Despite substantial developments of extraction and separation techniques, isolation of natural products from various sources is still a challenging task, and lead generation or validation of known natural products from plant extracts is fraught with false negatives (e.g., due to low concentrations, instability, or insolubility) and false positives (e.g., aggregates, and known, patented or nuisance compounds). Traditional and optimized solvent-based extraction methods, including those involving ultrasound, microwaves, and pressure-assisted extraction, supercritical fluids and solid phase extraction, ionic liquids and distillation have all been reported as a means to extract and separate components of different physicochemical properties, and de-replication of already known or nuisance compounds at the earliest stage of the fractionation procedure is desirable. We propose to utilize natural polymeric (different molecular weight chitosan and methylated derivatives) nanoformulations, typically employed in the lead optimization or drug development process to improve therapeutic efficacy and/or reduce toxicity, in the lead discovery process by employing them in the extraction process to solubilize and stabilize natural products for biological testing. A proof of concept will be demonstrated with green tea extracts through the isolation, quantification and assay of biological activity of the major known catechins, in particular EGCG, that react rapidly with reactive oxygen species (ROS), as compared with their non-nanoformulated counterparts. In addition, studies have demonstrated a modest cholesterol lowering effect with green tea catechin extracts, including EGCG. Although statins have been on the market as inexpensive and safe means to control cholesterol levels, nevertheless there remains a substantial market of unmet medical need for people who are statin intolerant or statin resistant. The long term goal of this work is to demonstrate the general utility of applying nanoformulations to natural products drug discovery, in particular extraction and isolation, and to develop, in a Phase II grant, an optimized EGCG nanoformulation and test for enhanced efficacy in an animal model of cholesterol lowering in a preclinical development phase.
The specific aims of this work are to: 1. Perform nanoformulation, stability and release kinetic studies with several novel natural polymer-based nanoformulation methods in the green tea extraction process to improve the extraction efficiency of the major catechins, especially EGCG, and perform stability and simulated release kinetic studies with the best nanoformulations as compared to their non-nanoformulated counterparts. 2. Run cell-based assays for measuring cholesterol absorption inhibition with the best three nanoformulated extracts from specific aim 1, again comparing the biological activity to their corresponding non- nanoformulated counterparts. 3. Confirm LDL and total cholesterol lowering in a mouse model with the best nanoformulation from specific aim 2 with purified EGCG in mice maintained on high fat diets to measure LDL and total cholesterol lowering to demonstrate improved efficacy as compared to its non-nanoformulated counterpart.

Public Health Relevance

Isolation of natural products from various sources is still a challenging task, and lead generation and validation of novel or known natural products is fraught with false negatives and false positives. Our goal is to improve the efficiency of natural drug discovery during the important extraction and isolation process through application of nanoformulations to better solubilize and stabilize the chemical constituents for testing.

National Institute of Health (NIH)
National Center for Complementary & Alternative Medicine (NCCAM)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1)
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Hopp, Craig
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Shifa Biomedical Corporation
United States
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