Background

For over 40 years, the time-tested Traditional Chinese Medicine formulas, in its raw form, were used in the clinics of YapChanKor for the treatment of musculoskeletal problems with great success. Over the past few years, a vast amount of clinical case studies and clinical data on the treatment and recovery of patients were systematically collected, however, scientifically-backed evidence on the efficacy of these formulations were still lacking. Therefore, the purpose for this anti-inflammatory study originated from the hypothesis that the proprietary herbal extracts derived from these time-tested formulations contained high levels of anti-inflammatory compounds since it has been shown that the use of these herbs significantly reduced the pain levels and improved recovery times of patients treated with these herbs.

Method

The herbal extracts used in this study were produced using a proprietary extraction method developed by Relivium Sciences. The in vitro toxicology and anti-inflammatory characteristics of the herbal extract were evaluated using Human Embryonic Kidney (HEK293) cells. In vitro toxicology of the herbal extracts was assessed through microscopy evaluation and the MTT assay.

 

To determine the inflammatory pathways inhibited by the herbal formulation, cells were treated with or without the extract for 24 hrs. To initiate an inflammatory response, Lipopolysaccharide (LPS, 100ng/mL) was introduced across 6 time points of 0, 1, 2, 4, 12, and 24 hours. Subsequently, using the Western Blot technique, 4 markers (P-P38MAPK, IL-6, IL-1β and TNF-α) were studied of which the expression of these markers would indicate the activation of an inflammatory response. Vinculin was used as the loading control and P38 MAPK, as an inactive form, was used as a control.

Results

The morphology of HEK cells following 24 hours exposure to the extracts was observed under light microscopy. Figure 1 shows a comparison between the control (no treatment of extract) and cells treated with the extract. No difference in cell morphology was observed. The MTT assay further demonstrated the herbal extract was non-toxic towards the HEK cells at the optimized concentration, with a high percentage of activity (> 90% viable cells) recorded when normalized against the control.  Through this study, the optimized concentration of the herbal extract was successfully determined.

 

Subsequently, investigation of the anti-inflammatory characteristics of the herbal extracts were carried out using Western Blot. The columns of the gels were labelled accordingly to the 6 time points and a positive (+) symbol denoted samples treated with the herbal extract for 24 hours, while a negative (-) symbol denoted samples without herbal extract treatment. Samples with LPS treatment were labelled accordingly.

 

Initially, as shown at the 0 hour time point, the herbal extracts itself did not elicit an immune response. In fact, the expression of endogenous TNF-α was reduced, as observed in Figure 2. Astonishingly, at the further time points of 2 and 4 hours, the herbal extract clearly inhibited the onset of inflammation by LPS, as evidenced in Figure 3, wherein, the expression of P-P38MAPK, IL-6, IL-1β and TNF-α was significantly lower than the controls. Interestingly, at 4 hours, it was observed that cells which were treated with the herbal extract demonstrated a reduced endogenous expression of P-P38, IL1-β, and IL-6. Continued exposure at 12 and 24 hours also showed that the herbal extract was capable of inhibiting the onset of inflammation by LPS (Figure 4).

Conclusion

This study successfully demonstrated that the herbal extract was highly efficacious in reducing inflammation.  More research is currently underway using different cell lines and additional methods to ascertain these results. We look forward to sharing more exciting results soon, as we go along this journey of unlocking the secrets of these herbs.