Diabetes mellitus is a chronic endocrine condition in which the pancreas either stops producing insulin or produces inadequate insulin. Diabetes affects about 425 million people globally and is defined by a rise in blood glucose concentration (>7 mmol/L) [95]. It has been associated with the development of various significant problems at cardiovascular, neurological, and renal levels, leading to increased morbidity and mortality [93]. The International Diabetes Federation anticipated that, by 2030, there will be 552 million diabetics globally [96].

To establish glycemic control, diabetics use insulin and other therapy drugs, such as metformin, sodium-glucose cotransporter-2 inhibitors, and glucagon-like peptide 1 [97]. Before the development of insulin, medicinal plants were used to treat this condition. Because of their low cost, availability, and lack of negative effects, the use of natural plants was and still is an alternative for many people. Various plant genera and phytochemical constituent types with anti-diabetic properties have been used in this context [31,45,97,98]. Therefore, it is not surprising that formulations using anti-diabetic plant extracts or phytocompounds have been derived. Additionally, nowadays, systems such as “Herbal-based anti-diabetic drug delivery systems” are largely used to provide herbal medicines to treat diabetes [98].

Certain regions of the world employ black mulberry leaves, fruits, and barks as anti-diabetic medications, believing in their efficacy in lowering blood glucose levels [31,99,100,101,102]. In accordance with this, Morus nigra has been shown to have a wide range of biological and pharmacological therapeutic benefits, including antidiabetic, anti-obesity, and anti-hyperlipidemic effects [103]. Hydroethanolic freeze-dried extracts of this fruit revealed potential for inhibiting pancreatic lipase, displaying a half maximal inhibitory concentration of 6.32 mg/mL [104].

Among both berries’ constituents, quercetin has been demonstrated to have considerable antioxidant and anti-inflammatory characteristics and the ability to interfere with a variety of antidiabetic activities, including insulin secretion and sensitization, glucose level improvement, and inhibition of intestinal glucose absorption. By activating adenosine monophosphate and preventing lipid peroxidation, this phenolic molecule promotes glucose transporter 4, the principal facilitative mediator of glucose uptake in skeletal muscles, adipose tissues, and other peripheral tissues. Given that, it is not surprising that quercetin can be used to stabilize blood glucose and body weight [105,106]. Furthermore, a single oral dosage of quercetin (400 mg) decreased α-glucosidase activity and reduced postprandial hyperglycemia in rats with type 2 diabetes [107].

Ferulic acid, another berry phenolic component, at 1000 mg per day for six weeks, showed the capacity to decrease total cholesterol, malonylaldehyde, TNF-α, and triglycerides by 8.1, 24.5, 13.1, and 12.1%, respectively, and increase HDL cholesterol by 4.3% [108]. These findings suggested that ferulic acid can also help diabetic patients with hyperlipidemia. Ferulic acid was found to be generally safe, with LD₅₀ values of 2445 mg /kg in male rats and 2113 mg /kg in female rats [109].

Additionally, diabetic male Wistar rats received injections of black mulberry fruit extracts at 150 and 300 mg/kg body weight for 4 weeks. After this time, microalbuminuria, albumin, glucose, insulin, creatinine, and creatine levels in the serum were measured. The study discovered that diabetic animals considerably improved in all of the measures tested. The activity of catalase activity was also improved. Furthermore, the histological examination of their kidney tissues revealed a significant reduction in degenerative anomalies and glomerular sclerosis. TNF-α, vascular cell adhesion molecule-1, and fibronectin mRNA expression were all downregulated in treated rats [101]. Therefore, the downregulation of TNF-α, VCAM-1, and fibronectin levels in diabetic rats avoids, or retards, the development of diabetic nephropathy. Altogether, these data support the evidence that mulberry fruit extract may be a potential agent in the treatment of diabetic nephropathy [103].

Plant-derived antimicrobial chemicals may limit the development of bacteria, fungi, viruses, and protozoa by different processes from those utilized by synthetic antimicrobials, and thus exhibit substantial therapeutic benefit in the treatment of resistant microbial strains. The antimicrobial activity of an agent is generally due its potential to chemically interfere with the manufacture or function of key components of bacteria and/or evade established antibacterial resistance mechanisms [45,110,111].

The majority of phytochemicals with therapeutic value found in fruits are secondary metabolites. Their antimicrobial activity varies depending on the structure, number, and position of substituent groups, the presence of glycosidic linkages, and the alkylation of hydroxyl groups [111,112]. As expected, blackberries’ antimicrobial properties differ among cultivars and ambient and soil factors. Furthermore, it is important to note that it is not possible to associate the antimicrobial activity with a specific compound due to the capacity of phenolic compounds to act synergistically [31,89,92,113,114,115,116,117,118].

Recent research has revealed that blackberries and mulberries have notable antimicrobial properties. The antimicrobial activity of different R. fruticosus extracts was investigated against Escherichia coli, Staphylococcus aureus, Bacillus cereus, B. subtilis, B. mojavensis, Salmonella Hartford, Proteus vulgaris, Pseudomonas baetica, Micrococcus luteus, and Saccharomyces cerevisiae. The inhibition zone diameter (mm) was measured, revealing that the ethanolic extracts are more competitive than the crude extracts, and show a notable antimicrobial potential against Proteus vulgari (20.53 mm). The lowest activity was observed against S. Hartford bacteria (9.54 mm). In this study, minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were not calculated [113].

Additionally, hydroethanolic extracts of R. ulmifolius proved to have bacteriostatic effects against three Gram-negative bacteria (E. coli, Morganella morganii, and P. mirabilis), four Gram-positive bacteria (MRSA-methicillin-resistant S. aureus, MSSA-methicillin susceptible S. aureus, Listeria monocytogenes, and Enterococcus faecalis), and one fungus (Candida albicans). The results obtained in this work revealed activity in some tested strains, with MIC values ranging between 5 and >20 mg/mL. To inhibit the growth of Klebsiella pneumoniae and Pseudomonas aeruginosa, a concentration above 20 mg/mL was necessary. For the remaining Gram-negative strains, the most effective results were shown against M. morganii (MIC = 5 mg/mL) and E. coli (MIC = 5 mg/mL), followed by P. mirabilis (10 mg/mL) [13]. In another study, methanolic extracts of R. ulmifolius showed antimicrobial potential against two Gram-negative bacteria (E. coli and Salmonella typhimurium), three Gram-positive bacteria (S. aureus, Enterococcus feacium, Streptococcus agalactiae) and one fungus (Candida albicans). The most notable values were observed against S. agalactiae and E. coli bacteria [114].

The antimicrobial effects of M. nigra were also evaluated, especially in S. aureus, P. aeruginosa, and E. coli, where the ability of its extracts to inhibit the production of proinflammatory cytokines and interfere with iNOS and NF-κB pathways was observed [114]. Considering the higher content of anthocyanins in this species, these effects could be attributed to these compounds. In fact, anthocyanins have potent antiviral and antibacterial properties, being already known for their antimicrobial potential against K. pneumonia, P. aeruginosa, S. aureus, E. coli, H1N1, SARS-CoV-2, and rabies and herpes simplex virus [45,112,114].

Additionally, the antibacterial efficacy of mulberry total flavonols was assessed against three bacteria (E. coli, P. aeruginosa, and S. aureus), revealing interesting MBC results against S. aureus and E. coli [115]. Another investigation demonstrated the potential of M. nigra ethanolic extracts to be used in acne-treatment beauty care products given their capacity to inhibit S. epidermis and P. acnes growth, revealing MIC values of 2.5% for both bacteria, and MBC scores of 2.5% and 5% against S. epidermidis and P. acnes, respectively [116].

Black mulberry juice also has antibacterial properties, with its ability against three Gram-negative strains (E. coli, P. aeruginosa, and S. typhimurium) and five Gram-positive strains (Bacillus spizizenii, B. subtilis, Corynebacterium diphtheriae, Enterococcus. faecalis, and S. aureus) being previously reported. The maximum zone of inhibition was against P. aeruginosa (19.87 mm), followed by Bacillus spizizenii (19.68 mm) and B. subtilis (18.46 mm). The minimum zone of inhibition was obtained against E. coli (9.98 mm). Among the Gram-positive species, Bacillus species exhibited the highest zones of inhibition while, regarding Gram-negative bacteria, P. aeruginosa had higher inhibition than S. typhimurium and E. coli [117].

Reactive species are products of normal cellular metabolism and play key roles in signal transduction pathways, growth regulation, gene expression, and immune responses. In the human body, various mechanisms are necessary to maintain redox homeostasis [45,119]. These mechanisms include non-enzymatic and enzymatic antioxidant defenses created in the body (endogenous), as well as those given by the food (exogenous). However, the overproduction and accumulation of free radicals can lead to oxidative damage [6]. This biological condition may be caused by a lack of antioxidant defense mechanisms, excessive reactive species production, and excessive activation of their systems, increasing aging and the pathology of many chronic diseases, such as cancer, cardiovascular disease, inflammation, diabetes, and Parkinson’s and Alzheimer’s disease [45,120]. Therefore, it is essential to reduce their levels. Flavonoids, stilbenes, and tannins are examples of exogenous antioxidants. For example, scavenging and detoxifying radical oxygen species and preventing their production, influencing the cell cycle, avoiding tumor suppression, and modulating signal transduction, apoptosis events, and metabolism, are all biologically relevant mechanisms attributed to phenolic compounds [11,45,58,72,121,122,123,124]. Their antioxidant diversity and concentration are greatly dependent on the species and cultivars. Pre-harvest practices, environmental conditions, harvest ripeness, postharvest storage, and processing operations are also key drivers of phytochemical profiles [11,17,40].

Blackberries are considered one of the richest sources of natural antioxidants due to their high content of phenolic compounds, such as anthocyanins, ellagitannins, flavonols, and flavanols [13,41,44,49,51,62]. In fact, they present an extraordinary capacity to scavenge chemically generated radicals, thus preventing a wide range of human disorders and maintaining a healthy balance between free radicals and antioxidant systems. In particular, blackberries have notable antioxidant abilities against superoxide radicals (O₂^●−), hydrogen peroxide (H₂O₂), hydroxyl radicals (^●OH), and singlet oxygen (¹O₂) [123].

The antioxidant capacity of blackberries was previously determined by in vitro assays, by the lipid peroxidation inhibition assays (TBARS), oxidative reactive oxygen and nitrogen species (ROS/RNS), hemolysis inhibition assay, the ORAC method, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS^•+), the ferric-reducing/antioxidant power (FRAP) method, 2,2-diphenylpicrylhydrazyl (DPPH^•), and Trolox equivalent antioxidant capacity (TEAC) assay [23,37,39,49,52,56,71,89,89,125,126,127,128,129,130,131,132].

In the TBARS experiment, R. fruticosus extract revealed a high antioxidant activity, displaying an IC₅₀ value of 100 ug/mL, which is substantially lower than that obtained with the positive control, Trolox (139 ug/mL) [49]. Additionally, using FRAP assay, ABTS^•+, and DPPH^•, the obtained results were between 4.45–14.16 for FRAP, 2.28–8.89 for ABTS^•+, and 2.63–9.35 mmol Trolox equivalents per⋅100 g fw for DPPH^• [71].

Furthermore, methanolic extracts of M. nigra at 76 µg showed the capacity to inhibit lipid oxidation by 28.7%, while ethanolic extracts exhibited lower inhibitory capacity (23.7–47.6%) [125]. The antioxidant abilities of its aqueous extracts were also evaluated, revealing lower abilities than the methanolic ones; at 100 µg/mL, the values obtained were 1.1% and 7.1% for aqueous and methanolic extracts, respectively, whereas at 300 µg/mL, the corresponding values were 7.1% and 21.6%, respectively [125].

However, when comparing wild blackberries (R. ulmifolius) with the cultivated ones (R. fruticosus), substantial differences were found, with the latter having higher antioxidant content [126].

To summarize, mulberries have lately gained a large amount of interest as prospective sources of functional foods due to a variety of biological benefits [103,133]. The obtained findings on the antioxidant activity of mulberry fruits support their incorporation in biological applications [100,103,125,130].

Inflammation is the immune system’s reaction to potentially damaging stimuli such as infection or injury. In the presence of stressors, immune cells release inflammatory substances, such as inflammatory cytokines, including TNF-α and interleukins (IL)-6 and 10, leading to increased nitric oxide (NO) levels and prostaglandins via the catalysis of cyclooxygenase-2 (Cox2) and NF-κB pathways [45,134,135]. Blackberry freeze-dried powders are capable of reducing mRNA expression of NF-kB and COX-2 in the liver [136].

A healthy lifestyle that includes physical activity, stopping smoking, and moderate alcohol intake, associated with a diet rich in fruits, vegetables, and whole grains, decreases the risk of developing chronic diseases. As expected, phenolic compounds, carotenoids, vitamins, and dietary fiber contribute to the anti-inflammatory and antioxidant effects of fruits and vegetables [45,48,137,138,139]. In particular, high quantities of dietary anthocyanins may be viewed as a feasible nutraceutical in the context of inflammatory disease. Among these, cyanidin 3-O-glucoside can reduce cytokine-induced inflammation in intestinal cells by inhibiting the production of NO, PGE2, and IL-8, and the expression of iNOS and COX-2 [112,138,139,140].

Focusing on blackberries and mulberries, anthocyanin-enriched fractions from fermented blueberry and blackberry beverages inhibited dipeptidyl peptidase-IV activity in LPS-stimulated murine macrophages. Computational docking demonstrated that this effect could be mainly attributed to delphinidin 3-O-arabinoside, which effectively inactivates dipeptidyl peptidase-IV by binding with a low interaction energy (−3228 kcal/mol). Additionally, anthocyanins and proanthocyanidins (100 µM cyanidin 3-O-glucoside and epicatechin equivalents, respectively) extracted from them reduced LPS-induced inflammatory response in mouse macrophages by stopping the NF-κB pathway [140]. Another study that used RAW 264.7 macrophages stimulated with LPS demonstrated that blackberry anthocyanin extract (0–20 µg/mL)-treated macrophages presented lower levels of IL-1 and TNF-α [141]. Once again, this reduction is mainly associated with the ability of anthocyanins to interfere with NF-κB signaling [140], particularly of cyanidin 3-O-glucoside, which previously showed potential to decrease pro-inflammatory mediators NO, PGE2, COX-2, and IL-8 generated by cytokine-stimulated HT-29 cells [139]. In accordance with this, R. fruticosus also showed capacity to inhibit the secretion of pro-inflammatory IL-8 cytokines in two cellular models (HT-29 and T-84 cells) in a dose dependent-manner in both cell lines [92].

Ellagitannins are another significant polyphenol that has displayed anti-inflammatory properties. Previous research [142] examined their anti-inflammatory efficacy of TNF-α, IL-1B, IL-8, and NF-κB on the AGS gastric cell line. Ellagitannins extracted from R. fruticosus suppressed TNF-α, showing an IC₅₀ value of 0.67–1.73 mg/mL. At 2 mg/mL, ellagitannins inhibited TNF-α and NF-κB nuclear translocation by 57% and 67%, respectively. At lower doses, ellagitannins reduced IL-8 secretion, revealing an IC₅₀ ranging between 0.7 and 4 mg/mL. Moreover, in a rat model of ethanol-induced stomach lesions, the protective effect of ellagitannins was also tested. Ellagitannins (20 mg/kg/day) were administered orally to rats for ten days, and ethanol was administered one hour before sacrifice. The mucosa of the stomach was separated and utilized to measure IL-8 release, NF-κB nuclear translocation, TEAC, and superoxide dismutase and catalase activities. This investigation demonstrates that the treatment with these compounds can decrease NF-κB nuclear translocation and suppress IL-8 production. The present work demonstrated that ellagitannins derived from Rubus berries definitively protect against the formation of gastric ulcers in rat animal models. In particular, ellagitannins can block the NF-κB cascade either directly on the cell response to pro-inflammatory cytokines or act as antioxidant agents by inhibiting reactive species generated in several inflammatory conditions [143].

The human brain is responsible for a wide range of cognitive, motor and behavioral functions that require significant amounts of energy. Neurons are responsible for transmitting information to and from the brain. Neurodegenerative illnesses are distinguished by progressive brain cell death and neuronal loss, which impair motor or cognitive function. Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and spinocerebellar ataxia are examples of common neurodegenerative disorders. These disorders are a major public health concern, particularly among the elderly [120]. These disorders develop because the brain is more sensitive to oxidative stress than other organs due to the poor activity of antioxidant defense mechanisms [144].

Many epidemiological studies are being conducted to study the potential of phenolics to be used to promote neuronal health and prevent neural cells from being damaged through their antioxidant and anti-inflammatory properties, and thus delay Parkinson’s and Alzheimer’s diseases, ischemic diseases, and aging effects [137,145].

The inclusion of blackberries in the diet has been shown to reduce brain degeneration [9,93,146,147,148]. The neuroprotective capacity of this fruit mainly comes from its antioxidant capacity, promoted by the presence of phenolic compounds, such as anthocyanins, caffeic acid, and quercetin [45,50,137,148,149,150]. Indeed, these compounds can penetrate the hematoencephalic membrane and have neuroprotective effects on various cerebral structures in the brain, including the hippocampus and cortex [122]. Flavonoids may also have important impacts on mammalian cognitive function, perhaps halting the aging-related declines in memory and learning. These benefits are mostly sought for preventing brain damage, such as neurodegenerative diseases, and improving memory, learning, and cognitive functions [148]. Blackberries from the north of Portugal can lower intracellular reactive species levels, alter glutathione levels, and inhibit the emergence of caspases during treatment, hence reducing oxidative stress and preventing neurodegeneration [147]. Mulberry fruit extracts and cyanidin 3-O-glucoside have shown the ability to inhibit reactive species production and, consequently, neuronal injury [151,152].

Neuroblastoma cells exposed to H₂O₂ and treated with raw, digested, and dialyzed blackberry extracts at physiological concentrations revealed lower age-related neurodegeneration [9]. In addition, animal research found that an intermediate dosage of blackberry juice (5.83 mg/kg anthocyanins, 27.10 mg/kg polyphenols) enhanced mechanisms of behavioral coping with diazepam l. The forced swim test supported these findings by demonstrating that blackberry juice, at moderate and high doses, improves the acute stress response [153]. These findings suggest that blackberry juice may have a therapeutic value in alleviating anxiety caused by stressful experiences.

M. nigra revealed a notable protective effect against Alzheimer’s disease, specifically by inhibiting amyloid-β-induced paralysis symptoms and suppressing over-sensitivity to exogenous serotonin by about 55.65% in transgenic Alzheimer’s disease Caenorhabditis elegans models, which were treated with up to 1.00 mg/mL. These effects are due to the capacity of this fruit to activate the DAF-16/SOD-3/GST-4 pathway, improve antioxidant capacity, delay aging, and alleviate the symptoms caused by the amyloid-β protein [154]. These findings suggest that functional foods, such as mulberry, can be used to lower the risk of Alzheimer’s disease.

Cancers are characterized by abnormal cell growth capable of invading other regions of the body, resulting in metastasis. A tumor is a complex multistage process that begins with the genesis of a cancer cell caused by DNA damage, followed by accumulation of mutations, progression to cell proliferation and tumor expansion, and, finally, progression to malignancy and metastasis. While new cancer incidence is expected to rise by 70% by 2034, approximately 35% of cancer deaths are attributed to behavioral and dietary risks, such as high body mass index, low fruit and vegetable intake, and lack of physical activity [155].

According to epidemiological and clinical research, a diet consisting of 400–800 g of various vegetables and fruits per day can prevent 20% or more of all cancer cases [2,48,137].

Phenolic berry content has shown the capacity to reduce inflammation, inhibit angiogenesis, protect against DNA damage, and influence apoptosis or proliferation rates in malignant cells. Indeed, they demonstrate the ability to interfere in all phases of cancer development, including initiation, promotion, progression, invasion, and metastasis [45,134,150,156]. Berry extracts also inhibited cancer-induced AP-1 and NF-κB, as well as decreasing the expression of the two proteins involved in tumor promotion and progression, i.e., vascular endothelial growth factor and COX2 [136,157]. These effects are intimately linked to the capacity of phenolics to alter the genomic stability at many phases in the cancer genesis process [137]. For example, anthocyanins have been shown to activate phase II enzymes, which may inactivate carcinogens triggered by phase I enzymes, and hence prevent DNA damage caused by the carcinogens [82,112,124,158].

Dietary bioactive compounds can also decrease telomerase activity by modifying histones or by inhibiting DNA methyltransferases. Telomerase activity has been detected in more than 80% of human malignancies, making the enzyme a promising target for anticancer treatment. According to research, the antiproliferative impact of blackberry fruits is mediated by their anti-telomerase activity [159]. Additionally, there have been no negative effects associated with the administration of blackberries, indicating that this fruit has the potential to be effective for a dietary plan to reduce cancer risk and assist cancer patients with illness prognosis [157].

Blackberries previously demonstrated significant chemo-preventative and antioxidant activities by inhibiting the growth, proliferation, and migration of the human A549 lung carcinoma cell line, and strong inhibitory effects on the cell growth of highly metastatic breast cancer HS578T cells, by inducing significant alterations in cell cycle regulators, causing G2/M arrests [160]. Blackberries and mulberries contain anthocyanin cyanidin-3-O-glucoside, which has promising qualities for usage in nutraceuticals, and has shown potential to limit cell proliferation, arresting the cell cycle in the G2/M phase, and inducing apoptosis in vitro [112,138,161]. In fact, in a recent investigation, rats were administered orally with purified cyanidin 3-O-glucoside (800 µmol/kg of body weight). After 30 min–2.0 h of delivery, this was detected in plasma, with a C_max value of 0.8 µM. This evidence represents added value regarding the incorporation of this anthocyanin in dietary supplements, aiding in the anticancer therapy of breast cancer [161].

Morus nigra extracts have also been the subject of much research. A three-month enriched diet applied in MUC2^−/− mice, with a model of spontaneous chronic intestinal inflammation and induced-intestinal tumors at three months, at 5% or 10%, resulted in a reduction in tumorigenesis and intestinal inflammation. Basically, mice aged 6 to 8 weeks that were supplemented with 5% or 10% M. nigra extracts for 10 days and there were observed improvements in their signs and symptoms caused by dextran sulfate sodium-induced acute colitis, preventing weight loss and bloody stools, and promoting positive changes in the histology of the colorectal lining [162].

Cardiovascular disorders affect the heart and blood vessels and are the major cause of death worldwide. People who have high blood pressure and cholesterol, as well as smokers, those who are sedentary or obese, and people who have a diet rich in salt, sugar, and fatty acids, are more susceptible to cardiovascular problems [163].

The current nutritional guidelines for the prevention of cardiovascular diseases include a Mediterranean-style diet rich in fruits, vegetables, and whole grains, as well as non-tropical vegetable oils, in order to reduce total cholesterol, oxidative stress, and inflammation [2,48,50,59,137,164].

Blackberry phenolic compounds have demonstrated the capacity to diminish LDL oxidation, quench free radicals by hydrogen molecule donation, and interfere with liposome oxidation systems [165,166]. In particular, anthocyanins from M. nigra showed the capacity to protect human primary endothelial cells by decreasing the production of the cytokine-induced chemokine monocyte chemotactic protein 1, a protein directly linked to atherogenesis, and which is mainly responsible for attracting macrophages to sites of infection or inflammation [167]. Moreover, although not directly shown in blackberry flavonoids, several flavonoids also revealed the capacity to protect platelet function, which is crucial in the pathogenesis of these diseases. In fact, flavonoids can minimize platelet aggregation, reduce platelet generation of superoxide anions, and increase platelet NO production [168].

In epidemiological studies, diets high in plant-derived phenolic compounds have been shown to reduce the incidence of coronary heart disease. The chronic antioxidant and hypolipidemic characteristics of these compounds play critical roles in the prevention of lipoprotein oxidation and the formation of atherosclerotic lesions [2,122,166,169].