Complement-fixing Activity of Fulvic Acid

By: Igor A. Schepetkin, Gang Xie, Mark A. Jutila, and

Complement-fixing Activity of Fulvic Acid from Shilajit and Other
Natural Sources
Igor A. Schepetkin, Gang Xie, Mark A. Jutila, and Mark T. Quinn*
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717
Abstract
Shilajit has been used traditionally in folk medicine for 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 physical and chemical properties of Shilajit
extracts, and nothing is known about their effects on the complement system. To address this issue,
we fractionated extracts of commercial Shilajit 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 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, we found a strong correlation between complementfixing
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.
Keywords
Shilajit; humic substances; fulvic acid; complement-fixing activity; carbohydrates
Introduction
Over the past three decades, research on medicinal properties of natural products has increased
significantly, and a large body of evidence suggests extracts from peat, sapropel, and shilajit
humus may represent a source of novel compounds with medicinal properties [reviewed in
(Schepetkin et al., 2002)]. Shilajit (common names: mumie, vegetable asphalt, mineral pitch)
is a semi-hard brownish black resin formed through long-term humification of several plant
types, mainly bryophytes, present in the vicinity of shilajit-exuding rocks (Ghosal et al.,
1991b;Agarwal et al., 2007). Shilajit is found in specific mountain regions of the world at
altitudes between 0.6 and 5 km (Ghosal et al., 1991b;Agarwal et al., 2007), and has been used
therapeutically for centuries as part of traditional systems of medicine in many countries
[reviewed in (Schepetkin et al., 2002;Agarwal et al., 2007)]. For example, Shilajit has been
used as a treatment for genitourinary diseases, diabetes, digestive disorders, nervous diseases,
tuberculosis, chronic bronchitis, asthma, anemia, eczema, bone fractures, and other diseases
(Acharya et al., 1988;Goel et al., 1990).
*Address for Correspondence: Dr. Mark T. Quinn, Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717,
Phone: 406-994-5721; Fax 406-994-4303, mquinn@montana.edu.
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Published in final edited form as:
Phytother Res. 2009 March ; 23(3): 373–384. doi:10.1002/ptr.2635.
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Although Shilajit samples from different regions of the world have similar physical properties
and qualitative chemical composition, they differ in the ratio of individual components
(Galimov et al., 1986). Shilajit humus consists of organic matter (60-80%), mineral matter
(20-40%), and ∼5% trace elements (Ghosal et al., 1991a;Frolova and Kiseleva, 1996). For
therapeutic applications, Shilajit has been used in the form of an aqueous extract, and extracts
of Shilajit have been shown to activate phagocytosis and cytokine release by murine peritoneal
macrophages (Bhaumik et al., 1993), stimulate osteoblastic differentiation of mesenchymal
stem cells (Jung et al., 2002), and induce proliferation of lymphocytes in the cortical thymus
layer and increased migration of these cells into thymus-dependent zones of the lymph nodes
and spleen (Agzamov et al., 1988).
The primary organic substance in aqueous extracts of Shilajit humus is fulvic acid (FA), and
it has been suggested that FA may account for many biological and medicinal properties of
Shalajit (Ghosal et al., 1988;Schepetkin et al., 2002). Indeed, FA has been used externally to
treat hematoma, phlebitis, desmorrhexis, myogelosis, arthrosis, polyarthritis, osteoarthritis,
and osteochondrosis. Likewise, FA has been taken orally as a therapy for gastritis, diarrhea,
stomach ulcers, dysentery, colitis, and diabetes mellitus [reviewed in (Schepetkin et al.,
2002;Agarwal et al., 2007)]. Despite the broad spectrum use of FA for a variety of medical
conditions, far less is known regarding the mechanisms of action of FA. The few reports
available have shown that humic substances can stimulate osteoclastic resorption of
transplanted bones as well as hydroxyapatite (Schlickewei et al., 1993), and FA/humic
substances isolated from soil and water reservoirs have been reported to stimulate neutrophil
and lymphocyte immune function (Joone et al., 2003;Schepetkin et al., 2003).
Since the complement system is involved in many disease syndromes that have been
traditionally been treated with extracts of Shilajit and other humic substances containing high
levels of FA (e.g., arthritis (Mizuno, 2006), asthma (Wills-Karp, 2007), eczema (Ferguson and
Salinas, 1984), and vascular disease (Acosta et al., 2004), we hypothesized that part of the
beneficial effects of these natural products might relate to their ability to modulate complement.
However, very little is known regarding the effects of FA/humic substances on the complement
system in vitro or in vivo. Thus, we performed studies to fractionate and characterize
physiochemical properties of humic substances extracted from Shilajit and then examined their
complement-fixing activity in comparison with standard FA samples obtained from the
International Humic Substances Society (IHSS).
Materials and Methods
Reagents
β-glucosyl Yariv reagent [1,3,5-tri-(4-β-D-glucosopyranosyloxyphenyl-azo)-2,4,6-
trihydroxybenzene] was purchased from Biosupplies Australia (Parkville, Australia). Gum
arabic was purchased from Fluka BioChemica (Buchs, Switzerland).
Cetyltrimethylammonium bromide (CTABr), diethylaminoethyl (DEAE) cellulose, Sephadex
G-50, galacturonic acid, galactose, arabinose, rhamnose, glucose, diphenylamine, aniline,
anthrone, thiourea, trifluoroacetic acid (TFA), lipopolysaccharide (LPS) from Escherichia
coli K-235, o-phenylene diamine, antibody-sensitized sheep erythrocytes, and gelatin veronal
buffer (GVB) were purchased from Sigma Chemical Co. (St. Louis, MO). Heparin sodium salt
from bovine lung was purchased from Calbiochem (San Diego, CA). The following fulvic acid
(FA) standards were purchased from IHSS: Suwannee river FA (SRFA; IHSS code, 1S101F),
Nordic Aquatic FA (NAFA; IHSS code, 1R105F), Florida (Pahokee) Peat FA (FPFA; IHSS
code, 2S103F), Pony Lake FA (PLFA; IHSS code, 1R109F), and Waskish Peat FA (WPFA;
IHSS code, 1R107F).
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Fractionation of Shilajit Humus
Crude Shilajit was obtained from Agada Herbs (St. Joseph, MI). This product is a water extract
of the raw resinous substance (Shilajit humus), collected in the Himalaya Mountains of Nepal.
The Shilajit from this company has been used successfully for medicinal purposes in for many
years. The isolation of humic substances from Shilajit was performed using the sequential of
precipitation by ethanol and adsorption on DEAE cellulose (Hejzlar et al., 1994). Briefly, 1 kg
of raw Shilajit extract was shaken for 2 hr at room temperature in 5 L of distilled H2O, and
any insoluble residue was separated from the supernatant by centrifugation. The supernatant
was precipitated by addition of a 4-fold volume of ethanol and incubation overnight at 4°C.
The precipitate was pelleted by centrifugation, re-dissolved in distilled H2O, sonicated, and
filtered through 0.2 μm membrane filters. The filtrate was concentrated in an Amicon
concentrator with a 5 kDa cut-off polyethersulfone membrane. The concentrate was diluted by
addition of a 10-fold volume of distilled H2O and ultra-filtered again. This procedure was
repeated at least 4 times to remove ethanol and H2O-soluble low-molecular weight compounds.
One aliquot of the final concentrated filtrate was lyophilized to give a crude extract of Shilajit
humic substances (designated as SHS), and the remainder of the extract was applied to a DEAE
cellulose column (500 ml) equilibrated with 50 mM Tris-HCl, pH 7.0. The column was washed
with 2 L of equilibration buffer to obtain the neutral, unbound fraction and then sequentially
eluted with 2 L of equilibration buffer containing 2 M NaCl and 2 L of 0.2 N NaOH. The
fractions were filtered through 0.2 μm membrane filters, concentrated in an Amicon
concentrator, and subjected to 6 rounds of dilution and concentration, as described above. The
final concentrated filtrates were lyophilized to give three fractions, designated as S-I (neutral
fraction, eluted by equilibration buffer), S-II (acid fraction eluted by 2 M NaCl), and S-III (acid
fraction eluted by 0.2 N NaOH).
Fraction S-II was further fractionated using size exclusion chromatography (SEC) on a
Sephadex G-50 column (2.5×92 cm) equilibrated with 10 mM Tris-HCl buffer (pH 7.4)
containing 150 mM NaCl at flow rate of 22 ml/hr. The elution profile was monitored by: (1)
measuring absorbance at 254 nm, (2) measuring fluorescence (λex=340 nm; λem=460 nm); and
(3) determining carbohydrate content, as described below. The three fractions obtained,
designated as S-II-1, S-II-2, and S-II-3, were pooled and concentrated using ultrafiltration (for
fraction S-II-1 and fraction S-II-2) or ion exchange chromatography on DEAE cellulose
column, followed by elution with 0.2 N NaOH and ethanol precipitation (for fraction S-II-3).
For analysis of biological activity, the fractions were diluted in Hanks balanced salt solution
(HBSS) to a concentration of 5 mg/ml and filtered through sterile 0.22 μm filters.
To evaluate the role of endotoxin, samples were applied to a column containing Detoxi-Gel
Endotoxin Removing Gel (Pierce, St. Louis, MO) and eluted with 0.05 M phosphate buffer
containing 0.5 M NaCl to decrease ionic interactions of sample molecules with the affinity
ligand. Concentrations of eluted samples were adjusted using absorbance at 254 nm, and the
samples were analyzed for biological activity as described below.
High Performance SEC (HP-SEC)
The homogeneity and average molecular weight of the polysaccharide fractions were
determined by HP-SEC using a Shimadzu Class VP HPLC and TSK-GEL G3000WXL column
(7.8 mm × 300 mm) eluted with 50 mM sodium citrate buffer, pH 7.5, containing 0.15 M NaCl
and 0.01% NaN3 at a flow rate of 0.3 ml/min. Peaks were detected using a refractive index
(RID-10A) detector (Shimadzu, Torrance, CA). The molecular weights of the fractions were
estimated by comparison with retention times of pullulan standards (P-800, 400, 200, 100, 50,
20, and 10; Phenomenex, Torrance, CA) or polyethylene glycol standards (PEG-11000, 5000,
3600, 1000, and 600; Pressure Chemical Co., Pittsburg, PA).
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Physical Characterization of Shilajit Fractions
For 1H-nuclear magnetic resonance (1H-NMR) analysis, samples (6 mg) were dissolved in 0.6
ml D2O, filtered through 0.2 μm filters, and spectra were recorded on a Bruker DRX-600
spectrometer (Bruker BioSpin, Billerica, MA) at 20°C using 3-(trimethylsilyl)-propionic
2,2,3,3,-d4 acid sodium salt as an internal reference. For 13C-NMR, samples (50 mg) were
dissolved in 1 ml D2O, filtered through 0.2 μm filters, and spectra were recorded on a Bruker
DRX-500 spectrometer (Bruker BioSpin, Billerica, MA) at 20°C.
UV–Vis spectra of samples dissolved in NaHCO3 (25 mM, pH 8.5) were recorded on a
SpectraMax Plus spectrophotometer (Molecular Devices, Palo Alto, CA) in a 1 cm quartz
cuvette by scanning from 200 to 800 nm. The E4:E6 ratio was determined at 465 and 665 nm,
as described by (Chen et al., 1977).
Fluorescence measurements were performed using an LS50 luminescence spectrometer
(Perkin Elmer). Samples were dissolved in NaHCO3 (25 mM, pH 8.5). Slit width for emission
and excitation wavelengths was 10 nm. For determination of the humification index (HIX), we
used the formula: HIX=(ΣI435→480)/(ΣI300→345), where I is the fluorescence emission intensity
with excitation at λex = 254 nm (Ohno, 2002). Since fluorescence intensity can be attenuated
by the solution itself (i.e., inner-filtering effect), we corrected for both primary and secondary
fluorescence inner-filtering effects in order to obtain an accurate measurement of the
fluorescence emission intensity (Ohno, 2002). For calculation of HIX values corrected for
inner-filter effects, we performed linear extrapolation on plots of HIX versus transmittance at
254 nm for 6-7 different concentrations of each fraction. The corrected HIX values correspond
to infinite dilution (i.e., approximating 100% transmittance) (Ohno, 2002). Synchronous
fluorescence spectra were recorded from 250 to 600 nm at a scan rate of 240 nm/min. The
excitation–emission wavelength difference (Δλ) was 20 nm (Chen et al., 2002).
Chemical Analysis of Shilajit Fractions
For elemental analysis, lyophilized samples were submitted to Desert Analytics (Tucson, AZ)
for analysis. Carbon, hydrogen, nitrogen, phosphorous, sulfur, halogens and metals were
measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Oxygen
content was taken as the difference from 100%.
The Bradford micro-protein assay was used to determine protein content, which bovine serum
albumin as the standard (BioRad, Hercules, CA).
Carbohydrate content was determined by the phenol-H2SO4 method [Dubois et al., 1956],
modified to a microplate format. Sample of 400 μl (500 μg/ml) were mixed with 200 μl 6%
phenol solution and 1 ml concentrated H2SO4. D-glucose was used as standard. The reactions
were incubated for 20 min at room temperature, and absorbance was measured at 488 nm.
The presence of arabinogalactan in the samples was detected by single radial gel diffusion in
a 1% agarose gel containing 100 μg/ml β-glucosyl Yariv reagent, which selectively interacts
with and precipitates compounds containing type II arabinogalactan structures (van Holst and
Clarke, 1985). Four μl of each Shilajit fraction (10 mg/ml) were loaded into the wells, and the
samples were incubated at room temperature for 24 hr in a humid atmosphere. A positive
reaction was identified by a reddish circle around the well, and arabic gum (4 mg/ml) served
as a positive control.
CTABr, a cationic detergent, was used to analyze carboxylic acid groups in Shilajit fractions
and standard FA (Denobili et al., 1990). Stock solution of the sample (1 mg/ml, pH 7.1) was
added to different amounts of 0.1% CTABr to produced 20 different CTA+/sample ratios.
Suspensions were left standing for 18 hr at 25°C in the dark before centrifugation at 17400 ×
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g for 30 min. Absorbance was measured at 400 nm, and the number of carboxyl groups was
determined to be at the minimum absorbance that coincides with quantitative precipitation with
the same number of CTA+ ions.
For monosaccharide composition analysis, samples were hydrolyzed at 100°C for 6 hr with 3
M TFA, and the resulting samples were separated by thin-layer chromatography (TLC) on
Whatman silica gel 60 plates with monosaccharide standards for reference (Dogsa et al.,
2005). The TLC plates were developed with butanol/acetic acid/water (3:1:1), and bands were
visualized by spraying the plates with aniline-diphenylamine reagent (2% aniline, 2%
diphenylamine, and 8.5% H3PO4 acid in acetone) and heating at 100°C for 10 min. Individual
monosaccharide bands were scraped from the plate, extracted with H2O, and quantified using
a colorimetric method with monosaccharide standards. Briefly, the extracts were mixed with
anthrone reagent (0.2% anthrone and 1% thiourea in H2SO4). After heating at 100°C for 10
min, absorbance was measured at 620 nm.
Complement-fixing Assay
The complement-fixing assay was performed as described (Diallo et al., 2001). Antibodysensitized
sheep erythrocytes were washed three times with GVB containing 0.5 mM Mg2+
and 0.15 mM Ca2+ (GVB2+) before use. The erythrocytes were resuspended in GVB2+ at a
concentration of 2×108 cells/ml, and human serum was diluted with GVB2+ to a concentration
giving about 50% hemolysis. Triplicate samples containing 50 μl of each serially-diluted
polysaccharide fraction were mixed with 50 μl diluted serum and added to microplate wells
and incubated at 37°C. After 30 min, sensitized sheep erythrocytes (50 μl) were added to each
well, and the samples were incubated for an additional 30 min at 37°C. After centrifugation
(900×g for 5 min), 50 μl of the supernatants were mixed with 200 μl distilled H2O in flatbottom
microplates, and absorbance was measured at 405 nm. 100% lysis was obtained by
adding distilled H2O to sensitized sheep erythrocytes. Samples containing GVB2+, serum, and
sensitized sheep erythrocytes were used as background controls (Acontrol), while heparin served
as a positive control. Inhibition of hemolysis induced by the test samples was calculated by the
formula: [(Acontrol - Asample)/Acontrol]×100%. A dose–response curve (6-7 points) was
constructed to calculate the concentration of test sample able to give 50% inhibition of
hemolysis (ICH50). Low ICH50 means high complement fixing activity. Heparin, a highly
sulfated glycosaminoglycan, was used as a positive control.
Statistical Analysis
Linear regression analysis was performed on the indicated sets of data to obtain correlation
coefficients, 95% confidence intervals, and statistical significance (GraphPad Prism Software,
San Diego, CA). Differences at P<0.05 were considered to be statistically significant.
Results and Discussion
Preparation and Partial Characterization of Shilajit Humic Substances
Shilajit humic substances (SHS) obtained from crude Shilajit humus were fractionated by ion
exchange chromatography, resulting one neutral fraction (S-I) and two acidic fractions (S-II
and S-III). The size distribution of molecules in these fractions and crude Shilajit humus was
characterized by HP-SEC, and the elution profiles are shown in Figure 1. Crude Shilajit humus
eluted over a broad range of molecular weights, extending from 1 to ∼1,000 kDa. The small
peak present at ∼1,000 kDa likely represents stable macromolecular aggregates in the sample.
The SHS elution profile contained four peaks, including three small, broad peaks with modes
corresponding to Mr of ∼800, 100, and 15 kDa and a major peak corresponding to ∼1.1 kDa.
The neutral fraction (S-I) elution profile had a bimodal profile, with peak modes corresponding
to Mr of ∼700 and 15 kDa. The elution profiles of acidic fractions, S-II and S-III, contained
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three peaks (modes corresponding to Mr of ∼700, 100, and 2 kDa) and two peaks (modes
corresponding to Mr of ∼700 and 2 kDa), respectively.
Fraction S-II, the most abundant fraction isolated by DEAE cellulose chromatography (>90%
of total yield), was further fractionated using Sephadex G-50 chromatography. Three subfractions
were obtained and designated as S-II-1, S-II-2, and S-II-3, based on total
carbohydrate, UV absorbance (254 nm), and fluorescence (λex=355 nm, λem=450 nm) elution
profiles (Figure 2A). The HP-SEC refractive index elution profile of sub-fraction S-II-1 was
similar to that of its parent fraction (S-II) in the Mr region of 20 to ∼800 kDa (Figure 2B). In
contrast, both fractions S-II-2 and S-II-3 eluted primarily as a single peaks, with modes
corresponding to Mr of ∼1.8 and 3.1 kDa, respectively (Figure 2B). Note that the average Mr
of sub-fractions S-II-2 and S-II-3 is less than the nominal Mr cutoff of the membrane used for
concentrating the crude SHS. Thus, it is likely that fraction S-II consisted of non-covalent
complexes or micelles that were dissociated under the buffer/salt/mechanical conditions of the
final Sephadex G-50 chromatography step. Indeed, it has been previously reported that
concentrated solutions of humic substances can form micelles that cannot be filtered even
through 100 kDa membranes (Benedetti et al., 2002;Brown et al., 2004). Furthermore, charge
effects, solution conditions, and membrane surface characteristics have also been shown to
impact ultrafiltration and SEC fractionation, with various organic components being affected
differently (Buffle and Leppard, 1995;Schafer et al., 2002;Benedetti et al., 2002).
Lyophilization of the fractions resulted in powders differing in color from white (S-I) to black
(S-III), and analysis of carbohydrate and protein content indicated a wide range in composition
between fractions (Table 1). In general, the primary fractions and sub-fractions with the lowest
carbohydrate content contained the highest levels of protein (e.g., fractions S-III and S-II-3).
Sugar composition analysis revealed that polysaccharides in all Shilajit fractions, except for
fraction S-II-3, consisted primarily of glucose (Glc), galactose (Gal), xylose (Xyl), and
rhamnose (Rha), with Glc and Gal being the dominant monosaccharides. In contrast, fraction
S-II-3 contained a minimal amount of Gal, but had a much higher level of glucosamine (GlcA)
in mol % than all other fractions (Table 2). Analysis of the Shilajit fractions using the Yariv
test showed that all fractions, except for fraction S-II-3, contained type II arabinogalactan
(Table 1). This finding supports the current hypothesis that Shilajit originates from a vegetative
source (Agarwal et al., 2007). Indeed, latex bearing plants (Euphorbia royleana Boiss,
Trifoleum repens) and bryophytes present in the vicinity of Shilajit-exuding rocks contain a
large amount of arabinogalactan (Saare-Surminski et al., 2000;Popper and Fry, 2003).
Fluorescence spectrometry was used to determine the extent of humification in the Shilajit
fractions and standard FA samples (Zsolnay et al., 1999;Ohno, 2002). We found that corrected
values of HIX and the E4:E6 ratios were lower for the Shilajit fractions, as compared to standard
FA obtained from IHSS (Figure 3). These lower HIX values indicate that the Shilajit fractions
are enriched in polysaccharides and probably other weakly chromophoric biomolecules (Ohno
et al., 2007).
Elemental Composition
The elemental composition of the primary Shilajit fractions (S-I, S-II, and S-III) is shown in
Table 3. We calculated the atomic ratios of H/C, O/C, and N/C, which have been commonly
used as indicators of structural characteristics of humic substances and their diagenetic history
(Kim et al., 2003). The van Krevelen diagram, created by plotting H/C vs. O/C, showed that
the humic substances from Shilajit were located clustered near the carbohydrate region,
suggesting that they could be products of polysaccharide degradation and/or contain native
polysaccharides (Figure 4). The average O/C ratio, which is indicative of carbohydrate content,
carboxylic groups, and the degree of oxidation, was higher in the Shilajit fractions than in
standard FA and humic acid samples (Figure 4). Conversely, the Shilajit fractions had lower
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C/N ratios than the standard FA and humic acid samples, except for Pony Lake FA, which is
derived primarily from carbohydrates and proteins of algae and cyanobacteria (Brown et al.,
2004;McKnight et al., 1994).
NMR Analysis
The 1H-NMR spectrum of crude Shilajit was close to that of Shilajit obtained from other
mountain regions (e.g., see Jung et al., 2002]) and contained much stronger signals in the
aliphatic (0.5-2.8 ppm) and aromatic (6-8 ppm) regions, as compared to spectra of the isolated
Shalajit fractions. In general, the spectra of crude SHS and primary fractions S-II and S-III
were similar to each other, and proton signals in the region from 0.0 to 5.5 ppm were more
separated than in the same region of spectra from fraction S-I (Figure 5A).
The chemical shifts of anomeric protons were evaluated according to data previously reported
for humic substances and polysaccharides (Gane et al., 1995;Dong and Fang, 2001). 1H-NMR
spectra of the Shilajit fractions indicated the presence of alkyl components (0.5-2.3 ppm),
including methylene groups from methylenic chains (0.94-1.38 ppm) and terminal methyl
groups (0.0-0.94 ppm). Spectra of the fractions indicated a significant amount of methylene
and methyl groups α to carbonyl groups and/or attached to aromatic rings, typically resonating
in the 1.8-2.7 ppm region. The spectrum of fraction S-II contained strong sharp signals at
1.8-1.9 ppm and 3.2 ppm, which can arise from protons belonging to repetitive chemical
fragments, such as protons on CH3–CO– and CH3–O– groups that are formed during
humification, possibly by oxidative degradation (Ruggiero et al., 1980). In comparison, these
peaks were absent in fraction S-I, which contains much more native arabinogalactan. The signal
at 2.1 ppm in the spectra of fractions S-II and S-III indicates the presence of α-methyl protons
in ketones. Fraction S-III exhibited small, unique proton signals in the region of 2.1-3.1 ppm.
For example, a pair small doublets at 2.55-2.68 ppm is consistent with the presence of
methylene protons (–CH2–) in acyl groups (R-CH2-C=O), such as in free or esterified
carboxylic groups (C=O). Spectra of all Shilajit fractions and the parent Shilajit sample showed
a broad proton resonance between 3.3 and 4.2 ppm, with a maximum near 3.6-3.7 ppm.
Resonances in this region derive from protons belonging to methyl and methylene groups
connected to electronegative atoms, primarily oxygen, which are present in carbohydrates,
methoxy compounds, carboxylic acids, and organic amines (Sciacovelli et al., 1977;Wilson et
al., 1983;Grasso et al., 1990;Yamauchi et al., 2004). The signals in the region of 4.0-5.5 ppm
are partially due to protons on alcoholic OH groups. Signals for aromatic protons at 5.6-6.1
ppm were barely visible in the 1H-NMR spectra and appeared as weak resonances in all three
samples. However, the spectrum of fraction S-III showed a broad resonance in the 5.8-6.9 and
6.9-8.6 ppm regions, which are normally attributed to protons in olefinic and aromatic moieties,
respectively. Spectra of fractions S-I and S-II showed very small broad signal at these regions,
possibly because the aromatic groups in these samples was highly oxidized. The sharp peak at
8.35 ppm in the spectrum of fraction S-III suggests the presence of formyl groups covalently
bonded the macromolecules of humic substances (Jokic et al., 1995).
The 1H-NMR spectra of sub-fractions S-II-1 and S-II-2 were similar to that of parent fraction
S-II, but with less prominent peaks at 1.9 and 3.2 ppm (data not shown). The spectrum of
fraction S-II-3 indicated the presence of aromatic protons and much stronger signals at 1.1 and
3.2 ppm (data not shown).
13C-NMR (500 MHz) spectra of fractions S-II and S-III are shown in Figure 5B. The chemical
shifts were evaluated according to data previously reported for humic substances and native
polysaccharides (Baddi et al., 2004;Jokic et al., 1995;Polle et al., 2002). These spectra were
characterized by the presence of many signals in the area of aliphatic carbons (0-50 ppm),
carbohydrate carbons (60-96 ppm), anomeric carbons (96-108 ppm), aromatic carbons
(108-145 ppm), and carboxyl and carbonyl carbons (163-190 ppm). However, signals for
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methoxyl carbons (50-60 ppm), phenolic carbons (145-163 ppm), and ketone carbons (190-220
ppm) were absent. The alkyl region (0-50 ppm) of fraction S-II showed a maximum at 18 ppm,
which can be attributed to acetate groups in carbohydrates and corresponds to the CH3–CO–
groups with sharp signals at 1.8 ppm in the 1H-NMR spectrum of the fraction. The intensity
of the peak at 160-200 ppm, attributed to carboxylic, amidic, and ester carbons, was greater in
fraction S-III, as compared to fraction S-II. This feature is likely due to an increase in carboxylic
groups and correlates with the strong anionic properties of this fraction. Signals in the region
of 60-108 ppm are usually assigned to O- and N-substituted carbons. Since the nitrogen content
of these fractions is relatively low and the C/N ratio is relatively high (Table 2), it is likely that
most carbon resonances this region arise from carbohydrates and carboxylic groups. Sharp
peaks at approximately 62 and 73 ppm could arise from carbon C6 in carbohydrates and
protonated ring carbons (C2-C5) of carbohydrates (Jokic et al., 1995).
In comparison with 13C-NMR spectra of natural FA from different sources (for example, see
(Baddi et al., 2004), fractions S-II and S-III contained lower levels of aromatic carbon.
Thus, 13C- and 1H-NMR data suggest predominantly carbohydrate-derived material in isolated
Shilajit fractions with low contribution of aromatic carbons.
Complement-fixing Activity of Shilajit Fractions and FA from IHSS
Crude SHS and all fractions isolated from the Shilajit showed dose-dependent fixation of
human complement in vitro with ICH50 values ranging from 15.4 to 273 μg/ml (Table 4). The
most potent complement-fixing ability was found in the relatively low-molecular weight
fraction S-II-3, which contained the lowest amount of carbohydrate. In contrast, the neutral
fraction S-I failed to fix complement, even at the maximal the concentration tested (500 μg/
ml) (data not shown). A plot of carbohydrate content in the Shilajit fractions versus the
reciprocal values of ICH50 (1/ICH50) demonstrated a good negative linear correlation (r=
-0.848; n=7; P<0.02) between these values (Figure 6).
Five FA standards from IHSS were also tested for complement-fixing activity. All samples
exhibited complement-fixing activity (ICH50) (Table 4). It is interesting to note that the least
active standard (Pony Lake FA) is formed in an Antartic lake without lignin sources and
presumably consists of diagenetic products from algae and bryophyte polysaccharides
(McKnight et al., 1994).
Plots of HIX versus ICH50 did not demonstrated any correlation when the plots contained the
Shilajit fractions together with FA standards (r= 0.201, n=11 for E4:E6 vs. ICH50; r=0.314,
n=11 for HIX vs. ICH50). However, plots of HIX versus ICH50 did show some correlation
when the plots contained the Shilajit fractions only (r=0.659). Furthermore, the integrated
emission in area of the red-shift (435-480 nm) of synchronous fluorescence spectra and 1/
ICH50 were significantly correlated in separated plots contained the Shilajit fractions and FA
standards, r=0.838 (P<0.02) and r=0.962 (P<0.01), respectively (Figure 7). Thus, preferential
complement-fixing activity is found in the material with higher levels of humification.
Since endotoxin can be a contaminant of isolated organic materials, we determined whether
endotoxin could be contributing to the biological activity of the Shilajit fractions. First, LPS
from E. coli was evaluated for complement-fixing activity; however, no activity was found
over the concentrations tested (2-250 μg/ml) (data not shown). To further verify that endotoxin
contamination of the samples did not contribute to complement-fixing activity, we applied
fraction S-II-3 to a column of endotoxin-removing gel and analyzed the eluted sample. As
shown in Figure 8, complement-fixing activity of fraction S-II-3 after removal of possible
endotoxin was essentially the same as that of control fraction S-II-3. Thus, these data clearly
demonstrate that endotoxin was not responsible for biological activity of the Shilajit fractions.
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Content of Carboxylic Groups in Shilajit Fractions and FA
As noted above, the highest complement-fixing ability was found in the relatively lowmolecular
weight fraction S-II-3, which contains the lowest amount of carbohydrate. During
humification, a progressive transformation of polysaccharides into other oxygenated
compounds, particularly carboxylic groups, takes place (Castaldi et al., 2005). Indeed, (poly)
carboxylic acids with a very limited number of hydroxyl groups are the major compound class
in FA (Reemtsma et al., 2006). Thus, we estimated content of carboxylic groups in the Shilajit
fractions and FA from HISS. The content of carboxylic groups in fraction S-II-3 was close to
the content in FA from other natural sources, such as Suwannee River, Waskish Peat, and
Nordic Aquatic FA (Table 4). Plots of carboxylic group content versus the reciprocal of
ICH50 (1/ICH50) demonstrated a good positive linear correlation r=0.880 (n=12; P<0.001)
between these values (Figure 9). Thus, these results suggest that carboxylic groups are
important for complement-fixing activity of the Shilajit fractions and FA from other natural
sources. One possibility is that carboxylic groups play a role in interaction with complement
components. For example, carboxylic groups have been reported to be the main molecular
fragments required for interaction of humic substances with cell membranes (Muscolo et al.,
2007), complexation with cationic species (Livens, 1991;Prado et al., 2006;Esteves da Silva
and Oliveira, 2002), and association with inorganic surfaces (Fu and Quan, 2006).
Additionally, fraction S-II-3 fraction contains the highest level of glucosamine among the
Shilajit fractions (Table 3), suggesting the possibility that glucosamine residues may also be
involved in the biological activity observed. Indeed, previous studies have shown that various
antigens with complement-fixing capacity were also enriched in glucosamine (Shinagawa and
Yanagawa, 1972;Hammerberg et al., 1980).
The potent complement-fixing activity of humic substances isolated from Shilajit may
contribute to the therapeutic potential of Shilajit extracts. The complement system plays an
essential role in innate immunity, contributing to inflammatory responses and the destruction
and removal of pathogens [reviewed in (Gasque, 2004)]. However, excessive or uncontrolled
complement activation can also contribute to host tissue damage, and therapeutic strategies
have been developed to inhibit this process (Mollnes and Kirschfink, 2006). Likewise, the
removal of complement by fixation has also been proposed to be a potential therapeutic strategy
for treating inflammatory diseases (Nergard et al., 2004). A number of reports have shown
polysaccharides from different plants can enhance wound healing, and some of these
polysaccharides also have potent complement-fixing activity (Table 5). For example,
Samuelsen et al. (Samuelsen et al., 1995) reported that the wound healing properties of
Plantago major L. polysaccharides were at least partly due to their ability to fix complement.
Similarly, wound healing properties of polysaccharides from Biophytum petersianum Klotzsch
were reported to be related to their effects on the complement system (Inngjerdingen et al.,
2006). Indeed, Wagner (Wagner, 1990) suggested that the anti-complement properties of plantderived
polysaccharides significantly contribute to their anti-inflammatory properties. Our
studies suggest that products of oxygenated degradation of plant polysaccharides also have
potent complement-fixing properties in vitro and are among the most active of the natural
products reported to date.
Acknowledgements
We would like to thank Dr. Andrei Khlebnikov (Department of Chemistry, Altai State Technical University, Barnaul,
Russia) for reviewing this manuscript and providing helpful suggestions. We would also like to thank Dr. Scott Busse
(Montana State University, Bozeman, MT) for help in running NMR samples. This work was supported in part by
Department of Defense grant W9113M-04-1-0001, National Institutes of Health grant RR020185 and contract
HHSN266200400009C, an Equipment grant from the M.J. Murdock Charitable Trust, and the Montana State
University Agricultural Experimental Station. The U.S. Army Space and Missile Defense Command, 64 Thomas
Drive, Frederick, MD 21702 is the awarding and administering acquisition office. The content of this report does not
necessarily reflect the position or policy of the U.S. Government.
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Abbreviations
FA
fulvic acid
IHSS
International Humic Substances Society
SEC
size exclusion chromatography
HP-SEC
high performance SEC
HIX
humification index
NMR
nuclear magnetic resonance
CTABr
cetyltrimethylammonium bromide
DEAE
diethylaminoethyl
GVB
gelatin veronal buffer
TFA
trifluoroacetic acid
LPS
lipopolysaccharide
SRFA
Suwannee river FA
NAFA
Nordic Aquatic FA
PLFA
Pony Lake FA
FPFA
Florida Peat FA
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WPFA
Waskish Peat FA
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Figure 1.
Analysis of Shilajit fractions by size-exclusion chromatography. Crude Shilajit, total humic
substances from the crude Shilajit (SHS), and the three primary fractions (S-I, S-II, and S-III)
isolated by ion exchange chromatography were analyzed by HP-SEC and monitored with a
refractive index detector, as described. Peak retention times of the indicated pullulan (PUL)
and polyethylene glycol (PEG) standards are shown for reference.
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Figure 2.
Chromatographic separation of Shilajit fraction S-II. Panel A. Shilajit fraction S-II was
separated by SEC on Sephadex G-50 and monitored for absorbance at 254 nm (■) and
fluorescence (●). Total carbohydrate content in each fraction was determined by the phenol-
H2SO2 method (detected at 488 nm) (□). Fractions were combined as indicated to obtain the
S-II sub-fractions selected for further analysis (designated S-II-1, S-II-2, and S-II-3). Panel
B. S-II sub-fractions were analyzed by HP-SEC and monitored with a refractive index detector,
as described. Peak retention times of the indicated pullulan (PUL) and polyethylene glycol
(PEG) standards are shown for reference.
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Figure 3.
Humification index (HIX) and E4:E6 ratio of Shilajit fractions and standard FA. Values were
determined for each sample, as described under Materials and Methods. The data are presented
as the mean ± SEM of 3 independent experiments.
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Figure 4.
The van Krevelen diagram of atomic ratios of H/C versus O/C for the Shilajit fractions. Plots
for humic acids are shown as solid circles (●), FA (except for Pony Lake FA) are shown as
open diamonds (◇), Pony Lake FA are shown as open circles (○), and the Shilajit fractions
are shown as stars (⋆). The positions of the Shilajit fractions were determined from data of
element analysis (Table 3). The positions of FA and humic acids from other natural sources
were using previously published data (Lawrence, 1989;Provenzano and Senesi, 1999;Ma et
al., 2001;Brown et al., 2004). The locations of regional plots for primary organic substances
on the diagram (lipids, proteins, carbohydrates, condensed hydrocarbons and lignin) were
reproduced from (Kim et al., 2003).
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Figure 5.
1H-NMR and 13C-NMR spectra of primary Shilajit fractions. 1H-NMR spectra of crude Shilajit
extract and SHS are shown for comparison.
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Figure 6.
Plot of complement-fixing activity of Shilajit fractions versus carbohydrate content in the
fractions. Complement-fixing activity is represented as inverse ICH50 (1/ICH50). Dashed lines
indicate area of the 95% confidence band.
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Figure 7.
Fluorescence spectra and plot of complement-fixing activity versus integrated fluorescence for
the Shilajit fractions. Panel A. Solutions of Shilajit fraction S-I, sub-fraction S-II-3, Pony Lake
FA (PLFA), and Florida Peat (FPFA) (20 μg/ml in 25 mM NaHCO3) were analyzed with a
scanning fluorometer, and the synchronous spectra (Δλ=20 nm) are shown. Panel B.
Complement-fixing activity, represented as inverse ICH50 (1/ICH50) was plotted versus
integrated fluorescence of the synchronous spectra from 435-480 nm. Dashed lines indicate
area of the 95% confidence band.
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Figure 8.
Evaluation of the role of endotoxin in complement-fixing activity of sub-fraction S-II-3.
Complement-fixing activity was determined in control samples of fraction S-II-3 (□) and
samples treated with endotoxin-removing gel (■). The data are presented as the mean ± SEM
of 3 samples from one experiment that is representative of 2 independent experiments.
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Figure 9.
Plot of complement-fixing activity versus carboxylic group content in the Shilajit fractions and
standard FA samples. Complement-fixing activity is represented as inverse ICH50 (1/ICH50).
Dashed lines indicate area of the 95% confidence band.
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Table 1
Chemical and physical properties of Shilajit fractions
Fraction Color (powder) Chemical features Carbohydrate content (%) Yariv test Protein content (%)
SHS Dark brown - 40 Positive 4.5
S-I White Neutral 47 Positive 0.2
S-II Brown Acidic 36 Positive 2.3
S-III Black Acidic 21 Positive 6.8
S-II-1 Brown Acidic 56 Positive 0.2
S-II-2 Brown Acidic 34 Positive 2.5
S-II-3 Pale brown Acidic 14 Negative 6.3
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Table 2
Monosaccharide composition (mol %) of the Shilajit fractions
Fraction Glc Gal Xyl Ara Rha GalA GlcA
SHS 41 32 8 3 7 5 4
S-I 57 26 10 2 5 5 4
S-II 34 35 8 3 10 5 4
S-III 43 24 13 3 13 <2 4
S-II-1 34 38 8 4 10 6 <2
S-II-2 37 31 10 3 9 10 <2
S-II-3 46 7 8 3 17 <2 20
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Table 3
Elemental analysis of the Shilajit fractions
Na C H O N S P Si Br Cl F I Metals H/C O/C C/N
S-I ND 41.55 6.02 40.16 0.69 0.10 <0.2 ND ND ND ND ND ND 1.72 0.73 70.25
S-II 4.05 40.59 5.59 46.86 1.54 0.17 0.11 0.55 <0.1 0.3 <0.1 <0.1 <0.98 1.64 0.87 30.75
S-III 8.78 38.69 4.27 36.32 1.41 0.15 <0.1 0.55 <0.1 8.5 <0.1 <0.1 <1.23 1.31 0.71 30.01
N.D., not detected.
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Table 4
Complement-fixing activity and carboxylic group content of the Shilajit fractions and standard FA from IHSS
Sample ICH50 (μg/ml) COOH (mM/g)
SHS 161.8±14.7 0.70±0.04
S-I N.A. N.P. (0)
S-II 126.1±11.3 1.35±0.06
S-III 30.6±4.2 1.67±0.07
S-II-1 272.9±29.4 1.26±0.09
S-II-2 58.2±6.7 1.62±0.11
S-II-3 15.4±3.1 3.07±0.23
Florida Peat FA 13.8±1.6 5.48±0.37
Nordic Aquatic FA 20.1±1.9 3.41±0.25
Suwannee River FA 39.5±4.3 3.07±0.23
Waskish Peat FA 41.5±3.8 3.23±0.24
Pony Lake FA 201.7±18.8 N.P. (0)
N.A., not active; N.P., not precipitated by CTABr. The data are presented as the mean ± SEM of 3 independent experiments.
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Table 5
Plant pectin polysaccharides with the highest reported complement-fixing activity
Plant Common Name Fraction Mr
(kDa)
ICH50
(μg/ml) Reference
Plantago major PMII 46-48 25 (Samuelsen et al., 1996)
Glinus oppositifolius
GOA1 70 34
(Inngjerdingen et al., 2005)
GOA2 30-39 60
Avicennia marina HAM-3-IIb-II 105 23 (Fang et al., 2006)
Vernonia kotschyana Vk2a 1150 2 (Nergard et al., 2006)
Biophytum petersianum BP100 III 31 9 (Inngjerdingen et al., 2006)
Trichilia emetica Te 100 acidic 4 223 <15 (Diallo et al., 2003)
AG-I/II – arabinogalactan type I or type II; RG-I – rhamnogalacturonan type I.
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