Source: UNIVERSITY OF RHODE ISLAND submitted to
BENEFICIAL EFFECTS OF MAPLE SYRUP PHYTOCHEMICALS AGAINST INFLAMMATION ASSOCIATED WITH METABOLIC SYNDROME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1011820
Grant No.
2017-67017-26784
Project No.
RI.W-2016-08934
Proposal No.
2016-08934
Multistate No.
(N/A)
Program Code
A1341
Project Start Date
Jul 1, 2017
Project End Date
Jun 30, 2020
Grant Year
2017
Project Director
Seeram, N. P.
Recipient Organization
UNIVERSITY OF RHODE ISLAND
19 WOODWARD HALL 9 EAST ALUMNI AVENUE
KINGSTON,RI 02881
Performing Department
Biomedical & Pharmaceutical Sc
Non Technical Summary
The United States is the world's largest consumer of sweeteners, including corn sweeteners and other edible syrups. Despite the availability of a wide variety of sweeteners, there is increasing consumer demand for natural sweeteners among which pure maple syrupis highly regarded. Also, while on a sheer volume basis, maple syrup production and availability is dwarfed by high fructose corn syrup, corn syrup, and other refined sugars, with growing consumer interest in local food and growing skepticism of artificial ingredients and flavors in food products, a great market opportunity exists in replacing the use of some percentage of artificial syrup with maple syrup. Unfortunately, there are limited studies to support the potential health benefits of maple syrup despite knowledge that this plant-derived natural sweetener contains multiple bioactive components. Studies from our group and otherssuggest that the complex matrix of maple syrup constituents in toto, including phytochemicals, minerals, vitamins, phytohormones and carbohydrates, impart positive biological effects to this whole food that are superior to refined sugar (e.g. sucrose) alone.Therefore, the major objectives of this project are to evaluate the anti-inflammatory effects of maple syrup constituents using an animal model of diet-induced-obesity and metabolic syndrome in combination withhuman adipose explants in culture. This project will produce meaningful new information on clinical markers of inflammation and insulin sensitivity and also on the mechanisms underlying the potential favorable effect of maple syrup constituents on obesity-induced metabolic disorders. Giventhe wide consumption of maple syrup worldwide, and the economic importance of this crop to the United States, establishing the anti-inflammatory effects of this food using preclinical animal models will have a positive overall impact by providing critical data necessary to guide the design of future human studies on this natural product.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70220991010100%
Goals / Objectives
The inhibition of inflammation and pro-inflammatory processes is associated with human health promotion and disease prevention. Data suggest that (poly)phenolics, whicharephytochemicals abundant in plant-derived foods, possess anti-inflammatory properties. Maple syrup is a plant-derived natural sweetener produced byconcentrating the sap collected from the sugar maple (Acer saccharum)species.Our group has conducted extensive phytochemical studies on maple syrup whichhaveled to the isolationand structure elucidationof over 60(new and known) phenolic compounds therein. We have developed a food-gradephenolic-enriched maple syrup extract (MSX) which retains all of the bioactive constituents found in the whole maple syrup food and is well tolerated and non-toxic to animals (at 1000 mg/kg/day for 1 week).Our published in vitro and preliminary unpublishedanimal studies support the anti-inflammatory potential of MSX but significant gaps still remain in current scientific understanding of the anti-inflammatory potential of this natural sweetener. This data is critical to accomplish our long term goal which is to identify bioactive food components relevant to human disease preventionand increase the science based knowledge of utilizing plant foods for human health. To this end, the objectives of the current proposal are to: 1) evaluate the anti-inflammatory effects of MSXin a mouse model of diet induced obesity and metabolic syndrome, and 2) investigate the impact of MSX on human adipocyte differentiation, function and response to insulin.Overall, this project willlead to the long-term improvement and sustainability of this economically important North American agricultural cropgiven that it is commercially produced only in theUnited States (mainly New England States) and Canada (mainly, Quebec).
Project Methods
Methods for Objective A: We will conduct animal feeding and molecular biology studies to evaluate the anti-inflammatory effects of MSX. Briefly, we will utilize a C57BL/6 mouse model of diet-induced obesity and inflammation by evaluating circulating markers of inflammation, as well as inflammatory mediators in key tissues associated with insulin resistance after exposure to MSX.Methods for Objective B: We will conduct cell culture studies to investigate the impact of MSX on adipocyte differentiation, function, and response to insulin. Human adipocytes derived from adipose tissue and differentiated 3T3-L1 cells will be incubated with MSX and glucose utilization, insulin sensitivity and cytokine secretion will be evaluated.

Progress 07/01/18 to 06/30/19

Outputs
Target Audience:Dr. Seeram and Dr. Slitt presented their results on this grant at the American Chemical Society national meeting in Spring 2020 in Orlando to over 50 attendees. Also, Dr. Seeram presented data as the Keyonote speaker at the annual meeting of t the International Maple Syrup Institute in October 2020 inMinnesota. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several undergraudate students worked on the grant. How have the results been disseminated to communities of interest?To themaple growers and producers at the natinonal meeting of the IMSI. Also,on the website www.uri.edu/maple What do you plan to do during the next reporting period to accomplish the goals?Plan to publish a manuscript

Impacts
What was accomplished under these goals? Background: Obesity is a diet-induced disease that stems into numerous pathologies such as metabolic syndrome, type II diabetes mellitus, Nonalcoholic Steatohepatitis, Fatty Liver disease and even cancer. Chronic low grade inflammation is linked in the etiology of these diseases. Polyphenol rich nutraceuticals such as Maple Syrup Extract (MSX) may prevent or reduce the severity of these inflammatory processes. Methods: MSX was evaluated in a murine diet-induced obesity model for a period of 18 weeks. Body weight and food consumption were monitored in addition to measuring Fasting Blood Glucose, Glucose, and Insulin Tolerance during this study. IL-6 was quantified from serum following necropsy. Liver and White Adipose Tissue were excised and weighed at the time of necropsy. Livers were sectioned, stained using H&E and scored for histological features associated with NASH/NAFLD. Livers were also subjected to bottom-up proteomic analysis for significant protein changes. Results: 18 weeks of high fat high fructose consumption paired with sedentary behavior caused significant increases in body weight, serum IL-6 concentrations, glucose and insulin intolerance as compared to low fat diet fed mice. The addition of MSX 0.5% w/w exacerbated liver steatosis whereas MSX 0.05% w/w failed to improve histological scoring as compared to high fat diet control mice. Conclusion: MSX supplementation in high fat diet feed did not ameliorate the progression of fatty liver disease in this rodent model as compared to our previous pilot study. The addition of MSX at 0.05% did significantly reduce pro-inflammatory serum IL-6 concentrations as compared to high fat diet control mice. The addition of fructose and glucose in drinking water combined with the duration of diet exposure may have masked the beneficial effects of MSX witnessed in previous studies.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 1. Maple-derived food bioactives in human health & diseases: The path forward. Seeram N.P. American Chemical Society (ACS) National Meeting, Orlando, FL, March 30, 2020. (Oral).


Progress 07/01/17 to 06/30/18

Outputs
Target Audience:We have had two principal audiences - scientists and the public. We had communicated our pilot animal studyfindings on MSXat the annual American Chemical Society meeting in April 2017 at the First Global Symposium on Chemistry and Biological Effects of Maple Food Products in the Division of Agricultural and Food Chemistry. Dr. Seeram (co-I) was the opening speaker at the International Polyphenol Congress held in Madison WI in July 2018 and he presented data on maple polyphenols incluiding MSX. Dr. Slitt (Co-I) also presented our MSX work at a local AAPS meeting in May 2018 and at the USDA Project Directors Meeting in Boston 2018. Dr. Seeram has also done multiple public speaking presentations at several RI Garden Clubs on maple phytochemicals and MSXincludingNewport Garden Club (November 2017), South Kingston Garden Club (September 2018)and Arbutus Garden Club in Charlestown (October 2018). Changes/Problems:The small deviations for the project were that we selected a slightly different diet than proposed. There were some concerns from the reviewers that we needed to have a lower percent fat diet. Our initial study used a 45% kCal diet, but this diet did not significantly induce inflammation. Therefore, we decided to switch to a American Life Style Obesity (ALIOS) diet, which is a western diet (22% hydrogenated vegetable oil, 0.5% cholesterol) along with high fructose-sucrose water (42g/L) to induce more inflammation and a non-alcoholic steatosis phenotype. We also adjusted the timing of our tissue collections, with the first collection being at week 18 instead of week 15 and our second time point at week 34. Basically, we measurements in body weight, glucose sensitivity, and insulin sensitivityto better gauge the appropriate timing. A second addition to the project was to include THP-1 human monocytes in addition to the primary monocytes proposed. A major unanticipated finding is that high concentrations of MSX in feed or culture media exert adverse effects. We have observed that lower MSX concentrations (1 ppm) that are more relevant to consumption exert much more significant anti-inflammatory properties than high concentrations (100 ppm). This suggests that there might be some constituents of MSX that have toxic properties, but only at high, non-relevant concentrations. What opportunities for training and professional development has the project provided?The project has funded 1 graduate research assistant. The graduate assistant (Nick DaSilva) has received an interdisciplinary training in medicinal chemistry and pharmacology/toxicology. The graduate student has participated in routine lab meetings in Dr. Seeram and Dr. Slitt's research groups. He has received training in culture of human monocytes, as well as conducting a large-scale rodent experiment. Since the start of this study, our group has trained approximately 4undergraduate students per semester who were exposed to a number techniques and skillsets such as basic cell culture, animal welfare/feeding, and experiment design/execution/data analysis. This study has also been used to initiate collaborations with several research groups (domestic and foreign) to further investigate the effects of MSX in various tissues from this rodent study namelykidney, small intestines and fecal microbial diversity. How have the results been disseminated to communities of interest?Results have been disseminated primarly through professional research societies such as the American Chemical Society and the American Society of Nutrition (ASN). What do you plan to do during the next reporting period to accomplish the goals?The final necropsy is to be performed withtissues harvested and analyzed as described in previous sections. Specifically, much of the tissue analysis that remains is to be performed by collaborating research groups on a contractualbasis. These results will then be compiledin manuscript form for submission into a suitable journal within the grant specified timeline (June 2019). We are currently preparing at least two resulting manuscripts from the funded work and will present the findings at the Spring ACS meeting in Orlando, Florida in a symposiumentitled " Global Symposium on Chemistry and Biological Effects ofMaple-Food Products". This session is being organized by a member of the PI's group (Dr. Hang Ma) and the PI (Seeram) and Co-I (Slitt) will be presenting research findings at the symposium. The PIs have also forged collaborations with other experts to expand some of the proposed endpoints to areas that are emerging and novel to Maple. For example, Dr. Seeram's group has collected fecal material from the funded mouse study and the impact of MSX feeding on gut microial diversity will be assessed by16S rRNA sequencing performed by Dr. Andree Marette's group at University Laval in Canada. Kidney samples from the resulting study will be analyzed by Dr. Nathan Cherrington's research group at the University of Arizona. Lastly, through collaborations at the Universit of Rhode Island, our group as acquired expertise in label-free proteomics methodology by MS-SWATH for untargeted proteomics.

Impacts
What was accomplished under these goals? We are about 75% completed performing the proposed studies for pre-adipocyte work with murine 3T3-L1 adipocyes, with some timepoints and modifications to the culture conditions remaining. We have observed that treatment with maple syrup extract (MSX) can slightly decrease lipid accumulation and decreases LPS-induced cytokine secretion into media. We have completed the proposed human monocyte experiments and were able to add an additional human monocyte/macrophage cell line. This work was able to demonstrate that LPS can stimulate Tnf-alpha and Il-6 secretion into cell culture media and this was significantly decreased by co-treatment with MSX (0.1 ppm and 1 ppm). We have completed week 17 of our in vivo mouse study and are currently on the week 30timepoint. We are getting ready to necropsy the mice. We have also measured and asessedfasting blood glucose,glucose and insulin tolerance. Liver samples will be examined and scored by a trained pathologist.

Publications