Fulvic Acid Induced H2O2 Production in Cartilage Cells (Ioannidis Et Al

Proceedings of the 8th Korea-Russia International Symposium on Science and Technology (KORUS-2004), Russia, Tomsk, June 26 - July 3, 2004, Vol.3, P.352-355

FULVIC ACID FRACTIONS FROM MUMIE

Byoung S. Kwon1, Andrei I. Khlebnikov2, Igor A. Schepetkin3, and Sang B. Woo1

1Immunomodulation Research Center and Department of Biological Sciences, University of Ulsan, Korea,
2Altai State Technical University, 46 Lenin Avenue, Barnaul 656099, Russia, and
3Cancer Research Institute, Tomsk, Russia

Phone: +7-3852-245513, Fax: +7-3852-367864, Email:

Abstract

Fulvic acids (FA) from mumie (shilajit) were fractionated by size exclusion chromatography and characterized by IR, NMR 1H and mass spectra. The presence of functional groups that are usually responsible for antiradical action was indicated. Special investigation of antiradical activity of the fractions has been undertaken. It was performed spectrophotometrically by superoxide radical generation with xanthine/xantine oxidase enzyme system using nitroblue tetrazolium chloride as a spectroscopic indicator. First order kinetics of superoxide radical formation allowed to easily determine the inhibition of the reaction by FA fractions from mumie. The antiradical activity of the fractions is discussed.

Keywords: mumie, fulvic acid, antiradical activity

Introduction

Mumie (shilajit) is semihard, brownish black to dark, greasy resin that has a distinctive coniferous smell and bitter taste. Mumie is found in mountains of China, Nepal, Pakistan, Tibet, Ural, Baykal, Sayan, Caucasus, Altai, Kazakhstan and other mountain regions of the world. Mumie humus consists of organic matter (60-80%) and mineral matter (20-40%) containing ≈5% of trace elements [1]. Main organic substances in water-soluble fraction of mumie consist of fulvic acids (FA) [2]. Recently we demonstrated that FA from mumie enhanced a production of reactive oxygen species and nitric oxide in murine peritoneal macrophages [3]. Besides, FA are powerful antioxidants and possess superoxide and hydroxyl radical scavenging properties [4]. FA can be accumulated in tissues as semiquinone radicals and behave as electron donors or acceptors, depending on the redox state of the system (for review see [5]). Superoxide radical plays many roles in inducing and maintaining diseases, including inflammation [6]. The antiradical activity of the FA from mumie has not been studied. Thus, the present investigation was undertaken to characterize FA fractions from mumie in terms of their antiradical activity and spectroscopic properties.

Materials and Methods

Reagents. Xanthine, xanthine oxidase grate 1 from buttermilk (EC 1.1.3.22), nitroblue tetrazolium chloride (NBT) were obtained from Sigma Chemical Co. (St. Louis, U.S.A.).

Mumie Fractionation and FA Subfractionation. The mumie investigated was a representative sample from a large deposit (>500 kg) of this substance in the mountains of Kazakhstan (Ust’-Kamenogorsk). Mumie from this deposit has been used successfully for medicinal purposes in Russia for many years. The isolation of humic substances from mumie was performed by the method based on different solubilities in water at different pH values and ethanol precipitation [3]. Briefly, 1 kg of mumie was shaken for 16 h at room temperature in 5 L of 0.5 M NaOH. The residue was separated from the supernatant by centrifugation (3900´g, 15 min). Then, the supernatant was acidified (HCl; pH 1.0) and humic acids were precipitated. After centrifugation, the FA solution was adjusted to pH 7.0, filtered through a 0.45 mm membrane filter, and mixed with ethanol (final concentration 66%, v/v) to precipitate the high-molecular-weight FA. The precipitate (FAtotal sample) was dissolved in water and fractionated into two fractions (G1 and G2) and subfractions (G1A1 and G1A2) of G1 by size exclusion chromatography on Sephadex G-25 column (96 cm length) with phosphate buffer saline (pH 7.0).

Evaluation of Superoxide Radical Scavenging Activity. The xanthine/xanthine oxidase enzyme system causes superoxide radical formation and is widely used for testing antiradical activity of plant extracts [7, 8] and FA of different origins [4]. Antiradical activity was determined spectrophotometrically using Specord M40 spectrophotometer (Carl Zeiss, Germany) monitoring the effect of mumie FA fractions on the reduction of NBT by superoxide radical observable at 550 nm. Superoxide radicals were generated following a described procedure [9]. The reaction mixtures in the sample quvette consisted of xanthine (230 mM), xanthine oxidase (6.7 mU), NBT (96 mM), FA fractions, and phosphate buffer (0.1 M, pH 7.2), in a final volume of 1900 ml. A stock solution of xanthine, 18mM, was made up by dissolving xanthine in 0.1M sodium hydroxide. The reaction was conducted at temperature 25°C for 5 min, and initiated by the addition of xanthine oxidase.

Effect on Xanthine Oxidase Activity. The effect of the FA fractions on xanthine oxidase activity was evaluated by measuring the formation of uric acid from xanthine with the use of Specord M40 spectrophotometer at 25°C. The reaction mixtures contained the same proportion of components as in the enzymatic assay for superoxide radical scavenging activity, except NBT, in a final volume of 1850 ml. The absorbance was measured at 295 nm for 5 min.

Spectroscopic Analysis. For IR analysis, dry samples (2 mg) were mixed with 800 mg of dry potassium bromide (KBr) and pressed into disks (2 mm in thickness). The IR spectra (500-4000 cm-1) were recorded on Nicolet Model 210 Fourier-transform IR spectrophotometer. Proton magnetic resonance spectra were recorded on Bruker NMR spectrometer (500 MHz) for 10% solutions of the fractions in D2O. Tetramethylsilane was used as a standard. Mass spectra were obtained with mild ionization by ionic spray method.

Results and Discussion

Superoxide Radical Scavenging Activity of FA fractions. The superoxide radical scavenging properties of mumie fractions were investigated with superoxide radicals generated under the catalytic activity of xanthine oxidase. The fractions strongly scavenged the superoxide radical in a concentration dependent manner. The values of the inhibitory effect of FA fractions at the concentration of 100 mg/ml are shown in Fig. 1. NBT reduction was decreased by 50% (IC50) in the presence of 93.7 mg/ml G1, 141.3 mg/ml G2, 69.6 mg/ml G1A1, or 231.6 mg/ml of G1A2 fraction. Thus, G1 fraction is more active than G2. Subfractionation of G1 allowed to obtain samples with enhanced (G1A1) and lowered (G1A2) antiradical activity.

Fig. 1. The effect of FA fractions from mumie on NBT reduction induced by superoxide radical generated in a xanthine/xanthine oxidase system (at concentrations of fractions 100 mg/ml)

Since an inhibitory effect on the enzyme itself would also lead to a decrease in NBT reduction, the effect of the fractions on the xanthine oxidase activity was checked. In this regard we evaluated the effect of the total FA fraction on the metabolic conversion of xanthine to uric acid (Fig. 2). The rate of uric acid production monitored at 295 nm demonstrated negligible inhibition of xanthine oxidase by total FA fraction.

Fig. 2. The effect of total FA from mumie on NBT reduction induced by superoxide radical generated in a xanthine/xanthine oxidase system (■) and xanthine oxidase activity (□).

IR Spectroscopy of FA fractions. Infrared spectra of the samples in KBr pellets (See example in Fig. 3) are very similar to each other in accordance with similar chemical nature of the fractions and subfractions. All the spectra contain strong band near 3400 cm-1 (vibrations of hydroxyl groups), show significant absorbance at 1630-1640 cm-1 (aromatic rings and/or carboxyl groups), 1400 cm-1 (deformational vibrations of hydroxyl groups), 1070-1150 cm-1 (CO bonds), 540-545 cm-1 (very characteristic for aromatic rings).

Fig. 3. IR spectrum of G1 fraction

Mass and NMR 1H Spectra. The results of mass-spectrometry allow to conclude that dozens of individual chemical compounds are contained in each fraction. In general, it is in agreement with the theory of humic substances formation by a random decomposition of organic matter. Although it is impossible to identify individual compounds in these mixtures, from NMR and IR spectra we can make some definite conclusions about the nature of substances in all the pools of compounds. All the NMR spectra show signals near 0.8 ppm (protons of methyl groups in the aliphatic chains), 6.5-7.5 ppm (protons of aromatic rings). The group of signals in the region 1.2-2.3 ppm testifies about the presence of CH2 groups in aliphatic chains. Weak resonance at 2.6 ppm can be related to methyl groups connected directly to the aromatic rings. Intensive signal at 3.4-4 ppm is rather interesting. These are the signals of protons at carbon atoms connected directly to strongly electron-acceptor atoms (for example, to oxygen). Shin et al. [10] assign this signal to carbohydrates. The strong off-scale peak at 4.7 ppm is related to water used as a solvent.

Thus, spectroscopic analysis show the presence of functional groups that are adopted to be responsible for antiradical activity of different substances [11]. It is seen from the spectra that G1 fraction contains major portion of carbohydrates and aromatic compounds. Mainly sub-fraction G1A1 is then enriched with carbohydrates. On the other hand, somewhat more aromatic substances pass into sub-fraction G1A2, as compared to G1A1. The fraction G2 contains little carbohydrates and aromatics, and contains much aliphatic compounds. Peaks in the spectrum of this sample are narrow, suggesting that it is composed of compounds with lower molecular weight than in other fractions. In this context, the enhanced activity of G1A1 as compared to G1 sample suggests that enrichment of G1A1 fraction with carbohydrates may be a significant reason of its higher antiradical properties.

Mumie (shilajit) has been used as a popular remedy in many countries as an anti-inflammatory agent, but the main active principles of the natural drug were not clearly identified yet. In this work, we report that FA from the mumie are potent scavengers of superoxide radical. As superoxide radical plays important role in inducing and maintaining diseases, including inflammation, our data seem to suggest that FA are active biological components in mumie.

References

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