Background
The use of thermal spring waters for health and recreation in Tunisia is a traditional activity dating back to Roman times. This tradition continues today through balneotherapy, also called spa therapy, which is practiced in a Turkish bath also known as a “
Hammam”, and is recommended as a therapeutic and prophylactic measure against many types of illness and toxicity [
1]. In Tunisian spa resorts, as in many countries in the world (Japan, New Zealand, France, Spain, Greece…etc.), the use of hot springs shows similarities. The spa guest can recover by bathing in or drinking thermal water, or by inhaling its vapors [
2]. Bathing is mainly recommended for skin care, joint and muscle problems and arthritis. Inhaling is used for the treatment of chronic diseases of the upper and lower airways. Drinking the water is beneficial for some specific diseases. The mechanisms by which broad spectrums of disease are alleviated by spa therapy have not been fully elucidated. [
3].
While the chemical concentrations in thermal waters are admittedly associated with their therapeutic effects [
4], the inclusion of efficient bioproduct additives produced by photosynthetic organisms and which act against oxidative stress may comprise a significant supplementary value for the increasingly competitive sector of balneotherapy. To accomplish this, these organisms must tolerate: 1) the thermal stress generated in hot thermal spring waters, and 2) an antibiotic additive for the prevention of bacterial proliferation. We hypothesized that the thermophilic microorganisms inhabiting thermal springs-especially cyanobacterial strains-might be likely candidates for bioproduct additives. Indeed, cyanobacteria are photosynthetic and gram-negative, capable of occupying roughly all environments on earth that are visible-light illuminated, with extremophile cyanobacteria thriving in many widely varying habitats such as the Dead Sea, deserts, snow and the outflow of geothermal springs [
5,
6]. Their adaptation to extreme conditions is mostly due to the modification of membranes, nucleic acid structure and to the production of efficient protective bioproducts including enzymatic and nonenzymatic antioxidants which combat oxidative stress [
7‐
11] through free radical scavenging that inhibits lipid peroxidation, and to the chelating of metal ions which induce oxidation [
7,
12]. For example, evidence is now accumulating as to the links existing between oxidative stress and various diseases including cancer, neurodegenerative disorders, diabetes, cardiovascular diseases, inflammation and rheumatoid arthritis [
13‐
15]. Enzymatic antioxidants include mainly superoxide dismutase (SOD), catalase and glutathione peroxidase, while non enzymatic antioxidants are composed of carotenoids, ascorbic acid, tocopherols, Mycosporine-like amino acids (MAAs) and phenolic compounds [
6,
16‐
18].
The subject of this study is thus the prophylactic and therapeutic potential of Tunisian hot springs in which the thermophilic cyanobacterium Leptolyngbya sp. proliferates. Our objective was twofold: 1) to determine the phytochemical constituents, the phenolic profile and the antioxidant activities of the strain’s methanolic extracts, along with both its capsular and releasing polysaccharides, and 2) to explore the possible advantages of the potential use of cyanobacterium in thermal baths in “salus per aquam” (SPA) resorts. The results may have a positive impact on Tunisian thermal tourism activity.
In this study we show for the first time, to the best of our knowledge, the presence of various phenolic compounds including hydroxytyrosol, oleuropein, naphtoresorcinol, catechin, luteolin 7 glucoside, naringenin, flavon, resveratrol and pinoresinol in the cyanobacterium biomass, capsular polysaccharides and releasing polysaccharides.
Discussion
This study confirmed the presence of diverse phytochemicals and antioxidant activities in the methanolic extracts of the biomass (BME), the capsular (CME) and the releasing polysaccharides (RME) of the Tunisian thermophilic cyanobacterium Leptolyngbya sp.
The
Leptolyngbya BME presented the highest concentrations of phenols, flavonoids and vitamin C, the highest scavenging ability of DPPH free radical and the highest hydroxyl radical scavenging ability. The concentrations of phenols, flavonoids and vitamin C were found in the
Leptolyngbya sp. BME and were higher than those reported by Ijaz and Hasnain [
33] for the genus
Leptolyngbya, and by Rai and Rajashekhar [
34] for other cyanobacteria strains (
Phormidium corium,
Oscillatoria fremyii, Spirulina major…)
. These differences may be attributed either to the cyanobacterial strains and their environmental origins or to the extraction methods and solvents used. Definitely, the high amount of phenols, flavonoids and vitamin C in our case may be considered as a way to avoid oxidative stress induced by the high temperature levels in thermal spring water [
6]. Furthermore, methanol is the most commonly used solvent for phenolic extraction due to its high polarity and its wide solubility properties.
The high level of DPPH radical scavenging activity of BME is mostly attributed to its high content in phenolic acids (particularly gallic, ferulic and vanillic) and in flavonoids (mainly luteolin 7 glucoside and naringenin). Phenolic acids and flavonoids are potent free radical scavengers and so possess antioxidative properties [
35‐
37]. The high-level accumulation of these phenolic compounds in the biomass of the thermophilic cyanobacteria
Leptolyngbya sp. may be an important mechanism for self-protection when under stressful conditions. This strategy has been well described by Dhananjaya et al. [
38].
Hydroxyl radical is one of the most reactive oxygen species in the body. It severely damages proximate bio-molecules (DNA, protein) resulting in mutagenesis, carcinogenesis and cytotoxicity [
39]. Removal of the hydroxyl radical from living organisms thus protects them from different illness and diseases. The results of the hydroxyl radical scavenging ability demonstrated that BME was the most powerful with an IC
50 = 0.38 mg/ml and exhibited a significant decrease in a concentration-dependent manner of the hydroxyl radical. This result is in accordance with an earlier published paper [
40] and leads us to believe that BME may be considered to be a potent quencher of the hydroxyl radical and that the Tunisian thermophilic cyanobacterium
Leptolyngbya sp. might help the human body to prevent oxidative damage. Moreover, the presence of hydroxytyrosol and oleuropein in BME, well known for its hydroxyl radical scavenging capacity [
41], must be reported.
In this study, we have also demonstrated that the highest content of MAAs was observed for CME which means that
Leptolyngbya sp. has the ability to accumulate MAAs in its capsular polysaccharides. The existence of MAAs in cyanobacteria has been reported since 1969 by several authors [
42‐
46]. However, despite this evidence the exact location of MAAs in cyanobacteria is not well known, except in certain cyanobacterial strains (
Nostoc commune,
Arthrospira platensis and Microcoleus sp.) in which they have been shown to be actively secreted and cumulated extracellularly [
22,
28]. These observations show good agreement with our results.
The DPPH radical scavenging capacity of CME and RME was moderate and did not exceed 22.3 ± 1.1%. According to Hajimahmoodi et al. [
47], the aqueous extract of
Chlorella vulgaris extracellular polysaccharides showed an activity in the area of 109.02 ± 8.25% of radical scavenging in the DPPH assay, a result in stark contrast to our data, the difference being essentially attributed to the biochemical composition of the extracts for each extracellular polysaccharide. Indeed, the EPS aqueous extract of
Chlorella vulgaris was rich in phenolic compounds whereas the capsular and releasing polysaccharides of the
Leptolygbya sp. methanol extracts were rich in MAAs. When compared with BME, CME and RME (the EPS aqueous extract of
Chlorella vulgaris) presented high ferrous ion chelating ability. This inevitably led us to predict that CME and RME contained polysaccharides enabling iron chelating ability. This prediction was verified in our laboratory (data not shown). In fact, the compound’s chelating ability is described by Melo-Silveira et al. [
30] as: “the formation of bonds between two or more binding sites within the same molecule and a single central atom”. This specificity was mainly observed in organic substances such as polysaccharides, which have the ability to bind to metal atoms from chelate [
48]. The hydroxyl radical scavenging capacity of CME and RME was considered moderate compared to BME, but promising compared to other cyanobacterial extracts [
49].
In HPLC analysis, 25 compounds were identified in BME while 21 were identified in CME and 23 in RME. According to numerous studies the most predominant phenolic compounds in cyanobacteria are gallic acid, vanillic acid, syringic acid, ferulic acid, chlorogenic acid, 3.4-dihydroxybenzoic acid, protocatechuic acid, caffeic acid, coumaric acids and rutin [
33,
50‐
52]. Only slight variability is observed compared to our data. This difference is essentially attributed to the presence of nine other phenolic compounds: hydroxytyrosol, oleuropein, naphtoresorcinol, catechin, luteolin 7 glucoside, naringenin, flavon, resveratrol and pinoresinol, and to the absence of caffeic acid and rutin. The absence of some phenolic compounds may be attributed to auto-oxidation, and especially to enzymatic oxidation by peroxidase and polyphenol oxidase [
53]. Dhananjaya et al. [
38] demonstrated that rutin and caffeic acid were mainly observed for cyanobacteria under salt stress but not under thermal stress. The existence of hydroxytyrosol and oleuropein—the major polyphenols in olives—in the
Leptolyngbya sp. BME at 4
.0 ± 0.1 and 2.0 ± 0.1 mg/g DW, respectively, must be emphasized. In fact, hydroxytyrosol prevents bone loss [
54], whereas oleuropein is considered as a medicinal compound with diverse biological properties such as antidiabetic, anti-cancer and anti-atherosclerotic properties [
55]. Furthermore, special attention must be paid to stilbene and resveratrol, also observed in the three methanol extracts of the Tunisian thermophilic cyanobacterium
Leptolyngbya sp. In fact, resveratrol has been reported to prevent atherosclerosis and to be useful in treating some chronic diseases such as neurodegenerative disorders and diabetes mellitus [
56].
Bathing in hot springs increases body temperature, which increases blood flow, resulting in the increased absorption capability of the intestines [
57]. It has been also reported that bathing below 40 °C stimulates parasympathetic activity and activates gastroenteric digestive functions [
58]. These findings lead us to hypothesize that bathing, along with thermal water consumption, may offer the appropriate level of absorption of the
Leptolyngbya phenolic compounds for therapeutic effect. Furthermore, the human epidermis barrier function is important to transdermal delivery of drugs, and its permeability to many phenolic compounds was proved by Roberts et al. [
59]. Phenolic compounds are widely used in topical preparations for their local anesthetic, antipruritic or antibacterial properties; they are generally applied to the skin either as preservatives or to obtain a local effect [
59]. Immersion in hot spring water and application of jet-water opens the skin pores and can facilitate the penetration of the
Leptolyngbya sp. antioxidant compounds.
Acknowledgements
This study was conducted by Lamia Trabelsi as a part of her HDR research, and is part of a collaborative effort between the Laboratory of Marine Biodiversity and Biotechnology, National Institute of Marine Sciences and Technology, Monastir, Tunisia and the National Center for Scientific Research CNRS 6249, Chrono-Environment Laboratory, Besançon, France.