1. A review: Using nanoparticles to enhance absorption and bioavailability of phenolic phytochemicals BILAL JAVED PhD BOTANY 09-ARID-1473 PMAS-ARID AGRICULTURE UNIVERSITY RAWALPINDI

2. Learning outcomes Introduction Importance/Functions Phenolic phytochemicals Health benefits Low absorption rate of phenolic phytochemicals Low solubility and poor stability Low permeation due to passive diffusion and active efflux Nanoparticle system for delivery of phenolic phytochemicals Nanoparticles enhance solubility of low-water-soluble phenolic phytochemicals Nanoparticles prevent phenolic phytochemicals against degradation in the gastrointestinal tract Nanoparticles enhance absorption of phenolic phytochemicals in epithelial cells via endocytotic cellular uptake Potential challenges of nanoparticle application Conclusion

3. INTRODUCTION Phenolic phytochemicals have been of particular interests in food and pharmaceutical fields because they have potential to reduce the incidences of coronary heart disease, diabetes, cancers, and other chronic diseases. However, extremely low absorption rate of phenolic phytochemicals restricts their bioactivity in vivo. Such low absorption and bioavailability are due to their low water solubility, poor stability, passive diffusion, and active efflux in the gastrointestinal tract. Nanoparticle delivery system has been widely applied in pharmaceutical field to enhance absorption of bioactive compounds.

4. Importance/Functions They are formed in plant as secondary metabolites to show multiple functions for plant growth, development, and defense. Phenolic phytochemicals are main components that contribute to flavor and color to plant. They are particularly accumulated in response to different growth stages, environmental changes to behave as signaling molecules to regulate physical function of plants.

5. Importance/Functions Phenolic phytochemicals also protect plant against insects, fungi, bacteria, and viruses. Epidemiologic and experimental studies have confirmed that consumption of phenolic phytochemical-rich foods was positively correlated to healthy benefits for human body.

6. Phenolic phytochemicals Phenolic phytochemicals represent a variety of compounds that are the largest category of phytochemicals and more than 8000 phenolic phytochemicals have been reported to exist in different fruits and vegetables. One of the major phenolic phytochemicals is flavonoids which are basically comprised of fifteen carbons, with two aromatic rings conjugated by a three-carbon bridge (C6-C3-C6 structure) (Fig. 1). Main flavonoids include flavonols, flavan-3-ols, flavanones and isoflavones.

7. Fig. 1. The basic structure of phenolic phytochemicals. (A) Flavonoids; (B) Phenolic acid; (C) Hydroxycinammates; (D) Stilbenes.

8. Health benefits Anti-cancer properties. Anti-inflammation. Anti-diabetes functions. Anti-aging function. Antioxidant capacity to induce apoptosis of cancer cells. Anthocyanins protected against UV-induced oxidative damages. Decreased the neurotoxicity to inhibit neurodysfunction.

9. Low absorption rate of phenolic phytochemicals Naturally low aqueous solubility, poor gastrointestinal stability, passive diffusion, and active efflux of phenolic phytochemicals in the gastrointestinal tract result in such low absorption.

10. Low solubility and poor stability Low solubility and poor stability of phenolic phytochemicals in the gastrointestinal tract play important roles in their low absorption rate. Aqueous solubility of phenolic phytochemicals is determined by their affinity to water molecules and molecular weight plays a primary role in aqueous solubility. It has been proposed that higher molecular weight compounds were not dissolved well in water. Different pH conditions in the gastrointestinal segments may also cause the degradation of phenolic phytochemicals.

11. Low permeation due to passive diffusion and active efflux No specific receptors on the surface of small intestinal epithelial cells have been found to carry phenolic phytochemicals into cells. Thus, the mechanism for phenolic phytochemicals to transport across epithelium was principally based on passive diffusion, including paracellular and transcellular diffusions. After absorbed, phenolic phytochemicals undergo active efflux process by which majority of phenolic phytochemicals are pumped back to lumen (Fig. 2).

12. Fig. 2. Passive diffusion, metabolism, and active efflux of phenolic phytochemicals on epithelial cells.

13. Nanoparticle system for delivery of phenolic phytochemicals Nanoparticles can interact with phenolic phytochemicals by hydrogen bonds and hydrophobic interactions to encapsulate phenolic phytochemicals in nanoparticles, which can enhance aqueous solubility of phenolic phytochemicals. Nanoparticles also can prevent against oxidation/degradation of phenolic phytochemicals encapsulated in the gastrointestinal tract.

14. Conti… Nanoparticles can be taken directly up by epithelial cells in small intestine, which significantly increases absorption and bioavailability of phenolic phytochemicals.

15. Nanoparticles enhance solubility of low-water-soluble phenolic phytochemicals Nanoparticles are established with hydrophobic groups inside and polar groups on surface of particles. Therefore, nanoparticles can remain stable in dispersion system due to their inter-particle repulsions and hydration. Phenolic phytochemicals can interact with hydrophobic sites of nanoparticles via hydrogen bonds and hydrophobic interactions.

16. Sufficient surface charges and suitable hydration property keep phenolic phytochemical encapsulated nanoparticles stable in aqueous system, which enhances the water solubility of phenolic phytochemicals.

17. Nanoparticles prevent phenolic phytochemicals against degradation in the gastrointestinal tract Phenolic phytochemicals encapsulated by nanoparticles lower the oxidation and/or degradation risks in the gastrointestinal tract.

18. Nanoparticles enhance absorption of phenolic phytochemicals in epithelial cells via endocytotic cellular uptake Nanoparticles have been reported to be penetrated across small intestinal epithelium by either paracellular or transcellular pathway. These transport ways enhance absorption of phenolic phytochemicals encapsulated in the gastrointestinal tract (Fig. 3).

19. Tight junctions are secreted by junctional protein among the epithelial cells. They are responsible for the integrity of the epithelium. Paracellular transport refers to a passive diffusion through intercellular spaces among the epithelium. Either nanoparticles or junctional protein mediators can control disruption of tight junctions for enhancing transport of bioactive compounds via paracellular way. After delivery, tight junctions return to normal state for function. Tight junction mediators, such as chitosan, thiolated chitosan, chitosan derivatives, and polyacrylate derivatives were reported to fabricate nanoparticles or coating on nanoparticles to enhance paracellular transport.

20. Uptake of nanoparticles by cells in transcellular pathways Cellular uptake of nanoparticles by cells in transcellular pathways is associated with energy input. Leroux et al. reported that an energy-requiring process was associated with the uptake of poly( D,L-lactic acid) nanoparticles labeled by a fluorescent dye because more uptakes of the nanoparticles were observed when the incubation temperature was elevated from 4 to 37 C. The energy-dependent uptakes of nanoparticles were also reported in PLGA nanoparticles, protein based nanoparticles, and other types of nanoparticles.

21. Macropinocytosis or endocytosis Cellular uptake process of nanoparticles is conducted by macropinocytosis or endocytosis containing clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis. Macropinocytosis is an actin-driven process that can internalize the nanoparticles without receptor mediation. Particles with an average size below 2 mm are enclosed by macropinosomes formed on the surface of enterocytes and internalized to cells. The internalized nanoparticles are further translocated to endolysosome.

22. Fig. 3. Cellular uptake of nanoparticles by epithelial cells.

23. Potential challenges of nanoparticle application Nanoparticles after entering the gastrointestinal tract are exposed to – different pH, – excess amount of ions, and – different kinds of digestive enzymes, which may affect efficacy of nanoparticles for delivery of phenolic phytochemicals.

24. Conti… pH plays an important role in affecting stability of nanoparticles in the gastrointestinal tract. For example, gelatin nanoparticles were aggregated at pH 6-7 because isoelectric point of gelatin significantly reduced the inter-particle repulsion. Large quantities of ions are present in the gastrointestinal tract to balance the pH condition (such as hydrochloride acid, sodium bicarbonates, etc), to provide the signals (such as sodium and potassium) to transport of nutrient compounds, and to activate the enzyme to proceed the digestion process.

25. Conclusion The extremely low absorption and bioavailability of phenolic phytochemicals is generally attributed to their low solubility, poor stability, low permeability and active efflux process, and metabolisms in the gastrointestinal tract. Nanoparticles may improve the aqueous solubility by encapsulating phenolic phytochemicals. Nanoparticles can also prevent phenoli phytochemicals against the oxidation/degradation in the gastrointestinal tract by nanoencapsulation. Future work would be focused on phenolic phytochemical encapsulated nanoparticles designed for oral administration, better gastrointestinal stability, mucus penetrating function, and intestinal epithelial cell targeting properties.

26. Reference Zheng Li, Hong Jiang, Changmou Xu, Liwei Gu, 2015. A review: Using nanoparticles to enhance absorption and bioavailability of phenolic phytochemicals. Food Hydrocolloids 43 (2015) 153e164.

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