Quantitative evaluation of the in vitro effect and interactions of active fractions in Yaotongning-based formulae on prostaglandin E2 production

Traditional Chinese medicine (TCM) formula of Yaotongning Capsules (YTNC) is a common remedy to treat rheumatism (RA) in China and possesses diverse biological activities including anti-inflammation. However the effects of component material medicines (CMMs) in YTNC and different combinations of the CMMs on the efficacy of YTNC and the interactions of these CMMs have been being unclear due to ten CMMs and too many compounds involved in YTNC. Moreover, many TCM formulae are available for treating RA according to TCM theory. It is unknown if the YTNC prescription is better than other TCM formulae for treating RA or better efficacy could be obtained when some CMMs in YTNC are replaced by other herbs. Quantitatively investigate the in vitro effect of active fractions from the CMMs of YTNC and other eight herbs commonly used in the TCM formulae for RA treatment on anti-inflammatory activity of different combinations of the active fractions, the interactions of the active fractions to evaluate the reasonability, advantage (or disadvantage) of the YTNC prescription and to see if the prescription could be improved from the point of anti-inflammation.Twenty-six active fractions, which were categorized as alkaloids, flavonoids, saponins, volatile oils/aqueous extracts and polysaccharides were prepared to design TCM samples by combining some of the active fractions, based on the YTNC formulating principle, combination chemistry concept and the importance of the active fractions in YTNC. The anti-inflammatory activities of the samples were evaluated by their half-maximal inhibitory concentration (IC50) values that inhibiting the production of prostaglandin E2 (PGE2) in ANA-1 murine macrophages (ANA-1 cells). The cells plated in 96-well plates were classified into blank group and test sample group. Each group was stimulated with lipopolysaccharides (LPS, 1 mg/mL) for 2h. ANA-1 cells were pretreated with different concentrations of test samples prior to the addition of arachidonic acid (10 μmol/L). The supernatants were collected and measured using PGE2 ELISA Kit, and the cytotoxicity was assayed by cell counting Kit-8 (CCK8)-based test. The interactions of the active fractions in YTNC were evaluated by comparing the experimental IC50 values of the samples derived from YTNC to their corresponding additive IC50 values. The effect of each active fraction on cellular anti-inflammation-PGE2 secretion inhibition activity, and the reasonability, advantages (or disadvantages) of YTNC were evaluated based on the comparison of IC50 values of the samples.The disassembled formulae consisted of some active fractions of YTNC and the whole prescription of YTNC consisted of the all active fractions from YTNC all demonstrate cellular anti-inflammatory activity, and there were no significant differences between these formulae. The vehicle of YTNC Chinese rice wine exhibits the ability to enhance the cellular anti-inflammation of YTNC. Synergistic effect exerts in the combination of alkaloids, flavonoids and saponins of YTNC, antagonistic or additive effects occur in the other combinations of active fractions from YTNC. The anti-inflammatory activities of some TCM samples which include some active fractions from the eight selected herbs are significantly higher than the samples derived from YTNC. The flavonoids of Carthamus tinctorius, the volatile oils of Cinnamomum cassia and Angelica pubescens perform better in cellular anti-inflammation than the flavonoids and volatile oils in YTNC.The prescription of YTNC is reasonable in the view of anti-inflammation. The saponins and polysaccharides from the CMMs of YTNC have better anti-inflammatory activities than the saponins and polysaccharides from the other eight herbs. Reducing the varieties of YTNC CMMs and replacing the flavonoids and volatile oils of YTNC with the flavonoids of Carthamus tinctorius and the volatile oils of Cinnamomum cassia (or Angelica pubescens) would improve the safety and anti-inflammatory activity of YTNC. Synthetically evaluating various pharmacological activities of TCM formulae designed in the present work may lead to develop more effective and safer TCM using YTNC as prototypes.