Boswellia carterii extract inhibits TH1 cytokines and promotes TH2 cytokines in vitro

Abstract

Traditional herbal formulas used to treat inflammatory arthritis in China and India include Boswellia carterii or Boswellia serrata. They both contain boswellic acids (BAs) which have been shown to exhibit anti-inflammatory and antiarthritic properties. This study tests the hypothesis that mixtures of BAs derived from B. carterii have immunomodulatory properties. B. carterii plant resin obtained from China was prepared as an ethanol extract, and the presence of seven BAs was confirmed by column chromatography, high-performance liquid chromatography, and UV laser desorption/ionization tandem mass spectroscopy. The extract was then tested for its ability to alter in vitro production of TH1 cytokines (interleukin-2 [IL-2] and gamma interferon) and TH2 cytokines (IL-4 and IL-10) by murine splenocytes. Delivery of the resin extract using ethanol as a solvent resulted in significant cellular toxicity not seen with the addition of ethanol alone. By contrast, delivery of the resin extract using a sesame oil solvent resulted in a dose-dependent inhibition of TH1 cytokines coupled with a dose-dependent potentiation of TH2 cytokines. These results indicate that a purified mixture of BAs from B. carterii plant resin exhibits carrier-dependent immunomodulatory properties in vitro.

FIG. 1.

Fractionation scheme of BCE. The initial ethanol extract was subjected to SiO₂ column chromatography, and fractions 33 through 68 and 69 through 85 were subpurified by HPLC. Yields shown are derived from a 60-mg column injection. Yields from fractions 86 through 98 and 99 through 118 are shown following recrystallization without subpurification. A, 11-keto-β-BA; B, acetyl-11-keto-β-BA; C, α-BA; D, β-BA; E, acetyl-11-dien-β-BA; F, acetyl-α-BA; G, acetyl-β-BA.

FIG. 2.

Identification of BAs present in the initial B. carterii ethanol extract by HPLC. Shown in the upper panel is a representative analytical HPLC tracing. The lower panel contains the molecular structure of the eluted compounds. Compounds (retention time [range] in min) are as follows: A, 11-keto-β-BA (2.6-3.4); B, acetyl-11-keto-β-BA (3.5-4.2); C, α-BA (6.6-7.9); D, β-BA (7.5-8.6); E, acetyl-11-dien-β-BA (9.5-10.4); F, acetyl-α-BA (9.9-10.6); G, acetyl-β-BA (11.5-12.2).

FIG. 3.

Identification of BAs present in the initial B. carterii ethanol extract by laser desorption MS/MS analysis. Spectra were performed on the BCE and fragmentation ions were determined in MS/MS (reflectron mode) to confirm their identification by observing characteristic fragment ions. The inset shows an example of the MS/MS confirmation of the 11-keto-β-BA. The ion current corresponding to the MNa⁺ distribution is gated and subjected to a second mass analysis, thereby demonstrating the relative selectivity of the technique. MNa⁺ predicted and observed masses are monoisotopic since these peaks were resolved to an isotopic distribution. The observed versus expected masses are shown in Table 1.

FIG. 4.

BCE in ethanol exhibits cellular toxicity not seen with SO as a carrier or with ethanol alone. Murine splenocytes from B6, DBA/2, or B6D2F1 mice were cultured in vitro with either ethanol (EtOH) alone (final concentration, 0.1%), SO alone (final concentration, 0.5%), or three concentrations of BCE in their respective solvents (final concentrations of 10, 50, and 200 μg/ml). Viability was determined by trypan blue exclusion at 24 h of culture (see Materials and Methods).

FIG. 5.

BAs in SO inhibit TH1 cytokine production in vitro. IFN-γ (A) and IL-2 (B) levels in culture supernatants were determined by ELISA at 24 h following stimulation with ConA as described in Materials and Methods. Similar results were seen in two additional independent experiments, and standard errors were calculated on replicate analyses. Untreated, SO without BCE (negative control); Stimulated, SO plus ConA without BCE (positive control). The final concentrations of BCE plus SO plus ConA medium are indicated as 10 μg/ml, 50 μg/ml, and 200 μg/ml. F1, B6D2F1 mice; DBA, DBA/2 mice.

FIG. 6.

BAs in SO promote TH2 cytokine production in vitro. IL-4 (A) and IL-10 (B) levels in culture supernatants from DBA/2 splenocytes were determined by ELISA at 48 h after stimulation with anti-CD3 as described in Materials and Methods. Similar results were seen in two additional independent experiments, and standard errors were calculated on replicate analyses. Untreated, SO without BCE (negative control); Stimulated, SO (without BCE) plus anti-CD3 (positive control). The final concentrations of BCE plus SO plus anti-CD3 medium are indicated as 10 μg/ml, 50 μg/ml, and 200 μg/ml.