as IL-1β) and chemokines (including macrophage inflammatory protein-1α in the cerebral cortex).

Lu Xin et al. [17] investigated the in vitro and in vivo antioxidant activity of hop polyphenol extracts. In vitro, hop polyphenol extracts effectively scavenged reactive oxygen species and significantly inhibited Cu-VC-induced DNA oxidative fragmentation damage; in vivo, oral administration of 200–800 mg/kg of hop polyphenol extract significantly inhibited the decrease in superoxide dismutase and glutathione peroxidase activity in mouse livers induced by bromobenzene, and also reduced the content of thiothiobarbituric acid products in the livers of bromobenzene-induced oxidative stress mice.

Huang et al. [18] evaluated the therapeutic potential of the isoprenylated flavonoid quercetin in hops for stabilizing the expression of human Swedish mutant amyloid precursor protein (N2a/APP: a cell model that can adequately characterize AD) in mouse neuroblastoma N2a cells. ELISA and Western blot analyses showed that humulone inhibited Aβ accumulation and APP processing; in N2a/APP cells, goldenseal extract improved the hyperphosphorylation of tau protein through the PP2A and GSK3b pathways; the improvement of tau protein hyperphosphorylation by goldenseal extract was validated in HEK293/Tau cells (another cell line with high tau protein hyperphosphorylation). These results suggest that humic acid may have potential therapeutic effects for AM and/or neurodegenerative diseases associated with neuropathology.

2.3 Antitumor

Humulone in hops exhibits antitumor activity. Jiang et al. [19] investigated the anticancer mechanism of humulone and found that the upregulation of pro-apoptotic proteins (Bax, PARP, AIF, protease-3, protease-8, and protease-9) and downregulation of anti-apoptotic proteins (Bcl-2) were involved in xanthohumol-induced cancer cell apoptosis. Additionally, reduced expression of Notch1, mTOR, and STAT3 mediated the inhibition of non-cancer cell proliferation. Furthermore, humic acid induces cell cycle arrest by regulating the expression of p53, p21, and cyclin D1. Downregulation of FAK and MMP-2 expression contributes to the migration and invasion of cancer cells by humic acid. MDR1, EGFR, and STAT3 are downregulated in MCF-7/ADR cells treated with humic acid, suggesting that humic acid may synergize with current traditional chemotherapy. Regarding the effects of humic acid on ERK1/2 phosphorylation, conflicting results have been reported. For example, humic acid treatment increased ERK1/2 phosphorylation in thyroid cancer cells while decreasing it in laryngeal cancer cells. This discrepancy may be due to the cell-specific nature of humic acid's effects on ERK1/2 phosphorylation. In summary, humic acid does indeed influence a series of key proteins associated with cancer cell proliferation, apoptosis, migration, invasion, and multidrug resistance.

2.4 Hypoglycemic effects

Recent studies have found that certain components in hops also possess hypoglycemic effects and can inhibit the onset of diabetes. Seliger et al. [20] reported that humic acid, isohumulone, and 8-PN are effective non-competitive tight-binding inhibitors of human aldose reductase AKR1B1 (a key enzyme in the treatment of diabetes complications) and AKR1B10 (a key enzyme in cancer treatment), while the activity of the related enzyme AKR1A1 is not affected by these three compounds.

Sumiyoshi et al. [21] investigated the effects of hop extract on obesity in mice fed a high-fat diet. Hops extract can inhibit the increase in fat tissue weight, fat cell diameter, and liver lipid levels induced by a high-fat diet, improve impaired glucose tolerance, and increase water excretion in mice with water overload; its various components can inhibit lipid accumulation and PPARγ expression in 3T3-L1 fat cells.

Obara et al. [22] randomly divided 94 diabetic patients into four groups and conducted a 12-week double-blind dose study. Participants consumed placebo capsules or capsules containing 16, 32, or 48 mg of isoxanthenol daily. After 4 weeks of treatment, fasting blood glucose levels decreased in the 32 mg and 48 mg groups, while no changes were observed in the placebo group. After 4 weeks of treatment with 16 mg and 8 weeks with 32 mg and 48 mg, glycated hemoglobin (HbA1c) levels significantly decreased; at 12 weeks, the body mass index (BMI) of the 48 mg group was significantly lower than that of the placebo group, and the reduction in total fat area was also significantly greater than that of the placebo group. The study demonstrated that the intake of isosapogenin reduced fasting blood glucose levels and confirmed that long-term blood glucose control was due to the decrease in HbA1c, while also confirming that daily intake of 32 mg of isosapogenin can improve hyperglycemia.

2.5 Hepatoprotective effects

Zhou et al. [23] further investigated the hepatoprotective effects of hop flavonoids (HF). The results showed that hop flavonoids significantly reduced serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), mouse tumor necrosis factor-α (TNF-α), and thiobarbituric acid reacting substances (TBARS) in liver tissue induced by CCl₄, while increasing the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and catalase (CAT) in liver tissue.

tissue. Histopathological studies confirmed that it has a protective effect against CCl₄-induced liver damage. Western blotting indicated that pretreatment with HF downregulated The expression of cleaved caspase-3, BAX, and TNF-α, while increasing the expression of BCL-2. Studies suggest that the hepatoprotective effects of HF may act through reducing oxidative stress, inhibiting inflammatory responses, and apoptosis.

Hege et al. [24] investigated the effects of isohumulone in hops on acute alcoholic fatty liver degeneration in mice. The study found that acute ethanol administration led to significant fat accumulation in the liver, increased nitric oxide production, and elevated levels of plasminogen activator inhibitor-1 protein compared to the control group, with a significant increase in inducible nitric oxide synthase protein levels. In mice pretreated with isohumulone, the effects of alcohol were significantly attenuated; in J774A.1 macrophages pretreated with isohumulone, the mRNA expression of inducible nitric oxide synthase and interleukin-6, as well as nitric oxide release, were significantly attenuated in response to lipopolysaccharide induction.

Fernández-García et al. [25] reported that ferulic acid has a protective effect against age-related liver changes. After treatment with ferulic acid, age-related oxidative stress, inflammation, and apoptosis were significantly reduced; in most cases, this effect was dose-dependent. Therefore, humic acid can significantly regulate inflammation, apoptosis, and oxidative stress in aged livers, exerting a protective effect on liver changes.

2.6 Estrogen-like effects

Menopause is an important stage in the physiological process of women, characterized by ovarian function decline, hormonal imbalances caused by reduced secretion of various hormones and gonadotropins, and decreased hormone levels, which can lead to a series of menopausal symptoms. 8-PN[26] in hops is an isomer of demethyl-humulone, a compound with the chemical formula C₂₀H₂₀O₅, and is currently the most effective plant estrogen isolated. 8-PN promotes the formation of osteoblasts in MC3T3-E1 cells, inhibits their absorption, increases alkaline phosphatase (ALP) activity, bone calcium concentration, and mineralization nodules, and also inhibits the differentiation of RAW264.7 osteoclast-like cells into osteoclasts. inhibits the expression of proteins and mRNA of nuclear factor κB co-activator receptors in osteoblasts; conversely, it promotes the expression of osteoprotegerin (OPG). The effect of 8-PN is stronger than that of two plant estrogens, genistein and daidzein [27].

Keiler et al. [28] investigated the safety of chemically standardized beer flower extract in the uterus and its effect on preventing bone loss in an ovariectomized rat model. They ovariectomized female Wistar rats and divided them into a control group with a diet free of plant estrogens, a treatment group with a diet containing 0.93 mg/(kg·d) of E2 benzoate, and a treatment group with a diet containing 60 mg/(kg·d) of standardized hops flower extract. The rats were fed for 8 weeks, and micro-computed tomography of the tibia and vertebrae, as well as histological changes in the uterus and tibia, were analyzed. The results showed that in the treatment group administered hops extract orally for 8 weeks, no uterine weight gain or hyperplasia was observed in the endometrium, but site-dependent skeletal effects were observed. Hops extract significantly reduced the number of osteoclasts at the metaphysis of the tibia and prevented the reduction in trabecular thickness caused by estradiol depletion; however, hops extract could not prevent the microstructural changes in the lumbar vertebrae induced by ovariectomy, and some parameters (such as trabecular thickness and number) were even lower than those measured in the ovariectomized control group.

2.7 Antiviral

Hops exhibit antiviral activity. Humulone in hops not only inhibits bovine viral diarrhea virus (a surrogate model for hepatitis C virus) but also has potential for hepatitis C virus research (due to its ability to inhibit hepatitis C virus replication in cell culture systems harboring replicative hepatitis C virus RNA replicons). Lou et al. [29] found that humulone at a concentration of 3.53 μmol/L significantly inhibited hepatitis C virus RNA levels, indicating that humulone has antiviral activity against hepatitis C virus.

2.8 Reversal of drug resistance

Liu et al. [30] further revealed another mechanism of geosmin's reversal of drug resistance. In the MCF-7/ADR cell line, geosmin was found to inhibit the efflux function of the multidrug resistance protein (ABCB1), and it was observed that geosmin is a substrate of ABCB1 and stimulates its ATPase activity. The results showed that yellow rot extract exhibits synergistic effects with colchicine, an ABCB1 substrate, in MCF-7/ADR cell lines. Yellow rot extract binds to the central transmembrane domain site, which overlaps with the binding site of doxorubicin. This mechanism is supported by molecular modeling and simulation data, which show that yellowish-brown polyphenol binds to the transmembrane domain of ABCB1, while doxorubicin also binds to the transmembrane domain of ABCB1. However, yellowish-brown polyphenol exhibits higher binding affinity than doxorubicin, resulting in smaller fluctuations in protein and ligand positions. The results indicate that humulone inhibits doxorubicin transport mediated by ABCB1, stimulates ABCB1 ATPase activity, and acts as a substrate for ABCB1 to achieve an effect of reversing drug resistance.

3  Outlook

In summary, hops exhibit a wide range of pharmacological effects. Among these, the α-acid, β-acid, and humulone components of hops have been extensively studied. α-Acid undergoes isomerization to form highly unstable isomers, which are easily decomposed upon exposure to light. To address these issues, the chemical structure of α-acid was modified to obtain more photostable α-acid derivatives: dihydro-, tetrahydro-, and hexahydro-iso-α-acid. β-acid generally does not undergo isomerization, but under certain conditions, β-acid can undergo hydrogenation reactions to form more stable and active hexahydro-β-acid.

Similarly, by modifying humic acid, isohumic acid and dehydrocyclic humic acid were obtained. Among these, humic acid and isohumic acid can inhibit the growth of certain specific human cancer cells without affecting the growth of non-cancerous cells in animals. Hops, as a traditional Chinese medicine, although some reports have been published on the active components and mechanisms of action of its active parts, there are few reports on its clinical application. In terms of its application, it has been widely used in food, alcohol manufacturing, sugar industry, and pharmaceutical industry both domestically and internationally. As a natural plant, hops have clear antibacterial, antioxidant, antitumor, and hypoglycemic effects. With the continuous deepening of research into the active components and effective constituents of hops, this traditional Chinese medicine will play an increasingly important role.

References

[1]Chinese Academy of Sciences. Flora of China [M]. Beijing: Science Press, 2013.

[2]Zhang Xia, Xu Jiping, Liu Haiying, et al. Geographic distribution of hops and wild hops in China [J]. Arid Land Resources and Environment, 2008, 22(1): 180-183.

[3] Zhu Dan, Niu Guangcai, Jiang Shujun, et al. Chemical Constituents and Pharmacological Effects of Hops [J]. Pharmaceutical Special Topics, 2008, 17(21): 1-2.

[4] Yan Jingjing, Liu Shumin, Guo Zeqing, et al. Study on the Influencing Factors of α-Acid Isomerization Reaction in Hops Extract [J]. Food Research and Development, 2016, 37(10): 28-29.

[5] Shi Jie. Research Progress on Acidic Compounds in Hops [J]. China New Technology and New Products, 2011, 11(12): 5-6.

[6] Zhang Weiku, Wang Shoubao, Li Ping, et al. Flavonoid Components of Hops [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 38(10): 1539-1541.

[7] Cao Yan, Feng Zuoshan, Wang Juan, et al. Effects of Hops Polyphenols on Lipid Metabolism in Ovariectomized Rats [J]. Food Science and Technology, 2015, 40(3): 221-223.

[8] Lin Liuyue, Jiang Yiping, Zhang Qiaoyan, et al. Research Progress on the Chemical Constituents and Pharmacological Activities of Hops [J]. Chinese Journal of Traditional and Herbal Medicine, 2017, 42(10): 1830-1834.

[9] Li Xianzhen, Wang Wei, Du Guocheng, et al. Research progress on the resistance mechanism of hops [J]. Journal of Microbiology, 2015(5): 1-5.

[10] Gao Zhiming. Antimicrobial activity and application expansion of hops [J]. Food Industry Science and Technology, 2012, 33(23): 428-430.

[11] Bogdanova K, Roderova M, Kolar M, et al. Anti-biofilm activity of bioactive hop compounds humulone, lupulone, and xanthohumol toward susceptible and resistant Staphylococcus aureus [J]. Research in Microbiology, 2018, 169(3): 127-134.

[12] BOGDANOVA K, KOLAR M, LANGOVA K, et al. Inhibitory effect of hop fractions against Gram-positive multi-resistant bacteria [J]. Biomed Papers, 2018, 162: 1-7.

[13] GU Xiaofeng. Overview of research on the natural product humulone [J]. Asia-Pacific Traditional Medicine, 2015, 11(10): 66.

[14] LI Yujing, LIU Yumei. Study on the correlation between active components of hops and antioxidant activity [J]. Food Science, 2018, 2(28): 1-7.

[15] Guo Miao, Yang Xiaolan. Lipid-lowering and antioxidant effects of hop polyphenols on hyperlipidemic mice [J]. Food Science, 2015, 36(3): 183-186.

[16] ANO Y, DOHATA A, TANIGUCHI Y, et al. Iso-α-acids, bitter components of beer, prevent inflammation and cognitive decline induced in a mouse model of Alzheimer's disease [J]. J Biol Chem, 2017, 9 (292): 3720.

[17] Lu Xin, Yang Xiaolan. Study on the antioxidant activity of hop polyphenol extracts in vitro and in vivo [J]. Food Science, 2015, 36(1): 13-17.

[18]HUANG X F , WANG J , CHEN X , et al.  The p renylflavonoid xanthohumol  reduces  Alzheimer-like  changes  and  modulates multiple  pathogenic  molecular  pathways in the Neuro2a/ App swe cell model of AD [J] . Front Pharmacol , 2018 , 9 : 199.

[19]JIANG C H , SUN T L , XIANG D X , et al. Anticancer activity and mechanism of xanthohumol : A p renylated flavonoid from Hops ( Humulus lupulus  L. )  [J] . Front  Pharmacol , 2018 , 9 (530) : 1-11.[20]SELIGER J M , MISURI L , MASER E , et al. The hop-derived compounds xanthohumol , isoxanthohumol  and 8-p renylnarin- genin are  tight-bindinding  inhibitors  of  human  aldo-keto  re- ductases 1B1and 1B10 [J] . J Enzyme Inhib Med Chem , 2018 ,  33(1) : 607-614.

[21]SUMIYOSHI M , KIMURA Y. Hop  ( Humulus lupulus  L. )  extract inhibits  obesity  in  mice  fed  a  high-fat  diet  over  the long term [J] . Br J Nutr , 2013 , 109(1) : 162-172.

[22]OBARA K , MIZUTANI M , HITOMI Y , et al.  Isohumulones ,  the bitter component of beer , improve hyp erglycemia and de- crease body fat inJapanese subj ects with p rediabetes [J] . Clin Nutr , 2009 , 28  (3) :278-284.

[23]ZHOU D , WANG C G ,  LI  X ,  et  al. Dietary  functional  fla- vonoids as natural hepatoprotective agents against acute liver injury from hop  ( Humulus lupulus  L. )  [J] . J  Func  Foods ,  2018 , 45 :471-479.

[24]HEGE M , JUNG  F , SELLMANN C , et al. An iso-α-acid-rich extract from hops (Humuluslupulus)  attenuates acute alco- hol- induced liver  steatosis  in  mice  [J] .  Nutrition , 2018 , 45 :  68-75.

[25]FERNANDEZ-GARCIA  C , RANCAN  L , PAREDES  S  D ,et al. Xanthohumol  exerts  p rotective  effects  in  liver  altera- tions associated with aging [J] . Eur J Nutr , 2018 , 3 : 1-10.

[26]Cui, Y., Chen, J., Yan, J., et al. Research progress on the biological activity and quantitative analysis of 8-PN, a phytoestrogen component in hops [J]. Journal of Nanchang University (Medical Edition), 2016, 56(5): 97-99.

[27]LUO D , KANG L , MA Y , et al.  Effects and mechanisms of 8- p renylnaringenin on osteoblast MC3T3-E1and osteoclast-like cells RAW264.  7  [J] . Food Sci Nutr , 2014 , 2(4) :341-350.

[28]KEILER A M , HELLE J , BADER M I , et al. A standardized Humuluslupulus  (L. )  ethanol  extract  partially prevents o- variectomy-induced bone loss in rat without induction of ad- verse  effects  in  the  uterus  [J] .  Phytomedicine ,  2017 ,  34 :  50-58.

[29]LOU S , ZHENG Y M , LIU S L , et al.  Inhibition of hepatitis C virus replication  in  vitro  b y  xanthohumol , a  natural  p roduct present in Hops [J] . Planta Med , 2014 , 80(2-3) : 171-176.

[30]LIU F M , YIN  H , DONG J J , et al. Experimental and simula- tion  identification  of  xanthohumol  as  an  inhibitor  and  sub- strate of ABCB1  [J] . Appl Sci , 2018 , 8(5) : 681.