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. 2011 Jul 22;74(7):1621-9.
doi: 10.1021/np200336g. Epub 2011 Jun 10.

Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis)

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Synergy-directed fractionation of botanical medicines: a case study with goldenseal (Hydrastis canadensis)

Hiyas A Junio et al. J Nat Prod. .

Abstract

It is often argued that the efficacy of herbal medicines is a result of the combined action of multiple constituents that work synergistically or additively. Determining the bioactive constituents in these mixtures poses a significant challenge. We have developed an approach to address this challenge, synergy-directed fractionation, which combines comprehensive mass spectrometry profiling with synergy assays and natural products isolation. The applicability of synergy-directed fractionation was demonstrated using the botanical medicine goldenseal (Hydrastis canadensis) as a case study. Three synergists from goldenseal were identified, sideroxylin, 8-desmethyl-sideroxylin, and 6-desmethyl-sideroxylin. These flavonoids synergistically enhance the antimicrobial activity of the alkaloid berberine (also a constituent of H. canadensis) against Staphylococcus aureus by inhibition of the NorA multidrug resistance pump. The flavonoids possess no inherent antimicrobial activity against S. aureus; therefore, they could have been missed using traditional bioactivity-directed fractionation. The flavonoid synergists are present at higher concentration in extracts from H. canadensis leaves, while the antimicrobial alkaloid berberine is present at higher levels in H. canadensis roots. Thus, it may be possible to produce an extract with optimal activity against S. aureus using a combination of goldenseal roots and leaves.

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Figures

Figure 1
Figure 1
Synergy directed fractionation. Synergy testing is used to identify extracts and fractions likely to contain combinations of compounds that work together. These extracts are then fractionated, and LC-MS profiles are compared to bioassay data to identify potential bioactive compounds. The process is repeated iteratively as necessary to obtain pure compounds (synergists), the structures of which are elucidated using NMR and other spectroscopic techniques.
Figure 2
Figure 2
Isobolograms for berberine, the tannin free Hydrastis canadensis extract (after liquid-liquid extraction, see Supporting Information, Figure S1), the most active fraction from the first stage of the separation (fraction 4) and the most active fraction from the second stage of the separation (sub-fraction 2). All were tested in combination with berberine. The crude extract and its two fractions synergistically enhanced the antimicrobial activity of berberine, as demonstrated by the convex shape of the isobolograms, and the FIC values reported in Table 1.
Figure 3
Figure 3
Panel A shows minimum inhibitory concentration (MIC) of starting material (sm), berberine alone, and berberine in combination with 11 fractions (each at a fixed concentration of 75 μg/mL) from the first stage of separation (flash chromatography over silica gel with CHCl3:MeOH gradient) of the Hydrastis candensis leaf extract. The MIC of berberine (75 μg/mL) was reduced significantly by combining with the starting material and with several of the fractions, with the most active fraction being fraction 4. Panels B and C show the distribution of flavonoids (measured with negative-ion LC-MS), and alkaloids (measured with positive-ion LC-MS), respectively in the fractions. Fraction 4 contains the highest levels of the 3 flavonoids. There is a moderate multiple correlation between MIC and flavonoid peak area (R = 0.77, p = 0.016).
Figure 4
Figure 4
Comparison between MIC (A) and distribution of flavonoids (B) and alkaloids (C) after the second stage of the separation (flash chromatography over silica gel with hexane:EtOAc gradient) of the Hydrastis canadensis leaf extract. Sub-fraction 2 contained the highest levels of the three flavonoids, and was one of the most active fractions. Note, however, that other compounds appear to play a role in the activity of the starting material (sm); several fractions with little to no flavonoid content have activity on the same order as sub-fraction 2. The presence of additional active compounds would also explain the weak multiple correlation between activity and flavonoid content after this second stage of separation (R = 0.55, p = 0.37).
Figure 5
Figure 5
The three flavonoids from Hydrastis canadensis inhibit the NorA efflux pump of Staphylococcus aureus. Decrease in fluorescence over time is due to efflux of ethidium bromide, which is blocked by the positive control (CCCP) and all three flavonoids, sideroxylin (1), 8-desmethyl-sideroxylin (2), and 6-desmethyl-sideroxylin (3). Sideoxylin is the least active of the three flavonoids. The effect is observed with wild type Staphylococcus aureus (NCTC8325-4) (A) but not a NorA deletion mutant (K1758) (B). All of the flavonoids and controls were tested at a final concentration of 100 μM in Müeller-Hinton broth containing 10% DMSO (vehicle). Each point is an average % fluorescence from three independent measurements, with error bars equal to ± standard error (SE).

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