Antioxidant and Antibacterial Properties of Extracts and Bioactive Compounds in Bryophytes

: Today global health problems such as increased risks of oxidative stress-related diseases and antibiotic resistance are issues of serious concern. Oxidative stress is considered to be the underlying cause of many contemporary pathological conditions such as neurological disorders, ischemia, cancer, etc. Antibiotic-resistant bacteria are a concerning issue in clinical practice, causing an increase in deadly infections. Bryophytes synthesize an outstanding number of secondary metabolites that have shown several potential therapeutic and nutraceutical applications. Research in the ﬁeld has led to the isolation and characterization of several compounds (ﬂavonoids, terpenoids, and bibenzyls). Some of these compounds have shown promising in vitro antibacterial activities and antioxidant potential comparable to known natural antioxidants such as ascorbic acid and α -tocopherol. However, the process of developing new drugs from naturally occurring molecules is often an impervious path. In this paper, the current state of research of bryophytic antioxidant and antibacterial applications is discussed.


Introduction
Bryophytes lack anatomical features in order to avoid both abiotic and biotic stresses. As a consequence, they have to adapt to counteract environmental stresses with a high degree of chemical diversity [1]. A large number of chemical entities (ca. 3000) have been isolated from bryophytes: aromatic compounds such as phenolic compounds, polyphenols, bibenzyls, (bis)bibenzyls, and terpenoids [2]. Extensive research has been carried out to screen the biological activity of bryophytic molecules. Several molecules and extracts from bryophytes have shown a wide range of biological activities (among them, antimicrobic and antioxidant activities) [3]. In contemporary days, two issues of global concern arise, namely the increased risk of oxidative stress-related diseases and antibiotic resistance [4,5].
Aerobic organisms physiologically produce reactive oxygen species (ROS). ROS are produced in low to moderate concentrations during cellular metabolism and serve a wide number of significant cellular functions such as gene activation, cell growth, signalling molecules, and physiological processes such as inflammation [5]. To balance ROS production, aerobic organisms have developed both enzymatic and non-enzymatic antioxidant systems capable of maintaining adequate balance of oxidants/antioxidants [6]. However, exposure to oxidative stress-inducing agents (e.g., ionizing radiations, heavy metals, etc.), may lead to the disturbance of such homeostasis. Oxidative stress arises when antioxidant systems cannot cope with ROS production and therefore the balance shifts in favour of oxidants [6]. Increased ROS concentrations cause damage to nuclear DNA, mitochondrial DNA, membranes, and proteins. The disturbances in the cellular redox homeostasis participate in the onset of several increasing pathological conditions such as ischemia, neurological disorders, cancer, diabetes, atherosclerosis, etc. [7][8][9]. For example, ROS are implied in several processes related to tumorigeneses such as cell motility, tumour proliferation, and metastasis [10]. Furthermore, oxidative damage to mitochondrial DNA might cause dysfunctions in the mitochondrial respiratory chain causing further ROS generation and, ultimately, oncogenicity [11]. As a consequence of the increased risks of diseases related to oxidative stress, extensive research has been conducted on non-toxic natural antioxidants that can help to cope with oxidative stress [12].
Antioxidant compounds can act directly and indirectly on the redox balance of cells (Figures 1 and 2) [13]. Some compounds are capable of directly antagonizing and reducing ROS. For example, compounds having phenolic groups in their structures' phenolic groups (e.g., flavonoids, phenols) act as hydrogen donors to free radicals, stabilizing the excess electron on the aromatic ring by resonance. Other compounds such as flavonols act indirectly by chelating metal ions that can alter the redox balance in cells (e.g., zinc, copper) [14]. Furthermore, it is known that the Keap1/Nrf2/ARE system is involved in the activation of antioxidant responses in cells [15]. Compounds such as polyphenols, bibenzyls and terpenoids are inducers of Nrf2-ARE system, enhancing the expression of the antioxidants cytoprotective proteins (e.g., gluthatione-S-transferse; glutathione reductase; gamma-glutamylcisteine; NAD(P)H:quinone oxidoreductase; superoxide dismutase; catalase), granting long-term protection from oxidative stress [16]. Due to its central role in redox homeostasis, dysregulations of the Keap/Nrf2/ARE system have been linked to oxidative stress-related diseases [17].
Furthermore, for decades antibiotics have been employed both therapeutically and prophylactically against human diseases as well as in agriculture and for animals [18,19]. As a consequence, several antibiotic-resistant strains have begun to spread, and bacterial infections have again become a threat [20]. Bryophytes, being rich in secondary metabolites that show several biological activities [3], might be a valuable source to discover novel drugs that aid coping with both prevention of oxidative stress-related diseases and antibiotic resistance issues. The present review is intended to revise the research state-ofthe-art of antioxidant and antibacterial compounds found in bryophytes and to outline future investigation needed in the field of applications of bryophytes in nutraceuticals and pharmaceutics.
Appl. Sci. 2022, 12, x FOR PEER REVIEW 2 of 17 DNA, membranes, and proteins. The disturbances in the cellular redox homeostasis participate in the onset of several increasing pathological conditions such as ischemia, neurological disorders, cancer, diabetes, atherosclerosis, etc. [7][8][9]. For example, ROS are implied in several processes related to tumorigeneses such as cell motility, tumour proliferation, and metastasis [10]. Furthermore, oxidative damage to mitochondrial DNA might cause dysfunctions in the mitochondrial respiratory chain causing further ROS generation and, ultimately, oncogenicity [11]. As a consequence of the increased risks of diseases related to oxidative stress, extensive research has been conducted on non-toxic natural antioxidants that can help to cope with oxidative stress [12]. Antioxidant compounds can act directly and indirectly on the redox balance of cells (Figures 1 and 2) [13]. Some compounds are capable of directly antagonizing and reducing ROS. For example, compounds having phenolic groups in their structures' phenolic groups (e.g., flavonoids, phenols) act as hydrogen donors to free radicals, stabilizing the excess electron on the aromatic ring by resonance. Other compounds such as flavonols act indirectly by chelating metal ions that can alter the redox balance in cells (e.g., zinc, copper) [14]. Furthermore, it is known that the Keap1/Nrf2/ARE system is involved in the activation of antioxidant responses in cells [15]. Compounds such as polyphenols, bibenzyls and terpenoids are inducers of Nrf2-ARE system, enhancing the expression of the antioxidants cytoprotective proteins (e.g., gluthatione-S-transferse; glutathione reductase; gamma-glutamylcisteine; NAD(P)H:quinone oxidoreductase; superoxide dismutase; catalase), granting long-term protection from oxidative stress [16]. Due to its central role in redox homeostasis, dysregulations of the Keap/Nrf2/ARE system have been linked to oxidative stress-related diseases [17].   Furthermore, for decades antibiotics have been employed both therapeutically and prophylactically against human diseases as well as in agriculture and for animals [18,19]. As a consequence, several antibiotic-resistant strains have begun to spread, and bacterial infections have again become a threat [20]. Bryophytes, being rich in secondary metabolites that show several biological activities [3], might be a valuable source to discover novel drugs that aid coping with both prevention of oxidative stress-related diseases and antibiotic resistance issues. The present review is intended to revise the research state-of-theart of antioxidant and antibacterial compounds found in bryophytes and to outline future investigation needed in the field of applications of bryophytes in nutraceuticals and pharmaceutics.

Methodology
The relevant literature was searched through Scopus and Web of Science using "article, abstract, and keywords" as the search field. Literature concerning the antibacterial activity was searched with both "antibacterial" and "antimicrobial" keywords since the two terms are used interchangeably. To investigate in greater detail the literature relating to compounds with known antibacterial activity, words such as "bibenzyls", "flavonoids", "terpenes", and "terpenoids" (from now on specific compound classes) were inserted. The literature on antibiotic-resistant bacteria research was searched typing "bacteria species" AND "bryophytes", "extracts" OR "specific compound class". The bacterial strains considered for this review were those listed as "serious concern" and gathered from [10]. The same search was carried out to find literature on antioxidant activity by searching terms related to antioxidant compounds: "antioxidant activity" AND "bryophytes" OR "specific compound class". For literature concerning the current state of in vivo research "bryophytes" AND "extracts" AND "animal models" OR "mice" OR "rats" OR "in vivo" were inserted. Regarding toxicological screening, "bryophytes" AND "extracts" OR "toxicity", "genotoxicity" OR "toxicological screening" were inserted.

Methodology
The relevant literature was searched through Scopus and Web of Science using "article, abstract, and keywords" as the search field. Literature concerning the antibacterial activity was searched with both "antibacterial" and "antimicrobial" keywords since the two terms are used interchangeably. To investigate in greater detail the literature relating to compounds with known antibacterial activity, words such as "bibenzyls", "flavonoids", "terpenes", and "terpenoids" (from now on specific compound classes) were inserted. The literature on antibiotic-resistant bacteria research was searched typing "bacteria species" AND "bryophytes", "extracts" OR "specific compound class". The bacterial strains considered for this review were those listed as "serious concern" and gathered from [10]. The same search was carried out to find literature on antioxidant activity by searching terms related to antioxidant compounds: "antioxidant activity" AND "bryophytes" OR "specific compound class". For literature concerning the current state of in vivo research "bryophytes" AND "extracts" AND "animal models" OR "mice" OR "rats" OR "in vivo" were inserted. Regarding toxicological screening, "bryophytes" AND "extracts" OR "toxicity", "genotoxicity" OR "toxicological screening" were inserted.

Antioxidant Activity
Secondary metabolites compounds function as a nonenzymatic defence that protects bryophytes against various environmental stresses [1,21]. In general, plants are known to produce several secondary metabolites that can act as antioxidant scavengers (e.g., phenolic compound; flavonoids; terpenes). Bryophytes have evolved the capacity to synthesize antioxidative molecules as a mechanism to deal with the formation of free radicals (e.g., reactive oxygen species, hydrogen peroxide) derived from abiotic stresses (i.e., light, desiccation, pollution). Several studies indicated that secondary metabolites in bryophytes are synthesized in response to oxidative stress-inducing agents such as UV radiations [22][23][24] and cadmium [24]. This feature has prompted researchers to deepen knowledge about the antioxidant potential of bryophytes for therapeutic purposes.
Cell models are a valuable tool in the selection of compound bioactivities prior to clinical trials in animals and humans [43]. Furthermore, in vivo testing of pure isolated compounds is a mandatory step in the drug discovery process, supporting in vitro and cell studies [44]. Currently, no research has focused on in vivo experimentations of bryophytic antioxidant compounds. At the present stage, only a few studies have investigated the antioxidant activity of bryophytes extracts and pure compounds in cell models (phagocytes; murine fibroblasts) [45][46][47][48][49][50]. Ielpo et al. [45] studied the antioxidant activity of acetonic extracts of Lunularia cruciata on phagocytes through the chemiluminescence inhibition test. In another study, Leptodictyum riparium acetonic extracts were used to treat whole blood phagocytes gathered from three healthy donors. Blood samples were treated with the ROS inducing agents opsonized zymosan (OZ) and phorbol myristate acetate (PMA). The authors found that the acetonic extracts of L. riparium inhibited luminol-dependent chemiluminescence in samples treated with OZ and PMA in a dose-dependent manner [46]. In a study by [47], the authors treated murine fibroblast NIH-3T3 cells with water extract from the moss Cryphaea heteromalla. NIH-3T3 cells exposed to 500 µM tert-butyl hydroperoxide (TBH) oxidative stress-inducing agent and treated with the water extract (0.5 µg/mL) showed a 50% inhibition of ROS generation for those treated only with TBH (500 µM) for 1 h.
Other researchers have found dose-dependent Nrf2-ARE system induction by diterpenoids isolated from liverworts (Table 2) [48][49][50]. Nrf2 (nuclear factor erythroid 2-related factor) is a transcription factor responsive to cell redox status via Keap (Kelch-like ECH-associated protein 1), that bound the enhancer sequence ARE (antioxidant response elements). The ARE sequence is associated with several antioxidant cytoprotective proteins such as the NAD(P)H: quinone oxidoreductase 1 (NQO1) [13]. An investigation by [48] demonstrated that the diterpenoid frullanian D isolated from the liverwort Frullania hamatiloba stimulated the nuclear translocation of Nrf2, inducing Nrf2-related enzymes (NAD(P)H:quinone oxidoreductase 1, γ-glutamyl cysteine synthetase) in MOVAS cells. Furthermore, 18 h pretreatment with frullanian D ameliorated the H 2 O 2 oxidative damage in the model cells. Another study found a NAD(P)H:quinone oxidoreductase 1 (NQO1) inducing activity in Diplophyllum taxifolium ethanolic extract. Researchers also isolated and characterized sixteen terpenoids and found that three of them, diplotaxifol A, diplotaxifol B, and atractynelonide III, induced NQO1 in a dose-dependent manner [49]. Similar results were obtained by [50] with the sacculatane diterpenoids epyphyllin A-D and pellianolactone B.
Therapeutic applications are also hampered by the need to collect large amounts of natural plant material, obtain pure material for high-value secondary metabolites, genetically stable populations and controlled growth conditions. As a result, in vitro cultures are the best way to develop large-scale production of bryophyte nutraceuticals [51]. At the current state of the research, methods to in vitro grow several bryophytes species have been well established [35]. Other studies have focused on the difference in antioxidant metabolites between in vitro grown and naturally grown bryophytes [30,33,37,52,53]. Interestingly, these studies have found no significant differences in antioxidant activity and metabolite composition in in vitro grown bryophytes.

Antibacterial Activity
Bryophytes, like other organisms, have to deal with pathogens. As a consequence, these plants have adapted their biochemistry to synthesize several compounds to contrast the presence of pathogenic bacteria and fungi [1,21]. Ref. [2] reported several studies that have led to the identification and isolation of a large number of antibacterial compounds from bryophytes. Since then, the antibacterial activity of bryophytic extracts has been extensively researched.
The main techniques used to test antimicrobial chemicals from bryophytes are the disk diffusion test and the broth dilution test. Disk diffusion is a simple and reliable test in which a bacterial inoculum is applied to a culture agar plate [55]. Before the incubation (16-24 h at 35 • C), disks imbued with fixed concentrations of tested compounds are applied on the inoculated agar. After the incubation, the zone of inhibition (i.e., millimetres around disks with no bacteria growth) is measured. Broth dilution consists of two-fold serial dilution of the tested compound in a liquid culture medium (e.g., 4, 8, 16 µg/mL). Standardized bacterial suspensions (1-5 × 10 5 CFU/mL) are inoculated in antimicrobial containing tubes. After the incubation (16-24 h at 35 • C), tubes are examined to spot evidence of bacterial growth (i.e., medium turbidity). The lowest concentration at which bacterial growth is not evidenced represents the minimal inhibitory concentration (MIC) [55].

Conclusions and Future Perspective
Bryophytes synthetize unique compounds that have shown a wide range of biological activities such as antimicrobial, antiviral, antifungal, anticarcinogenic, insecticidal, neurotrophic, muscle relaxing, cardiotonic, and anti-obesity activities [2]. To our knowledge, bryophytes extracts/pure compounds have only been in vivo tested for their anti-inflammatory and antinociceptive [93], wound healing [94], anticarcinogenic [95,96], nanoparticle pharmacokinetics [97], and antilipidemic activities [29]. Although extensive research has been conducted on in vitro antioxidant activity, no investigations have examined the antioxidant activity of bryophytic extracts and pure compounds in in vivo models. Antioxidant activity should not be concluded based on single or multiple in vitro tests [25]. Physiological processes such as absorption, distribution, metabolism, and excretion can affect the effectiveness of certain compounds [98]. Bryophytes' antibacterial compounds have shown promising activities against some human alarming pathogens. However, very little has been attempted in testing bryophytes against the most concerning pathogenic strains, apart from a few studies against MRSA. Furthermore, as for antioxidants, most of the data concerning the antibacterial activity were gathered from in vitro studies. In vitro antibacterial testing is based on two main techniques, namely the disk diffusion test and broth dilution test [99]. Both techniques involve the direct contact of the tested molecules with the bacterial cells, and therefore do not consider the pharmacokinetics and pharmacodynamics of the tested molecules [55]. Moreover, at the present stage only two studies have looked at the toxicity of pure bryophytic compounds and extracts [100,101]. Toxicity screening is an essential step in the drug development process [102] and should be carried out first with respect to in vitro and in vivo experimentations. Future research should be focused on the in vivo testing of antioxidant and antibacterial bryophytic compounds. This would help to circumscribe the huge number of molecules that have been discovered. Moreover, further experimentations should focus on the efficacy of bryophytic antibacterial against antibiotic-resistant strains. In conclusion, bryophytes can be exploited as a source of antioxidants and antibacterial compounds in perspective pharmaceutical and nutraceutical applications. Bibenzyls, terpenoids, phenols, and polyphenols from bryophytes are promising chemicals for the future development of novel antioxidant and antibiotic drugs. However, at the current stage, knowledge sustaining concrete applications of antioxidants and antibacterial from bryophytes is still fragmentary, and more in-depth multidisciplinary research is needed to select the safest and most effective compounds.