International Journal of Cosmetic Science, 2015, 37, 339–347
doi: 10.1111/ics.12204
Thermal mud maturation: organic matter and biological activity M. Centini*, M. R. Tredici†, N. Biondi†, A. Buonocore*, R. Maffei Facino‡ and C. Anselmi*
*Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, Via della Diana 2, 53100 Siena, Italy, †Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente – Sezione di Microbiologia Agraia, University of Florence, Piazzale delle Cascine 24, 50144 Florence, Italy and ‡Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
Received 15 October 2014, Accepted 24 January 2015
Keywords: antioxidant activity, chemical analysis, maturation process, Saturnia spa, thermal mud
Synopsis OBJECTIVE: Many of the therapeutic and cosmetic treatments offered in spas are centred on mud therapy, to moisturize the skin and prevent skin ageing and rheumatic diseases. Thermal mud is a complex matrix composed of organic and inorganic elements which contribute to its functions. It is a natural product derived from the long mixing of clay and thermal water. During its maturation, organic substances are provided by the microalgae, which develop characteristic of the composition of thermal water. METHODS: The aim of this study was to identify methods for introducing objective parameters as a basis for characterizing thermal mud and assessing its efficacy. Samples of thermal mud were collected at the Saturnia spa, where there are several sulphureous pools. The maturation of the mud was evaluated by organic component determination using extractive methods and chromatographic analysis (HPLC, GC-MS, SPME). We also studied the radical scavenging activity of mud samples at different stages of maturation, in a homogeneous phase, using several tests (DPPH, ORAC, ABTS). RESULTS: We identified several classes of compounds: saturated and unsaturated fatty acids, hydroxyl acids, dicarboxylic acids, ketoacids, alcohols and others. SPME analysis showed the presence of various hydrocarbons compounds (C11–C17) and long-chain alcohols (C12–C16). Six or seven months seemed appropriate to complete the process of maturation, and the main effect of maturation time was the increase of lipids. Six-month mud showed the highest activity. The hydrophilic extract was more active than the lipophilic extract. CONCLUSION: The results indicate that maturation of thermal mud can be followed on the basis of the changes in its organic composition and antioxidant properties along the time. They also highlight the need to develop reference standards for thermal muds in relation to assess their use for therapeutic and cosmetic purposes. sume Re OBJECTIF: Beaucoup de traitements therapeutiques et cosmetiques offerts dans les spas sont centres sur la therapie de boue, pour hydrater la peau et prevenir le vieillissement de la peau et les maladies rhumatismales. La boue thermale est une matrice complexe com ses posee d’elements organiques et inorganiques qui contribuent a fonctions. C’est un produit naturel derive du long melange d’argile et de l’eau thermale. Au cours de sa maturation, des substances orgaCorrespondence: Marisanna Centini, Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, Via della Diana 2, 53100 Siena, Italy. Tel.: +39 577 232072; fax: +39 577 232070; e-mail: marisanna. [email protected]
niques sont fournies par les micro-algues qui se developpent, caracteristiques de la composition de l’eau thermale. METHODES: L’objectif de cette etude etait d’identifier des methodes pour introduire des parametres objectifs comme base pour caracteriser la boue thermale et d’evaluer son efficacite. Des echantillons de boue thermale ont ete recueillies au spa Saturnia, o u il y a plusieurs piscines sulfureuses. La maturation de la boue a ete evaluee par la determination des composants organiques en utilisant des procedes d’extraction et l’analyse chromatographique (HPLC, GC-MS, SPME). Nous avons egalement etudie l’activite de differents stades de piegeage des radicaux d’echantillons de boue a maturation, dans une phase homogene, en utilisant plusieurs tests (DPPH, ORAC, ABTS). RESULTATS: Nous avons identifie plusieurs classes de composes: acides gras satures et insatures, les acides hydroxyles, les acides carboxyliques, cetoacides, les alcools et autres. L’analyse SPME montre la presence de divers composes hydrocarbures (C11-C17) et longue cha^ıne (C12-C16). Six ou sept mois semblaient d’alcools a appropries pour terminer le processus de maturation; l’effet principal du temps de maturation a ete l’augmentation des lipides. La boue de six mois a montre la plus forte activite. L’extrait hydrophile etait plus actif que l’extrait lipophile. CONCLUSION: Les resultats indiquent que la maturation de la boue thermale peut ^etre suivie sur la base de l’evolution de sa composition organique et des proprietes antioxydantes au cours du temps. Ils soulignent egalement la necessite d’elaborer des normes l’evaluation de de reference pour les boues thermales par rapport a leur utilisation a des fins therapeutiques et cosmetiques. Introduction The use of mineral clays for cosmetic and therapeutic purposes is almost as old as human race itself. They are used in pharmaceutical formulations (as active principles or excipients), in spas and in aesthetic medicine [1–6]. In spas, clay minerals are widely used to treat dermatological diseases and to alleviate the pain of chronic rheumatic inflammation. In aesthetic medicine, they are used as cosmetic products to clean and moisturize the skin, to treat acne or cellulite and to combat compact lipodystrophies [7]. They are mixed with water (geotherapy), with sea or salt lake water, or mineral-medicinal water, then matured (pelotherapy) or mixed with paraffin (paramuds) [7]. The main factors underlying the features of peloid mud are the composition and granulometry of the ‘virgin’ clay, the geoche-
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
339
M. Centini et al.
Thermal mud maturation
mistry of the mineral water, the procedure(s) of mixing and remixing and the maturation time [8]. Peloid muds should have suitable properties: water retention, consistency, adhesiveness, heat capacity, cooling rate, exchange capacity, handling and a pleasant sensation when applied to the skin [9]. Care must be taken about hazardous chemical elements that may be toxic (such as As, Cd, Cu, Hg, Pb, Se, Te, Tl, etc.); it is important to check for their presence in the clay–water mix used in spas and to understand their mobility in order to avoid their absorption through the skin, with the risk of toxicity [5, 7, 10]. The maturation procedure in spas is based on extremely complex processes involving not only the ‘virgin’ clay/mineral water interactions but also biological and biochemical processes related to the growth of micro-organisms and microalgae, depending on the habitats in the open-air tanks where the clay is left to mature, although it becomes enriched in organic compounds [8, 11]. Unfortunately, remixing-shaking and maturation times are largely empirical in many spa-centres and left to the discretion of their practitioners. Consequently, the factors governing the physico-chemical and biological reactions occurring during maturation are beyond scientific control and not repeatable in different spas [9]. During the last few years, some scientific studies have focused on characterization of the muds and evaluation of the maturation process. These investigations have looked at the changes in the physico-chemical properties of thermal mud induced by maturation, using various instrumental techniques [4, 8, 11–13]; the microflora has been examined [14–17] and the organic matter determined, particularly the lipid fraction [15, 18–27]. Tuscany is rich in thermal springs, so it appeared interesting to focus on this area, which has never been adequately investigated from a scientific point of view. For this purpose, we collected samples of thermal material at Saturnia spa, where there are several sulphureous pools that, according to some geologists, are a manifestation of other activities in the Monti Volsini mountain areas. Water springs at a constant temperature of 37.0°C form a small lake in a natural ‘crater’ [28]. Muds are prepared in tanks where clay are left to mature together with spring water constantly flowing at 37.0°C for 12 months. Hot muds are employed for the treatment of some rheumatic diseases, cold muds for face masks for greasy and impure skin. The aim of this study was to identify an experimental method (protocol) to introduce objective parameters for the characterization of thermal mud and assessment of its antioxidant efficacy. The study can be divided in two main sections: 1 evaluation of the mud maturation process by analysis and characterization of organic compounds, using several analytical techniques as follows: high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), solid phase microextraction (SPME); and 2 evaluation of radical scavenging activity using several in vitro tests as follows: 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC) assay and 2,20 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay.
Materials and methods Samples preparation Water and mud samples were collected monthly from June to January at Saturnia spa (Grosseto, Italy). We also took a sample of
340
12-month mud that is used as ‘mature’ mud in the spa. Each sample was collected in a high-density polyethylene (PH-HD) container with a cap and undercup to avoid contamination and water evaporation. Three extractions (100 mL three times) of 500-g monthly mud samples were carried out using solvents with different polarity so as to extract all the substances with different polarity in the matrix: chloroform, ethyl acetate and ethanol. The extracts, the first two white and the third yellow, were dried on sodium sulphate, then filtered and evaporated under pressure. All the organic solvents used were of analytical grade (Sigma-Aldrich, Milan, Italy). Samples of thermal water (500 mL) taken monthly from the spring (as well as the muds) were extracted with chloroform and ethyl acetate. The physico-chemical parameters and the components of Saturnia thermal water, as reported in previous studies [29, 30], are listed in Table I. HPLC analysis For HPLC analysis, we used a Shimadzu Chromatograph LC-10 ADVP equipped with a Shimadzu SPD-M 10 AVP photodiode array detector (Shimadzu, Milan, Italy). The extracts (mud and thermal water) were dissolved in acetonitrile (1 mg mL1), filtered through a 0.45-lm filter and analysed. Chromatographic determination was carried out using a Supelcosil LC-18 column (250 mm 9 4.6 mm, 5 lm particle size) in the following conditions: injection volume 20 lL; mobile phase acetonitrile : water (95 : 5, v/v); flow rate 1 mL min1; detector wavelength 254 nm. GC-MS analysis Gas chromatography-mass spectrometry analysis was carried out on a Gas Chromatograph 3800 and Saturn Mass Spectrometer 2000 Varian (Turin, Italy). Samples were dried at 80°C for 30 min and derivatized with bis(trimethylsilyl) trifluoroacetamide. ChroTable I Thermal water analysis
Parameters
Measurement
Results
Water temperature pH Electrical conductivity at 25°C Total hardness Fixed residue at 180°C Hydrogen sulphide (H2S) Carbon dioxide (CO2) Ammonium ion (NHþ 4) Nitrite (NO 2) Nitrate (NO3 ) Chloride (Cl) Fluoride (F) Sulphate (SO 4) Phosphorus pentoxide (P2O5) Sodium (Na+) Potassium (K+) Magnesium (Mg2+) Calcium (Ca2+) Iron (Fe) Alkalinity (HCO 3) Silicon dioxide (SiO2)
°C – lS cm1 °F mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1
36.9 6.25 2996 204 2990 14.5 674 26.8
doi: 10.1111/ics.12204
Thermal mud maturation: organic matter and biological activity M. Centini*, M. R. Tredici†, N. Biondi†, A. Buonocore*, R. Maffei Facino‡ and C. Anselmi*
*Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, Via della Diana 2, 53100 Siena, Italy, †Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente – Sezione di Microbiologia Agraia, University of Florence, Piazzale delle Cascine 24, 50144 Florence, Italy and ‡Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy
Received 15 October 2014, Accepted 24 January 2015
Keywords: antioxidant activity, chemical analysis, maturation process, Saturnia spa, thermal mud
Synopsis OBJECTIVE: Many of the therapeutic and cosmetic treatments offered in spas are centred on mud therapy, to moisturize the skin and prevent skin ageing and rheumatic diseases. Thermal mud is a complex matrix composed of organic and inorganic elements which contribute to its functions. It is a natural product derived from the long mixing of clay and thermal water. During its maturation, organic substances are provided by the microalgae, which develop characteristic of the composition of thermal water. METHODS: The aim of this study was to identify methods for introducing objective parameters as a basis for characterizing thermal mud and assessing its efficacy. Samples of thermal mud were collected at the Saturnia spa, where there are several sulphureous pools. The maturation of the mud was evaluated by organic component determination using extractive methods and chromatographic analysis (HPLC, GC-MS, SPME). We also studied the radical scavenging activity of mud samples at different stages of maturation, in a homogeneous phase, using several tests (DPPH, ORAC, ABTS). RESULTS: We identified several classes of compounds: saturated and unsaturated fatty acids, hydroxyl acids, dicarboxylic acids, ketoacids, alcohols and others. SPME analysis showed the presence of various hydrocarbons compounds (C11–C17) and long-chain alcohols (C12–C16). Six or seven months seemed appropriate to complete the process of maturation, and the main effect of maturation time was the increase of lipids. Six-month mud showed the highest activity. The hydrophilic extract was more active than the lipophilic extract. CONCLUSION: The results indicate that maturation of thermal mud can be followed on the basis of the changes in its organic composition and antioxidant properties along the time. They also highlight the need to develop reference standards for thermal muds in relation to assess their use for therapeutic and cosmetic purposes. sume Re OBJECTIF: Beaucoup de traitements therapeutiques et cosmetiques offerts dans les spas sont centres sur la therapie de boue, pour hydrater la peau et prevenir le vieillissement de la peau et les maladies rhumatismales. La boue thermale est une matrice complexe com ses posee d’elements organiques et inorganiques qui contribuent a fonctions. C’est un produit naturel derive du long melange d’argile et de l’eau thermale. Au cours de sa maturation, des substances orgaCorrespondence: Marisanna Centini, Dipartimento di Biotecnologie, Chimica e Farmacia, University of Siena, Via della Diana 2, 53100 Siena, Italy. Tel.: +39 577 232072; fax: +39 577 232070; e-mail: marisanna. [email protected]
niques sont fournies par les micro-algues qui se developpent, caracteristiques de la composition de l’eau thermale. METHODES: L’objectif de cette etude etait d’identifier des methodes pour introduire des parametres objectifs comme base pour caracteriser la boue thermale et d’evaluer son efficacite. Des echantillons de boue thermale ont ete recueillies au spa Saturnia, o u il y a plusieurs piscines sulfureuses. La maturation de la boue a ete evaluee par la determination des composants organiques en utilisant des procedes d’extraction et l’analyse chromatographique (HPLC, GC-MS, SPME). Nous avons egalement etudie l’activite de differents stades de piegeage des radicaux d’echantillons de boue a maturation, dans une phase homogene, en utilisant plusieurs tests (DPPH, ORAC, ABTS). RESULTATS: Nous avons identifie plusieurs classes de composes: acides gras satures et insatures, les acides hydroxyles, les acides carboxyliques, cetoacides, les alcools et autres. L’analyse SPME montre la presence de divers composes hydrocarbures (C11-C17) et longue cha^ıne (C12-C16). Six ou sept mois semblaient d’alcools a appropries pour terminer le processus de maturation; l’effet principal du temps de maturation a ete l’augmentation des lipides. La boue de six mois a montre la plus forte activite. L’extrait hydrophile etait plus actif que l’extrait lipophile. CONCLUSION: Les resultats indiquent que la maturation de la boue thermale peut ^etre suivie sur la base de l’evolution de sa composition organique et des proprietes antioxydantes au cours du temps. Ils soulignent egalement la necessite d’elaborer des normes l’evaluation de de reference pour les boues thermales par rapport a leur utilisation a des fins therapeutiques et cosmetiques. Introduction The use of mineral clays for cosmetic and therapeutic purposes is almost as old as human race itself. They are used in pharmaceutical formulations (as active principles or excipients), in spas and in aesthetic medicine [1–6]. In spas, clay minerals are widely used to treat dermatological diseases and to alleviate the pain of chronic rheumatic inflammation. In aesthetic medicine, they are used as cosmetic products to clean and moisturize the skin, to treat acne or cellulite and to combat compact lipodystrophies [7]. They are mixed with water (geotherapy), with sea or salt lake water, or mineral-medicinal water, then matured (pelotherapy) or mixed with paraffin (paramuds) [7]. The main factors underlying the features of peloid mud are the composition and granulometry of the ‘virgin’ clay, the geoche-
© 2015 Society of Cosmetic Scientists and the Societe Francßaise de Cosmetologie
339
M. Centini et al.
Thermal mud maturation
mistry of the mineral water, the procedure(s) of mixing and remixing and the maturation time [8]. Peloid muds should have suitable properties: water retention, consistency, adhesiveness, heat capacity, cooling rate, exchange capacity, handling and a pleasant sensation when applied to the skin [9]. Care must be taken about hazardous chemical elements that may be toxic (such as As, Cd, Cu, Hg, Pb, Se, Te, Tl, etc.); it is important to check for their presence in the clay–water mix used in spas and to understand their mobility in order to avoid their absorption through the skin, with the risk of toxicity [5, 7, 10]. The maturation procedure in spas is based on extremely complex processes involving not only the ‘virgin’ clay/mineral water interactions but also biological and biochemical processes related to the growth of micro-organisms and microalgae, depending on the habitats in the open-air tanks where the clay is left to mature, although it becomes enriched in organic compounds [8, 11]. Unfortunately, remixing-shaking and maturation times are largely empirical in many spa-centres and left to the discretion of their practitioners. Consequently, the factors governing the physico-chemical and biological reactions occurring during maturation are beyond scientific control and not repeatable in different spas [9]. During the last few years, some scientific studies have focused on characterization of the muds and evaluation of the maturation process. These investigations have looked at the changes in the physico-chemical properties of thermal mud induced by maturation, using various instrumental techniques [4, 8, 11–13]; the microflora has been examined [14–17] and the organic matter determined, particularly the lipid fraction [15, 18–27]. Tuscany is rich in thermal springs, so it appeared interesting to focus on this area, which has never been adequately investigated from a scientific point of view. For this purpose, we collected samples of thermal material at Saturnia spa, where there are several sulphureous pools that, according to some geologists, are a manifestation of other activities in the Monti Volsini mountain areas. Water springs at a constant temperature of 37.0°C form a small lake in a natural ‘crater’ [28]. Muds are prepared in tanks where clay are left to mature together with spring water constantly flowing at 37.0°C for 12 months. Hot muds are employed for the treatment of some rheumatic diseases, cold muds for face masks for greasy and impure skin. The aim of this study was to identify an experimental method (protocol) to introduce objective parameters for the characterization of thermal mud and assessment of its antioxidant efficacy. The study can be divided in two main sections: 1 evaluation of the mud maturation process by analysis and characterization of organic compounds, using several analytical techniques as follows: high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), solid phase microextraction (SPME); and 2 evaluation of radical scavenging activity using several in vitro tests as follows: 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, oxygen radical absorbance capacity (ORAC) assay and 2,20 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay.
Materials and methods Samples preparation Water and mud samples were collected monthly from June to January at Saturnia spa (Grosseto, Italy). We also took a sample of
340
12-month mud that is used as ‘mature’ mud in the spa. Each sample was collected in a high-density polyethylene (PH-HD) container with a cap and undercup to avoid contamination and water evaporation. Three extractions (100 mL three times) of 500-g monthly mud samples were carried out using solvents with different polarity so as to extract all the substances with different polarity in the matrix: chloroform, ethyl acetate and ethanol. The extracts, the first two white and the third yellow, were dried on sodium sulphate, then filtered and evaporated under pressure. All the organic solvents used were of analytical grade (Sigma-Aldrich, Milan, Italy). Samples of thermal water (500 mL) taken monthly from the spring (as well as the muds) were extracted with chloroform and ethyl acetate. The physico-chemical parameters and the components of Saturnia thermal water, as reported in previous studies [29, 30], are listed in Table I. HPLC analysis For HPLC analysis, we used a Shimadzu Chromatograph LC-10 ADVP equipped with a Shimadzu SPD-M 10 AVP photodiode array detector (Shimadzu, Milan, Italy). The extracts (mud and thermal water) were dissolved in acetonitrile (1 mg mL1), filtered through a 0.45-lm filter and analysed. Chromatographic determination was carried out using a Supelcosil LC-18 column (250 mm 9 4.6 mm, 5 lm particle size) in the following conditions: injection volume 20 lL; mobile phase acetonitrile : water (95 : 5, v/v); flow rate 1 mL min1; detector wavelength 254 nm. GC-MS analysis Gas chromatography-mass spectrometry analysis was carried out on a Gas Chromatograph 3800 and Saturn Mass Spectrometer 2000 Varian (Turin, Italy). Samples were dried at 80°C for 30 min and derivatized with bis(trimethylsilyl) trifluoroacetamide. ChroTable I Thermal water analysis
Parameters
Measurement
Results
Water temperature pH Electrical conductivity at 25°C Total hardness Fixed residue at 180°C Hydrogen sulphide (H2S) Carbon dioxide (CO2) Ammonium ion (NHþ 4) Nitrite (NO 2) Nitrate (NO3 ) Chloride (Cl) Fluoride (F) Sulphate (SO 4) Phosphorus pentoxide (P2O5) Sodium (Na+) Potassium (K+) Magnesium (Mg2+) Calcium (Ca2+) Iron (Fe) Alkalinity (HCO 3) Silicon dioxide (SiO2)
°C – lS cm1 °F mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1 mg L1
36.9 6.25 2996 204 2990 14.5 674 26.8
