General Protocol to Design Highly Effective Food Products against Chronic Degenerative Diseases by using Nutraceutical Combinations as Novel Element

Abstract

Chronic degenerative diseases (CDDs) are the leading causes of death worldwide. Nutraceuticals are compounds naturally found in foods that have preventive and therapeutic activities against CDDs. However, no single nutraceutical has been successful in preventing or curing CDDs. This can be attributed to the complexity of CDDs, which are multifactorial and multisymptomatic. Nutraceutical combinations that attack different targets of a CDD have the potential to exert a synergistic effect, i.e., an effect greater than the sum of the effects of the individual compounds. Therefore, synergistic nutraceutical combinations can be a powerful solution, as they can serve as a basis to design novel and highly effective food products against CDDs. However, many factors need to be considered to successfully formulate these products. Hence, to make the job easier and less overwhelming for academia and food industries, the main objective of this thesis was to develop a general protocol to design highly effective food products for the prevention/co-treatment of CDDs by using nutraceutical combinations as novel element, and partially validate it with colon cancer as model CDD. The method consists of a 10-step procedure that considers all factors to successfully design these products. Some steps are routinely applied by academia and industry, such as in vitro gastrointestinal digestion, animal, and clinical studies. However, novel steps were incorporated that cover the design and evaluation of synergistic nutraceutical and food ingredient combinations. To partially validate the protocol, the first five steps were undertaken, which cover from the selection of the target CDD (in this case, colon cancer) up to selecting and preparing the food ingredients based on the best nutraceutical combination. First, the effect of dihydrocaffeic acid (D, a chlorogenic acid metabolite) over the viability of several human cancer cell lines, including MCF-7 (breast), Hep-G2 (liver), PC-3 (prostate), and HCT-116 (colon), and healthy dermal fibroblasts (HDFa), was tested to determine its anticancer potential. Then curcumin (C), sulforaphane (S), and dihydrocaffeic acid were evaluated, individually and in combination (CD, SC, SD, and CSD at different doses and proportions), over the viability of HT-29 and Caco-2 colon cancer cells, and compared with healthy fetal human colon (FHC) cells. The cytotoxic concentrations to reduce cell viability by 50%, 75%, and 90% (CC50, CC75, and CC90, respectively) were obtained by using the MTS assay. Combination effects (i.e., synergy, additivity, or antagonism) were determined with the combination index (CI) method. Broccoli sprouts and carrot juice were chosen as the sources of sulforaphane and dihydrocaffeic acid, respectively. To elevate the levels of chlorogenic acid (and consequently, dihydrocaffeic acid) in carrot juice, wounding stress was applied to carrots. The effects of wounding intensity, storage, peeling, blanching, filtration, and pasteurization over physicochemical, nutritional, nutraceutical, and sensory properties of carrot juice were evaluated. Dihydrocaffeic acid was significantly more cytotoxic for most cancer cell lines, including MCF-7, PC-3, and HCT-116, but less cytotoxic for Hep-G2 compared with HDFa. Thus, dihydrocaffeic acid is a relevant candidate for cancer prevention and treatment. Furthermore, the SD(1:1) combination exerted synergistic effects against HT-29 at 90% cytotoxicity level (doses 90:90 µM), whereas CD(1:4) was synergistic at all cytotoxicity levels (9:36–34:136 µM) and CD(9:2) at 90% (108:24 µM) against Caco-2. SD(1:1) was significantly more cytotoxic for cancer cells than FHC healthy cells, while CD(1:4) and CD(9:2) were similarly or more cytotoxic for FHC. Consequently, SD(1:1) was chosen as the best combination. A model explaining SD(1:1) synergy was proposed. Moreover, juices from unpeeled carrots had 7–40% more minerals, 0.46–1.6 less °Brix, and 1.16x more titratable acidity. The carrot juice with the highest phenolic content was obtained by cutting unpeeled carrots into slices and storing them for 48 h at 15 °C before blanching (80 °C for 6 min) (stressed unpeeled carrot juice, SUCJ). SUCJ had 3600% more chlorogenic acid, 195% more total phenolics, and similar carotenoid content than conventional carrot juice. Sensory evaluation of SUCJ was acceptable and willingness-to-pay increased by providing information about health benefits. Mechanistic tests are needed to elucidate the anticancer mode of action of dihydrocaffeic acid, and to validate the proposed model of synergy between sulforaphane and dihydrocaffeic acid. Additionally, the remaining five steps must be carried out to fully validate the protocol, including in vitro gastrointestinal digestion and combination studies of broccoli sprouts and SUCJ, formulation of a synergistic beverage against colon cancer, and shelf-life, animal, and clinical evaluations. The study of nutraceutical and food ingredient combinations is an emerging field. Therefore, the solution to CDDs could be just a matter of finding the right combination, along with a healthy lifestyle.