A strategy for the identification of combinatorial bioactive compounds contributing to the holistic effect of herbal medicines

Abstract

It has been well claimed that herbal medicines (HMs) elicit effects via a multi-compounds and multi-targets synergistic mode. However, it lacks appropriate strategies to uncover the combinatory compounds that take effect together and contribute to a certain pharmacological effect of an herb as a whole, which represents a major bottleneck in providing sound evidence in supporting the clinic benefits of HMs. Here, we proposed a strategy to the identification of combinatory compounds contributing to the anti-inflammatory activity of Cardiotonic Pill (CP). The strategy proposed herein contains four core steps, including the identification of bioequivalent combinatorial compounds, chemical family classification-based combinatorial screen, interactive mode evaluation, and activity contribution index assay. Using this strategy, we have successfully identified six compounds in combination responsible for the anti-inflammatory effect of CP, whose anti-inflammatory activities were found comparable to that of the whole CP. Additionally, these six compounds take effect via an additive mode but little synergism. This study, together with our recent work in the identification of bioactive equivalent compounds combination, provides a widely applicable strategy to the identification of combinatory compounds responsible for a certain pharmacological activity of HMs.

Figure 1. The strategy used to screen combinatorial compounds that take effect via an additive mode in HMs.

(a) the identification of BECCs via a bioactive equivalence oriented feedback screening approach; (b) screening active compounds based on chemical family classification; (c) determining the pharmacological activity of individual compounds and their interactive mode; (d) designation of DCC based on activity contribution index and activity validation of DCC. BECCs: bioactive equivalent combinatorial components; DCC: dominant compounds combination.

Figure 2. Anti-inflammatory effects of Cardiotonic Pill in LPS-stimulated RAW264.7 macrophages.

Cells were pretreated with different concentrations of CP (0.1–0.4 mg/ml) for 1 h, followed by treatment with LPS (1 μg/ml) and incubation for indicated time. Concentration of (a) NO, (b) PGE₂ and (c) IL-6 in the supernatant. (d) Protein expression levels of iNOS and COX-2. β-actin was used as an internal loading control. The expression levels of mRNA for (e) iNOS, (f) COX-2, (g) IL-6 and (h) IL-1β were analyzed by quantitative real-time PCR. GAPDH served as internal control for normalization of mRNA expression. Data are presented as mean ± SD of three independent experiments performed in duplicate. ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus LPS group (one-way ANOVA, Dunnett test). CP: Cardiotonic Pill.

Figure 3. Chemical family classification-based screening of anti-inflammatory compounds from CP.

Cells were pretreated with tested samples (at a corresponding concentration in 0.4 mg/ml CP) for 1 h, followed by treatment with LPS (1 μg/ml) and incubation for indicated time. Concentration of (a) NO, (b) PGE₂ and (c) IL-6 in the supernatant. (d) Protein expression levels of iNOS and COX-2. β-actin was used as an internal loading control. The expression levels of mRNA for (e) iNOS, (f) COX-2, (g) IL-6 and (h) IL-1β were analyzed by quantitative real-time PCR. GAPDH served as internal control for normalization of mRNA expression. Data are presented as mean ± SD of three independent experiments performed in duplicate. ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus LPS group (one-way ANOVA, Dunnett test). CP: Cardiotonic Pill; PA: combination of 10 phenolic acids; GN: combination of 4 ginsenosides; TN: combination of 4 tanshinones; BECCs: bioactive equivalent combinatorial components (combination of PA, GN and TN).

Figure 4. Combination index and dose-dependent inhibition of LPS-induced inflammatory indicator production by single active compounds or compound combinations.

RAW264.7 macrophage cells were pretreated with different concentrations of tested samples in the presence of 1 μg/ml LPS for 24 h. Dose-effect curves of PA, TN, and their combinations in inhibition of (a) NO and (b) IL-6 production. Dose-effect curves of TN at higher concentrations in inhibition of (c) NO and (d) IL-6 production. Dose-effect curves of 10 phenolic acids in inhibition of (e) NO and (f) IL-6 production. Dose-effect curves of 4 tanshinones in inhibition of (g) NO and (h) IL-6 production. (i) Combination index. The combination index was calculated using CompuSyn software based on the data of 3 replicates. 0.9 < CI < 1.1 indicated an additive effect. CP: Cardiotonic Pill; PA: combination of 10 phenolic acids; TN: combination of 4 tanshinones; PA&TN: combination of PA and TN.

Figure 5. Anti-inflammatory effect validation of DCAC in LPS-stimulated RAW264.7 macrophages.

Cells were pretreated with tested samples (at a corresponding concentration in 0.4 mg/ml CP) for 1 h, followed by treatment with LPS (1 μg/ml) and incubation for indicated time. Concentration of (a) NO, (b) PGE₂ and (c) IL-6 in the supernatant. (d) Protein expression levels of iNOS and COX-2. β-actin was used as an internal loading control. The expression levels of mRNA for (e) iNOS, (f) COX-2, (g) IL-6 and (h) IL-1β were analyzed by quantitative real-time PCR. GAPDH served as internal control for normalization of mRNA expression. Data are presented as mean ± SD of three independent experiments performed in duplicate. ##p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus LPS group (one-way ANOVA, Dunnett test). CP: Cardiotonic Pill; BECCs: bioactive equivalent combinatorial components; DCAC: dominant combination of anti-inflammatory compounds.

Figure 6. Effects of compound removal on the anti-inflammatory activity of DCAC.

Cells were pretreated with tested samples (at a corresponding concentration in 0.4 mg/ml CP) for 1 h, followed by treatment with LPS (1 μg/ml) and incubation for 24 h. (a) NO and (b) IL-6 concentration in the supernatants of macrophages treated by DCAC with each compound removed respectively. Data are presented as mean ± SD of three independent experiments. **p < 0.01 versus LPS group; #p < 0.05, ##p < 0.01 versus DCAC group (one-way ANOVA, Dunnett test). CP: Cardiotonic Pill; DCAC: dominant combination of anti-inflammatory compounds; P1: danshensu, P2: protocatechuic aldehyde; P5: isolithospermic acid A; P6: salvianolic acid A; T1: tanshinone I; T2: dihydrotanshinone I.