HEALTH BENEFITS OF PATCHOULI ALCOHOL: MECHANISTIC STUDIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012768
• Project No.: MD-HNFS-9897
• Project Start Date: Oct 1, 2017
• Project End Date: Sep 30, 2021

Project Director
Lee, SE, .

Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742

Performing Department
Human Nutrition & Food Science

Non Technical Summary
Patchouli essential oils have been used for medicinal applications due to a variety of biological effects including antibacterial, antifungal, antiinfluenza and antioxidant activities. Patchouli is a species of plant from the genus Pogostemon and major component of patchouli oil. Recently we found that patchouli alcohol possesses anti-inflammatory activity in immune cells. The general aim of this proposal is to study if patchouli alcohol influences occurrence of metabolic disorders including inflammation, obesity and cancer, focusing on elucidating the mechanisms and biological targets. The expected results will expand our understanding on health benefit of using patchouli alcohol and the scope of action using essential oil prevention of human chronic diseases.

Animal Health Component

66%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	2239	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
2239 - Flavoring and beverage plants, general/other;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

essential oil

patchouli alcohol

inflammation

obesity

cancer

Goals / Objectives
Obesity is a worldwide epidemic associated with increased risk of various types of cancer. Chronic inflammation is a central characteristic of obesity, leading to many complications such as cancer. In particular, obesity-induced inflammation confers additional cancer risk beyond obesity itself.Patchouli alcohol is a tricyclic sesquiterpene and most abundant form in patchouli oil. Recently, we reported that patchouli alcohol possesses anti-inflammatory activity in mouse macrophages and colon cells (1). However, health benefits effects and reliable mechanisms by patchouli alcohol on metabolic disorders and human chronic diseases remain unanswered. The primary goal of this proposal is to explore if patchouli alcohol prevents inflammation, obesity and cancer. The second goal is to determine proposed mechanisms and molecular targets by which patchouli alcohol reduces inflammatory response in macrophages, differentiation of preadipocytes and proliferation of colonocytes.In the following specific aims we will investigate:Aim 1. Determine anti-inflammatory activity of patchouli alcohol Aim 2. Determine anti-obese and anti-diabetic activity of patchouli alcoholAim 3. Determine anti-cancer activity of patchouli alcohol

Project Methods
Aim 1. Determine anti-inflammatory activity of patchouli alcoholAim 1.1. In vivo study to exam anti-inflammatory activity of patchouli alcohol using DSS-induced colitis modelExperimental designThirty six experimental mice (C57BL/6 background) will be assigned to three groups and each will be receiving 1) vehicle (0.5% methylcellulose), 2) low dose of patchouli alcohol (20 mg/kg body weight) and 3) high dose of patchouli alcohol (40 mg/kg body weight) (twelve mice/each group). Patchouli alcohol suspension will be given by oral gavage every day for 2 weeks before DSS treatment. Then 5% (w/v) of DSS will be given to mice through autoclaved drinking water for one week with gavage of patchouli alcohol. Aftertreatment of DSS and patchouli alcohol, the mice will be euthanized.MeasurementsExtent of colitis, immunohistochemistry, MPO activity and gene expressionAim 1.2. In vitro studies to exam the mechanism(s) of anti-inflammatory activity using THP-1 monocytesExperimental designHuman monocytic cell line (THP-1) will be pretreated with different concentrations of patchouli alcohol (0, 12, 25, 50, 100 µM) and then co-treated with LPS or IL-6 for 30 minutes or 24hours.MeasurementsTranscriptional activity of NF-?B, nuclear localization of p65 and STAT3, proteasomal degradation of p65, phosphorylation of JNK/p38 MAPK, JAK1/STAT3 and ERK, and expression of target genesAim 2. Determine anti-obese and anti-diabetic activity of patchouli alcoholAim 2.1. In vivo study to exam anti-obese and anti-diabetic activity of patchouli alcohol using Ay/J mouse modelExperimental designAy/J mice in a B6 genetic background (KK.Cg-Ay/J, Jackson Laboratories) will be obtained and bred to produce enough numbers for experiment. At 6 weeks old, thirty six Ay/J mice will be assigned to three groups and each will be exposed to 1) vehicle (0.5% methylcellulose), 2) low dose of patchouli alcohol (20 mg/kg body weight), and 3) high dose of patchouli alcohol (40 mg/kg body weight) respectively (twelve mice/each group) for 4 weeks.MeasurementsBody fat pad, lipolysis/fatty acid oxidation, glucose tolerance test, macrophage infiltration, histological analysis and expression of chemokines and inflammatory cytokinesAim 2.2. In vitro studies to exam the mechanism(s) of anti-obese and anti-diabetic activity using 3T3-L1 preadipocytes and C2C12 myocytesExperimental design3T3-L1 preadipocytes will be cultured in DMEM containing 10% FBS until confluent. Two days after post-confluence, cells will be stimulated to differentiate with 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, and 5 μg/mL insulin in 10% FBS/DMEM for 2 days (day 0). Cells will be then maintained in 10% FBS/DMEM medium with 5 μg/mL of insulin for another 2 days, followed by culturing with 10% FBS/DMEM medium until analysis. Patchouli alcohol (0, 12, 25, 50, 100 µM) will be treated from day 0 to day 4 (for study of gene expression) or day 8 (for triglyceride accumulation). For culture of myocytes, C2C12 cells will be maintained in DMEM supplemented with 10% FBS and then differentiated using DMEM replacing 1% FBS in the absence or presence of different doses of patchouli alcohol (0, 12, 25, 50, 100 µM) for 7 days.MeasurementsLipid accumulation, in vitro glucose uptake and lipolysis, expression of adipogenesis and lipogenesis marker genes, phosphorylation of AMPKα and ACC, expression and translocation of β-catenin protein, and luciferase activity of TOP/FOP flashAim 3. Determine anti-cancer activity of patchouli alcoholAim 3.1. In vivo study to exam anti-cancer activity of patchouli alcohol using inflammation-induced colon cancer model Experimental designThirty six ApcMin/+ mice at 6 weeks of age will be assigned to three groups and each will be exposed to 1) vehicle (0.5% methylcellulose), 2) low dose of patchouli alcohol (20 mg/kg body weight), and 3) high dose of patchouli alcohol (40 mg/kg body weight) respectively (twelve mice/each group) by oral gavage for 4 weeks. Then 2% (w/v) DSS will be given to mice through autoclaved drinking water for one week and the patchouli alcohol will be gavaged every day for 4 weeks.Measurements Tumorigenesis in colon and histological analysisAim 3.2. In vitro studies to exam the mechanism(s) of anti-cancer activity using SW480 colonocytesExperimental designSW480 cells will be treated with different concentrations (0, 12, 25, 50, 100 µM) of patchouli alcohol for 0, 24 and 48 hours.MeasurementsApoptosis, activity of caspases and expression of apoptosis-associated genes, cell cycle distribution and expression of growth arrest genes, cell invasion/migration and expression of MMP-2/9, In vitro angiogenesis and expression of VEGF

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The results will be disseminated to academic institutions and industry and community of interest. Changes/Problems:There was a change of research schedule. We performed Aim 2 study first due to urgency of the study in this area (metabolic disease). We will perform Aim 1 study in the second year. In Aim 2.1, we changed obese mouse model from Ay/J (genetic model) to high fat-diet induced model. The reason is that 1) diet-induced obesity model shares many characteristics with the common form of adult obesity and 2) Ay/J model is not realistic in human population due to rare frequency of Ay mutation. There is no change in research goal and strategies. What opportunities for training and professional development has the project provided?PI trained graduate student with one-on-one work for in vivo and in vitro studies. The graduate student was trained for oral gavage, isolation of diverse fat pads/liver/muscle tissues, glucose tolerance test and in vitro experimental techniques including differentiation of 3T3-L1 and C2C12 cellsand ORO staining and measurement of glucose uptake. How have the results been disseminated to communities of interest?No action was taken for disseminating result. However, the results we are reporting on will be included in the manuscript for upcoming publication and conference abstract in 2019 American Society for Nutrition (ASN) meeting. What do you plan to do during the next reporting period to accomplish the goals?We will continue to perform in vitrostudiesto exam the mechanism(s) of anti-obese and anti-diabetic activity using 3T3-L1 preadipocytes and C2C12 myocytes (Aim 2.2). Wewill perform the studiesto determine anti-inflammation and anti-cancer activity of patchouli alcohol (Aim 1,3).

Impacts
What was accomplished under these goals? Impact Patchouli alcohol is a major component of patchouli essential oil. This study explored if patchouli alcohol possesses health benefit effect focusing on metabolic diseases including obesity and diabetes. For the first time, we observed that treatment of patchouli alcohol attenuated accumulation of body fat and development of obesity, and enhanced glucose uptake in high fat diet-induced obese mouse model. In addition, we observed that patchouli alcohol inhibited fat cell differentiation and enhanced glucose uptake in differentiated muscle cells. This is the first study demonstrating that patchouli alcohol could be a novel and promising agent to suppress the progression of obesity and glucose intolerance. Issue Incidence of obesity sharply increased in the 21st century. Obesity is highly correlated with incidence of type 2 diabetes and a primary risk factor for diverse metabolic diseases such as cardiovascular risk, stroke and cancer. Recently, the demand of diverse plants-derived compounds has been increasing rapidly to inhibit metabolic disorders. Patchouli alcohol is a sesquiterpene alcohol found in Pogostemon cablin which is broadly used in oriental medicine and perfume industry. Despite the significance, the effect of patchouli alcohol on human health has not been investigated. Approach: Aim 2.1. In vivo study to exam anti-obese and anti-diabetic activity of patchouli alcohol In vivo mouse study Forty eight C57BL/6Jmale mice (Jackson's laboratories) aged 5 weeks were purchased and housed in a temperature- and humidity-controlled environment with 12-h light dark cycle with food and water available ad libitum. The mice were randomly assigned into one of four groups (n=12/group) and fed with following diets for 12 weeks; 1) normal diet (control), 2) high fat diet, 3) high fat diet with low dose of patchouli alcohol (25 mg/kg body weight) and 4) high fat diet with high dose of patchouli alcohol (50 mg/kg body weight). The control group received a standard chow diet. High fat diet groups were fed with a diet providing 45% kcal from fat. Patchouli alcohol was suspended in 0.5% methylcellulose and fed via oral gavage three times per week. Body weight was measured weekly. At the end of the experiments, all mice were fasted overnight and sacrificed by CO2asphyxiation. Body fat pads were isolated and their weights were measured. All experiments were approved in advance by the Institutional Animal Care and Use Committee(IACUC) of University of Maryland. Intraperitoneal glucose tolerance test Twenty C57BL/6Jmale mice were assign to one of four groups and treated with patchouli alcohol as described above. At the end of the 12th week, the mice fasted for 12 h and the oral glucose tolerance test was performed after i.p. injection of anhydrous glucose. The blood were collected from tail at 0, 15, 30, 90 and 120 min after glucose injection and glucose concentration were analyzed using glucose meter. Aim 2.2. In vitro studies to exam the mechanism(s) of anti-obese and anti-diabetic activity using 3T3-L1 preadipocytes and C2C12 myocytes Differentiation of 3T3-L1 preadipocytes and measurement of fat accumulation The 3T3-L1 cells were grown using Dulbecco's modified Eagle's medium (DMEM) containing 10% BCS at 37°C under an atmosphere of 5% CO2 with replacement of new media every other day. After two days post-confluence, 3T3-L1 cells were incubated with media (DMEM containing 10% FBS, 1 µg/mL insulin, 0.5 mM dexamethasone, and 0.5 mM isobutylmethylxanthine) to induce differentiation for 48 hours and then maintained with media (DMEM containing 10% FBS and 1 µg/mL insulin). To examine if patchouli alcohol affect adipogenesis, 3T3-L1 preadipocytes were differentiated in the presence of different concentrations (0-100 µM) of patchouli alcohol for 8 days and fat accumulation was compared with Oil Red O (ORO) staining. For ORO staining, the cells were washing with PBS and fixed with 10% buffered formalin for 10 min and washed twice with distilled water to remove formalin. The cells were stained with ORO solution (0.2% final concentration) for 10 min at room temperature and washed with distilled water four times to remove unstained ORO. The ORO-stained cells were visualized under microscope and ORO dye was dissolved in 0.5 mL isopropanol and absorbance of the isopropanol extract was measured at 500 nm using a microplate reader. Differentiation of C2C12 myocytes and measurement of in vitro glucose uptake The C2C12 cells were differentiated using 2% horse serum for 5 days and maintained under serum starvation overnight and treated with different concentrations (0-50 µM) of patchouli alcohol for 24 h. For in vitro glucose uptake, the media was replaced into glucose-free-serum-free DMEM and 100 µg of 2-NBDG was added with 100 nM of insulin. After 18 h, cells were washed with cold PBS for three times and fluorescencewas measured at 485 nm (Ex) and 535 nm (Em). Results: Aim 2.1. In vivo study to exam anti-obese and anti-diabetic activity of patchouli alcohol There were no significant weight differences between the groups before the start of the experiments (5-week age). However, treatment of patchouli alcohol resulted in a significant decrease of body weight in dose-dependent manner. In order to determine whether reduced body weight by patchouli alcohol treatment resulted from reduced fat accumulation, we compared abdominal fat accumulation between control and patchouli alcohol-treated mice. Accumulation of abdominal fat was decreased in patchouli alcohol-treated mice compared to control mice. An analysis of individual fat pad weights (expressed as mg weight of fat pad / g body weight) revealed a significant decrease of epididymal and retroperitoneal fat pad in patchouli alcohol-treated mice whereas brown adipose tissue were not significantly altered. In glucose tolerance test, higher glucose tolerance was found in mice treated with patchouli alcohol compared to those fed high fat diet alone. Aim 2.2. In vitro studies to exam the mechanism(s) of anti-obese and anti-diabetic activity using 3T3-L1 preadipocytes and C2C12 myocytes Fat accumulation were significantly reduced in differentiated 3T3-L1 cells treated with 50, 75 and 100 µM of patchouli alcohol in dose-dependent manner. In addition, patchouli alcohol increased uptake of 2-NBDG into differentiated C2C12 cells. We also measured cell viability to see any toxicity. The results indicate that effective doses to suppress fat accumulation and enhance glucose uptake do not cause toxicity in 3T3-L1 and C2C12 cells. Translation The results of study identify a novel bioactive compound that could potentially be used for prevention and therapy of obesity and expand our understanding on translational scope using essential oil components for management of human chronic diseases.

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