When we are mentally and physically stressed, it is thought that biproduct of oxygen (active oxygen) increases, causing various diseases. First, we started a research based on a hypothesis that the mechanism of anti-stress action is to remove increased active oxygen in the body. Eleuthero was added to a system where active oxygen is artificially produced in a test tube, and the amount was measured using an electronic spin resonator. The study demonstrated that eleuthero has ability to scavenge two active oxygen species, O₂^-(superoxide) and OH^-(hydroxyl radical) that give damage to the living body (Figure1).

Figure1. Eleuthero's ability to scavenge active oxygen species

We studied the action of eleuthero by targeting rheumatoid arthritis (RA) among diseases in which active oxygen is involved. Chronic RA, whose cause remains to be clarified, is an autoimmune disease common in women, affecting the joints of upper and lower limbs, resulting in joint deformation. In RA, inflammation occurs in the synovium of the joints from early stage, and neutrophils, macrophages, T-cells, and B-cells infiltrate in synovial tissues. These cells produce tumor necrosis factor (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6), while neutrophils produce superoxide, resulting in cartilage and bone destruction and joint dysfunction. This study examined the anti-inflammatory action of eleuthero and efficacy of its combination with anti-TNF-α antibody (biological therapeutic agent of RA) using collagen-induced arthritis mouse model (hereinafter referred to as "CIA mice'') known as an RA model.

1. CIA mice and administration of eleuthero extract

A mixed solution of 100 μg of bovine type II collagen and complete Freund's adjuvant (emulsion) was intracutaneously administered twice at an interval of three weeks to DBA/1 J male mice. Arthritis occurred in lower limbs of mice seven to 14 days after administration, causing redness and swelling (Photo2). The symptoms of arthritis were evaluated according to clinical scores as measured by calcaneal thickness and severity of swelling of the extremities, erythema, and deformation and rigidity of the joints. Eleuthero 100% extract powder was orally administered by gavage at 500 mg/kg body weight/day daily from one week before the first administration of emulsion, from the day of the second administration, or the day of the onset of arthritis. Distilled water or ascorbic acid with antioxidant capacity (vitaminC 10g/kg body weight/day) was administered to the control group in the same manner.

Photo2. Arthritis symptoms in mice

【Results1】

Delayed onset of arthritis and reduced symptoms were observed in the group of CIA mice to which eleuthero was administered from one week before emulsion administration, as compared with the group receiving distilled water (Figure2). The administration from the day of the second administration of emulsion delayed the onset but did not reduce the symptoms. The administration from the day of onset of arthritis showed no apparent effect. The results indicated that eleuthero has prophylactic effect but showed no treatment effect after the onset.

Figure2. Efficacy of eleuthero in CIA mice

2. Combination of anti-TNF-α antibody and eleuthero

At present, antirheumatic agents and biologics targeting cytokines are used as therapeutic drugs for RA; among them, anti-TNF-α antibody is primarily used. We studied whether eleuthero enhances the therapeutic effect of anti-TNF-α antibody. Eleuthero or ascorbic acid as control was orally administered by gavage daily from the day of the onset of arthritis; in addition, 50 μg of goat anti-mouse TNF-α antibody was intraperitoneally administered four times every three days. Saline or goat IgG was used as the control of goat anti-mouse TNF-α antibody.

【Results2】

The administration of anti-TNF-α antibody and eleuthero after the onset of arthritis achieved higher therapeutic effect as compared with anti-TNF-α antibody alone or combination with ascorbic acid (Figure3).

Figure3. Efficacy of combination of eleuthero with anti-TNF-αantibody in CIA mice

3. Action of eleuthero in active oxygen involved in the pathogenesis of RA

Human neutrophils were stimulated with a substance called phorbol myristate acetate (PMA), and the level of superoxide in the cell culture supernatant was measured by the cytochrome C method.

【Results3】

The level of superoxide generated by human neutrophils stimulated with PMA decreased in a concentration-dependent manner in the presence of eleuthero and vitamin C as control (Figure4).

Figure4. Action of eleuthero in active oxygen

4. Action of eleuthero in inflammatory cytokines involved in the pathogenesis of RA

TNF-α and IL-6 have been reported to be important among inflammatory cytokines involved in RA. We studied whether eleuthero extract inhibits the production of TNF-α or IL-6. The levels of inflammatory cytokines produced were measured using human peripheral blood mononuclear cells (PBMC; monocytes, dendric cells, NK cells, T-cells, B-cells) stimulated with a substance called lipopolysaccharide (LPS); the expression levels of mRNA for TNF-α and IL-6 were determined by RT-PCR method and the expression level of protein in the cell culture supernatant by ELISA method.

【Results4】

In the presence of eleuthero extract, the level of mRNA in PBMC during LPS stimulation and the levels of protein in the culture supernatant for TNF-α and IL-6 markedly decreased(Figure5). Both also showed a decreasing trend for ascorbic acid, but the degree was very small as compared with eleuthero.

Figure5. anti-inflammatory action of eleuthero

1. The start of onset was delayed and symptoms were reduced as the period of administering eleuthero before the onset of arthritis became longer, suggesting that routine intake of eleuthero has prophylactic effect for arthritis.
2. The combination of anti-TNF-α antibody as a therapeutic drug with eleuthero reduced the symptoms of arthritis and enhanced therapeutic effect. Treatment with anti-TNF-α antibody is expensive and associated with adverse reactions such as serious infectious disease and active tuberculosis. The study suggests that the combination with eleuthero can reduce the dose of anti-TNF-α antibody, reduce medical cost, and suppress adverse reactions. This is expected to be a new usage method of medicinal plants.
3. Eleuthero inhibits the production of superoxide by neutrophils and has antioxidant capacity through scavenging active oxygen itself. Moreover, it inhibited inflammatory cytokine TNF-α and IL-6 in gene and protein levels. This indicates that eleuthero is expected to be applied to not only arthritis but also various inflammatory lesions.

This study was for my degree (Ph.D. in medicine), which was published as follows:
Prophylactic and Therapeutic Effects of Acanthopanax senticosus Harms Extract on Murine Collagen-induced Arthritis. Phytother Res.28:1513-1519.(2014).