To the best of our knowledge, this is the first study to demonstrate the presence of phenolic compounds in commercially available plant-based protein supplements. While the majority of studies have focused on the presence of phenols in raw plant material [16] and their derived products [17] or as additives to increase the bioactive properties of protein supplements [18], there has been a notable gap in the investigation of these bioactive compounds in the context of plant-based protein supplements. Therefore, this study uniquely presents the profile and content of phenolic compounds and antioxidant capacity of five different plant-based protein supplements (soy, rice, pea, hemp, and pumpkin seed proteins), with the aim of broadening our understanding of their nutritional value beyond their protein contribution.

In the current study, twenty-five polyphenols were detected and quantified in plant-based protein supplements (Table 1) using the UHPLC-DAD-MS method. Eleven of the phenols were phenolic acids (gallic, p-coumaric, gentistic, caffeic, syringic, vanillic, benzoic, m-coumaric, salicylic, ferulic, and o-coumaric acids), thirteen compounds belonged to flavonoids ((+)-catechin, isorhamnetn-3-O-glucosied, kaempferol, myricetin, quercetin-3-O-glucoside, rutin, isorhamnetn-3-O-rutinoside, quercetin-3-O-galactoside, quercetin-3-O-vicianoside, kaempferol-3-O-rutinoside, quercetin, naringenin, and apigenin), and one represented ellagitannins (ellagic acid). Among the identified phenolic acids, five belonged to hydroxycinnamic acids (caffeic, p-coumaric, m-coumaric, o-coumaric, and ferulic acids), while six compounds belonged to hydroxybenzoic acids (gallic, gentisic, syringic, vanillic, benzoic, and salicylic acids). Gallic acid, p-coumaric acid, gentisic acid, vanillic acid, benzoic acid, salicylic acid, ferulic acid, and quercetin were found in all examined protein samples. Interestingly, benzoic acid was found to be the predominant compound in protein isolates obtained from rice, pea, hemp, and pumpkin seeds. Benzoic acid is known for its antimicrobial properties, effectively inhibiting the growth of bacteria and yeast. Consequently, it is commonly used as a preservative to extend the shelf life of various food products [19]. While generally recognized as safe for consumption in limited amounts, the excessive intake of benzoic acid could potentially lead to health issues, such as allergic reactions [20]. The significant concentration of benzoic acid detected in plant-based protein supplements raises intriguing questions. One possibility is that benzoic acid may result from the dehydroxylation of p-hydroxybenzoic acid during protein isolate production under certain conditions (e.g., elevated temperatures or specific catalytic reactions). This hypothesis is supported by the previous literature reports from other researchers who have identified p-hydroxybenzoic acid in the raw material of these supplements [16,21,22,23]. On the other hand, benzoic acid could be intentionally added as a preservative in the examined protein isolates. However, none of the tested plant-based protein supplements included information about the presence of benzoic acid on their packaging labels despite its significant concentration in the products.

The highest number of phenolic compounds were identified in soy protein supplements (19 compounds), including 9 compounds of phenolic acids and flavonoids each and 1 ellagitannin. The most abundant polyphenol in this sample was ellagic acid, with a 19.7% contribution to the total phenolic index (TPI). Moreover, the ellagic acid content found in the current study for this sample was more than two and four times higher than rice and pumpkin seed proteins, respectively (Table 1). The significant contribution of gentisic acid and benzoic acid also characterized the soy protein.

It should be emphasized that soy protein isolate was characterized by more detected flavonoids than the other analyzed plant protein samples. The flavonoid profile of soy protein includes (+)-catechin, kaempferol and its derivatives, myricetin, rutin, quercetin, naringenin, and isorhamnetin derivatives. The concentration of (+)-catechin was more than three and seven times higher than pea and pumpkin seed protein samples, respectively. Flavonoids identified in soy-based protein isolates are known to improve cardiovascular health by enhancing blood circulation, reducing inflammation, and offering lipid-lowering benefits crucial for metabolic syndrome prevention [24]. The enriched flavonoid composition of soy protein highlights its potential as a multifunctional food source, offering broader health benefits than other protein supplements.

The following protein sample with a high number of detected phenols (18) was a pumpkin seed protein supplement. It contained nine phenolic acids, eight flavonoids, and ellagic acid (Table 1). The most abundant compound in pumpkin seed protein was benzoic acid (37.8% of the TPI), followed by gentisic acid (26.7% of the TPI) and salicylic acid (10.2% of the TPI). The contribution of the other identified phenols in pumpkin seed protein samples was below 5% of the TPI. Interestingly, two detected compounds, i.e., gentisic and salicylic acids, were found in significantly (p < 0.05) higher concentrations than the other analyzed samples. The pumpkin seed sample showed an average 94% higher concentration of gentisic acid than other analyzed protein samples and more than 90% higher salicylic acid content than soy, rice, and pea proteins. Both gentisic and salicylic acids possess notable antioxidant and anti-inflammatory properties. Gentisic acid is thought to promote cardiovascular well-being by modulating cholesterol levels and protecting against atherosclerosis [25]. Salicylic acid, in turn, is recognized for its wide-ranging benefits, including anti-inflammatory, anticancer, neuroprotective, and antidiabetic effects [26]. Given its rich content of both gentisic and salicylic acids, pumpkin seed protein offers a unique spectrum of protective benefits.

In the hemp protein supplement, we identified 17 phenols, consisting of 10 phenolic acids and 7 flavonoids, representing the highest phenolic acid count compared to all other examined samples. Similar to rice, pea, and pumpkin seed protein samples, hemp protein samples were dominated by benzoic acid. Rutin, salicylic acid, and kaempferol-3-O-rutinoside contributed significantly to the TPI (10.3%, 8.9%, and 7.2%, respectively), while the other identified phenols in hemp protein isolate collectively contributed less than 5% to TPI. Among the analyzed samples, hemp protein was characterized by the highest content of two phenolic acids (gallic and caffeic acids) and four flavonoids (kaempferol, rutin, isorhamnetin-3-O-rutinoside, and kaempferol-3-O-rutinoside). The most significant differences in concentration among the samples were observed for rutin. This flavonoid was found only in two samples (hemp and soy proteins), with the twenty-sixth highest concentration in hemp protein. Hemp protein was also a good source of kaempferol-3-O-rutinoside with more than two-, three-, and five-fold higher content than soy, rice, and pumpkin seed proteins, respectively. Additionally, hemp protein contained approximately twice the amount of gallic acid content compared to the other analyzed protein samples. The remarkable concentration of rutin and kaempferol-3-O-rutinoside in hemp protein enhances its value as a superior nutritional supplement with antioxidative and anti-inflammatory properties crucial for preventing chronic diseases and supporting overall health [27].

Pea-based protein supplements contained 15 phenolic compounds, including 9 phenolic acids and 6 flavonoids. Like the other analyzed samples, benzoic acid was the predominant compound in pea protein (59.3%). Syringic acid (6.6%), quercetin-3-O-vicianoside (8.0%), and quercetin (6.5%) made significant contributions to the TPI in pea protein. Other identified phenols contributed less than 5% to the TPI. It should be noted that syringic acid was exclusively identified in pea protein, distinguishing it from the other examined samples. This phenolic acid demonstrates antioxidant, anti-inflammatory, antimicrobial, and neuroprotective properties, contributing to the prevention of various conditions, including diabetes, cardiovascular diseases, cancer, and cerebral ischemia [28,29]. Pea protein was also characterized by the highest concentrations of quercetin glycosides (quercetin-3-O-glucoside, quercetin-3-O-galactoside, and quercetin-3-O-vicianoside) and apigenin. Among these, quercetin-3-O-vicianoside content was significantly higher in pea protein compared to pumpkin seed and hemp protein samples. Apigenin levels were 35% higher in pea protein than in rice protein. The results of the studies suggest that apigenin may reduce the secretion of key proinflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-10, and, therefore, shows promise for managing various inflammatory conditions like cancer, diabetes, cardiovascular, and neurodegenerative diseases [30].

The rice protein supplement had the lowest number of phenols (13 phenols), including 8 phenolic acids, 4 flavonoids, and 1 ellagitannin. As observed in the other samples, benzoic acid dominated, with the highest contribution among all analyzed protein samples, constituting 70.1% of the TPI (Table 1). The second most prominent compound observed in rice protein was quercetin, constituting 7.0% of the TPI. Other detected phenols comprised less than 5% of the TPI. Rice protein was characterized by a significantly higher ferulic acid and naringenin content compared to the other plant-based protein samples. Ferulic acid concentration was 36.43 µg/g and was approximately two times higher than the other analyzed protein plant-based supplements. The concentration of naringenin in rice protein was more than 10% higher than in soy and pumpkin seed protein samples. Ferulic acid presents notable therapeutic potential in addressing conditions like diabetes, cancer, and cardiovascular diseases, primarily due to its antioxidant and anti-inflammatory properties. It exhibits various biological activities including anticarcinogenic, antiallergic, antimicrobial, and hepatoprotective effects, making it a valuable compound for medical research and clinical applications [31]. Naringenin, in turn, exhibits a wide range of potential health benefits, including antioxidant, anti-inflammatory, antidiabetic, anti-hypertensive, neuroprotective, and cardioprotective properties. Although most of the evidence comes from in vitro and animal studies, clinical trials focusing on cardioprotective effects have shown promising results, particularly in patients with cardiovascular risk factors [32].

As mentioned above, to the best of our knowledge, there is no information on the profile and content of phenols in the analyzed protein plant-based supplements. Therefore, our results were compared with available data on phenols identified in row material (soybeans, rice, peas, hemp, and pumpkin seeds) or derived products. In the case of soybeans, the results of the study by Zhu et al. [23] pointed to the presence of 8 phenolic acids in 18 soybean cultivars. In the above research, the soybeans did not contain flavonoids, ellagitannins, and six phenolic acids (gentisic, vanillic, benzoic, m-coumaric, salicylic, and o-coumaric acids) identified in our study. On the other hand, four phenolic acids (protocatechuic, chlorogenic, p-hydroxybenzoic, and cinnamic acids), which were detected in soybeans from China, were not found in soy proteins [23]. The same authors showed that the primary phenolic acids in the analyzed soybeans were chlorogenic, p-hydroxybenzoic, and caffeic acids in the black soybean samples. In contrast, protocatechuic and p-coumaric acids were the major compounds in yellow soybean cultivars [23]. In comparison, 14 different soybeans also from China examined by Wang et al. [33] were characterized by the presents of 6 phenolic acids (gallic, p-coumaric, syringic, vanillic, ferulic, and protocatechuic acids) and 4 flavonoids (rutin, quercetin, epicatechin, and isoquercetin). The cited study did not detect the five phenolic acids and eleven flavonoids that were noted in our study. Ma et al. [34] analyzed nine brown and white japonica rice cultivars; results showed the presence of only six phenolic acids (caffeic, sinapic, ferulic, p-hydroxybenzoic, syringic, and p-coumaric acids). Moreover, as in soybeans, p-hydroxybenzoic acid was the predominant phenolic acid in the analyzed japonica rice. Results of a study by Li et al. [22] showed the presence of eleven phenolic acids in seven varieties of brown rice from southern China. In the above study, the rice samples did not contain the six phenolic acids (gallic, gentistic, benzoic, m-coumaric, salicylic, and o-coumaric), flavonoids, and ellagitannins that were detected in our study. On the other hand, four phenolic acids (protocatechuic, p-hydroxybenzoic, chlorogenic, and trans-3-hydroxycinnamic acids), which were found in rice samples [22], were not detected in rice protein isolate. Pumpkin seeds and their roasted products were characterized by the presence of seven phenolic compounds, including five phenolic acids (gallic, caffeic, p-hydroxybenzoic, ferulic, and p-coumaric acids) and two flavonoids (epicatechin and rutin) [17]. The results presented by Peng et al. [17] showed that p-hydroxybenzoic acid had the largest share in the content of polyphenolic compounds in the analyzed pumpkin seeds and pumpkin seed products. In turn, Foss et al. [16] showed the presence of 26 phenolic compounds in Indian hemp, including 12 phenolic acids, 8 flavonoids, and 7 stilbenes. In the cited study, the predominant compound was orientin, with approximately 25% contribution of TPI; moreover, this flavonoid was not detected in our samples. In comparison, Izzo et al. [35] detected twenty-two polyphenols in hemp from four different subclasses (phenolic acids, ligninamides, phenolic amides, and flavonoids) of these compounds. In the case of the pea, the available literature shows that a total of 115 structurally different phenolic compounds are present in peas, most of which are glycosylated flavonols (mainly 3-O-glycosides of kaempferol and quercetin) along with their biosynthetically related counterparts [21].

The total phenolic index (TPI) was found within 601.34–1620.69 µg/g (Table 1). The highest value of TPI was determined in the hemp protein sample, while the lowest value was pointed in soy protein. No statistical differences (p > 0.05) in the TPI values were found between the hemp and pumpkin seed proteins, as well as between rice and pea protein samples. The highest TPI values in hemp and pumpkin seed proteins can be attributed to the fact that these samples were characterized by the highest number of detected polyphenols (17 and 18 compounds, respectively). Hemp and pumpkin seed protein samples also showed more than 90% higher concentrations of salicylic acid than the soy, pea, and rice protein supplements. Moreover, as mentioned above, hemp protein demonstrated the highest content of rutin and kaempferol-3-O-rutinoside, while pumpkin seed protein was characterized by the highest concentration of gentisic acid (Table 1).

The dominant group of phenolic compounds in four out of five tested plant-based protein supplements were phenolic acids (Figure 1). Phenolic acids were dominated in rice, pea, hemp, and pumpkin seed protein supplements with an average contribution of 79% of the TPI. On the other hand, flavonoids were the leading group in the soy protein (46%), while phenolic acids constituted 12% less of the TPI. As mentioned above, ellagitannins were also found in examined samples (Table 1), which were represented by one compound (ellagic acid). This group was detected in three protein samples (soy, rice, and pumpkin seed proteins), with the highest contribution of ellagitannins found in soy protein (20%). Ellagic acid is recognized for its wide range of health benefits. It has been extensively studied for its antioxidant properties, attributed to its ability to scavenge free radicals and inhibit oxidative stress-induced damage to cells and tissues. It also exhibits anti-inflammatory, anti-carcinogenic, and anti-mutagenic effects, underscoring its potential as a multifaceted therapeutic agent. The results of the studies also suggest that ellagic acid supports gastrointestinal health [36,37]. The presence of ellagic acid in soy, rice, and pumpkin seed protein isolates further supports their health-promoting potential.