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reference
Gualtieri P, Marchetti M, Frank G, et al. Antioxidant-enriched diet on gene expression of oxidative stress and inflammation: a randomized controlled trial.Genes. 2023;14(1):206.
Study objective
To evaluate the effects of adding a whole juice supplement (apple plus bergamot) to the Mediterranean diet on body composition parameters, metabolic laboratory values, and on gene expression of oxidative and inflammatory proteins
Key to take away
Adding an apple/bergamot juice to an already healthy Mediterranean diet resulted in an improvement in the gene expression of several inflammation- and oxidation-related signaling pathways.
design
Randomized, crossover-controlled trial (not blinded)
Participant
24 normal-weight, healthy adult volunteers took part in this study (16 women, 8 men). The mean age was 30 (23–41) years.
The exclusion criteria included acute or chronic diseases such as diabetes, intestinal disorders, metabolic disorders, cardiovascular, kidney, liver or autoimmune diseases or cancer. Individuals who were pregnant, breastfeeding, or taking medications or supplements for any reason were excluded, as were those who frequently consumed flavonoid-rich beverages such as tea, herbal teas, coffee, cocoa, and fruit juices (more than 500 ml).
intervention
There were two arms, with all participants switching from one to the other: Arm 1 consisted of the Mediterranean diet only (MedDiet) and Arm 2 consisted of the MedDiet plus mixed apple-bergamot juice (MAB).
Subjects were randomly assigned to either Arm 1 or Arm 2. After two weeks of MedDiet with or without MAB juice, there was a three-week washout period, and each group was then assigned to the other arm for an additional two weeks.
Blood parameters were assessed at baseline and after each two-week intervention period.
The intervention in this trial consisted of mixed apple (Pyrus malus) and bergamot (Citrus bergamia, Risso) juice or MAB juice at a dose of 250 ml/day. The freshly squeezed juice consisted of an 80:20 volume ratio of apple to bergamot. Notably, the bergamot included the inner peel and not just the pulp. The MAB juice consisted of water (91%), carbohydrates (8.55%) and very little fiber (<0.5%). There were negligible amounts of protein or fat. The juice was considered low in calories: 35 kcal/100 g.
Analysis of the juice:
- Vitamin C=422,02 mg/L
- Gesamtphenole = 1.263 mg Gallussäureäquivalente (GAE)/L (Polyphenole = 484,87 mg/L)
- Flavonoide = 385 mg Quercetin-Äquivalente (QE)/L
- Wert der Sauerstoffradikal-Absorptionskapazität (ORAC) = 5.964,29 Mikromol Trolox-Äquivalente (TE)/L
This study conducted in Italy specified its MedDiet parameters as follows:"Carbohydrates 55-60% of total Kcal; protein 15-20% of total Kcal, of which 50% are of plant origin; total fats <30% of total Kcal; saturated fat <10% of total Kcal; polyunsaturated fatty acids (PUFA) 6-10% of total Kcal: 5-6% of total Kcal from n-6 PUFA and 1–2% of total Kcal from n-3 PUFA; MUFA about 15% of total kcal; Trans fatty acids 5000 µmolTe.”
Evaluated study parameters
Body composition was tracked using bioimpedance analysis (BIA).
Laboratory values included:
- Gesamtcholesterin (TC)
- High-Density-Lipoprotein (HDL)
- Lipoprotein niedriger Dichte (LDL)
- Glucose
- Insulin
- Berechnetes HOMA-IR (Homöostasemodell zur Beurteilung der Insulinresistenz)
The researchers analyzed the gene expression of the following:
- Katalase (KATZE)
- Chemokin-CC-Motivligand 5 (CCL5)
- Hemmfaktor für die Makrophagenmigration (MIF)
- Kernfaktor-Kappa-B-Untereinheit 1 (NFKB1)
- Peroxisom-Proliferator aktivierter Rezeptor γ (PPARγ)
- Hyperventilieren (SOD1)
- Vitamin-D-Rezeptor (VDR)
Primary outcome
The primary endpoint that this study aimed to measure was the effect of MedDiet on lipid and glycemic parameters with and without the addition of MAB juice.
Secondary outcomes included the effect of MedDiet with and without MAB juice on seven genes related to inflammation and oxidative stress.
Key findings
The addition of MAB juice resulted in an improvement in muscle mass (P<0.01) according to BIA.
Adding MAB juice to the MedDiet resulted in a significant reduction in the TC/HDL index (P<0.01)
There was a significant increase in the expression of 4 of the 7 genes involved in oxidation and inflammation:MIF (P≤0.05), PPARγ (P<0.001), SOD1 (P≤0.05) and VDR (P≤0.05) when comparing the MedDiet and MedDiet + MAB juice groups.
There were no effects on glycemia parameters, including glucose, insulin and HOMA-IR.
transparency
This study received no external funding. The authors declared that there were no conflicts of interest. Registered as NCT01890070.
Implications and limitations for practice
The Mediterranean diet is often touted as one of the best proven diets for reducing the risk of chronic noncommunicable diseases (CNCDs) such as type 2 diabetes, cancer and cardiovascular disease. A typical traditional Mediterranean diet is rich in olive oil, seafood, fruits, vegetables and wine, with limited amounts of meat and no processed foods. The combination of micronutrients, phytochemicals, fiber and the types of fatty acids in MedDiet are believed to contribute to its health benefits. The study examined here examined the physiological effects of a freshly squeezed mixed apple/bergamot juice (80/20) (MAB juice) in healthy Italian subjects who were already consuming what is touted as an ideal dietPattern.1
This crossover study took 7 weeks to complete. This included two weeks of MedDiet with or without MAB juice, a three-week washout period (normal diet, presumably close to MedDiet), and then another two weeks with or without MAB juice. Remarkably, significant changes in lean body mass and HDL levels were noted after just two weeks of MAB juice consumption.
Interestingly, the highest polyphenol content came from bergamot juice, even though it only made up 20% of the MAB juice
In this study, the expression of 7 genes was measured to track physiological changes in real time. The effects of diet on gene expression are expected to be immediate, so the short duration of the intervention was consistent with this secondary endpoint. The effects of MAB juice included activation of intracellular antioxidant systems (SOD1), balancing inflammatory mediators (VDR and MIF), and improved glucose regulation (PPAR gamma). These patterns of genetic expression suggest cellular changes consistent with metabolic profiles that reduce the risk of CNCDs over time.
It may be surprising that consumption of the MAB juice had no effect on glucose, insulin or HOMA-IR. This is despite the significant difference in the percentage of calories from carbohydrates when consuming the MAB juice (54.63 ± 11.01 vs. 60.52 ± 11.04;P=0.01), indicating that the MAB juice was actually added to the base MedDiet. According to previous research, the naturally occurring phytochemicals in fruits moderate the effects of their fructose content on glucose levels.2
It was also notable that no adverse signs or symptoms or negative laboratory parameters were noted in the subjects at any point in either arm.
There are thousands of phytochemicals in every fruit and vegetable. These compounds provide the net benefits of a plant-rich, fresh, local diet. Phytochemicals that have measurable physiological effects are called bioactive compounds or simply “bioactives.”
Phenols, particularly polyphenols, are perhaps the most studied class of bioactive compounds with proven health benefits, including reducing the risk of CNCD.3In this study, the polyphenolic composition of MAB juice was determined. Interestingly, the highest polyphenol content came from bergamot juice, even though it only made up 20% of the MAB juice. The most commonly found polyphenols were brutieridin, meltidin and naringin.
Apples are known for their fiber content, but they are also a rich source of polyphenols, including catechin, procyanidin, epicatechin, phloridzin, chlorogenic acid, quercetin glycosides, anthocyanins and hydroxycinnamic acid.4
So what led to the positive changes compared to MAB juice? This study can't tell us, and maybe that's not a bad thing.
Research often follows a reductionist path in the pursuit of precision. Although numerous studies detail the physiological effects of individual polyphenols, there is also a growing appreciation for the synergy of naturally occurring combinations. The most logical way to study nutrient synergies in natural foods is to use whole foods and measure their physiological effects. This may be simple and obvious, but the search for individual compounds from nature (e.g. bioactives) is a multibillion-dollar industry that continues to grow.5This study is commendable because of the simplicity of the design and the use of whole fruit juices. The precision of this study focused on determining the genes affected rather than the plant's chemistry - arguably a much more holistic design.
As clinicians, we know that while risk reduction is possible, some patients may have done “everything right” and still receive a diagnosis of CNCD. These patients are of normal weight, exercise regularly, eat a plant-rich diet, avoid toxic compounds, have healthy relationships, pursue their spiritual path, etc. Reducing risk in the absence of obvious risk factors represents a major challenge for preventive medicine practitioners. This study is the first to suggest that favorable metabolic genetic changes are the result of an apple/bergamot juice in addition to a healthy one nutrition are. This is intriguing and might be considered for the few patients where there isn't much room for improvement. Although this study is not intended to guide practical decisions, it does provide food for thought.