An in vitro and in vivo investigation of the
anti-inflammatory
properties of coal derived
humates.
C.E.J. van Rensburg, G. Jooné and A.D. Cromarty, Department of
Pharmacology, Faculty of Health Sciences, University of Pretoria, South
Africa.
|
INTRODUCTION:
Humic substances occur widely in nature.
The therapeutic properties of humates have been described as
antibacterial, antitoxic, anti-ulcerogenic, anti-arthritic,
anti-allergic, immunomodulatory and anti-inflammatory.
Various in vivo studies have been done to demonstrate the
anti-inflammatory activity of crude products prepared from humus
matter such as peat, mumie and sapropel.
No documentation could be found concerning the effects of
coal-derived humates on inflammatory reactions. These preparations,
especially products derived from brown coals, contain much higher
levels of high quality humate, are easier to produce under
controlled conditions and are more available in nature.
It has been documented that agents that block the adhesion
molecule, CR3, expressed on the surface of activated neutrophils,
are beneficial in the treatment of inflammation by inhibiting
recruitment of leukocytes into tissues. |
RESULTS:Contact hypersensitivity
Both the prednisilone and the leonardite derived humate product
caused a significant (P < 0.5) decrease in ear swelling at 3h as
well as 24 and 48h (Figure 2). Prednisilone proved to be superior to
the leonardite humate whereas the humate product derived from
bituminous coal had no significant effect on ear swelling.
No signs of toxicity were observed during the 7 days of
treatment with the humate products. However, the rats on
prednisilone lost weight compared to the control group (Figure 3).
Figure 1
Control rat after DNFB challenge.
Figure 2
Difference in ear
thickness between left and right ears of DNFB challenged rats either
without treatment or after a daily treatment for one week,
administered by gavage, of one of the following; 61mg/Kg bituminous
humate; 61mg/Kg leonardite humate; 1mg/Kg prednisilone.
The three
columns represent the differences in ear thickness at 3 hours, 24
hours and 48 hours post challenge. Significant differences compared
to untreated controls.
* p< 0.05; **
p< 0.01
Figure 3
The changes
in the body mass of rats after one week of treatment with 61 mg
/Kg/day bituminous coal derived and leonardite derived humate
products or 1 mg/Kg/day prednisilone compared to untreated
controls.
* p< 0.05; ** p<
0.01
 |
Measurement of CR3 expression:
Both potasium humate products caused a dose related inhibition
of CR3 expression by stimulated, but not resting neutrophils that
was significant (p< 0.05) at 10µg/ml and higher (Figure
4).
Figure 4
The effects of a 15 min treatment with
various concentrations of the bituminous coal derived and the
leonardite derived humate products on the expression of CR3 on
resting and PMA-stimulated human neutrophils determined by flow
cytometry.
|
AIM OF STUDY:To determine:
whether oral treatment with potassium humate over a period of a
week could decrease the inflammatory effects caused by a contact
hypersensitivity reaction in vivo and to compare the effects
with prednisilone.
the effects humates have on the expression of CR3 by activated
neutrophils. |
METHODS:Humate:
Two humate products were
dissolved in water and were tested.
| (i) |
one produced from brown coal
(leonardite) (contains 92% soluble humate
and fulvate). |
| (ii) |
one synthesized from bituminous
coal (contains 33% soluble humate).
|
Contact
hypersensitivity:
This experiment was done at the University of Pretoria
Biomedical Research Centre.
60 female Sprague Dawley rats of 8 to 10 weeks, divided into
four groups of 15 rats each.
On day 0, the rats were weighed and sensitized by painting the
shaved abdomen with 400µl of a 2.5% solution of
2,4-dinitro-fluorobenzene (DNFB) in acetone:olive oil (4:1) and
placed on one of the following oral treatments: (I) water only (2)
leonardite humate (61mg soluble humate /Kg) (3) bituminous coal
humate (61mg soluble humate /Kg) (4) prednisilone (1mg/Kg).
On day 6, the rats were challenged on the right ear by
application to upper surface of the ear, of 25µl of a 0.5% solution
of DNFB in acetone:olive oil (4:1) (Figure 1).
Three hours after challenge both ears were measured with an
engineering caliper across the ear at a distance of 3mm from the
tip. Each ear was measured 3 times and the average thickness
reported. The measurements of the ears were repeated after 24 and 48
hours.
Measurement of CR3 expression:
A suspension of neutrophils was treated with various
concentrations of the humate products for 15 min at 37°C, stimulated
with 100ng/ml PMA and incubated for a further 15 min. CR3
quantitation was done by flow cytometry analysis on a Coulter Epics
XL-MLC flow cytometer using an anti-CD11b FITC monoclonal antibody
(Beckman Coulter, Pallo Alto, California).
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DISCUSSION:
The antiinflammatory properties of oral potassium humate derived
from leonardite compared favourably with prednisilone
Both of the humate products inhibited CR3 expression on
activated neutrophils.
The difference in the activities between the two humate products
in the contact hypersensitivity model needs to be further
investigated.
An over-expression of CR3 is associated with the production of a
multitude of cytokines, reactive oxygen, nitrogen intermediates and
proteolytic enzymes that can cause tissue injury and lead to
inflammatory conditions.
No signs of toxicity was observed with the two humate products.
Potassium humate has been proven to be safe in humans, whereas
prednisilone is associated with serious side
effects.
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CONCLUSION:
The identification of a relative nontoxic compound such
as potassium humate that exerts its anti-inflammatory properties via
the blocking of an adhesion molecule that plays an key role in
inflammation, is therefore an exciting finding and merits further
evaluation in the treatment of patients suffering from inflammatory
conditions. | |
