Assessment of the impact of biopolymeric nanoparticles on the oral absorption of green tea catechins

Catechins are a class of flavonoids present in high amounts in green tea. Catechins are of pharmaceutical interest as several in-vitro and animal studies have indicated that they possess beneficial therapeutic properties which include antioxidant, anti-inflammatory, cardio-protective, neuro-protective and anti-cancer effects. Therefore, catechins are promising therapeutic agents for diseases and conditions such as hypertension, myocardial infarction, Alzheimer’s disease and cancer. However, following consumption, catechins are poorly absorbed into the systemic circulation. Studies indicate that at most, 5% of orally administered catechins are absorbed into the systemic circulation of rats. This poor oral absorption has been mainly attributed to their poor stability within the gastro-intestinal tract (GIT) and their poor permeability across the intestine. Nanoparticles (NPs), in particular those prepared from the biopolymers chitosan (CS) and alginate (Alg), have recently been used to enhance the oral absorption of compounds. The enhancement in oral absorption has been suggested to occur as a result of an enhancement in the stability of the compounds in the GIT and/or an enhancement in the permeability of the compound across the intestine by the NPs. Therefore, the hypothesis of this thesis was that NPs prepared from CS or Alg could enhance the oral absorption of catechins. To address this hypothesis, the aims were to: (i) encapsulate the catechin species (+)-catechin (CAT) and (-)-epigallocatechin gallate (EGCG) in CS-tripolyphosphate (CS-TPP) and CS-Alg NPs; (ii) assess the impact of encapsulation on the stability of CAT and EGCG in simulated gastro-intestinal fluids; (iii) assess the impact of encapsulation on the absorption of CAT and EGCG across intestinal tissue in-vitro; and (iv) assess the impact of encapsulation on the systemic absorption of EGCG following oral administration to mice. CAT was encapsulated in CS-TPP and CS-Alg NPs with the particles exhibiting mean sizes of 163.0 ± 2.0 and 469.2 ± 3.1 nm (mean ± s.d., n = 3), respectively. EGCG was encapsulated in CS-TPP NPs and the particles exhibited a mean size of 165 ± 2.0 nm (mean ± s.d., n = 3). Up to 5.4 and 3.7 µg of CAT and EGCG, respectively, were encapsulated per mg of the NPs. Encapsulation of the catechins in the NPs enhanced their stability. Encapsulation of CAT in CS-Alg NPs and CS-TPP NPs reduced its rate of degradation in simulated intestinal fluid pH 7.4 by a factor of 1.8 and 3.0, respectively, while encapsulation of EGCG in CS-TPP NPs reduced its rate of degradation by a factor of 5.5. Using excised mouse intestinal tissue (jejunum segment) mounted in Ussing chambers, it was observed that encapsulation significantly enhanced the absorption of EGCG. In the absence of encapsulation EGCG could not accumulate in the receptor chamber beyond 1.75 h, whereas in the presence of encapsulation, EGCG continued to accumulate in the receptor chamber beyond 1.75 h, and at 3 h the cumulative amount of EGCG absorbed was 143.8 ± 10.8 ng/cm2 (mean ± s.e.m., n = 3-4). However, the CS-TPP NPs could not significantly enhance the absorption of CAT (p > 0.05). The cumulative amount of CAT absorbed at 4 h was 334.0 ± 109.9 and 477.0 ± 165.1 ng/cm2 (mean ± s.e.m., n = 3-4), in the absence and presence of CS-TPP NP encapsulation, respectively. The mechanism by which absorption was enhanced was found to not be through an effect of the CS-TPP NPs on intestinal permeability (as shown by the transport of the marker compounds 14C-mannitol and 3H-propranolol) or an effect on intestinal efflux proteins (as shown by the transport of the P-glycoprotein substrate 3H-digoxin) but was likely due to the enhanced stability of the EGCG following encapsulation. In the absence and presence of NP encapsulation 1.3 ± 1.7 and 56.9 ± 3.0 % (mean ± s.d., n = 3) of the initial EGCG concentration was present on the donor side of the tissue after 2 h. The enhanced EGCG concentrations following encapsulation were likely to have driven flux of EGCG across the intestine over the 3 h experimental period. In the case of CAT, 94.9 ± 3.8 and 99.7 ± 0.7% (mean ± s.d., n = 3) of the initial CAT concentration was present in the donor chamber in the absence and presence of CS-TPP NP encapsulation, respectively. This small difference in CAT levels could explain the observation that the NPs could not significantly enhance the intestinal absorption of CAT. Following oral administration of EGCG to Swiss Outbred mice at a dose of 0.76 mg/kg, the area under the plasma concentration versus time curve (AUC) for non-metabolized and total EGCG was 114.3 ± 4.1 and 116.4 ± 4.1 nM.h (mean ± s.d., n = 3-5), respectively. However, following oral administration of EGCG encapsulated in the CS-TPP NPs, the AUC for non-metabolized and total EGCG was 166.0 ± 12.0 and 179.3 ± 10.8 nM.h (mean ± s.d., n = 3-5), respectively, indicating that encapsulation had enhanced the plasma exposure of EGCG by a factor of 1.5. Measurement of the concentrations of EGCG in the GIT of the mice indicated that encapsulation had significantly enhanced (p ˂ 0.05) the stability of EGCG in the stomach and jejunum of the mice. The enhanced stability resulted in an increase in the exposure of EGCG to the jejunum surface by a factor of 2.3, and was likely to be responsible for the observed enhancement in the plasma exposure of EGCG. In conclusion, the results of this research indicate that encapsulation of EGCG in CS-TPP NPs enhances its oral absorption in mice, attributed to enhanced stability of the catechin in the GIT following encapsulation. Therefore, the results suggest that these NPs could also enhance the absorption of EGCG in humans, which may lead to the use of catechins as therapeutic agents for the above mentioned diseases and conditions.