Potential molecular basis of the chondroprotective effect of Harpagophytum procumbens.
The study protocol was approved by the Veterinarian Ethics Committee of the University of Frankfurt and the governmental authority in Darmstadt. Twenty-three adult female white New Zealand rabbits (mean weight 4.6 [+ or -] 0.28 kg (SD)) underwent unilateral transection of the anterior cruciate ligament and removal of the medial meniscus of the right knee. The left knees served as intra-individual controls. The rabbits were divided into two groups. Group H (H, n = 11) received standard food pellets with added Harpagophytum extract FB9195 (14% harpagoside, Fig. 1) ad libitum based on 150mg extract per day with 0.1 mg harpagoside per gram pellets. The control group (C, n = 12) received standard pellets. The rabbits were placed in large individual cages and were forced to move every day. At sacrifice, after 27 weeks, both knee joints were removed to undergo macroscopic and histological evaluation (separate publication). Articular cartilage from the right and left heads of femur were removed manually and cut into three parts (1) to be stored in buffered formalin 4-6% for later histomorphometric investigations, (2) to be immersed in liquid nitrogen before embedding into Jung GmbH (020108926) medium for later histological analysis and, (3) for immediate extraction of RNA for reverse DNA transcriptase production and gene expression analyses of molecules known to be involved in cartilage metabolism and known rabbit gene sequences. These primer sequences were: biglykan forward: GCGCATCT CAGAAGCCAAGC; biglykan reverse: CGTTGT AGTAGGCCCGCTTC; aggrecan-2 forward: GCCTGTGGTGTGCGGTGGTG; aggrecan-2 reverse: GGACCCCAGGACCCCCAGTT; osteonectin forward: ACCCCCATGTGCG TGTGCCA; osteonectin reverse: ACGCA GTGGGGCCAGCTCAG; TIMP-2 forward: ACGGCAACCCCATCAAGAGGA; TIMP-2 reverse: GGAGTCCCAGGGCAC GATGAA. For further details of the methods see http://remed-chrubasik.uniklinik-freiburg.de.
[FIGURE 2 OMITTED]
During preparation of the cartilage specimens, the respective investigators (MFM, SC) discovered a macroscopical thickening of the cartilage of the right hip. However, the morphometric investigation did not correlate with per cell count or with cell density of the right and left hips of the rabbits or between the hips of Harpagophytum-treated and untreated rabbits (see webpage). Haematoxylin, safranin and elastin stainings showed a trend towards chondroid regeneration and increased elastic and collagen fibres in the hip cartilage of the surgically altered limb of the Harpagophytum-treated rabbits, although this was only statistically significant in the elastin stainings (Fig. 2). Of the examined genes, TIMP-2 mRNA showed a 2- and 5-times increase in two rabbits treated with Harpagophytum extract compared to the maximum TIMP-2 mRNA concentration in the control group.
In summary, these results suggest that one of the mechanisms of the therapeutic effect of H. procumbens may be a chondroprotective effect in which the tissue inhibitor of metalloproteinase-2 (TIMP-2) is involved.
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J.E. Chrubasik, E. Neumann, U. Muller-Ladner Department of Rheumatology and Clinical Immunology, Kerckhoff Clinic Bad Nauheim and Chair for Internal Medicine and Rheumatology, University of Giessen and Marburg, Benekestrasse 2-8, D-61231 Bad Nauheim, Germany
J.E. Chrubasik, M. Faller-Marquardt, S. Chrubasik
Institute for Forensic Medicine, Albert-Ludwig-University Freiburg, Albertstrasse 9, D-79104 Freiburg, Germany
E-mail address: sigrun.Chrubasik@klinikum.uni-freiburg.de (S. Chrubasik)
Department of Surgery, Wolfgang Goethe-University, Theodor Stern-Kai 7, 60590 Frankfurt/Main, Germany
Institute for Pathology, Albert-Ludwig-University Freiburg, Albertstrasse 9, D-79104 Freiburg, Germany
School of Medical Sciences, University of New South Wales, Sydney 2033, Australia
Figure 1. HPLC-fingerprint of the 14% Harpagophytum extract FB9195, Finzelberg, Andernach, Germany (references harpagoside HF-HO3 and methylcinnamate HF-MO6). Column: LiChroCART 100 Merck, Darmstadt, RP 18, end-capped, 0.2 [micro]m Solvent A: water; solvent B: methanol, isocratic: Min A B 0 0.75 0.75 20 Stop Flow rate: 1.5 ml/15 min, injection volume 10 [micro]l, detection 278 nm UVvis, temperature 40 [degrees]C.
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|Title Annotation:||LETTER TO THE EDITOR|
|Author:||Chrubasik, J.E.; Neumann, E.; Muller-Ladner, U.; Faller-Marquardt, M.; Chrubasik, S.; Lindhorst, E.;|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Sep 1, 2006|
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