1. Introduction
White mulberry (
Morus alba L.) is one of the plants that can grow in various climatic conditions. Its common application in traditional oriental medicine has resulted in numerous studies on its health-promoting properties [
1,
2,
3]. The leaves of white mulberry are commonly known and used in dried form for infusions. However, novel application methods are being sought for leaf extracts, e.g., as an ingredient in bread, dairy products, or candies [
4,
5,
6,
7,
8,
9], despite the legal difficulties related to adding
Morus leaves to food products in some regions, e.g., the European Union. Mulberry leaves differ in chemical composition, including in terms of polyphenols, depending on the cultivar or species used and growing conditions, which results in diverse functional properties [
10]. What is more, the processing of the leaves may also modify their properties due to the changes occurring during the treatment processes.
Polyphenols are currently becoming more widely available as components of dietary supplements or functional food products. Despite the increasing body of evidence regarding the pharmacological efficacy of their effects on human health, there is a parallel concern about their actual activity in living organisms. What is meant here is, first of all, the relationship between chemical structure and intestinal absorption, metabolism, and bioavailability of polyphenols [
11]. This is because it turns out that studies conducted
in vitro cannot fully reproduce
in vivo conditions. The process of digestion of polyphenols in a living organism is complex and depends on many factors, including the concentration of the components and the matrix in which they are delivered, the linkages with other structures, the profile of the gastrointestinal microflora (individually variable, depending, inter alia, on health status and diet), the current activity of digestive enzymes, etc. [
12,
13].
Therefore,
in vitro digestion is used at certain stages of the analysis of individual raw materials. It is pointed out that only
in vitro studies use experimentally standard microbiological material, the selected specific enzymes, and accurately described environmental conditions [
14].
The studies on the digestibility of polyphenols use different process conditions, depending on the character and concentration of the material, duration of each digestion step, presence and/or composition of intestinal microorganisms, etc. [
15,
16,
17].
Moreover, polyphenols play an essential role in many important diet-related mechanisms, e.g., by modifying glucose metabolism in diabetes mellitus [
2,
18], and even on the gut-brain axis [
19]. However, the amount of each phenolic compound can vary under different conditions, both at harvest and during processing [
20,
21,
22].
The authors have already proposed [
23] an interesting processing method—a simple conditioning processing of
Morus alba leaves. However, that study did not address the digestibility of selected biologically active compounds. This paper aims to fill this important gap based on European material. It also constitutes the next stage of chemical analysis of conditioned mulberry leaves, which involves the determination of key compounds (phenolic acids and flavonols) following
in vitro digestion, taking into account the specific stages and the chemical changes occurring at those stages (
Figure 1).
This research has led to a better understanding of the changes that occur in the Morus alba leaves during technological processing. This work contributes a considerable amount of information for future optimization of white mulberry leaf processing, for example, in the production of dietary supplements or as a food ingredient. Furthermore, the results provide a further argument for improving the processing of mulberry leaves by processing companies to get a valuable, high-quality intermediate product beneficial for consumers’ health.
4. Discussion
Phenolic acids and flavonols belong to the group of polyphenols, whose presence determines antioxidant activity. This study also analyzed benzoic and cinnamic acid derivatives, the amounts of which in leaf preparations increased during conditioning.
As already shown, leaf preparations produced in a laboratory and conditioned for three hours contained the most total phenolic acids and flavonols [
23], while preparations from non-conditioned leaves contained 70–80% less. On the other hand, on the semi-technical scale, the content of total phenolic acids and flavonols increased only after two hours of conditioning and then decreased. Chlorogenic acid and caffeic acid were the dominant acids in the leaf preparations. The dominant flavonols in all preparations were rutin, isoquercitrin, astragalin, and quercetin 3-(6-malonyl)-glucoside, similar to those in Korean leaves [
25]. Lee and Choi (2012) [
25] found rutin in the amount of 46.46–161.76 mg × 100 g
−1 d.m., isoquercitrin in the amount of 30.40–66.81 mg × 100 g
−1 d.m., and astragalin in the amount of 24.41–25.18 mg × 100 g
−1 d.m. of leaves harvested in May. The flavonol specific for mulberry leaves—quercetin 3-(6-malonyl)-glucoside—is a significant bioactive component with anti-atherogenic and anti-hyperglycemic effects [
26,
27]. The content of quercetin 3-(6-malonyl)-glucoside increased by 31% only in the course of two-hour conditioning. The changes in the raw material in the course of longer (three- and four-hour) conditioning resulted in a significant reduction, with some increase in quercetin content. Thus, as a result of the activity of endogenous esterases released during leaf crushing, hydrolysis of this flavonol took place, resulting in the release of the quercetin aglycone. It is suspected that up to 50% of the anti-diabetic effect of mulberry extracts may depend on the presence of the two main polyphenols of mulberry leaves, i.e., chlorogenic acid and rutin [
28]. Chlorogenic acid is credited with the ability to attenuate glycogenolysis and reduce glucose absorption, as well as having strong antioxidant properties [
29,
30]. On the other hand, rutin protects against tumorigenesis and inhibits peroxidation of the LDL fraction of cholesterol [
31].
The digestion process of the produced leaf preparations and their infusions was simulated in vitro. A multi-element gastrointestinal model—a mixture of bioactive compounds suspended in a plant matrix—was used to digest leaf preparations. A sequential arrangement was used, in which each successive step followed immediately the previous one at identical time intervals and in possibly identical experimental conditions. Thus, the biochemical conditions present in the mouth, stomach, and intestines were reproduced in the in vitro conditions of the bioreactor.
The first step of digestion was to simulate oral conditions, which was achieved by holding the sample in a bioreactor bowl for 10 min at 37 °C to homogenize it. Low pH conditions were then created, and pepsin was dosed, mimicking the gastric section of the GI tract. The study found that passing the samples through stages simulating the oral cavity and the stomach did not always result in a significant quantitative change in the concentration of phenolic acids and flavonols. Different trends were observed between formulations—polyphenol content decreased for the 0 h and 1 h formulations and increased for the 2 h, 3 h, and 4 h variants. In the case of infusions, polyphenol content decreased only in the variant conditioned for one hour. Such results of increasing or decreasing polyphenol concentrations at the initial stages of the simulated digestion process are supported by the literature. Some studies have pointed to the loss of polyphenols during the stages of oral and gastric conditions [
32,
33], while other authors have emphasized the stability of compounds during the passage through these stages. This was explained by the too-short exposure of the samples to the acidic environment of the stomach, which did not involve hydrolysis or the release of polyphenols from cellular structures. In addition, it is speculated that low pH has a protective effect on polyphenol structures [
34].
After incubation in simulated stomach conditions, the intestinal stage was conducted in the bioreactor. The greatest decreases in phenolic acids and flavonols were observed after the intestinal conditions were applied to both preparations and infusions of the leaves. This is fully justified because polyphenols that reach the colon are intensively processed into a simpler form by the microflora residing there. Their presence can also affect the growth of major strains of intestinal bacteria [
35]. It is increasingly emphasized that the antioxidant capacity of plant-derived foods is evidenced not only by the content of polyphenols but also by the activity of phenolic metabolites of bacterial origin, whose high concentrations are recorded precisely within the colon [
36].
Polyphenols are generally poorly absorbed during digestion, as they are converted through the action of digestive enzymes and intestinal microflora to lower molecular-weight compounds. Animal and human studies have shown this to be the case for many polyphenols, including chlorogenic acid, caffeic acid, ferulic acid, and rutin [
37].
As a result of the simulated digestion process, a significant reduction in chlorogenic acid content was observed in the digested leaf preparations, except for the 4 h preparation. A reduction in its content of at least 53% at the end of the intestine was recorded for conditioned leaves and 99% in the preparation from non-conditioned leaves. Losses of chlorogenic acid in a study by Siracusa et al. [
33] amounted to 58% after passage through the simulated stomach and 95% after passage through the simulated intestines. Caffeic acid content after the passage of the non-conditioned samples through the simulated intestines decreased by 73% compared to the stomach stage. However, in the conditioned variants, an increase in the content of this acid was observed in the subsequent stages of the digestion of the preparations. For chemically pure caffeic acid, Tagliazucchi et al. (2010) [
38] observed a ca. 25% reduction in its content and over 43% in gallic acid content at the intestinal stage.
In the intestines, as a result of bacterial esterase activity, the ester bond of chlorogenic acid is cleaved with the release of caffeic acid and chinoic acid. This generates caffeic acid, and later, after dehydroxylation, also 3-(3-hydroxyphenyl)-propionic acid [
39]. The amount of caffeic acid can therefore be considered an indicator of the progress of chlorogenic acid transformation in the preparation. In the analyzed preparations, caffeic acid was present in all samples, and the increasing content of caffeic acid at successive stages of digestion was attributed to the hydrolysis of chlorogenic acid, which was manifested by a decrease in its amount during the process of simulated digestion. During the digestion process, caffeic acid can be
O-methylated and converted to ferulic and dihydroferulic acids, and further to vanillic acid [
39]. In addition, the intestinal metabolism of caffeic acid leads to the formation of dihydrocaffeic acid and
m-coumaric acid [
39]. The confirmation of all these transformations, starting with chlorogenic acid and continuing with caffeic acid, was the presence of ferulic, isoferulic, dihydroferulic, and vanillic acids in the urine of people consuming coffee enriched with chlorogenic acid [
37]. Of those mentioned, ferulic acid and vanillic acid were identified in the preparations discussed. In the experiment conducted, a steady reduction in the amount of ferulic acid in the digestive content and an increase in vanillic acid were observed. In a study by Ekbatan et al. (2016) [
36], vanillic acid was detected in the transverse colon and descending colon, i.e., the terminal sections of the large intestine. In our study, the same correlation with a reduction in ferulic acid content was observed.
Protocatechuic acid content decreased with the progression of simulated digestion in all preparations, especially in the last section of the intestine, by 56–85%. Protocatechuic acid can be synthesized from caffeic acid as a result of microflora activity in the colon [
36] or from quercetin [
39]. Such transformations, however, were not dominant, as the amounts of caffeic acid and quercetin in conditioned preparations did not decrease during digestion. Also, the processes of quercetin release from more complex structures or other flavonols may have prevailed over the processes of degradation.
The content of
p-coumaric acid increased after digestion in the stomach and duodenum in the case of non-digested preparations, conditioned for two and three hours, followed by a reduction after simulating intestinal conditions. Ekbatan et al. (2016) [
36] observed an even greater reduction in the amount of coumaric acid than was observed in our experiment.
In this study, a decrease in phenolic acid content was mostly observed in the course of digestion, with the highest in the intestinal section. Such a reduction was also observed by other researchers [
40], who found a reduction of phenolic acids of ca. 36–46%. The reduction in the amount of phenolic acids may have been associated with changes in pH and the presence of bile acid salts in the intestine, leading to the precipitation of phenolic acids. In addition, the instability of caffeoylquinic acids in aqueous solutions may have contributed to the observed losses [
41].
Among the flavonols, rutin, isoquercitrin, astragalin, and quercetin 3-(6-malonyl)-glucoside predominated. During the simulated digestion process, it was found that the amount of rutin in the digestive content gradually decreased, both during the digestion of preparations and infusions. The effect of acidic gastric conditions was a reduction of ~3–15%, while after the application of intestinal conditions, the reduction was by 61–99%. In the digestive content obtained after the digestion of nondigested preparations at the intestinal stage, rutin was not found, as in a study by Siracusa et al. (2012) [
33]. Rutin, or quercetin 3-rutinoside, is a flavonoid glycoside. Its presence is believed to determine the health-promoting properties of the raw material. Due to the metabolism of gut bacteria, rutin and quercetin can be converted to 3,4-dihydrophenylacetic acid, a dopamine neurotransmitter with anti-inflammatory and cardioprotective properties. However, in the absence of free radicals, this acid inhibits cellular respiration in brain mitochondria, contributing to the neurodegeneration associated with Parkinson’s disease [
42].
In the course of bacterial decomposition of rutin in the gastrointestinal tract, quercetin is formed, which is quantified in the content of digested preparations and infusions. In the present study, the content of quercetin in the digestive content gradually increased by up to 144% until stage E, i.e., the terminal portion of the small intestine, or remained at a level similar to the earlier stages of digestion. Only after passing to the colon did its amount decrease, in the range of 23–70% depending on the sample variant and in the range of 1–63% depending on the infusion variant.
In a study by Tagliazucchi et al. (2010) [
38], the reduction in quercetin was 5.8% after the intestinal stage. Ekbatan et al. (2016) [
36] identified quercetin in their study only in the ascending part of the colon, probably released from complex chemical compounds. The increase in the content of quercetin was probably related to the breakdown of other flavonols in whose structure it is found, i.e., quercetin 3-(6-malonyl)-glucoside, rutin, isoquercetin (3-β-D-glucoside of quercetin), and isorhamnetin (3-methylquercetin). The content of these flavonols gradually decreased over the course of the simulated digestion.
As with quercetin, in the digested preparations, astragalin content also increased in the simulated small intestine. However, after implementing the large-intestine conditions, their amounts were reduced. The breakdown of astragalin, which contains kaempferol in its structure, was associated with an increase in the content of this particular flavonol during the digestion of the non-conditioned preparation.
In many cases, an initial increase in polyphenol content was observed due to its release from various leaf matrix structures [
43]. In general, in the gastrointestinal tract, there is a gradual release of compounds from different structures and an improvement in their availability through the conversion to other compounds to enable their appropriate activity or improve absorption. Determination of the specific antioxidant activity of the digestive content at different stages of digestion using analytical tests may be considered for future research.