Iso-α-acids in Nonalcoholic and Alcoholic Beer Stimulate Growth of Neuron-like SH-SY5Y Cells and Neuroepithelial Stem Cells

With the increasing popularity of nonalcoholic beer, the association between beer drinking and alcohol intake is lost. In the present study, we show that nonalcoholic beer can stimulate the expansion of neuron-like cell lines and neuroepithelial stem cells in culture, yielding an effect comparable to that of alcoholic beer. One ingredient in beer is hops, which is derived from the flower of hop plants. The female flower contains humulones, which are transformed into iso-α-acids during wort boiling and give beer its bitter taste. In this study, we tested the effects of these iso-α-acids and/or alcohol on the proliferation of neuron-like cells and neuroepithelial stem cells in culture. Iso-α-acids enhanced cell expansion, showing a bimodal dose–response curve with peaks around 2–30 nM and 2–5 μM, of which nanomolar concentrations are relevant in beer drinking. The more lipophilic trans-iso-α-acids, found to a greater extent in beer foam, are even more potent. Our results indicate that iso-α-acids, acting via peroxisome proliferator-activated receptors could be responsible for the observed effects. Altogether, our results indicate that nonalcoholic beer with ingredients such as iso-α-acids stimulate the proliferation of neuroepithelial stem cells.


■ INTRODUCTION
Beer has been part of the human diet for over 7000 years and is the most consumed alcoholic beverage in the world. There are risks with drinking too much alcoholic beer, and the national recommendations of a maximum 7 standard drinks per week for women and 14 for men is now even reduced in some countries. While too much alcoholic beer can give consequences such as alcoholism and addiction, low to moderate beer intake has on the other hand been considered healthy. 1 Whether these health effects come from the alcohol or from other ingredients in beer is, however, not completely understood, and the health effects of nonalcoholic beer compared with alcoholic beer are relatively unexplored.
During the 21st century, there has been a trend toward a healthier lifestyle, including the development and increased consumption of nonalcoholic beer. 2 Nonalcoholic beer has increased in popularity and is now also brewed by many microbreweries. Consumers prefer it not only because it is innovative and trendy but also because of increased health awareness. Nonalcoholic beer is low in calories, is considered safe, and can even be consumed during pregnancy. It has also become an attractive alternative to alcoholic beer in countries where the use of alcohol is restricted.
Beer contains a variety of nutrients and bioactive compounds with different health-promoting effects. 1 One of the main ingredients in beer is hops from the female flower of the hop plant, Humulus lupulus L. As a plant from the Cannabaceae family, hops contain many bioactive compounds. 3 Hops have been used in brewing since the Middle Ages and in traditional medicine since ancient times. 4,5 Among the bioactive compounds are α-acids, which are found in the glands of the flowers. During wort boiling, these α-acids are isomerized to iso-α-acids, the bitter component in beer 4 (Figure 1).
Many neurodegenerative disorders are characterized by neuroinflammation and cell death, where age is the main risk factor. In an aging society, there is an increasing need for compounds that protect against cell death and that stimulate neurogenesis. Ingredients in beer, other than alcohol, may have preventive effects on neurodegeneration. Iso-α-acids have been shown to be neuroprotective, can penetrate into the brain, 6 and have been shown to reduce levels of amyloid β, improve microglia function, suppress inflammation, and improve memory function. 6−9 Whether iso-α-acids also generate neurogenesis is not well-studied.
To determine whether the neuroprotective effects of alcoholic beer are due exclusively to ethanol or are due, in part, to nonalcoholic components, we performed cell proliferation assays with neuron-like cell lines and human neuroepithelial stem (NES) cells. The effects of nonalcoholic beer, with iso-αacids and very low levels of ethanol, were compared against alcoholic beer, with iso-α-acids at the same level. The potency of the more lipophilic trans-iso-α-acids, found in a greater extent in beer foam, was also studied. In addition, possible actions of trans-iso-α-acids via peroxisome proliferator-activated receptors (PPARs) were studied, as iso-α-acids previously have been shown to act via PPAR. 10 It has been shown that ligands of the cannabinoid receptor CB 1 act either directly or indirectly via PPAR. 11 As hops belong to the Cannabacea family, possible actions via the CB 1 receptor were also investigated.

Iso-α-acids
Isomerized hop extract, Hopsteiner Iso-Extract 30%, kindly provided by Hopsteiner (Mainburg, Germany), was purified at SciLifeLab, Stockholm, Sweden. Preparative high performance liquid chromatography (HPLC) was performed on a Gilson 305 HPLC system using an acidic eluating protocol. For acidic purification, the Gilson 305 HPLC system was equipped with an ACE 5 C8 (5 μm, 30 mm × 150 mm) column, and the compounds were eluted using a gradient system of acetonitrile and H 2 O containing 0.1% TFA. Analytical reversed-phase liquid chromatography−mass spectrometry (RPLC-MS) was performed using an Agilent/HP 1200 system 6110 mass spectrometer with electrospray ionization (ESI+). For the HPLC-MS method, the ACE C18 3.5 μm column (3.0 mm × 50 mm) was used. The mobile phase [0.1% TFA/CH 3 CN]/[0.1% TFA/H 2 O] and positive electrospray ionization was used for detection/characterization. The isomer fractions were combined, dried, and dissolved in 10 mM dimethyl sulfoxide (DMSO). The purity of the collected isomers were 87% (UV, 254 nm) considering peaks with a retention time between 0.7 and 3.5 min and with a height above 30 mAU (Supporting Information, Figures S1−S3). For comparison between different iso-α-acids, Thermo Scientific BeerNHop Solution (in methanol, MeOH) at a concentration of 276 μM was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The initial experiments were done with iso-αacids from Hopsteiner (purity 87%) and confirmed with Thermo Fisher (purity >95%). Both sources yielded equivalent results.
Trans-iso-α-acids, DCHA-Iso, ICS-I4, was purchased from Labor Veritas AG (Zurich, Switzerland) and dissolved in DMSO at a concentration of 100 mM. Isomers from different iso-α-acids are illustrated in Figure 1.

Cells and Cell Cultures
The human neuroblastoma SH-SY5Y cell line and Neuro-2a (N2a) cell line from mice, both from American Type Culture Collection (Manassas, VA, USA), were cultured in nontreated flasks in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (all from Gibco, Thermo Fisher Scientific) at 37°C in a humidified atmosphere containing 5% CO 2 . Cell culture medium was changed every second to third day. Trypsin-EDTA (Gibco) was used to dissociate adherent cells from the flask during routine cell culture passaging.
Carlsberg Alcohol Free (0.5% ABV, alcohol by volume) or Carlsberg Export (5.0% ABV) were used in beer-treatment experiments. Both Carlsberg Alcohol Free and Carlsberg Export are pilsner style beer containing 18 international bitterness units (IBU) or 18 mg of iso-αacids/L (Sweden). The volume of beer was added to reach a certain EtOH or iso-α-acid concentration. In beer-treatment experiments, 3.5 × 10 4 SH-SY5Y cells/well were plated and treated with Carlsberg Alcohol Free (0.5% ABV) or Carlsberg Export (5.0% ABV) for 4 days. N2a cells were seeded at a density of 1 × 10 4 cells/well in nontreated 24-well culture plates (Sarstedt) in 500 μL of DMEM/well supplemented as indicated above. N2a cells were cultured without treatment or with ethanol or iso-α-acids without/with ethanol; transiso-α-acids or cis-ρ-iso-α-acids for 4 days.
The NES cells were cultured in 20 μg/mL poly-L-ornithine and 2 μg/ mL laminin-coated 24-well culture plates (Sarstedt) in 500 μL of NES medium supplemented as indicated above. The NES cells were seeded at a density of 1.5 × 10 4 cells/well and cultured without treatment or with ethanol, iso-α-acids purified from Hopsteiner Iso-Extract 30% (Hopsteiner) without/with ethanol or trans-iso-α-acids for 7 days. In beer-treatment experiments, NES cells were seeded at a density of 5 × 10 4 cells/well and treated with Carlsberg Alcohol Free (0.5% ABV) or Carlsberg Export (5.0% ABV) for 3 days. Treatments are listed in Table  S1, Supporting Information. After the initiation of treatments, the cell culture medium was not changed. In control experiments with medium only and no treatment, the cell culture medium was replaced in the same way as in the treatment experiments.

Flow Cytometry
Cells were analyzed on a BD FACSVerse flow cytometer with BD FACSuite software (BD Biosciences, San Jose, CA, USA). Further analyses were performed using the software Flowjo version 10 (Flowjo, LLC, Ashland, OR, USA). SH-SY5Y cells were stained with 2 μM CFSE (Invitrogen, Carlsbad, CA, USA), incubated at 37°C for 15 min in a humidified atmosphere containing 5% CO 2 and washed twice in phosphate-buffered saline (Sigma) supplemented with 0.5% fetal bovine serum (Gibco). Cells stained with CFSE were then incubated as described above.

Evaluation of Cell Expansion and Proliferation
After completing the incubation, the cells were deattached with trypsin-EDTA (Gibco) for SH-SY5Y or N2a cells and TrypLE Express (Gibco) for NES cells, assessed for viability using Trypan blue (Sigma) and counted with a Blaubrand Burker hemocytometer (Sigma). Cell growth was quantified as the total number of cells on day 3 (NES), 4 (SH-SY5Y, N2a, NES), or 7 (SH-SY5Y, NES) divided by the number at the start and compared with the growth in the control wells assessed in the same way. CFSE-based cell proliferation was analyzed with flow cytometry described above and quantified as the percentage of CFSE low cells in treated cells compared with the percentage in control wells. The same evaluation procedure was also applied using the BrdU + in the proliferation assay.

Expansion Assay with Agonist and Antagonists
SH-SY5Y cells (1 × 10 5 cells/cm 3 ), cultured in nontreated 24-well culture plates (Sarstedt) for 48 h, were treated with trans-iso-α-acids in the concentration range of 276 pM to 2.76 μM without/with antagonists of PPARα, PPARγ, or cannabinoid-1 (CB 1 ) receptor at twice the concentration of trans-iso-α-acids. The cells were deattached with trypsin-EDTA (Gibco); the number of cells per well was determined with a hemocytometer, and expansion was calculated as described above. The following three antagonists were used: PPARα antagonist GW6471 (IC 50 0.24 μM) and PPARγ antagonist GW9662 (IC 50 3.3 nM) (both from Sigma) and CB 1 receptor antagonist Ibipinabant (IC 50 22 nM; Medchemtronica, Sollentuna, Sweden). In the experiments with the positive control, 1 × 10 4 SH-SY5Y cells/wells were seeded in nontreated 24-well culture plates and treated with bezafibrate (Sigma), a pan-agonist of PPAR with EC 50 of 50, 60, and 20 μM for PPARα, γ, and δ, respectively, for 7 days. The expansion was determined as described above.

Data Analysis
The number x of independently repeated experiments is designated by "n = x". Significant differences between mean values were determined by Wilcoxon's signed rank-test of paired t-test, two-sided, where p values (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) were considered statistically significant. Data are reported as means ± standard error of the mean (SEM). GraphPad Prism 9 (Graph Pad Software, San Diego, CA, USA) was used for analysis. Concentrations of iso-α-acids and ethanol varied independently or in combination are presented in 3D surface response or 2D contour plots using SigmaPlot versions 12−14.

Nonalcoholic and Alcoholic Beer Similarly Stimulate Growth of SH-SY5Y and NES Cells
Using equivalent volumes of Carlsberg Alcohol Free (0.5% ABV, resulting in 0.2−2 mM ethanol) and Carlsberg Export (5% ABV, resulting in 2−20 mM ethanol), we observed that nonalcoholic and alcoholic beer similarly stimulate growth (expansion) of SH-SY5Y and NES cells (Figure 2A,B). The nominal concentrations of ethanol are shown in Figure 2.
Compared to the untreated controls both nonalcoholic and alcoholic beer significantly increase cell expansion. There are, however, no differences between groups with equal iso-α-acid but different alcohol concentrations. This result indicates that nonalcoholic beer contains at least one substance that stimulates the expansion of SH-SY5Y and NES cells. To test whether iso-αacids could cause this effect, we examined the effect of iso-αacids on expansion of neuron-like cell lines and NES cells in culture. We also examined if such effects could be enhanced in combination with ethanol. EtOH (green bars), or alcoholic beer (Carlsberg Export, 5.0% ABV), equivalent to 2−20 mM EtOH (black bars). Bars represent mean ± SEM. Friedman's test shows that stimulation compared to controls was significantly different for both SH-SY5Y (p = 0.0253) and for NES cells (p = 0.0015). Using Wilcoxon's two groups test, we find no statistically significant differences between any two groups of equal iso-α-acids content for either SH-SY5Y cells or NES cells without alcohol (nonalcoholic beer) compared with 10 times more alcohol (alcoholic beer). Values normalized to control experiments where no iso-α-acids were added (white bars). Statistics performed by Wilcoxon's signedrank test, two-sided, comparing each group with controls, *p < 0.05.  Figure 3A). We performed repeated experiments and found that all treatments with iso-α-acids between 2.76 and 276 nM gave significantly higher expansion, p < 0.05 ( Figure 3B). The highest mean ratio was found for 2.76 nM (mean ratio 1.6 ± 0.14, p < 0.0001, n = 31) and for 27.6 nM (mean ratio 1.6 ± 0.3, p = 0.0006, n = 29). Iso-α-acid stimulation was statistically significant and higher compared to the effect of the known PPAR agonist bezafibrate ( Figure 3C). The kinetics of cell expansion using different treatments with iso-α-acids and ethanol showed increased cell expansion for 7 days ( Figure 3D). The solvents MeOH or DMSO showed no effect per se ( Figure 3E). NES cells cultured for 7 days with iso-α-acids at different concentrations showed a bimodal dose−response relation with peaks at low nM and μM concentrations, and the solvent DMSO showed no effect ( Figure 3F) .
To quantify proliferation, SH-SY5Y cells were stained with carboxyfluorescein succinimidyl ester (CFSE). The number of cell divisions after 7 days was increased at 2.76 nM iso-α-acids compared with that of the control ( Figure 4A). The increased number of SH-SY5Y cells in the S-phase after stimulation with iso-α-acids was also demonstrated with 5-bromo-2′deoxyuridine (BrdU) at 2.76 nM iso-α-acids ( Figure 4B). These results indicate that iso-α-acids stimulate the expansion of SH-SY5Y and NES cells.

Ethanol Stimulates Expansion of SH-SY5Y and NES Cells
SH-SY5Y cells were incubated for 7 days with different concentrations of ethanol. We found that ethanol alone also stimulates cell expansion. The dose−response curve is bellshaped, with a peak for ethanol concentration below 10 mM ( Figure 5A).
Treatment with ethanol at concentrations of 2−10 mM gave a significantly higher expansion of SH-SY5Y cells (p < 0.05) ( Figure 5B), and treatment with 7.5 mM gave the highest mean ratio 1.6 ± 0.2 (p = 0.0049, n = 11). To demonstrate proliferation, SH-SY5Y cells were stained with CFSE and treated with 2 mM ethanol for 7 days. The number of cell divisions measured as CFSE low was higher for cells treated with ethanol compared to that with the control ( Figure 4A). NES cells treated for 7 days with ethanol showed that the dose−response curve is bell-shaped with a peak at around 10 mM ethanol ( Figure 5C). These results indicate that ethanol on its own can stimulate expansion of both SH-SY5Y and NES cells.

Ethanol Enhances Expansion of Iso-α-acid-Treated SH-SY5Y and N2a Cells at Certain Concentrations
As both iso-α-acids and ethanol stimulate the expansion of SH-SY5Y and NES cells, we asked what the combined effects would be. Accordingly, we treated SH-SY5Y and N2a cells with combinations of iso-α-acids and ethanol. Figure 6 shows how independently varying the concentration of iso-α-acids and ethanol stimulates expansion of the SH-SY5Y and N2a cells, presented using both 3D surface plots and 2D contour plots.
The volumes of nonalcoholic beer (0.5% ABV) result in alcohol concentrations equivalent to 0.2−2 mM, and equivalent volumes of alcoholic beer (5% ABV) result in alcohol concentrations equivalent to 2−20 mM (Figure 2). The two beer types have the same bitterness of 18 IBU equivalent to 50 μM, and therefore, the different concentrations of iso-α-acids added with beer give the same concentrations of iso-α-acids, calculated to be between 117 nM to 1.17 μM.
As shown in Figure 6, the highest iso-α-acid-induced effect on SH-SY5Y and N2a cells is in the concentration range of 0.1 to 10 nM. Stimulation with iso-α-acids at 117 nM to 1.17 μM is more or less independent of the effects of alcohol at 0.1 to 10 mM EtOH ( Figure 6)concentrations relevant in beer drinking. This may also explain the observed lack of differences between   Groups with increasing alcohol and iso-α-acid concentrations showed significant stimulation compared to controls for both SH-SY5Y (p = 0.0081) and NES cells (p = 0.0081). Using Wilcoxon's test, we found no difference, however, between groups for either SH-SY5Y or NES cells when comparing the same amounts of iso-α-acids without alcohol (nonalcoholic beer) with 10 times more alcohol (alcoholic beer).

ACS Bio & Med Chem Au
pubs.acs.org/biomedchemau Article alcoholic beer and nonalcoholic beer following addition to SH-SY5Y and NES cells.

Iso-α-acids in Beer as an Explanation for the Expansion of SH-SY5Y and NES Cells with Beer
To further demonstrate the effects of iso-α-acids on cell expansion, SH-SY5Y and NES cells were treated with Carlsberg Alcohol Free (0.5% ABV) and Carlsberg Export (5% ABV) with volumes equivalent to iso-α-acid concentrations of 1 nM to 2.76 μM, yielding nominal EtOH concentrations of 0.002−4.8 mM for the nonalcoholic beer and 0.02−48 mM for the alcoholic beer. As shown in Figure 7, SH-SY5Y cells and NES cells expand when treated with either of the two beer types and with different concentrations of iso-α-acids, confirming our results in Figure  3A,F.

Effects of Trans-iso-α-acids on Expansion of SH-SY5Y and NES Cells
In beer, there are different iso-forms of iso-α-acids as well as reduced forms of iso-α-acids. To investigate the effects of αacids and their derivatives on cell growth, different cell lines were treated for 7 days with different forms of iso-α-acids, as listed in Table S1, Supporting Information.
Treatment with trans-iso-α-acids showed a bimodal bellshaped dose−response curve like those with iso-α-acids, although incubating with trans-iso-α-acids gave a peak at a lower concentration ( Figure 8A). Treatment with trans-iso-αacids resulted in significantly greater expansion of SH-SY5Y cells at 2.76 pM to 27.6 nM and 2.76 μM (p < 0.05) ( Figure 8B), and treatment with 276 pM gave the highest mean ratio of 1.5 ± 0.08 (p = 0.0005, n = 8). NES cells treated with trans-iso-α-acids also yielded a bimodal dose−response curve with a peak at a lower concentration, (Figure 8C). These results suggest that the transform has a higher potency compared with the cis-form.
PPARα, PPARγ, and CB 1 Antagonist Effects on Trans-iso-α-acid-Stimulated SH-SY5Y Cells Iso-α-acids act via the peroxisome proliferator-activated receptors, 10 and since trans-iso-α-acids most potently activate SH-SY5Y cells, we investigated if PPARα and PPARγ mediate the effects at the low concentration measured. As hops belong to the Cannabaceae family, we likewise investigated if the cannabinoid receptor CB 1 , upstream in PPAR signaling, 11 is involved in the observed cell expansion. SH-SY5Y cells were stimulated for 48 h with 276 pM to 2.76 μM trans-iso-α-acids together with twice the concentration of the PPARα antagonist GW6471, PPARγ antagonist GW9662, and the cannabinoid receptor CB 1 antagonist Ibipinabant. Treatment with 2.76 and 27.6 nM trans-iso-α-acid led to increased cell expansion as compared to media alone ( Figure 9A). Expansion was inhibited by either the PPARα antagonist GW6471, the PPARγ antagonist GW9662, or the cannabinoid receptor CB 1 antagonist Ibipinabant (p < 0.05) ( Figure 9B). These results indicate that PPARα, PPARγ, and CB 1 mediate the effects of trans-iso-α-acids at low nM concentrations.

■ DISCUSSION
Our study indicates that nonalcoholic beer can stimulate expansion of neuroepithelial stem cells in culture, and that isoα-acids from the hop plant could possibly explain this expansion. Alcoholic and nonalcoholic beer give effects similar to those of iso-α-acids alone, which supports our conclusions. We show further that iso-α-acids can stimulate SH-SY5Y and neuroepithelial stem cells and do so within a nanomolar concentration range relevant to concentrations reached in beer drinking. Concentrations of trans-iso-α-acids reach approximately 40 nM in blood 30 min after drinking two bottles of low-hopped beer 14 and to around 150 nM in post-mortem cases from people who had been drinking beer. 15 Iso-α-acids can also pass the blood− brain barrier. 6 An alcohol range of less than 10 mM, which on its own can expand SH-SY5Y and neuroepithelial stem cells, is also relevant to what is found with moderate alcohol intake. Thirty minutes after drinking two bottles of alcoholic beer (5.3% ABV), the blood alcohol concentration is 32 mg/100 mL, and two bottles would equal around 6−7 mM. 14 We have earlier shown that around 2.5 mM ethanol can enhance the expansion of γδ T cells stimulated with isopentenylpyrophosphate. 16 We expected to find an enhancing effect of alcohol on iso-α-acid-stimulated SH-SY5Y and NES cells, as well. Surprisingly, there was no enhancing effect in the concentration range relevant for beer drinking, and this can explain why the effects on cell expansion are similar for nonalcoholic and alcoholic beer ( Figure 6).
Once absorbed, alcohol is distributed in the body, including the brain, as ethanol passes the blood−brain barrier easily. The distribution is approximately 0.6 L/kg for women and 0.7 L/kg for men. 17 The volume of beer equivalent to iso-α-acid Iso-α-acids in beer are a mixture of at least six different stereoisomers (Figure 1), and there are also other forms of reduced iso-α-acids. We tried to analyze whether one or more of the forms of iso-α-acids, either the cis/trans stereoisomers or any of the reduced forms, have effects on expansion. We found that, compared with a mixture of iso-α-acids, the trans-form activates SH-SY5Y and NES cells at a lower concentration. Compared to cis-iso-α-acids, trans-iso-α-acids are less soluble in water and are therefore present to a greater extent in the foam of beer. 18 The more lipophilic trans-form may penetrate more easily through the cell membrane or bind with higher affinity to receptors involved. Both the cis-and trans-iso-α-acids are formed from isomerization of α-acids during wort boiling in the brewing process. The cis/trans ratio in wort is usually 68:32, 19 and this ratio is likely unchanged with consumption.
Iso-α-acids have been shown to be ligands of the nuclear receptors PPARγ/PPARα and to bind at 3−30 μM concentrations. 10 Here, we show that the PPARγ/PPARα and the cannabinoid receptor CB 1 may be activated by iso-α-acids at the lower (nM) concentrations (Figure 9), an iso-α-acid concentration present in beer drinking. Neurite outgrowth at 100 nM to 1 μM concentrations of PPARγ agonists has been found in SH-SY5Y cells. 20 Other studies have also found growth of neural stem cells at low μM concentration, 21 observations that support the physiological relevance of our findings.
Iso-α-acids as PPARγ ligands can cause a metabolic reprogramming of microglia to suppress inflammation. 6 Iso-αacids may also be important for metabolic reprogramming during adult neurogenesis. 22 Proliferation requires an upregulation of aerobic glycolysis, while differentiation needs a metabolic switch to the more efficient oxidative phosphorylation. Ligands of PPARγ have been shown to induce neural stem cell proliferation, 21,23 whereas ligands of PPARα enhance differentiation of astrocytes, 24 PPARγ increases oligodendrocytes, 25 and PPARα expands neurons. 26 As iso-α-acids in beer are both PPARα and PPARγ ligands, these compounds may not only generate proliferation of neuroepithelial stem cells but also induce differentiation.
The significance of consuming bitter compounds, as for instance hop products, has attracted renewed interest in recent years after characterization of the chemosensory tuft cells equipped with taste receptors TAS2Rs 27 present throughout the digestive tract. 28 It has recently been demonstrated that the bitter taste compounds, including iso-α-acids, are involved in hormone release from enteroendocrine cells, 29 and they signal through tuft cells to the immune system. 30 The stereoselectivity for iso-α-acids of TAS2Rs is, however, different from what we have observed here, where the cis-form rather than the transform has a higher potency at certain TAS2Rs. 31 It is intriguing that beer has recently been shown to protect against amyloid β aggregation in the brain. 32 In Alzheimer's disease (AD), it is not only the number but also the maturation of neurons that decline. 33 Nonalcoholic beer has been shown to stimulate the number of circulating endothelial progenitor cells. 34 If nonalcoholic beer also can stimulate expansion of neuroepithelial stem cells, this can be one explanation of the neuroprotective effect of beer. Other components in beer from hops such as xanthohumol improve cognitive flexibility 35 and stimulate neurite outgrowth. 36 Hop components have been used in traditional medicine for over a thousand years. These compounds are safe and without side effects at physiologically relevant concentrations, and they may therefore be suitable as food additives. 37 In conclusion, our results using neuroepithelial stem cells indicate that nonalcoholic beer with ingredients such as iso-αacids have many of the beneficial effects of alcoholic beer.
HPLC of Hopsteiner Iso-Extract, RPLC-MS, and UV of purified iso-α-acids; structures of the reduced iso-α-acids tested; concentration ranges of iso-α-acids and ethanol in experiments (PDF)