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Molecular Composition Study of Mumijo from Different Geographic Areas Using Size- Exclusion Chromatography, NMR Spectroscopy, and High-Resolution Mass Spectrometry

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Four mumijo samples originated from different mountain ranges were studied using size-exclusion chromatography (SEC), Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), and 1 H and 13 C NMR spectroscopy. All the used analytical techniques demonstrated that the mumijo samples studied consist of a "high-molecular" fulvic-like and a low-molecular components. The second component is shown using FTICR MS and NMR methods to be predominantly a range of vegetative and animal metabolites.
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... The molecular weight distribution of HAs was determined by gel permeation chromatography according to the described procedure [19][20][21][22]. An Abimed gel permeation chromatograph (Gilson, France) consisted of a column, a pump, an auto mated sampler, and a flow through ultraviolet detec tor. ...
... This technique allows for description of the distribution of carbon in HA molecules among their structural fragments with different chemical shifts. Thus, 13 C NMR spectroscopy is capable of esti mating the content of structural units in the carbon skeleton of HAs [4,17,19,23]. Therefore, the struc tural distinctions among the HAs isolated by sequen tial alkaline extractions can be detected by 13 C NMR spectroscopy. ...
... The distribution of carbon atoms among the dif ferent structural fragments was determined by the integration of the corresponding spectral regions. The following assignments according to [4,19] were made in the following spectrums ...
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The preparative yield, composition, and structure of humic acids obtained by sequential alkaline extractions from two soils (a soddy-podzolic soil under forest and a typical chernozem in treatment with permanent black fallow of a longterm experiment since 1964) have been studied. The preparative yield of humic acids from the first extraction is 0.40 and 0.94% for the soddy-podzolic soil and the chernozem, respectively. The preparative yield from the second extraction is lower by several times, and the yield from the third extraction is lower by an order of magnitude. The study of the obtained preparations by elemental analysis, size-exclusion chromatography, and C-13 NMR spectroscopy has shown insignificant changes in the elemental, molecular-weight, and structural-group composition of humic acids among the extractions. It has been supposed that this is related to the soil features: typical climatic factors for the formation of soil subtype in the case of soddy-podzolic soil and the land use in the long-term experiment in the case of typical chernozem. It has been concluded that that a single extraction is sufficient for the separation of humic acids and the preparation of a representative sample.
... Generally, Mumio contains about 14-20% moisture, 18-20% minerals; 13-17% proteins; 4-4.5% lipids; 3.3-6.5% steroids, 18-20% nitrogen-free compounds, 1.5-2% carbohydrates, and 0.05-0.08% alkaloids, amino acids and other compounds with nitrogen, EOs and vitamins etc (Konstantinov et al., 2013). This traditional medicine has proven to possess a wide spectrum of pharmacological activity and in combination with honey has been prescribed as an antiseptic agent (Garedew et al., 2004). ...
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In this study, an antimicrobial mumio-based hydrogel dressing was developed for wound healing application. The mechanism of gel formation was achieved via a double crosslink network formation between gelatin (GT) and polyvinyl alcohol (PVA) using polyethylene glycol diglycidyl ether (PEGDE) and borax as crosslinking agents. To enhance the mechanical integrity of the hydrogel matrix, bacterial cellulose (BC) was integrated into the GT-PVA hydrogel to produce a composite gel dressing. The obtained hydrogel was characterized by FTIR, SEM, TGA, and XRD. Gel fraction, in vitro swelling and degradation as well as compressive modulus properties of the gel dressing were investigated as a function of change in PVA and BC ratios. By increasing the ratios of PVA and BC, the composite dressing showed lower swelling but higher mechanical strength. Comparing to other formulations, the gel with 4%w/v PVA and 1%w/v BC demonstrated to be most suitable in terms of stability and mechanical properties. In vitro cell cytotoxicity by MTT assay on human alveolar basal epithelial (A549) cell lines validated the gels as non-toxic. In addition, the mumio-based gel was compared to other formulations containing different bioactive agents of beeswax and cinnamon oil, which were tested for microbial growth inhibition effects against different bacteria (S. aureus and K. pneumoniae) and fungi (C. albicans and A. niger) strains. Results suggested that the gel dressing containing combinations of mumio, beeswax and cinnamon oil possess promising future in the inhibition of microbial infection supporting its application as a suitable dressing for wound healing.
... A distinct group of sharp peaks at 127-135 ppm, which were characteristic of all spectra of shilajit, was attributed to aromatic carbon of hippuric acid. The latter is a typical component of shilajit (Agarwal et al., 2007;Konstantinov et al., 2013). A sharp signal at 165 ppm was attributed to amide structures of peptide chains and hippuric acid. ...
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Natural products, such as humic substances (HS) and shilajit, are known to possess antiviral activity. Humic-like components are often called as carriers of biological activity of shilajit. The goal of this study was to evaluate anti-HIV activity of well characterized HS isolated from coal, peat, and peloids, and compare it to that of water-soluble organic matter (OM) isolated from different samples of Shilajit. The set of humic materials included 16 samples of different fractional composition: humic acid (HA), hymatomelanic acid (HMA), fulvic acid (FA). The set of shilajit OM included 19 samples of different geographic origin and level of alteration. The HIV-1 p24 antigen assay and cell viability test were used for assessment of antiviral activity. The HIV-1 Bru strain was used to infect CEM-SS cells. The obtained EC50 values varied from 0.37 to 1.4 mg L⁻¹ for the humic materials, and from 14 to 142 mg L⁻¹ for the shilajit OM. Hence, all humic materials used in this study outcompeted largely the shilajit materials with respect to anti-HIV activity: For the humic materials, the structure-activity relationships revealed strong correlation between the EC50 values and the content of aromatic carbon indicating the most important role of aromatic structures. For shilajit OM, the reverse relationship was obtained indicating the different mechanism of shilajit activity. The FTICRMS molecular assignments were used for ChEMBL data mining in search of the active humic molecules. As potential carriers of antiviral activity were identified aromatic structures with alkyl substituents, terpenoids, N-containing analogs of typical flavonoids, and aza-podophyllotoxins. The conclusion was made that the typical humic materials and Shilajit differ greatly in molecular composition, and the humic materials have substantial preferences as a natural source of antiviral agents as compared to shilajit.
... NMR has been extensively used for decades to characterize the structure of the molecules in OM as it provides a detailed picture of its molecular architecture (Barron and Wilson, 1981;Hertkorn et al., 2016Hertkorn et al., , 2013K€ ogel-Knabner, 1997;Konstantinov et al., 2013;Matilainen et al., 2011;Wong et al., 2002;Zhong et al., 2011). Briefly, NMR is a physical phenomenon in which nuclei in a magnetic field absorb and re-emit electromagnetic radiation. ...
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Organic matter (OM) refers to the largest reactive reservoir of carbon-based compounds on Earth. Aside of its role as a source of carbon, OM is also actively involved in a wide range of ecological functions. It also plays an important role in the solubility, toxicity, bioavailability, mobility and distribution of pollutants. Therefore, OM is a key component in the local and global carbon cycle. About 12,000 articles containing organic matter in the title were published during the past decade, with a continuous increasing number each year (ISI Web of Science). Although this topic was widely explored and its interest has significantly increased, some limitations remain. These limitations can be technical (e.g., pre-treatment processes, low-resolution instrument, data handling) and can be related to the current approach. In this review, we first present the current strategies and tools to characterize the organic matter in the aquatic environment, then we tackle several aspects of current characterization limitations. Finally, we suggest new perspectives and priorities of research to improve the current limitations. From our point of view, simultaneous studies of particulate and dissolved OM fractions should be prioritized and multi-disciplinary approach, creation of databases, controlled experiments and collaborative works should be the next targets for future OM research priorities.
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Shilajit has a longstanding use as an anti-aging and memory enhancing drug. It is known to have excellent anti-bacterial effects and is believed to be effective for cognitive enhancement, but is difficult to standardize because of the lack of quality control standards. This study, for the first time, proposes a quality control standard using a simultaneous analytical method for the drug’s multi-compound content using high-performance liquid chromatography-ultraviolet detection (HPLC-UV) as an aid for the internationalization of Mongolian Shilajit. Phenolic compounds 1-6 were isolated from Mongolian Shilajit extract using bioassay-guided isolation, and the isolated compounds were evaluated for cognitive-related anti-Alzheimer’s disease (AD) activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and advanced glycation end-product (AGE) formation assays. The isolated compounds showed good effects for each activity. In addition, the isolated compounds were successfully quantified using a validated quantitative HPLC analysis method. As a result, the isolated compounds were suggested as standard marker compounds for Mongolian Shilajit. Also, we proved that the original material of Mongolian Shilajit is a lichen named Xanthoparmelia somloensis (Gyel.) Hale using HPLC-UV, ultra-high-performance liquid chromatography-electrospray ionization/hybrid linear trap-quadruple-orbitrap-high-resolution mass spectrometry (UHPLC-ESI/LTQ-HRMS).
Background: Mumijo (shilajit) has been well known in traditional medicine as a remedy for a number of diseases, such as bone fractures, wounds, inflammation, and headache. It is also widely used as an analgesic agent in folk medicine, but no scientific documentation exists concerning that effect. Objective: The current study was conducted to evaluate the ability of mumijo to reduce sensitivity to painful stimuli when compared with morphine sulfate and sodium diclofenac. Methods: A total of 176 animals were randomly and equally divided into 2 groups with 88 mice each-one for formalin test and the other for writhing test. For each test, the animals were allocated into 10 equal groups, based on the dosage of the analgesic, plus a negative control group, with 8 mice in each group. Intervention: The analgesic effect of mumijo extract in doses of 0.75, 7.5, 75, and 750 mg/kg was assessed and compared witha group receiving distilled water-the negative control group, and that for groups receiving 1, 2, or 4 mg/kg of morphine sulfate or 10, 20, or 30 mg/kg of sodium diclofenac-the positive control groups. Results: The results showed a significant decrease in pain intensity for all mice receiving doses of mumijo extract during a 1-h formalin test when compared with the distilled water group. For all the mumijo groups except the one receiving 750 mg/kg, the analgesic effect was significantly lower than that for the morphine sulfate group receiving 4 mg/kg. No significant differences existed between all mumijo and all diclofenac groups. In a writhing test, a significant inhibition of the pain response induced by acetic acid also occurred in all 4 mumijo-administered groups as opposed to the group receiving distilled water. No significant differences existed between the writhing response in groups receiving 75 and 750 mg/kg of mumijo and any doses of diclofenac or morphine. The comparison among the different doses of mumijo in the formalin test did not show any significant differences, but in the writhing test, the maximum dose showed a more effective analgesic action. Conclusion: The findings indicated a significant analgesic effect for mumijo extract on chronic pain in mice, occurring in a dose-independent manner.
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Shilajit is a pale-brown to blackish-brown exudation, of variable consistency, exuding from layers of rocks in many mountain ranges of the world, especially the Himalayas and Hindukush ranges of the Indian subcontinent. It has been found to consist of a complex mixture of organic humic substances and plant and microbial metabolites occurring in the rock rhizospheres of its natural habitat. Shilajit has been used as a rejuvenator and an adaptogen for thousands of years, in one form or another, as part of traditional systems of medicine in a number of countries. Many therapeutic properties have been ascribed to it, a number of which have been verified by modern scientific evaluation. Shilajit has been attributed with many miraculous healing properties.
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The present work describes the extraction of fulvic acids (FA) from Shilajit and its spectroscopic and mass spectrometric characterization. The spectral features obtained from FT-IR and 1HNMR were similar to those reported for humic substances from other sources. The molecular elemental composition analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in electrospray negative ion mode resulted in extraordinary high average O/C ratios (0.55) which might be caused by a significant contribution of carbohydrates in Shilajit. A very high average H/C ratio of 1.27 also points to dominant aliphatic or alicyclic structures and relatively low aromaticity. The average molecular formula of the nitrogen free elemental compositions measured by FT-ICR mass spectrometry is C18.2H23.0O10.0.
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Shilajit has been used traditionally in folk medicine for the treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding the physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, extracts of commercial Shilajit were fractionated using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are the products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, a strong correlation was found between the complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts.
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Recorded molecular weights (MWs) for humic substances (HS) range from a few hundred to millions of daltons. For purposes of defining HS as a specific class of chemical compounds, it is of particular importance to ascertain if this broad range of MWs can be attributed to actual variability in molecular properties or is simply an artifact of the analytical techniques used to characterize HS. The main objectives of this study were (1)to establish if a preferential range of MWs exists for HS and (2) to determine any consistent MW properties of HS. To reach the goal, we have undertaken an approach to measure under standardized conditions the MW characteristics of a large set of HS from different natural environments. Seventy-seven humic materials were isolated from freshwater, soil, peat, and coal, such that each possessed a different fractional composition: humic acid (HA), fulvic acid (FA), and a nonfractionated mixture of HA and FA (HF). Size exclusion chromatography (SEC) was used as the analytical technique to determine molecular weight characteristics. The MW distributions were characterized by number (Mn) and weight (Mw) average MW, and by polydispersity. The complete range of Mw values varied within 4.7-30.4 kDa. The maximum Mw values were observed for peat HF and soil HA, whereas the smallest weights were measured for river water HF. Maximum values of polydispersity (3.5-4.4) were seen for peat HF and soil HA, while much lower values (1.6-3.1) were found for all preparations isolated with XAD-resins. Statistical evaluation showed consistent Mw and Mn variations with the HS source, while polydispersity was mostly a function of the isolation procedure used. A conclusion was made that HS have a preferential range of MW values that could characterize them as a specific class of chemical compounds.