An in vitro and in vivo investigation of the anti-inflammatory properties of coal derived humates.

C.E.J. van Rensburg, G. Joonй 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.

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).

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.

IMAGE MISSING

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 2

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

Figure 3

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.

Figure 4

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.

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.