Gualtieri P, Marchetti M, Frank G, et al. Antioxidant-enriched diet on oxidative stress and inflammation gene expression: a randomized controlled trial. Genes. 2023;14(1):206.
To evaluate the effects of adding a whole-juice (apple plus bergamot) supplement to the Mediterranean diet on body composition parameters, metabolic laboratory values, and on the gene expression of oxidative and inflammatory proteins
Adding an apple/bergamot juice to an already healthy Mediterranean diet led to improvements in gene expression of several inflammation- and oxidation-related pathways.
Randomized, crossover, controlled trial (nonblinded)
This study included 24 normal-weight, healthy adult volunteers (16 female, 8 male). The average age was 30 (23–41) years.
Exclusion criteria included acute or chronic disease such as diabetes, intestinal disorders, metabolic disorders, cardiovascular, kidney, liver, or autoimmune conditions, or cancer. Anyone who was pregnant, lactating, or taking a medication or supplement for any reason was excluded, as was anyone who frequently consumed flavonoid-rich beverages such as tea, herbal teas, coffee, cocoa, and fruit juices (exceeding 500 mL).
There were 2 arms, with all participants crossing over from one to the other: Arm 1 consisted of only the Mediterranean diet (MedDiet) and Arm 2 the MedDiet plus mixed-apple-bergamot (MAB) juice.
Subjects were randomly assigned to either Arm 1 or Arm 2. After 2 weeks of the MedDiet with or without MAB juice, there was a 3-week washout period, and each group was then assigned to the other arm for an additional 2-week period.
Blood parameters were assessed at baseline and after each 2-week interventional 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 pressed juice consisted of an 80:20 ratio (by volume) of apple to bergamot. Notably, the bergamot included the inner peel, not just the flesh. The MAB juice was composed 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
- Total phenols=1,263 mg gallic acid equivalents (GAE)/L (polyphenols=484.87 mg/L)
- Flavonoids=385 mg quercetin equivalents (QE)/L
- Oxygen radical absorbance capacity (ORAC) value=5,964.29 micromol Trolox equivalents (TE)/L
This study, which took place in Italy, specified their MedDiet parameters as follows: “carbohydrates 55–60% of total Kcal; protein 15–20% of total Kcal of which 50% plant-derived; 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 <1% of total Kcal; 30 g of fiber; oxygen radical absorbance capacity (ORAC) > 5000 µMolTe.”
Study Parameters Assessed
Body composition was tracked using bioimpedance analysis (BIA).
Laboratory values included:
- Total cholesterol (TC)
- High-density lipoprotein (HDL)
- Low-density lipoprotein (LDL)
- Calculated HOMA-IR (homeostasis model assessment of insulin resistance)
Investigators analyzed gene expression of the following:
- Catalase (CAT)
- Chemokine C-C motif ligand 5 (CCL5)
- Macrophage migration inhibitory factor (MIF)
- Nuclear factor kappa b subunit 1 (NFKB1)
- Peroxisome proliferator activated receptor γ (PPARγ)
- Superoxide dismutase (SOD1)
- Vitamin D receptor (VDR)
The primary outcome this study was designed to measure was the effect of the MedDiet on lipid and glycemic parameters with and without the addition of MAB juice.
The secondary outcomes included the effect of the MedDiet with and without the MAB juice on 7 genes related to inflammation and oxidative stress.
There was an improvement in lean muscle mass with the addition of MAB juice (P<0.01) per the BIA.
There was a significant reduction in TC/HDL index with the addition of MAB juice to the MedDiet (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 MedDiet and MedDiet + MAB juice groups.
There was no effect on parameters of glycemia, including glucose, insulin, and HOMA-IR.
This study received no external funding. The authors declared no conflicts of interest. Registered as NCT01890070.
Practice Implications & Limitations
The Mediterranean diet is often hailed as one of the best-evidenced diets for reducing the risk of chronic noncommunicable diseases (CNCDs), such as type 2 diabetes, cancer, and cardiovascular diseases. A typical traditional diet from the Mediterranean region is high in olive oil, seafood, fruits, vegetables, and wine, with limited amounts of meats and no processed foods. It is thought that the combination of micronutrients, phytochemicals, fiber, and the types of fatty acids in the MedDiet contributes to its health benefits. The study under review here assessed the physiological effects of a mixed apple/bergamot (80/20) fresh-pressed juice (MAB juice) in healthy Italian subjects already consuming what is touted as an ideal dietary pattern.1
This crossover study took 7 weeks to complete. This entailed 2 weeks of MedDiet with or without MAB juice, a 3-week washout period (regular diet, which is presumably close to the MedDiet), and then another 2 weeks with or without MAB juice. Remarkably, significant changes in lean body mass and HDL levels were found with only 2 weeks of MAB juice consumption.
Interestingly, the highest levels of polyphenols were from the bergamot juice, even though it was only 20% of the MAB juice
This study measured the expression of 7 genes to track physiological changes in real time. Dietary effects on gene expression are expected to be immediate, so the short duration of the intervention was in keeping with this secondary endpoint. The effects of the MAB juice included activation of intracellular antioxidant systems (SOD1), balancing of inflammatory mediators (VDR and MIF), and improved glucose regulation (PPAR gamma). These patterns of genetic expression suggest cellular changes that are in keeping with metabolic profiles that lessen the risk of CNCDs over time.
It may be surprising that there was no effect on glucose, insulin, or HOMA-IR with consumption of the MAB juice. This is despite the significant difference in the percentage of calories from carbohydrates when taking the MAB juice (54.63 ± 11.01 vs 60.52 ± 11.04; P=0.01), which indicates the MAB juice was truly added to the base MedDiet. According to prior research, the naturally occurring phytochemicals in fruits mitigate the effects of their fructose content on glucose levels.2
Also of note was that there were no untoward signs or symptoms or negative laboratory parameters found in the subjects at any point in either arm.
There are thousands of phytochemicals in every fruit and vegetable. These compounds impart the net benefits of a plant-rich, fresh, locally sourced diet. Phytochemicals that have measurable physiological effects are called bioactive compounds, or simply “bioactives.”
Phenolics, especially polyphenols, may be the most well-studied class of bioactive compounds with proven health benefits, including reducing the risk of CNCDs.3 In this study, the polyphenolic composition of the MAB juice was determined. Interestingly, the highest levels of polyphenols were from the bergamot juice, even though it was only 20% of the MAB juice. The most abundant polyphenols found were brutieridin, meltidin, and naringin.
Apples are well-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 favorable changes from the MAB juice? This study cannot tell us, and perhaps that’s not a bad thing.
Research often follows a reductionist path in pursuit of precision. Although numerous studies detail the physiological effects of singular polyphenols, there is also a growing appreciation of the synergy of naturally occurring combinations. The most logical means of studying nutrient synergies in natural foods is to use whole food and measure its physiological effects. This may be simple and obvious, but the pursuit of individual compounds from nature (ie, bioactives) is a multibillion-dollar industry that continues to grow.5 This study is commendable in its simplicity of design and use of whole-fruit juice. The precision in this study was focused on determining which genes are affected rather than the chemistry of the plant—arguably, a much more holistic design.
As clinicians, we know that while risk reduction is obtainable, some patients have done “everything right” and still receive a diagnosis of a CNCD. These patients are normal weight, exercise regularly, eat a plant-rich diet, avoid toxic compounds, have healthy relationships, pursue their spiritual path, etc. Reducing risk when there are no overt risk factors presents a significant challenge for preventative 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 diet. This is intriguing and could be considered for those few patients who do not have much room for improvement. While not powered or designed to guide practice decisions, this study offers food for thought.