The term “ fatigue ” refers to a feeling of extreme tiredness caused by physical or mental stress, which leads to a temporary reduction in motor and psychological abilities. (Phillips, 2015) . Depending on the persistence of symptoms, fatigue can be classified into: i) acute fatigue , which is a natural response to intense effort that usually does not last long and can be resolved with rest (Kluger et al., 2013) , ii) prolonged fatigue , which is similar to acute fatigue, but lasts longer (30 days to 6 months) and can have a greater impact on daily activities (Esposito et al., 2022) , and iii) chronic fatigue syndrome ( Myalgic encephalomyelitis – ME , or chronic fatigue syndrome - CFS ) ) , which is a condition that is no longer physiological but pathological , which involves debilitating fatigue that does not improve even with rest and can seriously interfere with daily life (Davis and Walsh, 2010) .
Epidemiological studies indicate that approximately 5-8% of the general population experiences prolonged or temporary fatigue, while 3-4% are affected by CFS. A survey of a working population showed that prolonged fatigue affected 21.9 % of them and could reduce their productivity. (Kant et al., 2003) . Furthermore, fatigue is also a significant risk factor for accidents at work, as it can impair the ability to recognize dangers and react promptly to risky situations.
Although prolonged fatigue is a common problem, to date, there are no effective treatments, either pharmacological or non-pharmacological, that are widely recognized or recommended. Some approaches, such as exercise and cognitive behavioral therapy, have been studied, but both options have limitations and the results are not always satisfactory . (Yancey and Thomas, 2012) .
In terms of pharmacological treatments, some drugs have been tested in patients with CFS due to serious medical conditions (such as cancer or multiple sclerosis ) where the need to relieve fatigue outweighs the potential side effects. Drugs such as pemoline , amantadine , and modafinil have been used for fatigue related to multiple sclerosis, while donepezil and carnitine have been tested in patients with advanced cancers. However, the evidence for the efficacy of these drugs is weak and unconvincing. On the other hand, pemoline and methylphenidate have shown some efficacy in treating HIV-associated fatigue, although these results are from a study with a limited number of participants. ( Mücke et al., 2015) .
The condition of prolonged fatigue may represent the ideal target for the implementation of specific preventive strategies against conditions related to chronic fatigue.
To relieve fatigue, approximately 80% of people in the United States who suffer from prolonged fatigue turn to alternative therapies such as dietary supplements based on plant extracts .
The mechanisms underlying prolonged fatigue include chronic inflammation , mitochondrial dysfunction, sleep disturbance, and poor nutritional status. Vitamin deficiency is one of the possible causes of fatigue. Growing evidence suggests that fatigue can be improved through nutritional intervention aimed at reducing vitamin deficiency. The combination of a low- fat diet high in fiber, fruits, and omega-3 fatty acids with dietary supplements containing vitamins, minerals, and polyphenols could be a strategy to counteract prolonged fatigue.
Among the food substances most commonly consumed to relieve fatigue is caffeine, whose effect on physical and mental fatigue is well known. However , subjects with peptic ulcer , dyspepsia , hypersecretory gastritis , reflux disease , hypercholesterolemia , ischemic heart disease , arterial hypertension and cardiac arrhythmias , fibrocystic breast disease , who abuse alcohol or other psychoactive substances and pregnant women ( for whom no more than two cups a day are recommended ) cannot consume caffeine (Rodak et al., 2021) and this leads to the fact that the question of what is the best strategy to combat tiredness remains unanswered for many people .
More recently, some research has focused on the combination of pomegranate extracts and water-soluble vitamins to combat prolonged fatigue , preventing it from evolving into a pathological condition of CFS.
The pomegranate ( Punica granatum L . ) is a species of plant with an edible fruit that grows in many regions of the world. Pomegranate is known for its high consumption, industrial value and its nutritional and health properties . From a nutritional point of view, every 100 g of pomegranate arils contain approximately 72 kcal of energy, 16.6 g of carbohydrates, 1 g of protein, 13 mg of calcium, 1 mg of sodium, 379 mg of potassium, 12 mg of magnesium, 0.17 mg of copper, 0.7 mg of iron, 7 mg of vitamin C and 0.3 mg of niacin (Teixeira Da Silva et al., 2013) . Pomegranate is known to be rich in polyphenols, such as phenolic acids, flavonoids and tannins, which give it numerous beneficial effects on health. ( Viuda - Martos et al., 2013; Viuda - Martos et al., 2010) . Thanks to its bioactive compounds, the fruit can be considered a "functional food", useful for various problems, including inflammation, cardiovascular protection for its anti-hypertensive action , cancer prevention, liver protection, as well as for its antimicrobial, antiviral, antidiabetic, neuroprotective and dermatological properties. (Viuda-Martos et al., 2013) . Due to its health properties, pomegranate juice has been named a “superfood” (Fahmy et al., 2020) . In physically active subjects, pomegranate supplementation is beneficial for improving endurance and post-exercise physical performance, thanks to its antioxidant and anti-inflammatory action. ( Urbaniak and Skarpańska-Stejnborn , 2021) . Furthermore , a meta-analysis of clinical studies has shown that pomegranate juice intake leads to a reduction in some inflammatory markers (such as hs -CRP, TNF-α and IL-6) in adults. (Wang et al., 2020) .
Water-soluble vitamins, such as B vitamins and vitamin C, are organic compounds that the human body requires in small amounts to perform nutritional, biochemical, and physiological functions. physiological. The main sources of these vitamins are foods such as fruit, vegetables, legumes, cereals, meat and eggs . It is known that these vitamins contribute to the prevention of chronic and degenerative diseases. Among the biological benefits of water-soluble vitamins are antioxidant effects (Chakraborthy et al., 2014) , anti-inflammatories (Ueland et al., 2017) cardioprotective (Rodrigo et al., 2021) , neuroprotective (Monacelli et al., 2017) , metabolic regulation (Ragheb et al., 2020) and antianemics (Aljaadi et al., 2022) . A literature review conducted by Werbach et al. (Werbach, 2000) , highlighted the potential role of these vitamins and other nutrients in chronic fatigue symptoms, since vitamin deficiencies have been found in several studies of CFS patients.
The effects of the combination of a whole pomegranate fruit extract and vitamins C and B were investigated in a randomized clinical trial. In particular, the aim of the work conducted by Esposito C. et al., (Esposito et al., 2022) was to evaluate whether a food supplement based on pomegranate extract, B vitamins and vitamin C can help combat prolonged fatigue , measured through the administration of questionnaires that evaluate the level of fatigue before and after daily intake of the supplement for a month, also evaluating whether this food supplement is well tolerated. Furthermore, the metabolic profile of the whole pomegranate extract used as an ingredient in this food supplement was analyzed using a latest-generation LC-MS instrumentation to deepen the knowledge on the chemical composition of the phytocomplex present in this food supplement. RP-UHPLC-UV-MS analysis of the dry extract of the whole pomegranate fruit ( PunicaPLUS ®) indicated the presence of 59 main compounds belonging to different classes: ellagitannins , gallotannins , flavonolic acids and glycosides .
In the study , 78 subjects (21 men and 57 women) were recruited for one month, in order to evaluate the efficacy and tolerability of this food supplement. The results of the study clearly show that the daily intake of the food supplement based on pomegranate extract, B vitamins (at doses ranging from 50 to 500% of the daily reference intake for vitamins in adults) and vitamin C (at a dose equal to 250% of the daily reference intake for vitamin C) for a period of one month can produce a significant improvement in fatigue and quality of life. In fact, after the supplementation, the Fatigue Assessment Score scores ( FAS ) , Fatigue Severity Score ( FSS ) , and 11-point Numeric Rating Scales ( NRT ) significantly decreased between t0 and t1, and quality of life was consequently improved, as both scores Short Form 12 Health Survey ( SF-12 ) (in the physical and mental components) increased significantly. Furthermore, the food supplement was considered well tolerated by the investigating physician , as no subject reported adverse effects (AE) related to the consumption of this food supplement .
Supplementing with water-soluble vitamins may improve fatigue due to their role in a number of basic metabolic pathways that support energy production systems, oxygen transport, and protection from oxidative stress. Additionally, thiamine, pantothenic acid, folate, and vitamin C play a role in maintaining brain structure, enabling intercellular connections, and in the synthesis of neurotransmitters, resulting in improved cognitive and psychological function.
Although most of the pomegranate products studied for their health properties are derived from the edible part of the fruit (i.e. seeds and arils), the peel is rich in many health-promoting compounds (antioxidants and anti-inflammatory and immunomodulatory agents), which are often discarded as waste. (Lu et al., 2020) . The extract used as an ingredient in this food supplement ( PunicaPLUS ®) is obtained from the whole fruit and therefore contains the entire phytocomplex. It has been found to be particularly rich in ellagitannins , which are attributed to many health-promoting properties. Recent studies conducted on a wide range of athletes and physically active subjects have shown that pomegranate juice and extracts improve whole-body strength, the feeling of vitality and sports performance during intense physical exercise, thanks to the increase in elasticity of the vascular wall and, consequently, blood flow and oxygen supply to muscle needs ( Torregrosa -García et al., 2019) . In addition, Pomegranate supplementation has the potential to promote recovery from exercise and alleviate acute and delayed muscle fatigue, through attenuation of oxidative stress and inflammation by reducing free radical production, lipid peroxidation and inhibition of cellular inflammatory transcripts (TNF-alpha, COX-2 and NF- kB ) (Ammar et al., 2018) . The well-known anti-inflammatory activity of pomegranate may be one of the mechanisms of action through which this fruit helps relieve fatigue. In a literature review, Lee et al. reported that fatigued subjects exhibit higher levels of proinflammatory cytokines (e.g., interleukin-1 and -6 and tumor necrosis factor alpha), supporting the hypothesis that anti-inflammatory treatments could help reduce inflammation and somatic and physiological symptoms ( Haß et al., 2019) . Furthermore, pomegranate juice and extracts have been found to be active in decreasing the levels of pro-inflammatory cytokines in clinical studies involving patients with inflammatory diseases (e.g., hemodialysis patients, overweight and obese individuals, type 2 diabetes). (Barati Boldaji et al., 2020) . Another mechanism of action that could justify the positive results of pomegranate extract intake obtained against prolonged fatigue could be its ability to modify the intestinal microbiota, as demonstrated by recent studies. The association between intestinal microbiome and fatigue has been demonstrated in many pathological conditions, such as in cancer patients, in survivors of hematopoietic stem cell transplantation and in patients with multiple sclerosis, including those with myalgic encephalomyelitis/chronic fatigue syndrome ( Frémont et al., 2013) . In particular, subjects with chronic fatigue have low levels of bifidobacteria (Logan et al., 2003) and a significant increase in lactic acid produced by Enterococcus and Streptococcus in fecal samples (Sheedy et al., 2009) . In 2020, Lu et al. demonstrated that a pomegranate peel extract reshapes the composition of the gut microbiota, increasing eubiotic bacteria such as Lactobacilli , probably through the polyphenolic components that act as prebiotics as they are excreted unchanged in the intestinal lumen and concentrated in the colon, where they are metabolised by intestinal bacteria into low molecular weight substances such as urolithins , which modulate the intestinal microbiota and exert a systemic anti-inflammatory activity. (Lu et al., 2020) .
Furthermore, the clinical study conducted by Ullah H. et al., aims to evaluate the efficacy of a new food supplement ( Improve TM ) based on a chemically characterized pomegranate extract and water-soluble vitamins (B and C complex). The UHPLC-HRMS analysis of the pomegranate extract showed the presence of gallotannins and ellagitannins which are the most abundant phytochemicals. For the clinical study, 58 subjects were randomized into two groups, 1 and 2 (n = 29, each), which received the food supplement or the placebo. The effects of the food supplement on fatigue were assessed through validated questionnaires, recorded at time intervals t0 (at baseline), t1 (after 28 days), t2 (56 days) and t3 (after follow-up) in combination with the analysis of biochemical markers at t0 and t2. The FSS questionnaire scores decreased significantly at time intervals t2 and t3 in subjects treated with the food supplement , which however, limited to the experimental conditions applied in this study, did not significantly influence the biochemical parameters associated with fatigue and stress conditions. This study demonstrates that the tested food supplement reduces prolonged tiredness after two months of supplementation in healthy subjects with mild prolonged tiredness.
In conclusion, the literature data demonstrate that the combination of a pomegranate extract, B vitamins and vitamin C, reduces prolonged fatigue. The mechanism of action has not yet been fully clarified , but , considering the high content of ellagitannins of pomegranate extract , which are able to remodel the intestinal microbiota by increasing eubiotic bacteria such as L. actobacilli . ( probably through urolithins generated by the metabolism of polyphenolic components ) (Lu et al., 2020) , and , given the role of the intestinal microbiota in chronic and prolonged fatigue (Tuchman et al., 1991) , it can be assumed that the effect of this combination of ingredients is due to an improvement in the functionality and composition of the intestinal microbiota . An in vitro study on this aspect could provide useful information on the mechanism of action through which this combination of ingredients carries out the anti-fatigue effect demonstrated by scientific studies in the literature .
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