Source: The University of North Carolina at Greensboro submitted to NRP
EXPLORATION OF BIOACTIVE COMPONENTS IN HAWAIIAN MAMAKI TEA WITH POTENTIAL TO MODULATE VASCULAR INFLAMMATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032252
Grant No.
2024-67018-42460
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-08248
Multistate No.
(N/A)
Project Start Date
Jun 1, 2024
Project End Date
May 31, 2026
Grant Year
2024
Program Code
[A1343]- Food and Human Health
Recipient Organization
The University of North Carolina at Greensboro
1400 Spring Garden Street
Greensboro,NC 27412
Performing Department
(N/A)
Non Technical Summary
Atherosclerosis is a major cause of morbidity and mortality in the industrial world. Mamaki, a species of nettle endemic to Hawaii, has been used by natives for centuries for various health benefits. However, its bioactive components and the mechanisms of action remain largely unexplored. We hypothesize that the bioactive compounds in mamaki inhibit monocyte adhesion to human aortic endothelial cells (HAECs) by regulating chemokine and adhesion molecule expression, an important step in the initiation and progression of atherosclerosis. The goal of this study is to identify the bioactive constituents of mamaki leaves and elucidate the mechanisms of action. The results of this project could provide a basis for future clinical investigation into the efficacy of mamaki tea or its bioactive constituents in protecting against the development of vascular diseases.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7022234104040%
7012234101030%
7022234118030%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components; 701 - Nutrient Composition of Food;

Subject Of Investigation
2234 - Tea;

Field Of Science
1040 - Molecular biology; 1180 - Pharmacology; 1010 - Nutrition and metabolism;
Goals / Objectives
The goal of this study is to identify the bioactive constituents of mamaki leaves and elucidate the mechanisms of action. Objective 1 is to identify the bioactive compounds in mamaki tea extracts and to explore the mechanisms by which these bioactive compounds exert their anti-inflammatory effects in human aortic endothelial cells. Objective 2 is to examine whether dietary mamaki tea extracts can ameliorate the development of atherosclerosis in ApoE-/- mice and to explore the effects of dietary mamaki on the association between the gut microbiome and vascular inflammation.
Project Methods
1. Identify the bioactive compounds in m?maki tea extracts and to explore the mechanisms by which these bioactive compounds exert their anti-inflammatory effects in human aortic endothelial cells.1.1. Isolate the bioactive compounds in M-EtOH and determine the structures. Expt. 1. Bioassay-guided separation. Dried mamaki leaves will be extracted with ethanol. The dried ethanol extract will be submitted to chromatography, and fractions will be collected. Active fractions will be further separated by using high-performance liquid chromatography (HPLC) usually with a different solid phase from the previous one, until the compounds that account for the observed bioactivities of the parent fraction are purified and accounted for. Expt. 2. Dereplication: The spectroscopic and spectrometric analytical data including HPLC, Nuclear Magnetic Resonance (NMR) and Liquid chromatography-mass spectrometry (LC-MS) data collected on the active fractions or subfractions are then submitted to search in-house and commercially available databases of natural products to identify the structures responsible for the observed activity. Expt. 3. Structure elucidation of active molecules: Structure elucidation is the most challenge in natural product chemistry. We will use a combination of physico-chemical, spectroscopic and spectrometric analytical methods including NMR and MS to determine the structures of the active compounds.1.2. Determine whether bioactive compounds in M-EtOH inhibit TNF-α-induced expression of adhesion molecules and cytokines as markers of vascular inflammation.We will culture human aortic endothelial cells (HAECs), and cells will be pre-incubated with various concentrations of bioactive compounds isolated from M-EtOH (0, 1, 5, 25, and 50 µg/mL) for 24 h, followed by the addition of TNF-α (0, 10 ng/mL) for 24 h. The cell culture supernatants will be collected, and the production of VCAM-1, ICAM-1, E-selectin, MCP-1, IL-8, IL-1β, IL-6, and CD40 Ligand on the surface of HAECs will be determined by ELISA kits (R&D systems, MN). We will also test whether the effect of bioactive compounds on the production of adhesion molecules and inflammation markers is mediated at transcriptional levels using qPCR. 2: Examine whether dietary m?maki tea extracts can ameliorate the development of atherosclerosis in ApoE-/- mice and to explore the effects of dietary m?maki on the association between gut microbiome and vascular inflammation.ApoE-/- mice (6-week-olds) will be used for the studies. All mice will be fed with a modified AIN-93G rodent diet containing with or without 0.5% or 2.0% m?maki tea extracts (Bio-Serv, NJ). Control mice will receive a normal chow diet without m?maki tea extracts. The diet will be adjusted to compensate for the additional sugars and fiber provided by the m?maki tea extracts. At the beginning of the experiment and weekly after that, body weight and food and water intake will be recorded weekly throughout the study to determine whether m?maki tea extracts have any effect on these parameters. At the end of treatment, blood samples will be withdrawn by cardiac puncture under anesthesia. Aortic vessels will be harvested and frozen at 80°C for the biochemical assays. Plasma concentrations of chemokine JE (the mouse homologues of human MCP-1), VCAM-1, ICAM-1, E-selectin, IL-1β IL-6, and CD40 Ligand will be analyzed by ELISA kits and blood triglycerides, total cholesterol, LDL-cholesterol, and HDL-cholesterol will be determined using colorimetric kits (BioVision, CA, and Roche Applied Science, IN). The atherosclerotic lesions will be evaluated by imaging and analysis of Oil Red O-Stain. We will also determine F4/80-positive monocytes-derived macrophages and VCAM-1 expression in mouse aorta by immunohistochemistry. High throughput 16S rRNA amplicon sequencing and microbial profiling will be performed to determine the relationship between specific microbial families and the vascular activity of m?maki extracts.

Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Graduate/undergraduate students, staff, and scientists in the fields of biological sciences, nutrition, and pharmacy. 1). Undergraduate and graduate students participated in the project and received training. 2). Research data were presented at local meetings/conferences, including departmental lab meetings and the Carolyn & Norwood Thomas undergraduate research & creativity expo at the University of North Carolina at Greensboro. 3). Research data were presented at state and national conferences, including the 121st North Carolina Academy of Sciences Meeting and the National McNair Scholars and Undergraduate Research Conference. 4. We interacted with Mr. Grant Ferrier of Hawaii Forest Farm and scientists at the Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center, Agricultural Research Service, USDA. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Undergraduate and graduate students at the University of North Carolina at Greensboro were trained in various techniques related to this project. These techniques included but were not limited to, cell culture, flow cytometry, RNA isolation, cDNA synthesis, qPCR, and various ROS assays used to measure vascular inflammation. A postdoc from the University of Hawaii at Hilo participated in this project and was trained in the operation of grinding machines, mamaki extract techniques, use of rotary evaporators, SpeedVac, and lyophilizer, and operation of HPLC, LC/MS, NMR, etc. With the help of Ms. Mary Martinez, UNC Greensboro Animal Facility Operations Manager, the students listed in the approved?IACUC protocol for this project also received and completed the animal procedures and techniques training for the animal studies proposed under Aim 2. How have the results been disseminated to communities of interest?We meet with local farmers, such as those at Hawaii Forest Farm, about once a month to share our research. In these informal meetings, we discuss topics/findings, such as which types of Mamaki leaves show the strongest anti-inflammatory/oxidative activity.Data from this project have been presented at several local/state and national conferences/meetings, including the 2025 Carolyn and Norwood Thomas Undergraduate Research and Creativity Expo at UNC Greensboro, the 121st North Carolina Academy of Sciences Meeting, and 2025 National McNair Scholars and Undergraduate Research Conference. What do you plan to do during the next reporting period to accomplish the goals?We are planning to identify the bioactive compounds in mamaki tea extracts and investigate whether dietary mamaki tea extracts can ameliorate the development of atherosclerosis in ApoE-/- mice and explore the effects of dietary mamaki on the association between gut microbiome and vascular inflammation.

Impacts
What was accomplished under these goals? We collected the Mamaki leaves (Pipturus albidus), the primary type of mamaki leaves, at the Hawaii Forest Farm and extracted them after grinding. We further separated with high-performance liquid chromatography (HPLC). We have isolated 12 compounds from Mamaki leaves (Pipturus albidus). We performed a total sugar analysis of mamaki leaves (Pipturus albidus) using the Scientific Multiskan GO instrument. The results revealed the following total sugar content in the Mamaki leaves is: 74.82 mg/g. We demonstrated that the anti-inflammatory effects of Mamaki extract were almost comparable to those of green tea extract. Epigallocatechin-3-glucose (EGCG) and theophylline (e.g., caffeine, theophylline, and theobromine) are major components of green tea leaves. To explore whether the mamaki leaves contain EGCG or theophylline, we collected Mamaki leaves (Pipturus albidus) and performed liquid chromatography/mass spectrometry analysis. The results showed that Mamaki leaves (Pipturus albidus) did not contain these compounds, suggesting that the potential anti-inflammatory activity of Mamaki leaves may be attributed to specific active compounds different from those in green tea. We have investigated whether the anti-inflammatory effect of mamaki extract is due to its ability to free radicals. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays were used to quantify the antioxidant capacity of mamaki extract. Our data showed that mamaki extract at a concentration of 50 μg/ml could significantly scavenge DPPH radicals compared to the control. FRAP analysis demonstrated that Mamaki extract at concentrations as low as 25 µg/ml showed significant ferric-reducing capacity in a dose-dependent manner. The H2O2 scavenging ability of Mamaki extract was examined using a luminol-derivatized chemiluminescence assay. The results showed that Mamaki extract had a strong H2O2 scavenging ability in a dose-dependent manner. Peroxyl radicals play an important role in vascular inflammation. The oxygen radical absorbance capacity (ORAC) assay is a common and popular tool used to determine the antioxidant capacity of compounds through the inhibition of peroxyl radicals. Our ORAC results showed that 5, 25, 50, and 100µg/ml Mamaki extract had a significant dose-dependency compared with the control, indicating that Mamaki extract has a strong ability to peroxyl radicals. This study provides evidence for further consideration of mamaki tea as an affordable strategy for preventing and managing vascular disease. A manuscript with these findings is in preparation and is planned for submission to the Journal of Nutritional Biochemistry. We have presented these findings at local, state, and national meetings/conferences, including the Carolyn & Norwood Thomas undergraduate research & creativity expo at the University of North Carolina at Greensboro, the 121st North Carolina Academy of Sciences Meeting, and the National McNair Scholars and Undergraduate Research Conference.

Publications