Dietary Black Raspberry Supplementation as Natural Polyphenol Source Against Mild Dementia Patients with Overweight and Helicobacter pylori Infection

doi:10.21203/rs.3.rs-3899398/v1

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Dietary Black Raspberry Supplementation as Natural Polyphenol Source Against Mild Dementia Patients with Overweight and Helicobacter pylori Infection

https://doi.org/10.21203/rs.3.rs-3899398/v1

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Alzheimer's disease (AD) is the most common form of dementia. H. pylori infection and overweight have been implicated in AD via the gut-brain axis (GBA). This study aimed to determine supplementation of BRBs has a meaningful effect on the H. pylori infection, overweight and AD development in a clinical trial setting. We conducted a randomized placebo-controlled clinical trial in patients with mild clinical dementia who also had H. pylori infection and overweight. The study was carried out over 10 weeks, consisting of an 8-week intervention period (25g powder of black raspberries, BRBs, or placebo twice a day, morning and evening) and a 2-week follow-up. The primary outcomes were changes in Clinical Dementia Rating (CDR), Urea Breath Test (UBT), and Body Mass Index (BMI). Consumption of BRBs improved cognitive functions (p<0.00001), compared to the placebo group (p>0.05). Besides, BRB ingestion decreased H. pylori infection and BMI (p<0.00001 and p<0.05 respectively) while placebo group stay statistically the same (p=0.98 and p=0.25 respectively). BRBs significantly decreased inflammatory markers, improved oxidative index, adiponectin (p<0.05) compared to placebo group, while adenosine monophosphate-activated protein kinase (AMPK) and leptin did not significantly change. BRBs modulated the abundance of several fecal probiotics, particularly, Akkermansia muciniphila. Our results provided that BRBs suppressed H. pylori infection and decreased BMI and rebalancing the gut microbiome, which could improve cognitive functions in AD patients. Longer and larger randomized clinical trials of BRB interventions targeting H. pylori infection, overweight, or AD are warranted to confirm the results from this pilot trial.

Trial Registration: ClinicalTrials.gov identifier: NCT05680532

Health sciences/Health care

Biological sciences/Biological techniques

Black Raspberry

Helicobacter pylori

Obesity

Dementia

Alzheimer's disease (AD) is a neurodegenerative condition associated with dementia, memory and cognition impairment, and behavioral changes. AD is estimated to affect over 40 million people globally, with the prevalence of the condition anticipated to increase over the next several decades, especially in low socioeconomic status countries^1,2. The pathophysiology of AD is not clearly understood, but genetic and environmental risk factors appear to contribute to the condition.

One condition potentially related to AD development is infection with the bacterium Helicobacter pylori. AD affects nearly 4.5 billion people worldwide. This condition disproportionately affects those from disadvantaged, low socioeconomic status, or immigrant backgrounds. Lack of adequate sanitation and nutrition are among the environmental risk factors for developing infection^3–5. H. pylori infection frequently presents as general dyspepsia. Still, it has the potential to lead to more severe conditions such as peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma, which makes it imperative to diagnose and treat H. pylori infection, given its significant global prevalence⁶. H. pylori has been implicated in AD via the gut-brain axis (GBA), a network connecting the gastrointestinal and central nervous systems via signals such as metabolites from gut microbiota, gastrointestinal hormones, and immunologic modulators⁷. As bacteria such as H. pylori infect the gastrointestinal tract, they may affect immune and metabolic signaling within the GBA and thereby lead to CNS changes. Recent studies indicate a possible link between AD and H. pylori infection, evidenced by factors such as increased anti-H. pylori IgG in AD patients^8,9. However, the research on H. pylori in concurrence with AD remains inconclusive.

Another gastrointestinal factor implicated in AD development is obesity. As with H. pylori infection, obesity may be related to AD via the gut-brain axis, especially when considering immunological factors. For instance, obesity is associated with increased susceptibility to bacterial infection, reduction in gut microbiome diversity, and subsequent pro-inflammatory profiles, a cascade that may increase the risk of developing AD¹⁰. Interestingly, several studies demonstrate that this risk appears to be highest when obesity is present in middle age; the reason for this remains unclear^11,12.

Together, H. pylori infection, obesity, and AD form a triad of epidemiologically overall conditions that may be linked via immunological and metabolic pathways of the GBA. However, the literature remains inconclusive regarding definitive relationships, and no previous studies have attempted to connect all three of these conditions. This merits exploration regarding treatments that can target these pathways, providing more favorable inflammatory profiles and hopefully reducing AD incidence. One potential treatment route may be a dietary modification, which has been demonstrated to affect both obesity and H. pylori infection by modulating the gut microbiome and inflammatory cascades^13,14. Our previous research has highlighted the role of black raspberries (BRBs) in autoimmune and malignant conditions and found that in certain situations, BRBs and their metabolic components suppress pro-inflammatory markers and foster gut microbiota with less inflammatory profiles. For instance, in a murine model of ulcerative colitis, diet supplementation with BRBs was associated with reduced levels of phospho-IκBα, cyclooxygenase-2 (COX-2), toll-like receptor and prostaglandin E₂, all of which are associated with inflammation^15–19. It is possible that the gastrointestinal effects demonstrated to result from BRBs may extend to conditions such as obesity and H. pylori, both of which are also strongly associated with inflammation.

The purpose of this study was to determine whether supplementation of diet with BRBs has a meaningful effect on the gastrointestinal conditions of H. pylori infection and overweight and the development of AD in a clinical trial setting.

Clinical trial

The clinical trial protocol was approved by the IRB at Chung Shan Medical University, Taiwan (protocol number CS2-21103, Title: The improvement of black raspberry in obese and mild AZ patients infected with H. pylori). Inclusion criteria for this study were as follows: BMI ≥ 27; waist circumference for men ≥ 90 cm, women ≥ 80 cm; body fat for men ≥ 25%, women 30%; a ¹³C urea breath test (UBT) test value > 10% indicating H. pylori positivity; and clinical dementia rating (CDR) = 0.5. Exclusion criteria were as follows: BMI < 27; severe chronic disease (liver disease, cardiovascular disease, kidney disease); taking medications known to affect lipid metabolism or possess anti-inflammatory effects; gastrointestinal disorder; surgery; alcohol abuse; smoking; pregnant or lactating; taking berry-related supplementation or having known allergies or hypersensitivity to berries, including BRBs; taking antibiotics/ corticosteroids in the last 4 weeks; taking NSAIDs or COX-2 inhibitors due to external clinical conditions; and/or prior history of H. pylori infection treated with triple therapy.

In total, 21 subjects were recruited for the study; 11 (8 males and 3 females) in the berry group and 10 (4 males and 6 females) in the placebo group. Subjects were aged 60–85 years old. Those in the BRB group received 25 g of black raspberry powder dissolved in 240 mL of water twice a day, once after breakfast and once after dinner, resulting in total consumption of 50 g of black raspberry powder per day. Subjects in the placebo group received 25 g of dextrin twice per day, administered in the same way as the black raspberry powder to a total of 50 g of dextrin per day. Each 50 g of black raspberry powder was estimated to contain (mean ± SD) 1926.50 ± 547.00 mg/g of phenols, 336.50 ± 11.00 mg/g of flavonoids, 1101.50 ± 51.00 mg/g of anthocyanins, 5581.00 ± 715.00 mg/g of condensed tannins, and 10.49 g of fiber (Supplementary Table 1).

The study was conducted over 10 weeks, consisting of an 8-week intervention period and a 2-week follow-up. At week 0, anthropometric measurements were taken of weight, height, BMI, waist circumference, hip circumference, blood pressure, body fat, and triceps skinfold thickness. Patients were asked to maintain their regular diets while on trial, and they kept a 3-day dietary record consisting of 2 weekdays and 1 weekend day. Blood sampling was also taken to assess lipid profile, cytokine levels, inflammatory markers, antioxidant index, thyroid function, liver function, kidney function, and blood chemistry. A ¹³C UBT, fecal sampling, and CDR were also performed at week 0. At week 2, anthropometric measurements and dietary recall were repeated. At week 4, anthropometric measurements, dietary recall, and blood sampling were performed. At week 6, anthropometric measurements and dietary recall were performed. At week 8, anthropometric measurements, dietary recall, blood and fecal sampling, and ¹³C UBT and CDR were performed. At the week 10 follow-up, anthropometric measurements, dietary recall, and blood sampling were performed.

Fecal Sample Analysis

Fecal samples from the subjects consumed BRBs were collected at baseline and end of the intervention. The fecal sample is prepared in the Colon Heal bacterial detection kit (Aging and Disease Prevention Research Center, Fooyin University, Kaohsiung, Taiwan) following manufacturer's instruction. 16S library pool was performed using Illumina MiSeq system library preparation instruction. Forward (F)/reverse (R) primers (F: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’/R: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3') with overhang sequence were used for metagenome analysis to generate Illumina 16S library by two-step polymerase chain reaction. 16S libraries generated were pooled and sequenced using MiSeq with MiSeq V3 reagent kits and analyzed with MiSeq Reporter Software (MSR). Classification of the result is based on the Greengenes database (http://greengenes.lbl.gov/). The mean of relative abundance was compared at the phylum, genus, and species levels.

Preparations of different BRB extracts

Procedures to prepare BRB crude extract, crude extract treated with gelatin, and tannins fraction are shown in Supplemental Fig. 1. Phenolic contents in different BRB extracts are listed in Supplemental Table 1.

AGS cell model

Human gastric adenocarcinoma cell culture (AGS cell) was purchased from American Type Culture Collection (ATCC) and cultured in F12 (Ham’s media Kaighn Modification, Cytiva) medium containing 10% of Fetal Bovine Serum 37 ^oC humidified incubator with 5% CO2.

Cell viability

The cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. AGS cells (2 × 10⁵ /mL) were seeded into a 96-well plate and incubated overnight. Increasing concentrations of BRB crude and gelatin treated crude extracts (0, 50, 100, 250, 500, 1000 µg/mL in serum-free medium with 0.05 Dimethyl Sulfoxide (DMSO)) or H. pylori (OD₆₀₀ 0.3 (1 x 10⁸ CFU/ mL)) were co-cultured with AGS cells and incubated at 37°C for 24 hours. After 24 hours, the medium was removed and changed with 100 µL (0.5 mg/mL) of MTT in phosphate-buffered saline (1X PBS) and cultured at 37°C for 3 hours. Finally, the cells were mixed with 100 µL DMSO and shaken for 10 min (to dissolve formazan crystals), and the absorbance was measured using a microplate reader at 570 nm. The blank wells were prepared with no cells and positive control wells were 50 µg/mL Amoxicillin. The cell viability (%) was calculated with the following equation:

Cell viability (%) = Absorbance (Sample – Blank)/ Absorbance (Control – Blank) x 100%

H. pylori Adhesion

AGS cells were seeded in 24-well plates and incubated overnight. Increasing concentrations of BRB crude and gelatin treated crude extract (0, 50, 100, 250, 500, 1000 µg/mL) or amoxicillin (0.05 mg/mL) and H. pylori suspension were added to each well and incubated for 6 hours in a humidified CO2 incubator. The nonadherent bacteria were washed off with 1X PBS solution. The final suspension was collected after 6 hours of incubation to quantify the bacterial viability in the urease reagent and read at 560 and 600 nm. Adherent bacteria were evaluated with urease reagent (3 mM PBS pH 5.8; 2% urea and 4 µg/mL phenol red, pH 5.0) and read absorbance at 560 nm and calculated by the formula:

Anti-adhesive activity (%) = (control absorbance − sample absorbance) ∕ control absorbance × 100%

CagA, VacA western blot

Analysis of CagA and VacA expression detected using western blot analysis. AGS cells were seeded in 10 cm dish plates and incubated overnight. Increasing concentrations of BRB crude and gelatin treated crude extract (0, 0.05, 0.1, 0.25, 0.5, and 1 mg/mL) or amoxicillin (0.05 mg/mL) and H. pylori suspension were added to each plate and incubated for 6 hours. All cells were collected and lysed using ice-cold lysis buffer, and cell protein was collected after centrifugation (3000 RPM, 10 minutes, 4^oC). Cell proteins were separated in 8% polyacrylamide gel and blotted to polyvinylidene difluoride membrane (PVDF). The membrane was blocked in 5% skim milk in TBS buffer containing 0.2% Tween-20. The membrane was incubated with anti-beta actin monoclonal antibody (SC-47778, Santa Cruz Biotechnology, USA), anti-CagA monoclonal antibody (SC-28368, Santa Cruz Biotechnology, USA) and anti-VacA monoclonal antibody (SC-32746, Santa Cruz Biotechnology, USA) overnight at 4^oC. After the removal of the primary antibody, the membrane was soaked in HRP-conjugated anti-mouse secondary antibody (Invitrogen, Thermo Fischer, USA) for 1 hour at room temperature. Reacted band of the targeted protein was revealed using enhanced chemiluminescence (ECL) using ECL commercial kit (G-Biosciences, USA).

IL-8 secretion

IL-8 content was determined using a commercialized IL-8 ELISA kit (Biolegend, California, USA) by following the manufacture's protocol. Each sample was tested in triplicate

3T3-L1 pre-adipocyte and mature 3T3-L1 adipocytes

3T3-L1 was purchased from American Type Culture Collection (ATCC) and cultured in DMEM (Gibco) medium containing 10% bovine calf serum and differentiated into mature adipocytes by DMEM containing 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 1 mM DEXA and 10 mg/mL insulin at 37 ^oC humidified incubator with 5% CO2.

Cell viability of 3T3-L1 pre-adipocytes

The cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. 3T3-L1 pre-adipocytes were seeded into a 96-well plate and incubated overnight. Increasing BRB crude extract concentration, gelatin treated crude extract, and tannin fraction (0, 0.1, 0.5, 1, 2, 5, 10, 15, and 20 mg/mL) were added and incubated at 37°C for 24 hours. After 24 hours, the medium was removed and changed with 100 µL (0.5 mg/mL) of MTT in phosphate-buffered saline (1X PBS) and cultured at 37°C for 4 hours. Finally, the cells were mixed with 100 µL DMSO and shaken for 10 min (to dissolve formazan crystals), and the absorbance was measured using a microplate reader at 570 nm. The blank wells were no cells. The cell viability (%) was calculated with the following equation:

Cell viability (%) = Absorbance (Sample – Blank)/ Absorbance (Control – Blank) x 100%

Oil-red staining in mature 3T3-L1 adipocytes for lipid accumulation

Mature adipocytes in 24 wells were washed twice with PBS and fixed using 10% formaldehyde solution for 1 hour. After 1 hour, cells were washed twice using PBS, and 0.5% oil red solution (in isopropanol) were added and incubated for 15 minutes to stain the lipid droplets. Cells were washed with dd H₂O to remove unbound oil red. Isopropanol was added to dissolve the oil red and shake for 10 minutes. 100 µL of the solution was transferred to 96 wells, and read absorbance at 490 nm and lipid accumulation were calculated with the following equation:

Lipid accumulation (%) = Absorbance (Sample)/ Absorbance (Control) x 100%

Statistical analysis

The sample size was calculated to assess difference among groups (α: 0.05) with 5% reduction of UBT value using 95% confidence interval and 80% power (1-β) and according to calculation each group need at least 6 subjects and considering dropout rate of 20%, 10 subjects in each group is used. Repeated measure ANOVA and chi-squared test is used to assess difference within the group (intervention vs baseline) as appropriate. Results obtained from cells were analyzed with one-way ANOVA of SPSS (version 26, SPSS Inc). Significance difference is identified with p < 0.05

BRB consumption improves cognitive function in patients with mild dementia due to Alzheimer's disease associated with decreased inflammation and oxidative stress.

We conducted a placebo-controlled clinical trial in patients with mild clinical dementia due to AD who also had H. pylori infection and were overweight (aged 60–85 years old.). The baseline patient demographics show that age, Clinical Dementia Rating (CDR), body mass index (BMI), and urea breath test (UBT) are statistically the same (Table 1). Patients were randomized to either BRB or placebo group. BRB group consumed 8-week BRBs (25g, twice a day, morning and evening, n = 11, 8 males and 3 females). The placebo group consumed a placebo powder (25g, twice a day, morning and evening, n = 10, 4 males and 6 females). After the 8-week intervention, all patients were followed up for 2 weeks.

Table 1

Baseline patient demographics
	Placebo	BRBs	p value
Age	78.9 ± 3.0	77.5 ± 3.0	0.32
Gender
Male	4	8	0.20
Female	6	3
BMI	28.5 ± 0.9	29.5 ± 1.9	0.14
UBT	23.0 ± 1.8	22.1 ± 4.2	0.50
CDR	0.5 ± 0.0	0.5 ± 0.0	1.00
The results are expressed as mean ± SD.
BMI: body mass index; UBT: urea breath test; CDR: clinical dementia rating

Clinical Dementia Rating (CDR) values did not change in the placebo group, but those in the BRB group had an average CDR of 0 at the end of the 8-week intervention (Fig. 1A). It should be noted that neither amyloid beta nor p-tau was detectable in plasma from this patient population using an ELISA-based assay (data not shown). We then determined the effects of BRBs on Aβ-induced neurotoxicity in cultured neurons. Aβ42 oligomer treatment led to neurotoxicity in HT22 neuron cells, and BRB ethanol extract protected against Aβ-induced

damage (Fig. 1B). It is possible that ethanol extract of BRBs contains anthocyanins that have been shown to cross the blood-brain barrier to protect the brain from Aβ toxicity (35).

BRBs decreased Proteobacteria and Firmicutes to Bacteroidetes ratio in the feces (Fig. 2A).

Further, BRBs decreased fecal Helicobacter bacteria abundance (Fig. 2B), but increased fecal probiotics such as Lactobacillus reuteri, Bifidobacterium longum (Fig. 2C). Most interestingly, Akkermansia muciniphila abundance increased in all subjects who consumed BRBs (Fig. 2D).

¹³C UBT was significantly lower at week 8 in the BRB group when compared to baseline (Table 2). However, this difference was resolved in week 10. Inflammatory markers, reflected by tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were measured at weeks 0, 4, and 8. At week 4, levels of TNF-α, IL-8, COX-2, and iNOS were significantly lower than at baseline, whereas levels of IL-1β were significantly higher. At week 8, levels of TNF-α, IL-8, and COX-2 were significantly lower than baseline, with IL-1β continuing to be significantly higher than levels at both baseline and week 4. The oxidative index was quantified by superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS), and Trolox Equivalent Antioxidant Capacity (TEAC). At weeks 8 and 10, SOD, CAT, and TEAC levels were significantly higher than at baseline, but TBARS levels were significantly lower than at baseline.

Table 2

BRB consumption decreased UBT and inflammatory markers, and oxidative indexes
		Week 0		Week 8		Follow up
UBT
BRBs		22.12 ± 4.14		9.36 ± 6.36*		16.82 ± 10.46*
Placebo		22.98 ± 1.78		22.99 ± 2.065		22.88 ± 3.07
Inflammatory markers
TNF-α (pg/mL)
BRBs		19.76 ± 4.65		6.85 ± 3.27*		7.97 ± 2.17*
Placebo		19.81 ± 2.21		19.59 ± 2.31		19.41 ± 2.39
IL-1β (pg/mL)
BRBs		0.16 ± 0.07		0.12 ± 0.05*		0.18 ± 0.05*
Placebo		0.19 ± 0.08		0.19 ± 0.05		0.18 ± 0.05
IL-8 (pg/mL)
BRBs		7.61 ± 5.10		4.63 ± 3.63*		5.94 ± 3.09
Placebo		7.67 ± 4.25		7.86 ± 4.90		7.79 ± 4.23
COX-2 (ng/mL)
BRBs		1.74 ± 0.71		0.87 ± 0.56*		0.88 ± 0.50*
Placebo		1.68 ± 0.65		1.64 ± 0.72		1.69 ± 0.62
iNOS (ng/mL)
BRBs		21.15 ± 8.50		20.27 ± 10.28		22.37 ± 11.95
Placebo		21.73 ± 7.44		22.00 ± 7.75		21.21 ± 7.24
Oxidative indexes
SOD (U/mL)
BRBs	696.42 ± 35.30		784.86 ± 46.13*		743.84 ± 55.59*
Placebo	690.72 ± 34.84		695.44 ± 39.65		691.54 ± 41.35
CAT (U/mL)
BRBs	346.11 ± 14.60		360.84 ± 8.26*		360.21 ± 9.22*
Placebo	352.58 ± 14.16		351.95 ± 11.30		351.14 ± 9.03
TBARS (µmol/L)
BRBs	4.14 ± 1.20		1.20 ± 0.35*		1.97 ± 0.62*
Placebo	4.20 ± 0.57		4.25 ± 0.74		4.30 ± 0.90
TEAC (µmol Trolox/L)
BRBs	1319.77 ± 191.90		1840.68 ± 188.73*		1740.09 ± 155.99*
Placebo	1250.90 ± 185.14		1232.00 ± 199.59		1238.40 ± 237.70
The results are expressed as mean ± SD.
*Difference between different times and week 0 within the same group, P < 0.05.

Regarding changes in BMI, at week 4, the BRB group had a significantly lower body weight and BMI (p < 0.05) compared to their week 0 measurements (Table 3). Adenosine monophosphate-activated protein kinase (AMPK) and leptin did not significantly change from baseline across the course of the study, but adiponectin was significantly increased from baseline at both weeks 8 and follow-up (p < 0.01) (Table 3).

Table 3

Body weight, BMI, body fat, AMPK, adiponectin, and leptin levels
	Week 0	Week 4	Week 8	Follow up
Body weight (kg)
BRBs	83.41 ± 10.39	82.65 ± 9.82*	82.90 ± 10.37	83.15 ± 10.09
Placebo	74.14 ± 8.47	73.90 ± 7.72	73.45 ± 7.49	73.83 ± 7.96
Body Fat (%)
BRBs	30.61 ± 5.88	30.33 ± 6.17	30.14 ± 6.16	30.17 ± 5.76
Placebo	32.34 ± 5.70	31.92 ± 6.53	32.59 ± 5.72	32.70 ± 5.87
BMI (kg/m²)
BRBs	29.47 ± 1.92	29.21 ± 1.97*	29.30 ± 2.06	29.41 ± 2.01
Placebo	28.46 ± 0.92	28.38 ± 0.99	28.23 ± 1.12	28.35 ± 1.06
AMPK (pg/dL)
BRBs	9142.50 ± 6734.93	9408.57 ± 5844.41	9694.09 ± 7408.22	9641.23 ± 6664.65
Adiponectin (µg/mL)
BRBs	459.33 ± 318.14	651.27 ± 546.30	2394.65 ± 1418.26*	2134.89 ± 1153.37*
Leptin (µg/mL)
BRBs	5055.98 ± 2969.55	4845.42 ± 2049.15	4704.60 ± 2689.98	4866.47 ± 3584.98
The results are expressed as mean ± SD.
*Difference between different times and week 0 within the same group, P < 0.05.
AMPK: adenosine monophosphate-activated protein kinase

Further, neither the BRB nor placebo groups had significant changes in blood lipid profile from their respective baselines at these time points (Supplemental Table 2). Insulin, Homeostatic Model Assessment for Insulin Resistance score (HOMA-IR, quantified as [FBG * insulin]/405), and hemoglobin A1c (HbA1C) did not significantly change from baseline in the BRB and placebo groups (Supplemental Table 3). However, FBG was significantly higher compared to baseline at week 4 and week 8 in the BRB group (p < 0.05, p < 0.01, respectively). Blood chemistry of subjects, reflected by glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), blood urea nitrogen (BUN), creatinine, uric acid (UA), and creatinine phosphokinase (CPK), were not significantly different in either group across time points (Supplemental Table 3). Mineral content was reflected by sodium (Na), potassium (K), chloride (Cl), and calcium (Ca) levels. K levels were significantly higher at week 4 compared to baseline in the BRB group (p < 0.01) (Supplemental Table 4). Na was significantly lower at follow-up compared to baseline in the BRB group (p < 0.05).

At week 4, those in the BRB group had a significantly lower rump circumference (RC) and triceps skin fold (TSF) (p < 0.05) compared to their week 0 measurements (Supplemental Table 5). At week 8, those in the BRB group had a significantly lower body fat percentage, RC, and mid-arm circumference (MAMC) compared to their week 0 measurements (p < 0.05). Aside from these differences, waist circumference (WC), RC, MAMC, and TSF were not significantly different within the two groups at various recording times. At week 4, DBP was significantly lower than the baseline in the BRB group (p < 0.05). While this difference resolved, SBP became significantly lower from baseline in the BRB group at follow-up (p < 0.05) (Supplemental Table 6).

BRB extracts rescued H. Pylori-induced damages in cultured AGS cells

An MTT assay was performed on applying crude BRB extract to AGS cells and H. pylori to assess for protective effects of BRBs against H. pylori, with cell viability assessed at 24 hours (Fig. 3A). When H. pylori were added with no BRB extract or amoxicillin, cell viability decreased at 24 hours. However, when increasing concentrations of crude BRB extract were added to H. pylori and the AGS cells, cells were rescued. A similar effect was seen with the gelatin-treated crude extract. H. pylori adhesion was measured with varying doses of crude and gelatin-treated crude extracts. Increasing doses of crude and gelatin-treated crude extract reduced H. pylori adhesion (Fig. 3B).

Analysis was performed on proteins associated specifically with H. pylori. Western blot was performed to assess the expression of cytotoxin-associated protein A (CagA), associated with the induction of intestinal metaplasia and gastric adenocarcinoma²⁰. CagA expression was significantly reduced by both crude and gelatin-treated crude extract (Fig. 3C). Western blotting was also utilized to determine the expression of vacuolating cytotoxin A (VacA), another H. pylori product that modulates the function of immunologic cells and is associated with H. pylori colonization, peptic ulcer disease, and gastric adenocarcinoma²¹. VacA expression was

significantly decreased by crude extract and crude extract treated with gelatin (Fig. 3C). Lastly, interleukin-8 (IL-8) expression has been shown to be induced by H. pylori infection; the upregulation of IL-8 related to H. pylori is associated with gastritis and possibly gastric malignancies^22,23. When H. pylori was added to the AGS cell culture, IL-8 expression was significantly increased (Fig. 3D). Both extracts significantly decreased H. pylori-induced IL-8.

Overall, both crude extract and crude extract-treated with gelatin decreased H. pylori-induced damages, including increasing cell viability, decreasing H. pylori adhesion and expression of CagA, VacA, and IL-8 in cultured AGS cells (Fig. 3A-3D).

BRB extracts promoted 3T3-L1 pre-adipocyte viability and decreased lipid accumulation in 3T3-L1c mature adipocytes

3T3-L1 pre-adipocytes were incubated with crude extract, gelatin-treated crude extract, and tannin fraction. A significant increase in viability was seen in 3T3-L1 pre-adipocytes treated with all three extracts (Fig. 3E). Oil-red staining was then performed to assess the effects of those three extracts on lipid accumulation in 3T3-L1 mature adipocyte cells. All three BRB extracts significantly decreased lipid accumulation (Fig. 3F).

The purpose of this study was to assess whether BRBs had meaningful effects on markers of H. pylori infection and obesity, thereby impacting AD dementia. We conducted a randomized, placebo-controlled clinical trial, which examined the effects of BRBs on obese subjects who were H. pylori positive and had AD as measured by a CDR of 0.5. BRBs significantly improved cognitive function by moving CDR from 0.5 to zero and decreased UBT and BMI. These effects are associated with significant anti-inflammatory and antioxidant capacity.

Although neither amyloid beta nor p-tau was detectable in plasma from this AD patient population using an ELISA-based assay (data not shown), our results could suggest that they could be at an early stage of AD. More sensitive ultrasensitive platforms, including SIMOA, IMR, MSD, and Elecsys immunoassays, that overcomes the complex interferences of blood proteins, heterophilic antibodies, and low target biomarkers²⁴, will be used to confirm the levels of blood amyloid beta and p-tau in this AD population in the future.

Gut microbiota is a complex microorganism in the human GI tract that plays a crucial role in maintaining health²⁵. Gut microbiota can help to metabolize some indigestible dietary components and to prevent pathogen colonization in the GI tract²⁶. Gut microbiota mainly comprises two major phyla, Firmicutes and Bacteroidetes, and some minor phyla, such as Proteobacteria, and Actinobacteria²⁷. Alteration of gut microbiota can influence different metabolic disorders, the immune system, and also the central nervous system through the gut-brain axis²⁸. Recent studies show that there are links between gut microbiota and Alzheimer’s disease. Gut microbiota is affected by lifestyle, including food intake and habits. Host dietary components are used as substrates and energy sources to prevent pathogenic bacteria and produce beneficial metabolites²⁹. Proteobacteria is one of the gut microbiota phyla that comprises several human pathogens³⁰. Pathogenic infections such as Chlamydophila pneumoniae, Helicobacter pylori, Toxoplasma gondii, and others might contribute to the pathogenesis of Alzheimer’s disease³¹. Besides Proteobacteria, gut dysbiosis is linked to AD progression. Gut dysbiosis is a phenomenon where gut microbiota is altered that mainly showed by increasing ratio of Firmicutes to Bacteroidetes^32,33. Gut dysbiosis is associated with several AD pathology, such as accumulation of intestinal amyloid precursor protein (APP), induction of systemic inflammation by proinflammatory neurotoxin and bacterial lipopolysaccharide (LPS), and alteration of blood-brain barrier (BBB)^33,34,35,36.

BRBs contain high amounts of polyphenols which can act as antimicrobial agent, especially for H. pylori^37,38. Helicobacter, particularly Helicobacter pylori is a gram-negative bacterium that mainly resides in the gastric and induces several inflammatory responses due to its virulence factor³⁹. H. pylori infection can cause disruption of tight junction, and this disruption can lead to increased proinflammatory cytokines and harmful metabolites (e.g. bacterial amyloids and trimethylamine-N-oxide (TMAO)) ⁴⁰. These harmful substances produced by pathogens enter the circulatory system, disrupt BBB and induce an immune response in the central nervous system³³. H. pylori can induce a neuroinflammatory response in the C57BL6 WT mice through circulating proinflammatory cytokines⁴¹. Eradication of H. pylori from AD subjects result in improved cognition compared with AD patients who did not receive H. pylori eradication therapy⁴².

On the other hand, BRB intervention also increased probiotics such as Lactobacillus and Bifidobacterium. Probiotics play an essential role in maintaining gut health due to their ability to metabolize undigested food components and produce metabolites such as gamma-aminobutyric acid (GABA) and short-chain fatty acids (SCFA)⁴³. GABA is an inhibitory neurotransmitter that reduces stress and anxiety⁴⁴. In Alzheimer’s patients, GABA concentration is significantly decreased and causes cognitive impairment⁴⁵. SCFA are the metabolites from dietary fiber fermentation derived from gut microbiota and linked to gut-brain interaction⁴⁶. SCFA can cross the BBB and repair neuronal by upregulating cyclic-AMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF), inhibiting inflammatory response in the central nervous system, reducing Aβ aggregation into neurotoxic oligomer, reducing tau protein hyperphosphorylation and reducing the cognitive impairment in AD subjects^46,47. Our study result show that BRB supplementation increase Bacteriodetes and probiotic concentration. This result might indirectly link to increase production of GABA and SCFA due to Bacteriodetes such as Bacteriodes and Parabacteroides, and Bifidobacterium ability to produce GABA^{48, 49} and lactic acid bacteria (Lactobacillus and Bifidobacterium) to produces SCFA⁵⁰ but further study is needed to support this statement.

Akkermansia muciniphilla is a gram-negative bacterium belonging to Verrucomicrobiota, constitutting 1–4% of the total fecal microbiome^51,52. Akkermansia muciniphilla can degrade intestinal mucin and produce acetate and propionate as substrates for other gut microbiome and the host⁵³. Akkermansia muciniphilla abundance is decreased in obesity, inflammation, and diabetes in the human GI tract ⁵⁴. BRBs are rich in polyphenols, and several studies have shown that polyphenols promote the growth of Akkermansia muciniphilla in the mice GI tract^55–57. Interestingly, we showed that BRBs increased the abundance of Akkermansia muciniphila, a bacterium associated with reduced inflammation, and decreased in patients with mild cognitive decline⁵⁸, that could delay the cognitive decline and improved CDR in patients with Alzheimer's disease.

UBT values and inflammatory markers provided indices of efficacy against H. pylori infection. Overall, UBT values were meaningfully improved by BRB intervention, but this effect did not persist when BRB supplementation was discontinued. While most of the inflammatory markers, excluding iNOS, did indicate less inflammatory profiles with continued BRB supplementation, the post-intervention impacts were not explored, so additional study is needed to assess whether the alterations in inflammatory profile continue to benefit even after BRB treatment. These results indicate that BRBs may exert some degree of defense against H. pylori infection, but the results only support these benefits so long as BRBs continue to be consumed in the diet.

Assays were performed first on AGS and 3T3-L1 pre-adipocyte cell models to explore whether BRBs might have tangible results in human patients. While BRBs significantly improved the viability rates of AGS cells exposed to H. pylori. Further, both crude and gelatin-treated crude extracts reduced the adhesion rates of H. pylori in the AGS cell cultures. However, here it is important to note that the amoxicillin control also showed the same level of a significant reduction in adhesion rates as BRBs, suggesting that BRBs were similarly, but not more or less effective, than amoxicillin alone in reducing adhesion rates of H. pylori. Both crude extract and crude extract treated with gelatin decreased CagA and VacA protein expressions. These results suggest that while BRB extract can provide statistically comparable effects when compared to amoxicillin in reducing the expression of these H. pylori genes, it must be administered at sufficiently high doses. However, considering that amoxicillin is currently part of status quo treatment for H. pylori based on extensive research demonstrating its effectiveness, further research is needed to determine whether BRBs are a reasonable alternative to amoxicillin, seeing as they do appear to provide a significant additional benefit against H. pylori infection in AGS cells. Another route of exploration may involve seeing if the effects of amoxicillin and BRBs are cumulative; that is, whether the supplementation with BRBs of a pre-established amoxicillin regimen to treat H. pylori might be more effective than amoxicillin alone.

The 3T3-L1 pre-adipocyte and mature adipocyte models, crude extract, gelatin-treated crude extract, and tannins fraction were all effective. This result supports the notion that crude BRB extract is sufficient to show the effects. Tannins in the extract are essential. Gelatin-treated crude extract or tannins fraction and that more components than just tannins are likely active in reducing lipid accumulation. It is expected that the effects of crude BRB extract cannot be attributed to tannins alone. Still, more research will be needed to determine which additional components confer the other advantage to crude BRB extract.

Given the activity of BRBs against H. pylori, as demonstrated by the AGS cells, and against obesity, as shown by the 3T3-L1 pre-adipocyte cells, it was anticipated that they might exert similar effects on actual patients. Although BRBs did affect physiologic markers of obesity, including body weight, BMI, body fat, RC, MAMC, and TSF, these effects did not persist into the follow-up stage. Interestingly, while body fat, RC, and MAMC did a downtrend throughout the intervention, reaching significance by week 8, the other markers did not show trends with continued intervention. This finding suggests that adding BRBs to the diet provided variable benefits, with some lasting only temporarily despite additional BRB consumption and others taking some time to manifest. Overall, all benefits were lost within 2 weeks after the intervention was concluded, so continued consumption of BRBs appears to be necessary. BRBs did not confer any significant disadvantage as assessed by the obesity markers.

Interestingly, SBP only showed a significant reduction at follow-up, 2 weeks post-intervention, while DBP was only reduced at week 4. BRBs did not exert meaningful effects on blood lipid profiles. They did not affect markers of diabetes other than fasting blood glucose, which was significantly increased with an upward trend throughout the intervention. This trend merits further exploration as increased FBG is not a desirable result; while the average levels within subjects were maintained below pre-diabetes levels, it is uncertain whether continued consumption of BRBs would have breached that threshold. One of the most significant changes related to obesity was seen in adiponectin levels, which substantially increased after both 8 weeks of treatment and 2 weeks post-intervention. Low adiponectin levels are associated with obesity, diabetes, hyperlipidemia, and other conditions that place patients at high risk for cardiovascular disease, while high adiponectin levels are associated with a healthier metabolic profile, so it appears that BRBs resulted in a meaningful benefit here^59,60.

In conclusion, our results provide the first evidence that BRBs enhanced fecal Akkermansia muciniphila abundance and protectively altered parameters of H. pylori infection and obesity, leading to dampened inflammation and oxidation, which could result in improved cognitive functions in AD patients. Longer and larger randomized clinical trials of BRB interventions targeting H. pylori infection, obesity, or AD are warranted to confirm the results from this pilot trial.

Consent Statement:

The authors confirmed that all human subjectslprovided informed consent.

Acknowledgments

This work was supported by NIH grants CA148818 and USDA/NIFA 2020-67017-30843 (to L.-S. Wang) , and also supported by National Science and Technology Council of Taiwan grants 111-2320-B-040-003-MY3 (to C.-K Wang).

Conflicts of Interest

All authors declare the absence of any competing interests.

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Dietary Black Raspberry Supplementation as Natural Polyphenol Source Against Mild Dementia Patients with Overweight and Helicobacter pylori Infection

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Clinical trial

Preparations of different BRB extracts

AGS cell model

3T3-L1 pre-adipocyte and mature 3T3-L1 adipocytes

Statistical analysis

Results

BRB extracts promoted 3T3-L1 pre-adipocyte viability and decreased lipid accumulation in 3T3-L1c mature adipocytes

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