Bioactivity-Guided Identification of Botanical Inhibitors of Ketohexokinase.

OBJECTIVE: In developed countries with westernized diets, the excessive consumption of added sugar in beverages and highly refined and processed foods is associated with increased risk for obesity, diabetes, and cardiovascular diseases. As a major constituent of added sugars, fructose has been shown to cause a variety of adverse metabolic effects, such as impaired insulin sensitivity, hypertriglyceridemia, and oxidative stress. Recent studies have shown that ketohexokinase isoform C is the key enzyme responsible in fructose metabolism that drive's fructose's adverse effects. The objective of this study was to identify botanical ingredients with potential for inhibitory activity against ketohexokinase-C and fructose-induced metabolic effects by using a series of in vitro model systems. METHODS: Extracts from 406 botanicals and 1200 purified phytochemicals were screened (initial concentration of 50 µg/mL and 50 µM, respectively) for their inhibitory activity using a cell free, recombinant human ketohexokinase-C assay. Dose response evaluations were conducted on botanical extracts and phytochemicals that inhibited ketohexokinase-C by > 30% and > 40%, respectively. Two different extract lots of the top botanical candidates were further evaluated in lysates of HepG2 cells overexpressing ketohexokinase-C for inhibition of fructose-induced ATP depletion. In addition, extracts were evaluated in intact Hep G2 cells for inhibition of fructose-induced elevation of triglyceride and uric acid production. RESULTS: Among the botanical extracts, phloretin (Malus domestica) extracts were the most potent (IC50: 8.9-9.2 µg/mL) followed by extracts of Angelica archangelica (IC50: 22.6 µg/mL-57.3 µg/mL). Among the purified phytochemicals, methoxy-isobavachalcone (Psoralea corylifolia, IC50 = 0.2 µM) exhibited the highest potency against ketohexokinase isoform C activity followed by osthole (Angelica archangelica, IC50 = 0.7 µM), cratoxyarborenone E (Cratoxylum prunifolium, IC50 = 1.0 µM), and α-/γ-mangostin (Cratoxylum prunifolium, IC50 = 1.5 µM). Extracts of Angelica archangelica, Garcinia mangostana, Petroselinum crispum, and Scutellaria baicalensis exhibited ketohexokinase inhibitory activity and blocked fructose-induced ATP depletion and fructose-induced elevation in triglyerides and uric acid. CONCLUSIONS: Angelica archangelica, Garcinia mangostana, Petroselinum crispum, and Scutellaria baicalensis were the top four botanical candidiates identified with inhibitory activity against ketohexokinase-C. Future studies are needed to show proof of mechanism and the efficacy of these botanical extracts in humans to blunt the negative metabolic effects of fructose-containing added sugars.

Identification of a botanical inhibitor of intestinal diacylglyceride acyltransferase 1 activity via in vitro screening and a parallel, randomized, blinded, placebo-controlled clinical trial.

BACKGROUND: Diacylglyceride acyltransferase 1 (DGAT1) is the enzyme that adds the final fatty acid on to a diacylglyceride during triglyceride (TG) synthesis. DGAT1 plays a key role in the repackaging of dietary TG into circulating TG rich chylomicrons. A growing amount of research has indicated that an exaggerated postprandial circulating TG level is a risk indicator for cardiovascular and metabolic disorders. The aim of this research was to identify a botanical extract that inhibits intestinal DGAT1 activity and attenuates postprandial hypertriglyceridemia in overweight and obese humans. METHODS: Twenty individual phytochemicals and an internal proprietary botanical extract library were screened with a primary cell-free DGAT1 enzyme assay that contained dioleoyl glycerol and palmitoleoyl Coenzyme A as substrates plus human intestinal microsomes as the DGAT1 enzyme source. Botanical extracts with IC50 values < 100 µg/mL were evaluated in a cellular DGAT1 assay. The cellular DGAT1 assay comprised the analysis of (14)C labeled TG synthesis in cells incubated with (14)C-glycerol and 0.3 mM oleic acid. Lead botanical extracts were then evaluated in a parallel, double-blind, placebo-controlled clinical trial. Ninety healthy, overweight and obese participants were randomized to receive 2 g daily of placebo or individual botanical extracts (the investigational product) for seven days. Serum TG levels were measured before and after consuming a high fat meal (HFM) challenge (0.354 L drink/shake; 77 g fat, 25 g carbohydrate and 9 g protein) as a marker of intestinal DGAT1 enzyme activity. RESULTS: Phenolic acids (i.e., gallic acid) and polyphenols (i.e., cyanidin) abundantly found in nature appeared to inhibit DGAT1 enzyme activity in vitro. Four polyphenolic rich botanical extracts were identified from in vitro evaluation in both cell-free and cellular model systems: apple peel extract (APE), grape extract (GE), red raspberry leaf extract (RLE) and apricot/nectarine extract (ANE) (IC50 = 1.4, 5.6, and 10.4 and 3.4 µg/mL, respectively). In the seven day clinical trial, compared to placebo, only GE significantly reduced the baseline subtracted change in serum TG AUC following consumption of the HFM (AUC = 281 ± 37 vs. 181 ± 30 mg/dL*h, respectively; P = 0.021). Chromatographic characterization of the GE revealed a large number of closely eluting components containing proanthocyanidins, catechins, anthocyanins and their secondary metabolites that corresponded with the observed DGAT1 enzyme inhibition in the cell-free model. CONCLUSION: These data suggest that a dietary GE has the potential to attenuate postprandial hypertriglyceridemia in part by the inhibition of intestinal DGAT1 enzyme activity without intolerable side effects. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov NCT02333461.

Global assessment of select phytonutrient intakes by level of fruit and vegetable consumption.

Despite dietary recommendations that have repeatedly underscored the importance of increasing consumption of fruits and vegetables, intakes worldwide are lower than recommended levels. Consequently, the diets of many individuals may be lacking in nutrients and phytonutrients typical of a diet rich in a variety of fruits and vegetables. In the present study, we estimated phytonutrient intakes by adults categorised by sex, level of fruit and vegetable consumption (< 5 v. ≥ 5 servings/d), and geographic diet cluster. Intakes of nine select phytonutrients were estimated from the 2002-4 World Health Survey fruit and vegetable servings intake data (n 198,637), the FAO supply utilisation accounts data, and phytonutrient concentration data obtained from the US Department of Agriculture databases and the published literature. Percentage contributions to each phytonutrient intake from fruit and vegetable sources were also estimated. Estimated intakes of phytonutrients from fruits and vegetables varied across the thirteen geographic diet clusters, reflecting regional differences in both numbers and proportions of fruit and vegetable servings consumed, and the specific types of fruits and vegetables available in the diet. The mean phytonutrient intakes by adults consuming ≥ 5 servings/d of fruits and vegetables were approximately 2- to 6-fold the mean phytonutrient intakes by adults with low fruit and vegetable consumption (< 5 servings/d). In some cases, phytonutrient intakes by adults consuming ≥ 5 servings/d of fruits and vegetables in one geographic diet cluster were lower than the intakes by adults reporting < 5 servings/d in another cluster. The findings from this assessment provide important information regarding the major dietary patterns of phytonutrient intakes across geographic diet clusters.

Phytonutrient intake by adults in the United States in relation to fruit and vegetable consumption.

BACKGROUND: Individuals consuming diets dense in fruits and vegetables consume an array of phytonutrients as well as recognized nutritional components, including vitamins, minerals, and fiber. There is a growing body of evidence that phytonutrients may play positive roles in health. OBJECTIVE: The purpose of this research was to estimate usual intakes of nine individual phytonutrients by Americans consuming recommended levels of fruits and vegetables compared to intakes by adults not meeting these recommendations, and to identify contributions of food sources to total phytonutrient intakes. The phytonutrients examined in this study are found predominantly in fruits and vegetables. DESIGN: Food consumption data from the National Health and Nutrition Examination Surveys 2003-2006 and phytonutrient concentration data from US Department of Agriculture databases and the published literature were used to estimate energy-adjusted usual intakes. Student's t tests were used to compare mean energy-adjusted phytonutrient intakes between subpopulations who consumed recommended amounts of fruits and vegetables vs those who did not. Percentage contributions of each phytonutrient by food source were estimated for all adults. RESULTS: Energy-adjusted intakes of all phytonutrients other than ellagic acid were considerably higher among both men and women meeting dietary recommendations for fruit and vegetable intakes compared to those not meeting the recommendations; energy-adjusted intakes of ellagic acid were higher only among women meeting vs not meeting the recommendations. For five of the nine phytonutrients (α-carotene, ß-cryptoxanthin, lycopene, hesperetin, and ellagic acid), a single food accounted for 64% or more of the total intake of the phytonutrient. CONCLUSIONS: Energy-adjusted intakes of carotenoids and flavonoids are higher among men and women whose diets conform to dietary guidance for fruits and vegetables. A limited number of foods provide the majority of these phytonutrients. Findings from this research provide important reference information on the phytonutrient contributions of a diet rich in fruits and vegetables.

Clinical and biochemical effects of a combination botanical product (ClearGuard) for allergy: a pilot randomized double-blind placebo-controlled trial.

BACKGROUND: Botanical products are frequently used for treatment of nasal allergy. Three of these substances, Cinnamomum zeylanicum, Malpighia glabra, and Bidens pilosa, have been shown to have a number of anti-allergic properties in-vitro. The current study was conducted to determine the effects of these combined ingredients upon the nasal response to allergen challenge in patients with seasonal allergic rhinitis. METHODS: Twenty subjects were randomized to receive the combination botanical product, (CBP) 2 tablets three times a day, loratadine, 10 mg once a day in the morning, or placebo, using a randomized, double-blinded crossover design. Following 2 days of each treatment and during the third day of treatment, subjects underwent a nasal allergen challenge (NAC), in which nasal symptoms were assessed after each challenge dose and every 2 hours for 8 hours. Nasal lavage fluid was assessed for tryptase, prostaglandin D2, and leukotriene E4 concentrations and inflammatory cells. RESULTS: Loratadine significantly reduced the total nasal symptom score during the NAC compared with placebo (P = 0.04) while the CBP did not. During the 8 hour period following NAC, loratadine and the CBP both reduced NSS compared with placebo (P = 0.034 and P = 0.029, respectively). Analysis of nasal lavage fluid demonstrated that the CBP prevented the increase in prostaglandin D2 release following NAC, while neither loratadine nor placebo had this effect. None of the treatments significantly affected tryptase or leukotriene E4 release or inflammatory cell infiltration. CONCLUSION: The CBP significantly reduced NSS during the 8 hours following NAC and marginally inhibited the release of prostaglandin D2 into nasal lavage fluid, suggesting potential clinical utility in patients with allergic rhinitis.

Interleukin-1 genotype-selective inhibition of inflammatory mediators by a botanical: a nutrigenetics proof of concept.

OBJECTIVE: Although observational studies have shown that genotype may influence nutritional effects on target outcomes, there are few reported studies that stratified subjects by genotype before a nutritional intervention. This proof-of-concept trial determined whether specifically formulated botanical mixtures reduced inflammation in individuals with genetic variations that predispose to overexpression of interleukin-1beta (IL-1beta) and early heart disease. METHODS: Healthy adults with elevated C-reactive protein (CRP) were stratified into genetic groups based on being positive (IL1(Pos)) or negative (IL1(Neg)) for the at-risk IL-1 gene variations. IL1(Pos) (n = 39) and IL1(Neg) (n = 40) subjects were then randomized to the candidate botanical formulation or placebo. The botanical formulation included rose hips, a blueberry and blackberry mixture, and a grapevine extract. RESULTS: At 12 wk of dosing with the botanical formulation, IL-1beta gene expression by stimulated peripheral blood mononuclear cells was significantly lower than at baseline and significantly lower than placebo in IL1(Pos) and IL1(Neg) subjects. Mean IL-1beta gene expression treatment effect over the 12-wk period was greater in IL1(Pos) than in IL1(Neg) subjects. At 12 wk of dosing the botanical mixture produced no mean change in serum CRP levels. However, in IL1(Pos) subjects, significantly more subjects achieved a reduction in CRP with the botanical mixture than with placebo. No CRP effect was observed in the IL1(Neg) subjects. CONCLUSION: This study represents one of a few prospective clinical trials in which genetic variations were shown to differentially influence nutrient effects on outcomes.