Source: ALABAMA A&M UNIVERSITY submitted to
TOXICOLOGICAL RISK AND SAFETY ASSESMENT FOR HERBAL MEDICINAL PRODUCTS AND APPROACHES TO ISOLATE AND CHARACTERIZE THEIR ACTIVE COMPONENTS AND METABOLITES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
EVANS-ALLEN
Reporting Frequency
Annual
Accession No.
0228275
Grant No.
(N/A)
Project No.
ALAX-012-0812
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2011
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Boateng, J.
Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762
Performing Department
Food & Animal Sciences
Non Technical Summary
PROJECT SUMMARY Plant preparations are marketed as herbal medicines or dietary supplements for a variety of health problems. The demand for these preparations is on the rise and as such, there is an urgent need to investigate their potential for toxicity or safety, especially when consumed in high doses over a long period of time. This study proposes to evaluate the biological and pharmacological activities (toxicokinetics/biokinetics, biodistribution) of selected herbal medicinal preparations in order to establish a scientific platform on their biological activities; address concerns with respect to contaminants (heavy metals, pesticides and mycotoxins) and adulterations of these herbal preparations. The planned objectives are: (1) determine toxicological and safety issues of selected herbal medicinal products and their metabolites using in vitro cell culture and in vivo model systems to assess acute, sub-chronic and chronic toxicity; (2) examine toxicity of selected herbal medicinal products and their metabolites on changes in toxicological biomarkers, such as microsomal drug metabolizing enzymes, biochemical, hematological and histopathological modifications and oxidative stress related enzymes; (3) determine: (i) the occurrence of mycotoxins and pesticide residues utilizing biosensor arrays and molecularly imprinted polymers (MIP)-based biosensors; (ii) heavy metal contamination by Inductively coupled plasma atomic emission spectroscopy (ICP-AES) technique; (4) to study mechanisms (molecular and cellular) associated with selected herbal medicinal products using systems biology approach (omics technologies) such as metabolomics, proteomics and genomic array techniques and (5) isolation and characterization of active compounds and metabolites of selected herbal medicinal products using chromatographic fingerprinting analysis such as high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry(LC-MS)
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3142220115020%
7112235104020%
7122220115020%
7122220110220%
7122235110320%
Knowledge Area
314 - Toxic Chemicals, Poisonous Plants, Naturally Occurring Toxins, and Other Hazards Affecting Animals; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic; 2235 - Herbs and spices;

Field Of Science
1102 - Mycology; 1150 - Toxicology; 1040 - Molecular biology; 1103 - Other microbiology;
Goals / Objectives
Objective 1: To determine potential toxicity of herbal medicinal products (to be determined); an in vitro cell culture system and will be utilized to assess acute systemic toxicity and an in-vivo rodent model will be used to determine the subacute, subchronic and chronic of oral toxicity and safety assessment.Objective 2: To examine toxicokinetics and biokinetics of herbal medicinal products and assess potential biochemical changes in major drug metabolizing and antioxidant enzymes such as Phase I P450 and Phase II detoxification enzymes and oxidative stress related enzymes. Objective 3: Determine the presence of contaminants such(a) toxigenic fungi and mycotoxins by utilizing rapid and accurate immunochemical array Biosensor methods and (b) heavy metals using Inductively coupled plasma atomic emission spectroscopy (ICP-AES) technique.Objective 4:To study mode of action or mechanisms (at the molecular level) associated with herbal medicinal products on toxicological markers such as cytotoxicity endpoints, apoptosis and membrane changes in an in vitro model including the use of genomic microarray and proteomics.Objective 5: To characterize and indentify/profile herbal medicinal bioactives and metabolites using chromatographic fingerprinting techniques using high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). EXPECTED ACCOMPLISHMENTS/GOALS Key accomplishments are:Goal 1: Provide crucial data on toxicokinetics and biokinetics mechanisms of herbal medicinal products which will add to or increase the knowledge base for risk/safety assessment. Goal 2: Generate information on the various types of chemical compositions contained in medicinal herbs and provide chemical, bioactive and toxic markers. This information will contribute to setting standards for safety, efficacy of medicinal herbal products.Goal 3: Fulfill NIFA priority need, which is to train African-American or minority students and/or open up a pipeline of students that are poised to fill needs in the food safety area.Goal 4: The utilization of state of the art detection methods in training students will place students in high demand positions in the Food industry and ultimately give the U.S. a competitive edge on the global food market.Goal 5: Increase visibility of the research and teaching capacity of food/nutrition toxicology and safety program at AAMU.
Project Methods
Objective1: In vitro toxicity study: Primary human cell lines will be cultured in appropriate /recommended medium supplemented with fetal bovine serum (FBS) and antibiotics. Cells will be exposed to dietary-relevant doses/concentrations of herbal medicinal products under investigation. Acute and Chronic exposure levels will be measured. Medium containing test compounds will be collected for analysis of toxicological markers and the cells will be harvested, lysed and used for the determination of toxicological endpoints. In vivo toxicity study: This study will be conducted according to OECD guidelines (TG 407, 453 408) (OECD, 1998, 2001, 2009). Characterization of the dose/response relationship, identification of a no-observed-adverse-effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD), extrapolation of carcinogenic effects to low dose human exposure levels and prediction of chronic toxicity effects at human exposure levels will all be determined. Clinical parameters including feed observation, weight change, changes in skin, fur, eyes, and mucous membranes, will be noted. Histopathological studies will also be conducted.Objective 2: Enzymatic and non-enzymatic assays: Catalase (Hakan Borg 1988), reduced glutathione (GSH) forms (Griffith, 1985), glutathione peroxidase (GPx) activity (Jaskot et al., 1983), Glutathione s- transferase (GST) activity Habig et al. (1974) and measurement of SOD activity (Folhe and Otting 1984) will be performed on liver and cell lysates. Phase I enzymes will be also determined. For hermatological determinations blood samples will be collected and examined for haemoglobin concentration, packed cell volume, erythrocyte, leucocyte and differential counts. Biochemical analysis of serum and liver will also be conducted. Objective3: Determine the presence of contaminants such as fungi and mycotoxins in herbal products: Detection of mycotoxin and contaminants from herbal products will be determined using immune array biosensor and LS-MS/MS. Determination will be based on the methods by Lattanzio et al. (2007), Sapsford et al. (2005) and Feldstein et al. (1999) with modifications. Heavy metals will be determined by the (ICP-AES) technique. Objective4:Mechanisms (at the molecular level) associated with herbal medicinal products on toxicological markers using genomic microarray and proteomics To analyze gene expression in cells following exposure to test compounds microarray experiment outlined by de Longueville et al. (2003) which includes mRNA extraction, synthesis of labeled cDNA and Microarray analysis and hydridization will be performed with some modifications. Proteomic Analysis of livers from rodent will be analyzed following Wang et al. (2007) and Neuhoff et al., (1988).Proteins will be fixed onto IPG-strips, by silver staining or Coomassie Brilliant Blue.Protein identification will be determined using LC-MS/MS as described by Wang et al. (2007; 2010). Objective 5: Chromatographic finger printing of herbal products bioactives. Bioactive compounds will be determined by HPLC and LC/MSD as previously analyzed in our lab (Chukwumah et al. 2007; 2008; 2010).

Progress 10/01/11 to 09/30/15

Outputs
Target Audience:Support the training of Students, Underrepresented minorities; particularly African-Americans in food safety and toxicology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has benefited (four) 4 M.S. graduate students. This included research support towards their degrees, funding for their research training and forattending national meetings. For the PD, the project provided support to conduct summer research to generate data for application of federal grants (USDA/NIFA) and to attend annual Institute of Food Technology (IFT) meetings. The outcomes have strengthened her research and improved peer collaborations. How have the results been disseminated to communities of interest?The results from these projects were presented as a poster, oral presentation and published abstract to three communities of interest. (1) Alabama A&M University STEM Day student presentations and competition, (2) Institute of Food Technology (IFT) meetings. Four (4) publications have resulted from this grant (one journal publication, three published abstracts),and twopublished Master's theses. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Several opportunities have been provided to students and PD. Data from this grant was used to train (three) 3 M.S.; two (2) thesis and one (1) M.S. report. Data the grant provided PD with start up funds to equip lab with essential equipments required to perform experiments in order to generate preliminary data for application of federal grants (USDA/NIFA). Grant fulfilled NIFA's priority need, i.e. to train African-American or minority students and/or open up a pipeline of students that are poised to fill needs in the food safety area.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Ashly Kelly (2016). In Vitro Toxicity And Antimicrobial Properties Of Bitter Melon Extracts- ALABAMA A&M UNIVERSITY Master Thesis
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: S. Willis, J. Boateng, K. Busambwa, L. Shackelford and M. Verghese. Bitter melon and Blueberry leaf teas on endogenous antioxidant enzymes. - Journal of Pharmacology and Toxicology (2016)
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Shantrell Renee Willis (2014). Effects of Herbal Tea Consumption on Adult Rats: A Toxicological and Biochemical Study- ALABAMA A&M UNIVERSITY Master Thesis


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Support the training of Students, Underrepresented minorities particularly African-Americans in food safety and toxicology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This study has trainedthree (MS)students and one undergraduate student. One of the students is currently pursuing herPhD. in food science in Food Safety and Toxicology. One other student will be applyingto the program her PhD this fall , also in Food Safety and Toxicology. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To study mode of action or mechanisms (at the molecular level) associated with herbal medicinal products on toxicological markers such as cytotoxicity, apoptosis and membrane changes in an in vitro model. Human liver cell line (Hep2G) were exposed to different doses of ginger and basil extracts for 12 and 24h. Toxicity determinations were assessed by Lactate dehydrogenase (LDH) leakage, neutral red and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Apoptosis and membrane changes were also assessed via DNA fragmentation and Morphological changes by Hoechst staining. The effect of ginger and basil extracts on endogenous antioxidants glutathione-s-transferase (GST), glutathione (GSH) and superoxide dismutase (SOD) and catalase (CAT) enzyme activities were also determined. Results indicated ginger and basil extracts induced cytoxicity by inhibiting Hep G2 cell proliferation, apoptosis and modification to endogenous antioxidants. As previously noted these data suggest antioxidant capacity and polyphenol content in herbal products could significantly impact their bioactivities. These results contribute to current knowledge implying that herbal and spice products could be effective as an anticancer agent, and could indicate a cost effective way to develop health foods and to increase its range of applications in food and agriculture industries.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: S. Willis, J. Boateng, K. Busambwa, L. Shackelford, and M. Verghese Hepatoprotective effects of Bitter Melon and Blueberry leaf teas on endogenous hepatic antioxidant enzymes. Manuscript in review- Journal of Pharmacology and Toxicology.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: A. Kelly, M. Verghese and J. Boateng. In vitro toxicity and Modulation of Endogenous Antioxidant Defense Enzyme Activity by Bitter Melon Extracts in Hep G2 Cells. Presented at Institute of food Technologists meeting (IFT) 2015 in Chicago IL, July 11-14, 2015


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: TARGET AUDIENCES: Support the training of Students, Underrepresented minorities particularly African-Americans in food safety and toxicology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Trained one graduateand two undergraduate students to conduct research on the cytotoxic effects of selected herbs and spices; cinnamon, rosemary and basil. How have the results been disseminated to communities of interest? Data from this study was used for M.S. thesis document. One undergraduate who is assisting with the study will present data from study at AAMU Stem Day 2015. . What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? (Objective 5A). To identify herbal medicinal bioactives and metabolites using chromatographic techniques using high performance liquid chromatography (HPLC). The first objective for this experiment was to perform simulated in vitro digestion on selected culinary herb and spice products and to compare antioxidant activities and phenolic contents to non-digested culinary herbs and spices (Basil, oregano, ginger and cinnamon). Total phenolics, flavonoids and antioxidants capacity, ferric reducing antioxidant power (FRAP), Oxygen radical absorbance capacity (ORAC), Total Radical Trapping Antioxidant Parameter (TEAC) were determined. Results showed total phenolics, flavonoids content were higher in non-digested culinary herb and spice products compared to in vitro digested herbs and spices. FRAP, ORAC and TEAC values were also reduced after in vitro digestion. Current data suggest digestion may impact antioxidant capacity and polyphenol profile in herbs and spices.

Publications


    Progress 10/01/12 to 09/30/13

    Outputs
    Target Audience: TARGET AUDIENCES: Support the training of Students, Underrepresented minorities particularly African-Americans in food safety and toxicology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This study has provided data used for 2 M.S. thesis and data presentations at national meetings (IFT, and ARD). One student Ms. Shantrell Willis has completed and presented MS thesis and her thesis document is in publication process. She is also preparing a manuscript(s) for publication. After completing her master's project, Ms. Willis applied and was accepted to the PhD. Program at AAMU to further her studies in Food Safety and Toxicology. Another student Ms. Ashly Kelly is preparing to present data from this research in a thesis defense, summer 2014. Two abstracts resulting from this project has been submitted to Institute of food technologist (IFT) 2014 meeting: Effects of Sub Chronic consumption of Herbal Teas on Bone Mineralization. S. Willis*, J. Boateng, L. Shackelford, K. Busambwa, L. T. Walker and M. Verghese. In Vitro Toxicity and antimicrobial activity of Bitter Melon (Momordica charantia) Extracts. Ashly Kelly, J. Boateng, J. Patterson & M. Verghese, Ph.D. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? OUTPUTS: (Objectives 1and 2). This study examined the effect of herbal leaf teas on toxicological biomarkers, such as microsomal drug metabolizing enzymes, Glutathione-S-Transferase (GST), and redox enzymes including Superoxide dismutase (SOD), Glutathione (GSH). The study also examined several parameters and biomarkers of toxicity including (Alanine amino transferase (ALT), Creatinine, Gammaglutamyl Transferase (GGT), glucose, triglyceride after a 28 day repeated oral dose toxicity design in Fisher 344 rats. Also, the effects of herbal teas (blue berry leaf, gun powder green and bitter melon leaf teas) supplementation on bone and mineral metabolism and were evaluated. Bone parameters (length, weight, diameter, volume) were recorded and selected minerals (Ca, P, Mg, Fe and Zn) was analyzed. Results from these studies showed High dose consumption ofherbal leaf teas had no significant effect on the activities of drug metabolizing and redox enzyme activities .Plasma creatinine and ALT levels were decreased in rats administered high doses of herbal teas. This study suggests that high dose consumption of herbal teas (gunpowder green, blueberry leaf, and bitter melon leaf) showed no adverse toxic effect on organ weights. Results also showed there were no significant (p≤.05) differences in bone parameters between the control and rats administered herbal leaf teas. Also, there were no significant differences in Fe (mg/g) concentration in the control and rats administered herbal teas at all doses. Mg (mg/g) levels in the treated groups were significantly (p≤.05) higher compared to the control (untreated) as were Ca (mg/g) and Zn(µg/g) levels. (Objective 1A) In Vitro Toxicity of Bitter Melon (Momordica charantia) Extracts. The aim of this study was to determine the in vitro toxicity and antimicrobial activity of processed (steamed (SBME), boiled (BBME) oven dried (DBME)) and fresh bitter melon extracts (FBME) in hepatocarcinoma cells (HepG2). For toxicity evaluations, cellular morphology, Lactate Dehydrogenase (LDH) and mitochondrial function MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay were determined. Apoptosis was assessed by DNA gel electrophoresis. Results indicated that bitter melon extracts significantly (p<0.05) increased LDH leakage by comparison to the negative control. After 12 h and 24h exposure of Hep G2 cells to bitter melon extracts. Preliminary data showed inhibitory effects on HepG2 cell proliferation as determined by MTT viability assay. These results were evidenced by morphological appearance of cells and nuclei fragmentation.

    Publications

    • Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: 1. S. Willis, J. Boateng, L. Shackelford, K. Busambwa, L.T. Walker and M. Verghese. Sub-acute toxicity study of herbal tea supplementation and effects on toxicological biomarkers: oxidative stress and drug metabolizing enzymes. Presented at 17th Biennial Research Symposium, April 6-10, 2013 Jacksonville, Florida 2. S. Willis, J. Boateng, L. Shackelford, K. Busambwa, L.T. Walker and M. Verghese. Sub-acute toxicity study of herbal tea supplementation and effects on toxicological biomarkers: oxidative stress and drug metabolizing enzymes. Presented at the IFT Annual meeting, Chicago, July 13- 16, 2013. 3. A. Kelly, J. Patterson, M. Verghese and J. Boateng. Antimicrobial Effects of Bitter Melon (Momordica charantia) extracts. Presented at the IFT Annual meeting, Chicago, July 13- 16, 2013. 4. S. Willis, J. Boateng, M. Verghese, L. Shackelford, J. Patterson, B. Bright. Comparing Antioxidant Potential of Gunpowder Green, Blueberry Leaf, and Bitter Melon Teas. Presented at the IFT Annual meeting, Chicago, July 13- 16, 2013.


    Progress 10/01/11 to 09/30/12

    Outputs
    Target Audience: PROGRESS: 2011/09 TO 2012/08 TARGET AUDIENCES: Support the training of Students, Underrepresented minorities particularly African-Americans in food safety and toxicology Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Several opportunities have been provided to students and faculty. In addition to providing preliminary data for future research, data from this study was used for M.S. thesis document. Data is being compiled to be used as preliminary data for application of federal grants (USDA/NIFA, NIH). Fulfill NIFA’s priority need, i.e. to train African-American or minority students and/or open up a pipeline of students that are poised to fill needs in the food safety area. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? OUTPUTS: (Objective 1). The aim of this study was to evaluate the effect of high dose consumption of herbal leaf teas (gunpowder green (GPGT), bitter melon leaf (BMLT), and blueberry leaf teas (BLUT)) using a 28 day repeated oral dose toxicity design in Fisher 344 rats. To achieve this, rats (n=5) were administered once daily by gavage different dosages of herbal leafy teas for 28 days and daily feed intakes, weight were recorded. Also, total phenolics, flavonoids and antioxidants capacity (1,1-Diphenyl-2-picrylhydrazyl (DPPH?), ferric reducing antioxidant power (FRAP) in herbal leaf teas were determined. Results from these experiments indicated administration of herbal teas in rats had no adverse effects on weight, weight gains, and feed intake and relative organ weights. As such, the NOAEL for gunpowder green, bitter melon, and blueberry leaf teas was established at 6000mg/kg body weight. The total phenolic and flavonoid content of the whole leaves GPGT, BLUT, and BMLT indicated GPGT had the highest phenolic content and greater antioxidant potential. The tasks outlined under objective 1 of this project was initiated in February 2012. Protocol for the in-vivo study was submitted to the Institute of Animal Care and Use Committee (IACUC) committee at AAMU for approval and inspection of animal facility conducted before any animal students could be performed

    Publications