CHARACTERIZATION AND ORGANIC COMPOUNDS IN PELOIDS FROM MONGOLIA

The peloids are the important natural remedy all over the world. The physicochemical properties and some organic matters of peloids from 12 lakes in Mongolia were examined. These peloids belong to the continental hydrosulfi de sticky peloid except Gurvan nuur peloid. Peloid from Lake Gurvan nuur was classifi ed as a sapropel with high ash content. The peloid organic matters were investigated using chemical analysis and several analytical techniques, such as IR, and 13C NMR spectroscopy, GC/MS, gas chromatography. The concentration of total organic carbon in all continental hydrosulfi de sticky peloids is ranged from 0.4% to 3.1%, but 15.3% in the sapropel. It shows that the majority of the peloids in Mongolia are hydrosulfi de sticky peloid with lower organic matters than the sapropel and peat. In this study, no signifi cant difference of chemical composition was observed between dissoluble and insoluble organic matters from peloids. Some classes of biomolecules, including lipid and carbohydrate, were identifi ed. Biogenic stimulator humic acid (HA) is the major part of the peloid organic matter. HA concentration varied between 11.2% and 55.9% of total organic matter of peloids. The macromolecule of the sapropel HA exhibited a lower aromatization degree and more functional groups than other HAs, as revealed by the 13C NMR data and C/H ratio. The genesis of peloid organic matter is discussed in this paper. Our results suggest that the presence of known bioactive organic compounds, such as humic acid, lipid, and carbohydrate, as well as hydrogen sulfi de causes the balneological value of peloids in Mongolia.


Introduction
Peloids are a multi-component system, which consists of mineral water, clay minerals, organic matter and organo-mineral complex, and applied for healing procedure.In 1807, the fi rst chemical analysis of peloids was performed by French chemist Desser (Ivanov and Malakhov, 1963).Various types of peloids differ in concentration of organic matter and its composition.Recently, the use of healing mud in pharmaceutical formulations, spas and aesthetic medicine increased due to increasing interest and success of natural remedies (Veniale et al., 2007).Nowadays, manmade peloids that obtained by mixing mineral water and clay (Veniale et al., 2004), as well as maturated peloids after its application for therapy are widely used in many sanatoriums (Veniale et al., 2007).In this text, the term peloid refers to the natural healing mud, which is collected from the lake-bottom.
According to the conventional point of view, the therapeutic action of peloids is due to their thermal-, chemical-and mechanical properties, and hence, the thermal behavior of clay materials has been intensively studied (Cara et al., 2000;Legido et al., 2007;Ferrand and Yvon, 1991).During pelotherapy, a certain part of chemical components penetrate the skin by diffusion and electrophoresis.This concept had been demonstrated by using the Franz-type diffusion cells (Tateo et al., 2009).The mechanical particles in peloid induce mechano receptors in derma, which cause the recipient to experience the thermal and chemical effects (Vaisfeld and Galina., 1996).However, this is a limited approach to understanding the healing effects of peloid and usually emphasizes it due to heat.A lack of the fundamental research material of peloids in Mongolia results in scarce scientifi c background in the peloid application.
Organic matter represents 82-94% of peat, 15-80% of sapropel, 1-5% of hydrosulfi de sticky mud and less than 0.5% of pseudo-volcanic mud (Ivanov and Malakhov, 1963;Tsarfi s and Kiselev, 1990).Humic acid (HA) is a major part of peloid organic matter.In sapropel, HA stimulate the macrophage defense reaction, promote nervous tissue regeneration, stimulate tissue reparation, and produce anti-infl ammatory effect in case of tissue burns and cornea diseases (Degtyarenko and Mokulkin, 1997).In addition, HA is the known complexing ligand of trace elements, this is also possible to form complexes with amino acids, peptides, carbohydrates and steroids, which may be responsible for some of the effects occurring in tissues, including the elimination of heavy metals, desmutagenic effects, antioxidant and anticoagulant activity.
There are more than 40 lakes with healing mud in Mongolia.The physiochemical characteristics and chemical nature of peloid may be differ due to their various origins and -5 -healing effects.The study of the organic matters from hydrosulfi de sticky peloid is poorly addressed.The geochemical and mineralogical composition, the radioactivity of several natural nuclides of the analyzed peloids, and trace element concentration in the organic extracts were investigated earlier (Tserenpil, 2005).
The main objective of this paper is to determine the chemical composition of organic matters from natural healing mud in Mongolia, and to provide some fundamental data which are useful to explain the therapeutic effects of peloid.On the other hand, it is necessary to defi ne their chemical composition, particularly, organic matters for peloid maturation process.This process is strongly infl uenced by microbial activities and the availability of organic components (Carretero et al., 2006;Tateo et al., 2009).
Peloids from Lake Avarga toson, Uud and Khujirt are used in the sanatoriums by preparing a mixture of mud and natural mineral water called-Rashaan.

Methods
Sediments were collected separately into pre-cleaned wide-mouth glass bottles of 500-1000 ml volume and lake surface water was retained at the top to prevent an oxidation of sediments.The sediments were refrigerated (4 0 C) at laboratory until analysis.
The characteristics of peloids were determined according to Shukarev method (Bakhman and Ovsyanikova, 1965).Data were normalized to dry weight after desiccation at 105°C, until weight stabilization.Biogenic silica was measured by spectrophotometer after samples were digested in a 2 M Na 2 CO 3 solution at 85 0 C for 5 hours in plastic vessels.For the determination of dissolved sulfi de, sulfi de was fi rst volatilized adding acid by converting it to gaseous hydrogen sulfi de (H 2 S).H 2 S was purged from samples by carbon dioxide and trapped in an iodine solution, which was reduced by hydrogen sulfi de.Sodium thiosulfate was used as a titrant to verify the sulfi de concentration iodometrically.The concentration of total organic carbon (TOC) was determined using rapid dichromate oxidation of organic matter (Nelson and Sommers, 1996).The concentration of total organic matter (TOM) was evaluated by TOM=TOC•1.72equation.The pore water was -6 -separated from peloids by a simple pressing device.
Distilled water and organic solvents were used for extraction of organic matter.The solvents were evaporated by a rotary vacuum evaporator.HCl was used to release the organic matters associated with carbonate minerals.The lipid fraction was extracted with a mixture of chloroform/ethanol (1:1) (Dolmaa et al., 2004).Humic acid (HA) was extracted with 0.2 N NaOH aqueous solution after demineralization of peloid.The carbohydrate extract was obtained by water extraction at 40-50 0 C and purifi ed using a dialysis bag.The extract was freeze-dried for future analyses.This study involved identifi cation of the different fractions of organic matter from peloids by IR and 13 Ñ NMR spectroscopy, GC/MS, gas and gel chromatography.IR spectra were recorded on spectrometer IR-20 using a KBr pellet.GC/MS, of the organic extracts of peloid, are taken on HP 5971A mass spectrometry using capillary columns packed with BD5, OV70-280, SE-54, and SE-30 25 or 60 m long in a automatically programmed temperature schedule at 4 K/min.Individual organic compounds were identifi ed with reference to standard electronic libraries of mass spectra (chromatographic retention indices): NIST 21, NIST 98, NIST 107, PMW tox 3, Wiley 229, Wiley 138, and NBS 75K.Spectra with at least 85% similarity were chosen.For dialkyl phthalates identifi cation we selected spectra with a resemblance coeffi cient no lower than 95%. 13Ñ NMR spectra were acquired on spectrometer Varian VXR 500S with working frequency of 125.1 MHz for nuclei 13 C in solution dimethyl sulfoxide, mode INVGAT and time of scanning 24 hours.For the identifi cation of monosaccharide composition, we used gas-liquid chromatography.Trichloroacetic acid was used for protein analysis as a precipitating agent.

The general characteristics of peloids
The physicochemical characteristics of sampled peloids are reported in Table 1.Based on their properties, peloids were classifi ed by the national standard (Dolmaa et al., 2008) due to the lack of normative regulations about quality standards and non-existing international classifi cation for peloids (Veniale et al., 2004).The moisture content varied from 10.48 to 55.95%, which was lower compared to organic rich peloids (60-80%).Most peloids are dense 1.43-1.9g/cm 3 (excluding those from Gurvan nuur), and healing materials for pelotherapy may be prepared by mixing with mineral water.The concentrations of TOM were usually lower than the peat and sapropel, but higher than pseudo-volcanic peloid.For instance, TOM concentration varied between 0.74-5.4% in peloid samples, while the concentration was 26.32% in peloid from Gurvan nuur.Peloid H 2 S level was high (up to 0.89%) and resembles to the concentration for continental hydrosulfi de sticky peloids.
From Table 1, the studied healing muds are typical of the continental hydrosulfi de sticky peloids (except peloid from Lake Gurvan nuur) and were classifi ed into the following subgroups based on by mineralization and sulfi dation.Peloids from Lake Olziit, -7 -Khotont and Lake Ikh tsaidam were highly mineralized (pore water mineralization was 67.8-215.5 g/L) and medium sulfi dation type, Lake Zegstei, Bus, Darday and Khujirt's peloids are low mineralized (15.8-19.7 g/L) and high sulfi dation; peloids from Lake Avarga toson and Utaat minjuur are low mineralized (10.7-12.1 g/L) and low sulfi dation.Peloid from Lake Gurvan nuur dramatically differed from other mud samples in moisture content (55.95%), high concentration of TOC (15.30%), lower content of particles with diameter more than 0.25 mm (0.46%), and pore water mineralization (3.8 g/L).The results indicate that Gurvan nuur peloid was formed in low-mineralized water and therefore was classifi ed as sapropel peloid with high ash content (67.46%).H 2 S possesses important physiological and pharmacological functions in the regulation of blood pressure (Rui Wang., 2002), and its content varied between 0.03 and 0.89% in dry peloids.The increase in the availability of reactive organic matter promotes H 2 S formation through anoxic bacterial reduction of salt water sulfate (Allison et al., 1991).This positive correction between organic matter and sulfi de was also observed in these lakes, with higher organic content associated with higher sulfi de (Table 1).The maximum concentrations of H 2 S were 860 mg and 890 mg/100 g peloid in Lake Uud and Bus, respectively.The TOM concentrations were 4.47% and 5.4% in above mentioned peloids.

Dissoluble and insoluble organic matters from peloid
Dissoluble organic matter (DOM) and insoluble organic matter (IOM) isolated from peloids ranged from 0.37-15.51mg/g and 0.84-12.93mg/g, respectively (Table 2).The distribution and bruto-formula of individual organic compounds in DOM and IOM are summarized in Table 3.
Dialkyl phthalates (with various alkyl substitutes) were found in both DOM and IOM fractions as shown in Table 3.The range for peloid DAP concentration was comparatively high (3.3-99.1%).There was no relationship between their abundance and concentration of TOM in peloids.Dibityl-and dioctyl phthalates were more abundant than other DAPs.A high fraction of DAPs were observed in both DOM and IOM from Avarga toson peloid (91.4% and 94.76%, respectively).

Lipid
Lipid concentrations were 0.1-2.2mg/g in dry hydrosulfi de peloids, but elevated concentrations were appear (2.9%) in the sapropel.The chemical composition of lipid was further studied by GC/MS.From Table 4, hydrocarbons (16.0-41.2%),carboxylic acids (0-1.9%),esters of aliphatic acids (16.7-53.3%)and steroids (9.3.6%) were dominant in lipid from the hydrosulfi de peloid.n-Alkanes from C 16 to C 25 were more abundant among the lipid hydrocarbons.Alkanes were detected by GC/MS on fragment ions m/z 57+71+85+99+113....The high content of n-alkanes in lipid from marine sediments indicates that their genesis correlated with seaweed.Marine organisms such as diatomic seaweeds produce n-alkanes from C 15 to C 32 (Afonina, 1998).The concentration and composition of alkanes indicate their biogenic genesis.

Humic acid
HA concentration varied from 11.2 to 55.9 percent of TOM in peloids.The highest content of HA (14.7%) was identifi ed in the sapropel from Lake Gurvan nuur.HA concentration (2%) in the peloids from Lake Avarga toson, Bus and Khujirt was higher than other hydrosulfi de peloids.
Further study was focused on the sapropel HA.The elemental composition of isolat-ed humic acid was investigated in order to obtain general information about the sapropel HA molecular structure (Table 5).The sapropel HA from Lake Gurvan nuur had lower atomic ratio C/H (7.58) and is more aliphatic in nature than coal HA.The C/N ratio of the sapropel was 8.28.HA chemical properties were examined by IR and 13 C NMR spectroscopy.In the IR spectrum of sapropel HA, a broad intense band registered at 3440 cm -1 was assigned to OH and NH groups.The C-H stretch of aromatic ring or alkene group appears from 3040-3100 cm -1.The intense bands at 2921 and 2954 cm -1 were assigned to the C-H stretch of methyl-and methylene groups.The bands at 1720, 1682 and 1634 cm -1 were assigned to C=O stretch of carboxyl groups, and 1540 and 1506 cm -1 bands due to C=C stretch in condensed aromatic systems, respectively.The 799 and 751 cm -1 bands were assigned to aromatic compounds with aliphatic side assistants.
13 C NMR spectrum is shown in Fig. 2; a carbon atom (3.84%) for the carbonyl group assigned to 226 and 186 ppm, and the signals at 186-175 ppm corresponded to the carbon atom (11.19%) of carboxyl, ester, and amide.The chemical shifts at 92-50 ppm relate to the carbon atoms (13.88%), which are found in methoxyl, alcohol, and ether groups.The integrated area of above mentioned peaks showed that sapropel HA has a more functional groups than soil and lignite HA

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-15 -(data reported by Drosos et al., 2009).The signals at 162-140 ppm, due to aromatic carbon (5.47%) linked to the oxygen atom, at 140-106 ppm, due to aromatic carbon (18.38%) attached to carbon or hydrogen atoms, and the signals at 0-50 ppm due to a carbon (45.66%) in alkyl groups.The aromaticity of HA was calculated by expressing the level of aromatic carbon as a percentage of total contents of aliphatic and aromatic carbon atoms (28.62%), which resembled the value for HAs from river and lake sediments (Mengchang et al., 2008).

Carbohydrate
Carbohydrates account for a substantial amount of the dissolved and particulate organic carbon in the sediments of marine environments.Carbohydrates were isolated in peloids from Lake Darday, Ikh tsaidam, Utaat minjuur, Uud, Khujirt, and Gurvan nuur, and the concentrations were largely varied (0.02-1.60 mg/g dry hydrosulfi de peloid and 4.80 mg/g dry sapropel).
IR spectrum of carbohydrates from peloid showed a broad band at 3600-3280 cm -1 which was assigned to -OH group.The O-H stretch of the unassociated hydroxyl group in alcohols appears as a narrow band at 3670-3580 cm -1 .The 2924 cm -1 and 2855 cm -1 sharp and intense bands were assigned to valence vibrations due to CH 2 group.The carbonyl stretch C=O appears as an intense bands at 1680-1620 and 1590 cm -1 , also the C-O-C stretch in the region the 1090-1050 cm -1 .
Monosaccharide composition was studied by gas chromatography.Its compositions were not largely varied in the hydrosulfi de sticky muds from Lake Darday, Ikh tsaidam and Uud (Fig 3).Glucose, which is present at higher levels in vascular plants, was the most abundant monosaccharide in peloid carbohydrate extract (20.48-25.52mol%).Galactose, xylose -16 -and mannose contents were 13.50-16.91mol%, 8.57-11.54mol% and 11.07-13.55mol%;respectively.In order to verify the reliability of our data, we compared our results with values reported for marine and estuarine sediments.The concentration of total uronic acids varied between 13.12-24.07mol%, and those are carboxylated acidic of polysaccharides.
Free and associated carbohydrates were isolated from Gurvan nuur sapropel.Their contents were 0.27%, 0.21%, respectively.The chemical nature of the sapropel carbohydrate was studied by the elemental analysis and molecular weight distribution.The dissoluble carbohydrate consists of 39.8% C, 4.9% H and 3.4% N, insoluble carbohydrate 37.3% C, 4.9% Í and 5.3% N.These results showed that these fractions may contain protein.Protein was identifi ed in both free (18.3%) and associated (12.2%) carbohydrate fractions by analysis.

Discussion
Peloids were classifi ed according to their physochemical characteristics including peloid color, moisture content, dissoluble sulfi des and total organic matters concentration, as well as material properties related to therapheutic appliciation (such as particles with diameter more than 0.25 mm and elasticity).The analyzed peloids have the smell of hydrogen sulfi de, they are dark gray or black color, and possess elastic and adhesive characteristics (the latter assessed by touch).Moisture content, concentration of TOM, and hydrogen sulfi de indicated the peloids from 11 lakes belonged to the contanental hydrosulfi de sticky peloids.The hydrosulfi de sticky peloids from Lake Avraga toson, Bus, Utaat minjuur, Uud and Khujirt are rich in organic matter (3.7-5.4%),compared to others from the same class of peloids.In general, hydrosulfi de sticky peloids formed in the mineralized water that is rich in sulfate.The increase in the availability of reactive organic matter promotes H 2 S formation through anoxic bacterial reduction of salt water sulfate (Allison et al., 1991).Those two processes, including oxidation of organic matter and reduction of sulfate, are linked through the microbial ecosystem.Likewise, the level of H 2 S in peloids Bus and Uud was slightly higher than the level for contanental hydrosulfi de peloids (reported data by Shinkarenko and Milenina., 1981).This was likely due to their high organic content.
The sapropel from Lake Gurvan nuur in Khentii province is suggested for healing procedure of children and the elderly due to their low mineralization and high concentration of total organic matter.
A certain part of the sediment organic carbon is loosely or tightly associated with mineral particles or held together by interaction between organic molecules.Therefore, organic content may be associated with dissoluble and/or insoluble aggregates.The compositions of isolated DOM and IOM extracts were compared using GC/MS data.Major constituents were identifi ed as hydrocarbons, carboxylic acids, aliphatic and aromatic acid esters.There were distributed saturated and unsaturated hydrocarbons with C 12 -C 36 carbon atom.The distribution of carboxylic acids in DOM and IOM fractions is character--17 -ized by the predominance of the odd carbon numbered compounds (maximum at C 15 and C 17 ), which are considered acids produced by bacterial activities in the surface sediments (Ficken et al.,2000).
DAP concentrations in dissoluble and insoluble organic matter of peloids from Lake Avarga toson, Khujirt, Zegstei and Utaat minjuur were high.All DAPs in the ion chromatogram were dominated by peak with m/z 149, which was due to the C 6 H 4 (C=O) 2 O + H ion. The mechanism of formation of fragment ion (m/z 149) has been shown by the scheme in Fig. 4 (Vulfson et al., 1985).DAFs are used by chemical and polymeric industries as a plasticizer, therefore, DAPs in the environment are associated with anthropogenic sources (Afonina, 1998).However, the studied lakes are located far away from industrial sources and developed areas, and are not polluted according to Mongolian national tradition.Hence phthalates in organic extract from Mongolian peloids may not be from industrial products or waste.
A number of references specify that DAP is found in many plants.Recently, bis(2ethylhexsil) phthalate was found in an extract from fl ax cannabacea (Jamyansan, et al., 2003), o-phthalic acid and phthalates were found in greens, poppy-seed, and in the oil fraction from roots Levisticum offi cinalic koch (Giam et al., 1984).DAP from peloids in Mongolia probably derived from decayed remnants of plants.Higher concentration of DAPs are not very-toxic, cancerogenic or irritating to the skin (European Chemical Industry Ecology and Toxicology Centre, 1985).As a plasticizer, DAPs can be result in giving peloid's an elastic and sticky nature.
Lipid fraction isolated from geological sediments contains a broad group of naturallyoccurring molecules, which includes hydrocarbons, fatty acids and their esters, steroids, inorganic sulfur and others (Degens., 1967).Lipids penetrate through the skin more easily than water solution due to their oil solubility, and are considered anti-infl ammatory (Tolomio et al., 1999;Bruno et al., 2005).The n-alkanes with carbon atom C 16 to C 25 , esters of saturated and unsaturated fatty acids with even carbon number C 14, C 16 , C 18 and C 20 , and cholestan-3-one; cholestan-3-ol and 22,23-dihydrostigmasterol and ergosterol were abundant in most lipid extract from peloids.Lipids from the hydrosulfi de peloids contain more fatty esters and steroids than the sapropel (Figure 5).
Humic substances are not found in the tissue of living plants and animals, but they formed by secondary synthesis of microbially decomposed products from the remnant of plants and animals (Manskaya and Drozdova, 1964).Generally the microbial decomposition process is more developed in sapropel compared to hydrosulfi de sticky peloids.The sapropel HA from Lake Gurvan nuur (C/H=7.58) is more aliphatic than coal HA, which was also reported by Mengchang et al., (2008).The C/N ratio of the sapropel was 8.28, indicating that aquatic plants might be the dominant contributors to these humic substances (Meyers and Ishiwatari, 1993).Characterization of HA demonstrated that peloid HA was generated from the initial biomass of reservoirs, mainly from zoo-and phytoplankton (Puntus, 1998).
According to the 13 C NMR data the aromaticity of the sapropel HA exhibited lower (28.62%)value as HAs from river and lake sediments (Mengchang et al., 2008), while aromaticity of coal HA varied between 39.7-62.6%(Batuev et al., 2005).Data from this study indicated that sapropel HA differs due to lower molecular weight and high content of functional groups compared to other HAs.At present, HAs from sapropel are extensively used in manufacturing of cosmetic products.
The carbohydrates in peloids are the nourish, which enable numerous microorganisms to live and occupy the mud environment.Monosaccharide composition is a useful tool for identifying the sources of carbohydrates (Cowie and Hedges, 1984;D'Souza and Bhosle, 2001).Monosaccharide compositions did not vary in the hydrosulfi de sticky muds from Lake Darday, Ikh tsaidam and Uud (Fig 5).In order to verify the reliability of our data, we compared our results with those reported in literature for the marine and estuarine sediments.Monosaccharide composition and ratios in peloids were similar to -19 -the reported data (Khodse et al., 2008); that is, monosaccharides may have been derived from a mixture of phytoplankton, marine bacteria and terrestrial plants.
The distribution and concentration of organic matters in peloids chiefl y refl ects the relative contribution of terrestrial and marine plant inputs and bacterial activity.
During pelotherapy, chemical components, including mineral cations, many essential elements, and organic compounds, are available to penetrate the human body across the skin by diffusion and electrophoresis.

Conclusion
The physicochemical characteristics and some organic matter of peloids from 12 lakes in Mongolia were investigated.The majority of the studied peloids from Mongolian lakes were classifi ed as continental hydrosulfi de sticky peloid.But peloid from Lake Gurvan nuur were classifi ed as sapropel.
A variety of analyses were used to determine the amount and type of organic matter present in peloid samples.We isolated the organic matter (dissoluble and insoluble organic matter, lipid, humic acid, and carbohydrate) from the peloids and determined their composition.There were dominated by saturated and unsaturated hydrocarbons with C 14 -C 26 carbon atoms, aliphatic carboxylic acids and their esters, and esters of aromatic acids in dissoluble and insoluble organic matter.
Lipid extracts from peloids were dominated by n-alkanes with C 16 -C 25 , esters of fatty acids and steroids.We conclude that lipid of hydrosulfi de sticky peloid differs in high concentration of fatty esters and steroids from sapropel lipid.There was dominated hexose in carbohydrate extract from the analyzed peloid.The values of monosaccharide composition and ratios suggested they derived from a mixed source of phytoplankton, marine bacteria and terrestrial plants. 13C NMR results showed that sapropel HA has a low aromaticity than coal HA, and high content of reactive functional groups like soil HA.
Our results suggest that the presence of known bioactive organic compounds, such as humic acid, lipid, carbohydrate, protein, as well as hydrogen sulfi de, may be responsible for the balneological value of peloids in Mongolia.

Fig. 2 .
Fig.2. 13 C NMR spectrum of humic acid from the sapropel in dimethyl sulfoxide

Table 1 .
The general characteristics of peloids in Mongolia

Table 2 .
The concentration and elemental composition of DOM and IOM

Table 3 .
The chemical composition of DOM and IOM of peloids in Mongolia

Table 4 .
The concentration of lipid and predominant compounds in the extracts from peloids

Table 5 .
The elemental composition of humic acid from the sapropel