Tea derived galloylated polyphenols cross-link purified gastrointestinal mucins

Abstract

Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm) heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria.

Figure 1. The ensemble averaged MSD curves as a function of lag time obtained from a 10 mg/ml Muc5ac solution before and after treatment with EGCG and EC.

Treatment with 0.5% w/w EC had little effect on the rheological properties. Treatment with as little as 0.1% w/w EGCG induced a dramatic effect, causing the mucin solution to become viscoelastic. At concentrations above 0.5% w/w EGCG the solution has undergone a sol-gel transition and no further change could be detected using PTM.

Figure 2. The ensemble averaged MSD curves obtained from a 10 mg/ml Muc5ac solution before and after the addition of (a) green and black tea extracts and (b) the EGCG-rich green tea extract Sunphenon 80SK.

The addition of up to 1% w/w green tea extract caused the solution to become viscoelastic, whereas at 2% w/w the solution has undergone a sol-gel transition. Similarly, at 0.5% w/w black tea extract the solution is viscoelastic, whereas at 1% w/w the solution appears to have transformed into a weak gel. The addition of 0.1% w/w Sunphenon extract caused the purely viscous liquid to become viscoelastic, with a power law of 0.77±0.01. At a concentration of 0.3% w/w Sunphenon extract the solution undergoes a sol-gel transition, evident by the 0.03±0.03 power law exponent of the MSD curve. At 0.5% w/w Sunphenon there is an increase in the viscoelastic characteristics of the gel and no further change could be detected using PTM at higher concentrations.

Figure 3. The ensemble averaged MSD curves as a function of lag time for Muc2 solutions treated with (a) EC and EGCG and (b) green and black tea extracts.

There is little change in the viscoelasticity of the mucin solution when treated with EC. Treatment with 0.2% w/w of EGCG induced a sol-gel transition, whereas treatment with 0.5% w/w or higher EGCG results in a stronger gel.

Figure 4. The ensemble averaged MSD curves as a function of lag time for a 10 mg/ml Muc2 solution treated with (a) black and green tea extracts and (b) the EGCG-rich green tea extract Sunphenon 80SK.

The addition of green tea extract causes the solution to become viscoelastic, with a monotonic correlation observed between the extract’s concentration and viscoelasticity. Similarly, 0.5% and 1% w/w black tea extract cause the solution to become viscoelastic. At a concentration of 0.1% w/w Sunphenon extract the solution becomes viscoelastic, whereas at a concentration of 0.5% w/w the solution undergoes a sol-gel transition. Increasing the concentration of the extract results in a stronger gel, which is at the limits of what PTM can detect.

Figure 5. The scattering profiles of 10 mg/ml (a) Muc5ac and (b) Muc2 solutions treated with EC, EGCG and tea extracts.

In both types of mucin, the scattering profile of the stock solution is described in its entirety with a I(q)∼q^−1.7 power law and the addition of 0.5% w/w EC had very little impact on it. Treatment with 0.5%, 1% w/w EGCG and the tea extracts induced a large increase in the low-q regime in both types of mucin, a sign of formation of large length scale structures (>10 nm) within the sample. In the intermediate q-range there is a universal I(q)∼q^−1.7 power law, observed in all the solutions. In the low-q range, there was a I(q)∼q⁻³ power law observed for treated Muc5ac solutions, whereas a I(q)∼q⁻⁴ power law was observed in Muc2 solutions.

Figure 6. A schematic diagram of the proposed mechanism that leads to gelation of mucin solutions upon treatment with EGCG.

(a) Mucins are naturally secreted as oligomers, which are connected end-to-end through disulphide bonding at the two termini. The amount of internal cys- domains along the peptide backbone differs depending on the mucin type. (b) Clusters of EGCG bind to the exposed domains by hydrogen bonding on amino acids such as valine or leucine, which increases their size and creates sites for cross-links with other mucin molecules. (c) Untreated mucin solutions at pH 7 form semi-dilute unentangled networks, which behave as a purely viscous liquid. (d) Mucin/EGCG complexes in solution form cross links (blue circles), which form aggregates and given enough EGCG concentration eventually form a gel. (e) A magnification of the enlarged domains which act as crosslinks between mucin oligomers. The exposed domains along the peptide backbone can potentially contribute to the cross-linking process.

Figure 7. A schematic representation of the cross-linking process after the addition of EGCG to Muc2 and Muc5ac solutions.

The Muc2 molecule has only two cys-domains along its peptide backbone, which are saturated by EGCG easier than Muc5ac, which has at least 8 cys-domains. The presence of a large number of cys domains provides for a larger number of available sites for cross-linking, which lead to the formation of smaller aggregates compared to Muc2 (observed with SANS). Additionally, the presence of less cys-domains in the molecular structure of Muc2 leads to a more complete phase separation within the solution, with distinct boundaries (q⁻⁴ scattering), whereas Muc5ac has less distinctive boundaries between phases (q⁻³ scattering) as a result of a number of cys-domains remaining uncomplexed. Blue circles represent aggregation at the two termini of the mucin molecules, whereas red circles show cross-linking occurring at the cys-domains of the molecule.