3.2. Identification and Quantitative Analysis of the Phenolic Compounds and Iridoids of Smoothie Components and Smoothies
Table 3,
Table 4,
Table 5,
Table 6 and
Table 7 show the results of quantitative analysis of anthocyanins, flavonols, phenolic acids, flavan-3-ols and iridoids in all smoothie components and smoothies.
The abundance of phenolics and iridoids in the smoothies depended on the quantity and the origin of the ingredient used. The final products contained around 40% of an apple juice, 42% of strawberries, 18% of chokeberries and between 0.25–0.50% of a honeysuckle berry extract, which corresponded to the amount of bioactive compounds in the smoothies. The content of apple juice, being the liquid base of smoothies, was chosen to vary where a honeysuckle berry extract was added.
Both chokeberries and strawberries contained four different anthocyanins. Chokeberries were especially rich in cyanidin 3-galactoside (349.03 mg/100 g FW) and cyanidin 3-arabinoside (161.72 mg/100 g FW). Strawberries did not contain high concentrations of anthocyanins (24.27 mg/100g FW), compared to chokeberry (539.19 mg/100 g FW) or a honeysuckle berry extract (8808.30 mg/100 g FW). The main anthocyanin found in strawberries was pelargonidin 3-glucoside (21.04 mg/100 g FW). Apple juice did not contain any anthocyanins. As previously documented, a honeysuckle berry is a great source of various phenolics, including anthocyanins [
56]. Anthocyanins are the most abundant compound in honeysuckle berry fruit, ranging between 36 and 51% of all phenolics [
57]. The six out of 11 anthocyanins identified in all fruit together were found in the honeysuckle berry extract, while in chokeberries and strawberries only four were found. Cyanidin 3-glucoside was the major anthocyanin found in the honeysuckle berry extract (7433.40 mg/100 g FW). The mentioned anthocyanins, together with cyanidin 3-galactoside, were dominant ones in smoothies 2 and 3. Smoothie 1 contained only 2.29 mg/100 g FW of cyanidin 3-glucoside, and the amount significantly increased in the case of Smoothie 2 and Smoothie 3 (23.75 and 102.78 mg/100 g FW, respectively). A significant difference between smoothies 2 and 3 was additionally observed in the case of cyanidin 3,5-diglucoside, pelargonidin 3-glucoside and peonidin 3-glucoside. Cyanidin-3-
O-sophoroside, cyanidin 3-rutoside, pelargonidin 3-glucoside and peonidin 3-rutoside were found in the extract but were not found in the smoothies. They possibly might react with other compounds or were degraded during smoothie preparation. The addition of a honeysuckle berry extract resulted in a significant difference in total anthocyanin content between all of the smoothies. In fruit smoothies developed by Nowicka et al. [
58], the anthocyanins were found to be within the range of 23.59 mg/100 g FW to 189.02 mg/100 g FW. Anthocyanins cause the red fruit colour and possess numerous health-promoting properties [
59]. Anthocyanins were proved to show cardioprotective, antitumor, vasodilatory and glucoregulatory effects [
60,
61,
62]. Prevention of hyperglycaemia and thus the anti-diabetic properties of anthocyanins are based on the ability to reduce lipid absorption [
63]. Cyanidin 3-glucoside, which was highly concentrated in smoothies 2 and 3, plays a crucial role in counteracting diabetes [
58]. The compound shows a positive impact on insulin resistance by influencing transcription factor forkhead box O1 [
63].
Single flavonols were not found in high concentrations in the smoothies (not exceeding 15.34 mg/100 g FW) compared to other phenolics. In this study, five flavonols were found in the smoothies; however, a total of eight types of flavonols were found in all used fruit combined. Quercetin 3-
O-galactoside, quercetin 3-
O-arabinoside and quercetin 3-
O-xyloside were undetectable in the smoothies, because of their occurrence at very low concentrations (below 1 mg/100 g) and were only found in the apple juice. Quercetin 3-
O-glucoside was the only flavonol found in all the tested fruit. In strawberries, only quercetin 3-
O-glucoside and quercetin 3-
O-glucuronide were found, in concentrations of 2.67 and 1.49 mg/100 g FW, respectively. In apple juice, flavonols ranged between 0.28 and 1.10 mg/100 g FW, which included quercetin 3-
O-rutoside, quercetin 3-
O-glucoside, quercetin 3-
O-galactoside, quercetin 3-
O-arabinoside and quercetin 3-
O-xyloside. Chokeberries and the honeysuckle berry extract were the richest sources of flavonols. In chokeberries, quercetin-
O-dihexoside, quercetin 3-
O-vicianoside, quercetin 3-
O-rutoside and quercetin 3-
O-glucoside were identified, and their concentrations ranged from 2.06–13.49 mg/100 g FW. The flavonols in a honeysuckle berry extract were much higher: between 315.01 and 1792.40 mg/100 g FW. Quercetin 3-
O-vicianoside, quercetin 3-
O-rutoside and quercetin 3-
O-glucoside were identified in the extract. The dominant flavonol in all of the smoothies was quercetin 3-rutoside. Quercetin is ubiquitously distributed throughout the plant kingdom [
64]. It shows an antiviral potential and was recently reported to be the most effective natural compound against influenza [
65]. A recent study also reported a therapeutical effect of quercetin against COVID-19 [
66]. The total content of flavonols was significantly different in all of the smoothies and ranged between 7.27 (Smoothie 1) and 26.26 mg/100 g FW (Smoothie 3). In another study, where smoothies with sea buckthorn were analysed [
67], the concentration of flavonols in smoothies ranged between 25.46 mg/100 g FW and 65.26 mg/100 g FW, depending on the ingredients added. Flavonols exert functional effects in numerous cellular targets playing a role in carcinogenesis [
65]. Additionally, they are known for their antibacterial action, possibly via cell walls and membrane damage and ATP metabolism [
65].
A total of six phenolic acids were found in the smoothie ingredients, while three of them were present in the final product. Low concentrations of phenolic acids were found in strawberries and apple juice, at 5.69 and 7.02 mg/100 g FW, respectively. In strawberries, only p-coumaryl-beta-d-glucose was present; however, its concentration was as low as 5.62 mg/100 g FW, and it was not found in the final product. Similarly, p-coumaroylquinic acid found in apple juice at a concentration of 2.39 mg/100 g FW was not identified in the smoothies. Chlorogenic acid was another phenolic acid in apple juice (4.70 mg/100 g FW). In chokeberries, neochlorogenic and chlorogenic acids were identified, and their total concentration was 72.41 mg/100 g FW, being significantly higher than the acid concentration in all the smoothies, apple juice and strawberry. A honeysuckle berry extract was rich in neochlorogenic (304.96 mg/100 g FW), chlorogenic (2324.25 mg/100 g FW), and two dicaffeoylquinic acids (114.14 mg/100 g FW and 412.63 mg/100 g FW), and the total acid content was equal to 3155.98 mg/100 g FW. In Smoothie 1, only neochlorogenic (6.65 mg/100 g FW) and chlorogenic acids (11.93 mg/100 g FW) were identified. In smoothies with the honeysuckle berry extract, both of these acids were also present; however, their concentrations were significantly higher than that of Smoothie 1. Additionally, Smoothie 2 and Smoothie 3 contained a dicaffeoylquinic acid isomer originating from the honeysuckle berry extract. The total content of phenolic acids in all the smoothies was significantly different. Phenolic acids are the second most abundant phenolics found in honeysuckle berry fruit and contribute around 24% of total polyphenols [
57]. Phenolic acids are associated with antimutagenic, chemopreventive, cardioprotective and probiotic effects [
68]. Chlorogenic acid, which was the most abundant acid in all the smoothies, lowers liver glucose synthesis by activating adenosine monophosphate-activated protein kinase, having a positive effect on Type 2 diabetes mellitus [
68]. The mentioned acid is able to suppress the expression of cyclooxygenase-2 and inducible nitric oxide synthase related to osteoarthritis. Moreover, phenolic acids inhibit angiogenesis and also inhibit enzymes related to Alzheimer’s disease. They are able to decrease acetylcholine and butyrylcholine breakdown in the brain.
The richest source of flavanols was the honeysuckle berry extract (1200 mg/100 g FW), while they were not found in chokeberry. In strawberries, procyanidin B1, catechin, procyanidin B2 and epicatechin were detected. All the mentioned flavanols were found in apple juice; however, their concentrations were significantly lower than those observed for strawberry. Additionally, procyanidin C1 (3.17 mg/100g FW) was present in the apple juice. The honeysuckle berry extract was especially rich in procyanidin C1 (1034.66 mg/100g FW), followed by procyanidin B1 (130.24 mg/100g FW) and catechin (35.10 mg/100g FW). The total content of flavanols was significantly different in all the smoothies and ranged between 13.42 and 28.76 mg/100g FW. Smoothies did not contain epicatechin and procyanidin C1, and the most abundant was procyanidin B1 (6.39–14.43 mg/100g FW). The lack of some of the flavanols in the smoothies could be caused by the low amounts of these compounds in the fruit (between 3.17 and 16.53 mg/100g FW) and thus they were too low in the final product to be observed. Flavanol-rich food shows positive health impacts, e.g., improvement of insulin sensitivity, reduced platelet aggregation, recovery of endothelial function, decreased blood pressure. The average flavanol intake by an adult has been estimated to be between 50 and 100mg/day, corresponding to approximately a glass of Smoothie 3.
Iridoids are rarely found in fruit, and a honeysuckle berry is one of the exceptional sources of them [
56]. In the smoothie with no supplementation of the honeysuckle berry extract, no iridoids were detected. In the honeysuckle berry extract, the main iridoid was sweroside, at 3825.96 mg/100g FW, while an isomer of loganic acid was detected in the lowest amount, at 317.46 mg/100g FW. The amount of the latter was too low to be found in the final products. The reason for such a low concentration of loganic acid is the removal of acids and sugars during the dry extract preparation and separation in the resin column. The content of all iridoids in Smoothie 3 was significantly higher than in Smoothie 2; therefore, the addition of honeysuckle berries can serve as a rich source of this unique compound. The two dominant iridoids in smoothies with the honeysuckle berry extract were sweroside and loganin. Sweroside (found in the highest concentration) protects from myocardial ischemia by inhibiting apoptotic cascades and prevents non-alcoholic steatohepatitis by decreasing the synthesis of caspase-1 and interleukin-1 β [
69]. Iridoids result in the bitter taste of the fruit and have been found to contribute to numerous antioxidant and anti-inflammatory properties [
70,
71]. Moreover, they show a neuroprotective effect, including prevention of Alzheimer’s disease, as well as a hepatoprotective effect, including antifibrotic properties [
30]. Iridoids also act as anticonvulsants, show a sedative effect and could minimize irritable bowel syndrome.
The most abundant in all the smoothies were anthocyanins (109.28–253.86 mg/100g FW) followed by iridoids, highly concentrated in Smoothie 2 and Smoothie 3 (49.36 and 177.79 mg/100g FW, respectively). The highest values of flavonols, anthocyanins, phenolic acids, flavan-3-ols and iridoids were all recorded for Smoothie 3. Therefore, Smoothie 3 shows the highest health potential, including neuro- and cardioprotective, anti-inflammatory, anti-cancer and anti-diabetic effects associated with the presence of polyphenols and iridoids [
72].
The honeysuckle berry extract shows a significantly higher content of bioactive compounds than other smoothie components. Similarly, the smoothie with the highest addition of the extract contained higher concentrations of the analysed compounds than the other two. Some compounds are not present in the final product, which might be the effect of pre-treatment or the processing method of the components used. The lack of a direct dependency between the sum of smoothie components and the total content of polyphenols in smoothies might be the result of partial degradation of the compounds during the technological process or possible conversion of them. Polyphenols are very unstable, highly reactive, and are prone to transformations into various products during post-harvesting and processing [
53]. As Michalska and Łysiak [
73] have reported, mechanical and chemical processing negatively affects phenolic content. Polyphenols are highly prone to degradation and reaction with other food constituents [
74]. The stability of phenolics can be affected by structural changes resulting from the impact of light, pH, temperature, interactions or metal ions [
53]. When it comes to temperature, anthocyanins are susceptible to degradation at high temperatures, contrary to phenolic acids being much more stable in such conditions. Moreover, compounds like ascorbic acid and sugars are claimed to affect polyphenol stability [
75]. Phenolic compounds could be hydrolysed to aglycones [
76]. They are also vulnerable to heat. Anthocyanins may undergo changes involving polymerization or degradation to hydroxybenzoic acids. Flavan-3-ols are prone to conversion to tannins. Therefore, properties of obtained products might differ from those of their precursors. On the other hand, polyphenolic compounds show the ability of synergistic activity, unlike other bioactive compounds [
77].
The results present a rich content of diverse bioactive compounds in the smoothies, which further corresponds to their bioactive properties.
3.5. Physicochemical Parameters and Organic Acids of the Smoothies
Smoothie 1 and Smoothie 2 were analysed in terms of chemical composition, including dry matter, ash content, viscosity, extract, acidity, pectin, sugar and vitamin C content (
Table 10).
The dry matter content did not differ significantly between the two smoothies, reaching values between 13.10–13.16%. Similarly, the differences in ash content were not significant among the smoothies and ranged from 0.30–0.35%. In terms of viscosity, the higher value was observed for Smoothie 2 (2839 Pa·s). The parameter was affected by the addition of the honeysuckle berry extract. In terms of soluble solid content, no significant difference was found between Smoothie 1 and Smoothie 2. The titratable acidity values were similar for Smoothie 1 and 2 (0.73% and 0.74%, respectively).
Smoothies are mostly composed from widely consumed fruit, such as bananas and apples, but often red fruit such as blackberries and strawberries are added [
9]. The previous studies reported the pectin content for blackberries in the range of 0.40–1.19%, for raspberries in the range of 0.10–0.88%, while the highest values were in apricots, 0.42–1.32% [
86]. A study by Tkacz et al. [
67] revealed that 100% sea buckthorn juice contained 0.61% pectin. Both smoothies analysed in the current study showed values of pectin ranging from 0.80 to 0.89%, making them a good source of dietary fibre. Pectins are proved to counteract obesity, diabetes and cardiovascular diseases [
87].
Regarding vitamin C, its level was similar in both smoothies (14.02–14.12 mg/100 g). A slightly higher amount of vitamin C in raspberry sorbet was measured in an analogical study, at 20 mg/100 g [
88]. The values observed in the same study were 60 mg/100 g for a strawberry sorbet and 10 mg/100 g for a bilberry sorbet. A recommended daily intake of vitamin C is 90 mg/day for men and 75 mg/day for women [
89], which is equal to almost 2.5 glasses of Smoothie 1 or Smoothie 2. A half cup of raw red pepper contains 95 mg of vitamin C [
90], showing that the presented smoothies are not a very rich source of this vitamin.
The analysed sugar content ranged between 8.34 and 8.73%. Similar outcomes were presented in another study analysing two smoothies produced from cherry juice combined with a peach and an apricot puree, and its content ranged between 8.19 and 9.86% [
58]. The data presented regarding commercial smoothies showed the sugar content equal to 13% [
91].
The analysis of total polyphenol content determined the substances reacting with the Folin–Ciocalteu reagent, thus providing indirect information about the antioxidant capacity. The results increased significantly with the addition of the honeysuckle berry extract and ranged from 346.95 to 426.81 mg GAE/100 g. The total polyphenol content reported for banana pulp varied from 38 to 128 mg GAE/100 g, depending on the cultivar [
92]. Smoothies composed from strawberries, whole apples, bananas, orange and apple juice showed polyphenol content equal to 44 mg GAE/100 mL. The results measured for the smoothie with the honeysuckle berry extract are almost 10 times higher. In a study by Ribeiro et al. [
93], the measured bioaccessibility of phenolic compounds from smoothies differed from 20 to 47% between gastric and intestinal digests, showing high bioavailability of these phenolics.
Apart from the viscosity and polyphenol content, the other parameters did not show significant discrepancies between the smoothies. Compared to other studies, the analysed smoothies could help counteract metabolic diseases due to a high pectin content and enhance the immune system, associated with the presence of vitamin C [
90]. The higher polyphenol content of the smoothie with honeysuckle berry extract confirms earlier results showing high levels of phenolic compounds in the extract, corresponding to important biological activities such as anticancer, anti-inflammatory and anti-microbial effects [
94].
The organic acid content in the smoothies is presented in
Table 11. Among all the acids, quinic, citric, malic, shikimic and oxalic acids were identified. The most abundant one was quinic acid, followed by malic, citric and shikimic acids. The oxalic acid was only present in a trace amount. The level of analysed acids was not significantly different between the smoothies, which is associated with the addition of the honeysuckle berry extract being purified from organic acids and sugars. The dominant one in all smoothies was quinic acid, and the recorded values ranged from 0.840 g/100 g FW for Smoothie 1 to 0.868 g/100 g FW for Smoothie 2. Quinic acid shows numerous health-promoting properties, such as hepatoprotective, choleretic, antibacterial, antiviral, and anti-inflammatory activities [
95]. The lowest concentration identified was of oxalic acid, with values in the range of 0.005–0.006 g/100 g FW. Another study focused on smoothie analysis reported values of quinic acid ranging between 0.65–1.38 g/100g FW, and oxalic acids between 0.05–0.18 g/100g FW [
96]. A high concentration of oxalic acid is associated with decreased mineral bioavailability and formation of calcium oxalates in urinary stones; therefore, decreasing its amount is highly desired. The conducted studies prove that sweet taste and flavour depend not only on a high sugar content but also on a low concentration of acids [
97]. The level of sugars and organic acids in the products determine consumer perception and thus acceptance. As was also presented in
Table 10, the sugar content and titrable acidity of both smoothies were similar and thus equally accepted by the consumers.