Source: ALABAMA A&M UNIVERSITY submitted to NRP
INTERSPECIFIC VARIATIONS IN GROWTH AND DEVELOPMENT, MODE OF ACTION OF ANTIHYPERGLYCEMIC ACTIVITY AND BIOACTIVE COMPONENTS OF GENUS OCIMUM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1007502
Grant No.
2015-38821-24337
Cumulative Award Amt.
$493,892.00
Proposal No.
2015-05370
Multistate No.
(N/A)
Project Start Date
Sep 1, 2015
Project End Date
Feb 28, 2021
Grant Year
2015
Program Code
[EQ]- Research Project
Recipient Organization
ALABAMA A&M UNIVERSITY
4900 MERIDIAN STREET
NORMAL,AL 35762
Performing Department
IRPSP
Non Technical Summary
Type II Diabetes is a major disease currently afflicting an estimated 30 million Americans and costs the US an estimated $245 billion annually. Currently, there is a growing interest in herbal remedies due to high costs and the side effects associated with pharmceutical drugs andinsulinfor the treatment of diabetes. Among several plant species with purported antidiabetic properties, basil (Ocimum spp.) havebeen validated for their antidiabetic effectsusing laboratory animals. However, the mode of action nor the active compound(s) associated with such activity has/have been determined. This project aims to screen a large number (>94) of Ocimum genotypes for growth and development and potential antidiabeticactivity followed by an in-depth evaluation and identification of bioactive components, and development of organic production systems for selected genotypes. The objectives are to i) assess variations between species forgrowth and development, and antidiabeticactivity; ii) determine the mode of action of antidiabeticactivity of selected Ocimum spp. using animal models; iii) identify compound(s) in Ocimum spp.associated with antidiabetic activity;and iv) develop sustainable organic production systems to enable commercial cultivation of basil varieties with known bioactivity. The project has a multidisciplinary approach and multiinstitutional participation. This project is based on the success of similar research on a berry crop (Amelanchier alnifolia) by the investigators. The outcomes of this project include: an understanding of mode of action of antidiabetic effect and availability of a medicinal herb for potential commercial cultivation.
Animal Health Component
20%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5022220100050%
2042220106025%
2042220103025%
Knowledge Area
204 - Plant Product Quality and Utility (Preharvest); 502 - New and Improved Food Products;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic;

Field Of Science
1060 - Biology (whole systems); 1030 - Cellular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
Objectives: 1. Assess genotypic variations in adaptation, growth and development, and antihyperglycemic activity among Ocimum species;2. Determine the mode of action of antihyperglycemic activity of Ocimum spp. using proven cell culture techniques and animal models;3. Identify compound(s) in Ocimum spp.associated with antihyperglycaemic activity through bioassay-guided isolation procedures; and4) Develop sustainable organic production systems for potential commercial production.
Project Methods
Approach: A broad and comprehensive set of approaches encompassing agronomy, molecular and cellular biology, and analytical chemistry will be used to address potential interspecific variation among 94 Ocimum accessions belonging to 7 species, africanum, americanum, basilicum, campechianum, gratissimum, tenuiflorum, and selloi for growth, antihyperglycemic mechanisms; and identify and characterize chemicals that confer such an activity, and the development of sustainable organic production package for potential commercial cultivation.MethodsObjective 1: Seedlings of 94 Ocimum genotypes will be transplanted on to field plots and replicated thrice. Plants from 1-m row length from each plot will be harvested at peak vegetative phase to determine leaf area index, biomass and partitioning pattern among genotypes. Fresh leaves of each genotype will be extracted with methanol, at AAMU and shipped to Auburn University and to USDA-ARS-NPURU, MS for carrying out objectives 2 & 3, respectively.Objective 2: Using a combination of in vitro, intact cells, and animal studies, the 94 Ocimum genotypes will be analyzed initially for improved insulin action: alpha-glucosidase inhibition, glucose uptake, suppression of hepatic glucose production, PPARg activation, and DPP-IV inhibition. Extracts that demonstrate multiple positive effects from the above panel will be further characterized for additional mechanisms. [a] Modulation of insulin signaling: The effect of basil extracts on IR activation, signaling through Akt, MAPK, and glucose uptake will be assayed using Western blotting, in vitro kinase assays, and published methodologies by the Co-PD (Mathews et al, 2000; Kim et al, 2009); [b] Gluconeogenic gene expression and hepatic glucose production: The efficacy of basil extracts in suppressing dexamethasone-induced gene expression of PEPCK and G6Pase in H4IIE rat hepatoma cells will be determined; [c]Adipocyte differentiation and activation of PPAR receptors: Basil's effects on transcriptional activation of PPARalpha and PPARgamma, and on adipocyte differentiation in 3T3-L1 adipocytes will be explored (Amin et al, 2010); [d] Alpha-glucosidase inhibition: As reported previously by us (Zhang et al, 2012), purified rat intestinal acetone powder (Sigma) will be used as a source of mammalian alpha-glucosidase enzyme. Acarbose will be used as a positive control; [e] Potentiation of insulin release and DPP-IV inhibition: We propose to confirm this potential mechanistic aspect using both in vitro testing (Kim et al, 2009), and in animal studies; and[f] Animal Studies: Anti-diabetic effect of basil extracts in streptozotocin-induced diabetes: Potential bioactive components from basil extracts will be assessed for their bioavailability, toxicity, and anti-diabetic effect in both healthy and streptozotocin-induced diabetic rat model. [i] Bioavailability will be assessed using standard dosing methodology in male Wistar rats and by monitoring over 2 days (-1h, 0, 15', 30', 1h, 2h, 5h, 12h, 24h, and 48h) in serum, urine, and feces, using HPLC analysis. Toxicity studies will be conducted by histological examination of liver morphology and liver enzyme activities of alanine and aspartate amino transferase using standardized methodology. [ii] Combination of high-fat diet-fed and low-dose streptozotocin-induced diabetic rat: Male, Wistar rats (~250 g), will be fed a chow diet (10% kcals from fat D12450J, Research Diets) or high-fat diet (60% kcals from fat, D12492, Research Diets) for 2 weeks. On day13, rats will be fasted overnight, and a single ip dose of 30 mg/kg STZ dissolved in 0.1 M fresh cold citrate buffer pH 4.0 will be given the next morning.The high-fat diet feeding (or chow diet for controls) will be continued for another week. At the end of the third week, rats will be fasted for 6 h and blood taken from the dorsal pedal vein to assess glucose levels. For acute studies, basil extracts will be orally administered (250, 500, and 1000 mg/kg bw) and hypoglycemic effects monitored. For chronic studies, basil extracts (200 mg/kg) will be administered orally to test for (a) prevention of diabetes and (b) management of diabetes. For the prevention studies, basil extracts (200 mg/kg body wt.) will be administered orally during the high-fat diet and STZ-induction period (total of 3 weeks). To test for hypoglycemic effects following the induction of diabetes, basil extracts (200 mg/kg body wt.) will be administered orally for 30 days to HFD-STZ diabetic rats and fasting blood glucose, insulin, incretin and gut hormones will be measured. At the end of the study, animals will be euthanized, and truncal blood and liver, skeletal muscle, liver, and fat tissues will be excised, snap frozen in liquid nitrogen, and stored at -80C for future metabolic and gene expression analysis.Objective 3: Methanolic leaf extracts of selected Ocimum species will be concentrated in a rotavap, and partitioned between water and ethyl acetate. The water and ethyl acetate fractions will be tested in the cell assays (a-e), as described in Objective 2. Depending on the results from the assays, either the water or ethyl acetate fraction(s) will be subjected to fractionation by flash column chromatography. Subfractions obtained will be further assayed to determine the active subfraction. Chromatography-guided by assay will be repeated until a pure compound is isolated. The structure of the active compound(s) will be determined using various spectroscopic methods.Identification of the bioactive compound. The structure of the antihyperglycaemic constituent will be determined using a variety of spectroscopic techniques such as UV and Infrared spectroscopy, mass spectrometry (Electrospray Ionization mass spectrometry, Electron Impact mass spectrometry), and one- and two-dimensional nuclear magnetic resonance experiments (such as Correlation spectroscopy, Heteronuclear Single Quantum Coherence, Heteronuclear Multiple Bond Correlation, C; Nuclear Overhauser Enhancement Spectroscopy, Heteronuclear Single Quantum Coherence-Total Correlation Spectroscopy). LC-MS will be performed for the isolated compounds (as necessary) for molecular weight determination. Compounds isolated will be analyzed by GC-MS as needed on a JEOL GCMate II Instrument. Objective 4: Experiment 1: Effects of organic manures on leaf area index and biomass production: Experimental details: In fall of each year, a cover crop mixture of rye (Secale cereale) and Crimson clover (Trifolium incarnatum) will be planted on the experiment area marked out for this project research. In mid-April, the cover crop will be mowed and residue left on the field surface. In mid-May, 1.8 m wide and 4.5 m long plots will be rototilled so that 45 cm wide raised beds about 20 cm high can be prepared.Treatments: i) pelletized poultry litter; ii) composted manure; and iii) vermicompost/earthworm castings. Each treatment will be replicated four times. Organic manures containing the equivalent of 150 Kg of nitrogen/ha will be applied in two split doses of 50 Kg/ha at planting and 100 Kg/ha 45 days after planting. Planting will be same as described for Objective 1. Experiment 2. Determination of tillage method for optimal growth and biomass production Ocimum species. Three tillage systems: i) tilled flat rows: about 45 cm wide rows will be tilled up to approximately 18 cm deep; ii) tilled raised beds: tilled raised beds will be as described for experiment 1; and iii) no-till flat rows: seedlings will be transplanted using a no-till plug planter into plots having cover crop residue. Each treatment will be replicated thrice and planting of genotypes will be same as described for expt. 1.Statistical Analyses: All data generated in various experiments will be subject to statistical analysis using SAS procedures (SAS, 2010). The mean separation to determine significance of treatment effects will be done by Duncan's Multiple Range Test.

Progress 09/01/15 to 02/28/21

Outputs
Target Audience:Several farmers, extension service professionals, faculty, students, and community visited the experiment plots and learned about the basil research and its objectives, and implications.Undergraduate and graduate students presented postersbased on results of research on antimicrobial activity of basil accessionsat professional meetings such as the Annual Conference of the American Council for Medicinally Active Plants, the American Society for Horticultural Science, STEM Days, etc.This is in accordance with the dissemination of research results generated in this project described in the proposal narrative. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Ph.D. student from Alabama A&M University and one MS student from Auburn University were trained in this project. Two undergraduate students at AAMU were trained in raising greenhouse seedlings, field preparation, agronomic research, planting, and caring for Ocimum genotypes. They learned data collection, including recording flowering dates, biomass production, and tabulation. The Ph.D. student used the leaves of 16 accessions planted as part of this project to determine the antibacterial activity of basil species. She published four peer-reviewed journal articles and several conference papers based on the research findings. Additionally, one undergraduate student from Soil Science and two high school students learned about simple greenhouse research techniques and presented posters at the university STEM Days. How have the results been disseminated to communities of interest?A field day was organized to show a wide range of basil genotypes to medicinal plant growers, organic farmers, extension service professionals, faculty, and students. A group of undergraduate students from Auburn University and one group of high school students were given tours and knowledge of the culinary and medicinal value of Ocimum genotypes. One high school student presented a poster on water stress effects on growth and elemental content of leaves of seven Ocimum species as part of her summer apprenticeship program during the summer of 2016. As part of this project, the research was an integral part of field tours, university research Open Days, AgWeek, and extension and framers who visited the research station as part of the Professional Agricultural Workers Conference (PAWC) held each year at Tuskegee University. In addition to tours and such, research findings were presented at the local (Botanical Gardens), regional, national, international meetings/conferences, and peer-reviewed journals. Two more manuscripts are in preparation. 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 1.One hundred and fourOcimumgenotypes belonging toO. africanum,O. americanum, O. balsilicum, O. campechianum, O. gratissimum, O. selloi,andO. tenuiflorumwere planted in a randomized block design with four replications. All of the genotypes were scored for insect and disease, fresh and dry biomass production was determined, and leaf samples were collected for extraction and determining antihyperglycemic activity. The whole plant biomass and partitioning to stems and leaves at harvest. The total above-ground biomass ranged from 492g/Plant forO. basilicumlines to 768 g/Plant forO. tenuiflorumlines. Among species,gratissimumlines produced the highest fresh and dry leaf biomass of 313 and 0.65 g/Plant, respectively. TheO. gratissimumlines with taller plants (average 53 cm/Plant) with thick woody stems and a more number of bigger leaves produced greater biomass among the species studied. WhereasO. basilicumlines were short-statured (24.3 cm/Plant) with more branches (15.2/Plant) but produced the lowest amount of whole plant and leaf biomass. TheO. x. africanumlines flowered much earlier and had a greater amount of inflorescence than other species. The O. gratissimum lines had a more prolonged vegetative phase as they flowered later than all other lines. The accessions were planted in single-row plots each year to showcase and educate visitors about rich variation among basils for aesthetic and aromatic diversity. Objective 2:Leaf extracts of seventeen accessions belonging to the seven species were accomplished by the Co-PI at the USDA-ARS, Oxford, MS, and fractions were sent to the Co-PI at Samford University to conduct assays for determining antidiabetic activity using cell cultures.Yeast alpha-glucosidase inhibitory activity: Sixteen accessions belonging to O. africanum, O. americanum, O. basilicum, O. campechianum, O. gratissimum, and O. tenuiflorum were assessed for yeast alpha-glucosidase inhibitory activity compared to Acarbose. Two O. tenuiflorum, three O. gratissimum, one O. basilicum, and one O. americanum accessions showed 12 to 30% inhibitory activity. O. tenuiflorum accessions showed a slightly greater percentage of inhibitory activity and were about 50% of that by Acarbose. All accessions exhibited dose-related inhibition. The IC 50 values for O. tenuiflorum accessions were similar to that for Acarbose. In the subsequent years, sub-fractionates were tested. In summary, methanol extracts of 16 samples from six different basil (Ocimum) species,viz.O. basilicum(2 samples from Turkey, three samples from Macedonia, USA, and Brazil, respectively),O. gratissimum(2 samples from Taiwan and Zambia, respectively),O.tenuiflorum(3 samples from Cuba, Denmark, and India, respectively),O. africanum(3 samples from Zambia),O. americanum(2 samples from Togo and Tanzania, respectively), andO. campechianum(1 sample from Brazil) were evaluated for the antihyperglycemic activity; an in-house bioassay was developed. Yeast alpha-glucosidase activity for all of theOcimumspecies, along with dose-response and IC50 values for each sample, was determined; extracts of five samples fromO. basilicum(Brazil),O. gratissimum(Taiwan),O. gratissimum(Zambia),O. tenuiflorum(Cuba), andO. tenuiflorum(India) were discovered to possess the greatest inhibition of alpha-glucosidase activity; fractionation was completed using silica gel column chromatography on the five lead extracts, which yielded about 40 fraction samples. Objective 3. Identify compound(s) in Ocimum spp. associated with antihyperglycaemic activity through bioassay-guided isolation procedures: SixteenOcimumaccessions consisting of three O. africanum, twoO. americanum, fourO. basilicum, oneO. campechianum, threeO. gratissimum, and threeO. tenuiflorumspecies were analyzed for antidiabetic properties to identify the compound(s) that may be best associated with its antihyperglycemic effects. Methanol extracts of the fiveOcimumspeciesO. gratissimun(Brazil),O. gratissimum(Taiwan),O. gratissimum(Zambia),O. tenuiflorum(Cuba), andO. tenuiflorum(India), were discovered to be the most bioactive extracts, which possessed the greatest α-glucosidase inhibition activity.O.tenuiflorumdemonstrated comparable IC50 inhibitory concentrations as acarbose, a widely-prescribed antidiabetic medication. A UHPLC/PDA-QToF method was developed for the simultaneous quantitation of the 15 constituents present in 16 differentOcimumspecies. Meanwhile, we have initiated a targeted bioassay-guided fractionation process in order to obtain bioactive constituents. Further fractionation has been completed using silica gel column chromatography on the five lead extracts. Six phenolics, three each of triterpenes and fatty acid methyl esters, have been isolated from the accessions. The COVID-19 pandemic delayed and prevented any progress in 2020. However, in early 2021, a UHPLC/DAD/Q-ToF METHOD was developed for the analysis of different basil samples. The developed method was optimized and validated based on ICH guidelines. The results indicated good method performance with values of R2 (>0.99) for the calibration curves with sensitive LOD (0.04 - 0.6 µg/mL) and LOQ (0.08 - 1 µg/mL). The results were precise (RSD < 6%), with recoveries between 87.66-103.53%. The application of Q-ToF provided a high level of confidence for compound identification. The method enabled the quantification of 14 bioactive compounds (6 flavonoids, four phenolics, and four terpenoids) present in basil samples; from preliminary studies, several samples, especiallyO. gratissimumfrom Zambia, showed promising α-glucosidase inhibitory activities; thus, these samples were evaluated for their major compounds, such as rosmarinic acid and caffeic acid;O. gratissimumfrom Zambia which had a higher α-glucosidase inhibitory activity than other accessions also had the highest rosmarinic acid (57.015 + 0.610 mg. g) and caffeic acid (5.484 + 0.014 mg/g) content. This research is in progress despite the completion of the project. Objective 4: Develop sustainable organic production systems for potential commercial production: In 2018, a replicated field experiment was conducted to assess the effects of three manures on plant growth and biomass production of threeO. tenuiflorumgenotypes, 93B, LB, and 96. Three manures, cow manure, chicken manure, and vermicompost, were applied at rates to provide 100 kg N/ha in two split doses of 50Kg/ha at planting and the peak vegetative phase. Response of three Ocimum genotypes to three organic manures. Genotypic variation for the whole plant and leaf fresh biomass in response to the type of manure was significant. Application of vermicompost or Poultry manure resulted in significantly high total and leaf biomass in genotype 96 compared to other genotypes; LB gave higher biomass in response to Cow manure; Plants receiving vermicompost were in general shorter, had more branches/plant, and produced greater fresh and dry biomass than plants receiving chicken manure or cow manure. Plants receiving cow manure had a greater flower weight, whereas plants receiving chicken manure had fewer flowers and thus lower flower weight. Overall, vermicompost promoted more significant growth and biomass production than cow manure or poultry manure. A graduate student completed a comparison of extraction methods using the essential oil, chloroform, and methanol to determine the best extraction methods for profiling phytochemicals in seventeen genotypes belonging to seven species. Methanolic leaf extracts were the most optimal based on the number of compounds elicited.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, Leopold Nyochembeng, Vijaya Kumar Rangari, and Salam Khan. 2020. The chemical composition, characterization, and combination effect of Ocimum campechianum leaf essential oils and bio-produced silver nanoparticles against Listeria monocytogenes and Escherichia coli. International Journal of Current Engineering and Technology, 10 (4): 518-526, DOI: https://doi.org/10.14741/ijcet/v.10.4.4.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lokesh Mishra, Dipayan Sarkar, Srinivasa Rao Mentreddy, and Kalidas Shetty. 2019. Evaluation of Phenolic Bioactives Linked Anti-Hyperglycemic and Helicobactor pylori Inhibitory Activities of Asian Basil (Ocimum spp.) varieties. Journal of Herbal Medicine. DOI: 10.1016/j.hermed.2019.100310
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, L. Nyochembeng, W. Setzer, and B. Vogler. 2019. Comparison of Methods of Extraction and Antimicrobial Activity of Six Ocimum Species against Human Pathogens. Journal of Agriculture and Life Sciences Vol. 5, No. 2. doi:10.30845/jals.v5n2p10.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Suresh Mathews, Charles Cantrell, and Srinivasa Rao Mentreddy. 2019. Inhibition of alpha?glucosidase activity by extracts of Ocimum species. Nutrition 2019, June 8  11, 2019. Baltimore, MD.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bomma, M., F. A. Okafor, S.R. Mentreddy. 2019. The Antioxidant and Antibacterial Activity of Ocimum basilicum Against Human and Plant Pathogenic Bacteria. ACMAP - 9th Annual Conference, Dehradun, India. "ACMAP- 9th Annual Conference, India." Journal of Medicinally Active Plants 8, (2). https://scholarworks.umass.edu/jmap/vol8/iss2/1
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, Leopold Nyochembeng, William Setzer. 2017. The Antimicrobial Activity of Crude Leaf Extracts of Ocimum spp (O.basilicum and O.tenuiflorum) accessions on Bacterial Pathogens. . "ACMAP - 8th Annual Conference, Clemson, SC." Journal of Medicinally Active Plants 6, (1):21. http://scholarworks.umass.edu/jmap/vol6/iss1/1
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Monday Mbila, Sampson Hopkinson, and Srinivasa Mentreddy. 2017. Effects of Alabama Soil Types on the Plant Biomass and Elements in Holy Basil. International Journal of Agriculture, Environment and Bioresearch, 2 ( 03): 413-433. www.ijaeb.org
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Kaylyn Steger, Manjula Bomma, & S.R. Mentreddy. 2017. Effects of Water Stress on the Growth of Basil. High School student presentation. Summer Research, Department of Biological and Environmental Sciences, Alabama A&M University.


Progress 09/01/20 to 02/28/21

Outputs
Target Audience:Several farmers, extension service professionals, faculty, students, and the community visited the experiment plots and learned about the basilresearch and its objectives, and implications. Since teh inception of the project, five papers were published in peer-reviewed journals thathave extensive circulation among professionals. Changes/Problems:A six-month extension was granted until Feb 28 to enable the proper conclusion of the project. Bioassay guided trials are in progress to narrow down the bioactive compounds are in progress at collaborating institutions. What opportunities for training and professional development has the project provided?One undergraduate student conducted a preliminary trial to assess the potential of low-temperature plasma for promoting early germination and speeding seed germination rate using the five Ocimum accessions that showed promise as having antidiabetic activity in cell culture studies. Inter-specific variation was observed for seed germination. The undergraduatestudent was not paid from this project funds. How have the results been disseminated to communities of interest?Five peer reviewed journal publications and poster/oral presentations were made at professional and farmer meetings over teh project duration since its inception. There has not been any activity since March 2020. What do you plan to do during the next reporting period to accomplish the goals?We plan to properly conclude teh project and submit a final report.

Impacts
What was accomplished under these goals? Due to COVID-19 policy, teh labs at the three partnering institutions were closed which precluded any research until September 2020. The research is still in progress.

Publications


    Progress 09/01/19 to 08/31/20

    Outputs
    Target Audience:Several farmers, extension service professionals, faculty, students, and community visited the experiment plots and learned about the basil research and its objectives, and implications. Three papers were published in peer-reviewed journals that have extensive circulation among professionals. Changes/Problems:Due to COVID-19, we were unable to carry out the proposed research. TheUniversitiesmoved to 100% online delivery of instruction in March 2020. We have been unable to go to our research labs since this closure. Both, Samford University and Alabama A&M University have resumed quasi-normal operations with restricted access to the campus facilities including labs from Monday, August 24, 2020. This closure has significantly affected our ability to complete the work we had proposed earlier. Thus, a no-cost extension is requested. We need to complete the characterization of the bioactivity from extracts and subfractions of Ocimum spp. We also propose to carry out animal studies to characterize the glucose-lowering effects ofOcimum sppextract in a mouse model of diet-induced obesity and insulin resistance. What opportunities for training and professional development has the project provided?One graduate student whograduated with a Ph.D. in Plant and Soil Science is currently working as post-doctoral fellow and working on advanced research based on her Ph.D. dissertation research. She is being trained in writing manuscripts and proposals. How have the results been disseminated to communities of interest?Three high-quality journal papers were published. ? What do you plan to do during the next reporting period to accomplish the goals?A six-month no-cost extension has been sought to complete project objectives 2 and 3. Completion of in vitro and cell culture studies characterizing the activity of Ocimum spp. extracts and subfractions,Animal studies, data analysis, prepare manuscripts.

    Impacts
    What was accomplished under these goals? Objective 1: Evaluation of 105 varieties in year one and two and then sixteen varieties in year two and then three selected varieties in 2019 were completed and the field trials were not repeated in 2020 due to lack of access to the university facilities due to COVID-19 related Lockdown. Objective 2, 3 & 4: Due to COVID-19, we were unable to carry out proposed research. Our University moved to 100% online delivery of instruction in March 2020. We have been unable to go to our research labs since this closure. It is anticipated that we will resume normal work from Aug.24, 2020. This closure has significantly affected our ability to complete the work we had proposed earlier. Thus, a no-cost extension is requested. We need to complete the characterization of the bioactivity from extracts and subfractions of Ocimum spp. We also propose to carry out animal studies to characterize the glucose-lowering effects ofOcimum sppextract in a mouse model of diet-induced obesity and insulin resistance.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2020 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, Leopold Nyochembeng, Vijaya Kumar Rangari, and Salam Khan. 2020. The chemical composition, characterization, and combination effect of Ocimum campechianum leaf essential oils and bio-produced silver nanoparticles against Listeria monocytogenes and Escherichia coli. International Journal of Current Engineering and Technology, 10 (4): 518-526, DOI: https://doi.org/10.14741/ijcet/v.10.4.4.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, L. Nyochembeng, W. Setzer, and B. Vogler. 2019. Comparison of Methods of Extraction and Antimicrobial Activity of Six Ocimum Species against Human Pathogens. Journal of Agriculture and Life Sciences Vol. 5, No. 2, December 2018 doi:10.30845/jals.v5n2p10


    Progress 09/01/18 to 08/31/19

    Outputs
    Target Audience:Several students, farmers, extension service professionals visited the research plots and learned about the project, its objectives, potential outcomes. Research findings were presented at farmers meetings and professional scientific meetings. Changes/Problems:Premature demise of one of the collaborators, Late dr. Agnes Rimando slowed down research related to Objective 3 - bioassay-guided identification of bioactive compound(s). The USDA-ARS has replaced late dr. Rimando with dr. Charles cantrell to continue research where she left off. Other objectives are progressing well. What opportunities for training and professional development has the project provided?Three doctoral students are using leaves of basil genotypes being grown in this project for their dissertation research in Plant Science and Food Science. Thus, besides the research aimed at achieving the objectoves of this project, application of basil in other medicinal properties are also being assessed. How have the results been disseminated to communities of interest?One student graduated with a doctoral degree in plant and soil science. She has presented four papers at various meetings and has published one peer-reviewed journal paper, and has two manuscripts which are in review; The results have been presented at professional meetings. What do you plan to do during the next reporting period to accomplish the goals?Conduct field experiments aimed at the development of best management practices. Scale of mode of action research from cell culture to animal model studies Continue to assay-guided identification of bioactive compound(s).

    Impacts
    What was accomplished under these goals? Objectives 1: Assess genotypic variation for adaptation, growth, and development: About 91 accessions belonging to four Ocimum genus, O. africanum, O. basilicum, O. gratissimum, and O. tenuiflorum were establsihed in single replication field plots and assessed for growth, biomass, and insect and disease tolrance. The total above ground biomass ranged from 492g/Plant for O. basilicum lines to 768 g/Plant for O. tenuiflorum lines. However, O. gratissimum lines produced the highest amount of fresh and dry leaf biomass of 313 and 0.65 g/Plant, respectively. The O. gratissimum lines with taller plants (average 53 cm/Plant) with thick woody stems and larger number of biggerl leaves produced a greater biomass among teh four species studied. Whereas, O. basilicum lines with short statured (24.3 cm/Plant) with more branches (15.2/Plant) produced teh lowest amount of whole plant and leaf biomass. The O.x. africanum lines frlowered much earlier and had a greater amount of inflorescence than other species. The O. gratissimum lines had a longer vegetative phase as they flowered later than all other lines. Objective 2. Determine the mode of action of antihyperglycemic activity of Ocimum spp. using proven cell culture techniques and animal models; Sixteen Ocimum samples consisting of three O. africanum, two O. americanum, four O. basilicum, one O. campechianum, two O. gratissimum, and three O. tenifolium accessions were analyzed for the presence and levels of compounds with anti-hyperglycemic effect that have been reported in Ocimum, with the objective of identifying an accession that is most effective against diabetes. Analysis of crude extracts of the above 16 Ocimum samples demonstrated that O.tenuiflorum and O.gratissimum exhibited 28% and 22% inhibition, respectively, of yeast alpha-glucosidase activity, compared to 64% inhibition by acarbose, a widely-prescribed anti-diabetic medication, at comparable concentrations, suggesting that O. tenuiflorum and O.gratissimum species have potential antidiabetic properties. This research is in progress and will sacled up to animal model studies in 2019. Objective 3. Identify compound(s) in Ocimum spp.associated with antihyperglycaemic activity through bioassay-guided isolation procedures: This research is in progress. The premature demise of the collaborator, Late Dr. Agnes Rimando has necessitated suspedning research related to this objective until Dr. Charles Cantrell at the USDA-ARS-NPURU, Oxford, MS was identified for replacing Late Dr. Rimando. Objective 4: Develop sustainable organic production systems for potential commercial production: In year 2018, a replicated field experiment was conducted to assess the affects of three manures on plant growth and biomass production of three O. tenuiflorum genotypes, 93B, LB, and 96. Three manures, cow manure, chicken manure and vermi compost were applied at rates to provide 100 kg N/ha in two split doses of 50Kg/ha at planting and at the beginning of reproductive phase, respectively. Response of three Ocimum genotypes to three organic manures. Genotypic variation for whole plant and leaf fresh biomass in response to type of manure was significant. Application of vermicompost or Poultry manure resulted in significantly high total and leaf biomass in genotype 96 compared to other genotypes; LB gave a higher biomass in response to Cow manure; Overall, vermicompost promoted greater growth and biomass production than cow manure or poultry manure. Th eeffects of manures on essential oil content and composition is in progress. Plants receiving cow manure had a greater flower weight whereas plants receiving chicken manure had fewer flowers and thus lower flower weight.

    Publications

    • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Manjula Bomma, F. Okafor, S.R. Mentreddy, and L. Nyochembeng. 2018. The Characterization, of the Synergistic Antibacterial Activity of Bio- produced Silver Nanoparticles by Using Ocimum campechianum species. In Review
    • Type: Conference Papers and Presentations Status: Submitted Year Published: 2019 Citation: Manjula Bomma, F. Okafor, and S.R Mentreddy. 2018. Comparative Study of Antibacterial Activity of Seven Ocimum Species on Human and Plant Pathogenic Bacteria.
    • Type: Journal Articles Status: Published Year Published: 2019 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, L. Nyochembeng, W. Setzer, and B. Vogler. 2019. Comparison of Methods of Extraction and Antimicrobial Activity of Six Ocimum Species against Human Pathogens. Journal of Agriculture and Life Sciences Vol. 5, No. 2, December 2018 doi:10.30845/jals.v5n2p10


    Progress 09/01/17 to 08/31/18

    Outputs
    Target Audience:Several student groups, extension service professionals, and a few farmers visited the research plots at the research station. The project objectives and current research results were discussed. Three abstracts were publihsed based on research results by one of the co-PIs and one graduate student. Thus, targt audience comprised of professionals was reached, besides students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two Ph.D students and tree MS students are being trained. Two undergraduate students are being mentored and trained in research methods. How have the results been disseminated to communities of interest?Through presentations by graduate students and project PIs at professional and STEM meetings. What do you plan to do during the next reporting period to accomplish the goals?COnitnue agronomic trials. Scle up antidiabetic research from cell culture to mice-model studies. Continue to fractionate the extracts for determining the bioactive compound.

    Impacts
    What was accomplished under these goals? Two major experiments were conducted at teh University Research Station as part of Objective 1. In Expt. 1: Eighty five accessions belonging to seven Ocimum species were planted in single plot trials were planted in early June. Measurements included Plant height, total plant fresh and dry weights, leaf biomasss, and number of branches per plant. The experiment is in progress. In Experiment 2: The treatments ocmprised of three accessions and three manures, Chicken Manure, Vermicompost, and Cow manure applied in three split applications of 50kg of N/ha each. Plants receiving vermicompost were in general shorter, had more branches/plant and produced greater fresh and dry biomass than plants receiving chicken manure or cow manure. Objective 2: 1. Yeast alpha glucosidae inhibitory activity: Sixteen accessions belonging to O. africanum, O. americanum,O. basilicum, O. campechianum, O. gratissimum, and o. tenuiflorum were cpmapred for yeast alpha glucosidase inhibitory activity compared to Acarbose. Two O. tenuiflorum, three O. gratissimum, one O. basilicum, and one O. americanum accessions showed 12 to 30% inhibitory activity. O. tenuiflorum accessions showed a slightly greater percentage inhibitory activity and were about 50% of that by Acarbose. All accessions exhibited dose-related inhibition. The IC 50 values for o. tenuiflorum laccessions were similar to that for Acarbose. Sub-frantonates are now being tested. Objective 3. Six phenolics, three each of triterpenese and fatty acid methyl esters have been isolated from the accessions. The research is in progress. Besides research related to project objectives, the ocimum species being grown as part of the project are being tested for antimicrobial activity against human pathogens and plant pathogens, anti-liver cirrohsis, anti-breast cancer cells, and compputational modeling.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy. Leopold Nyochembeng, William Setzer, and Vogler Bernhard. 2018.Comparison of Antimicrobial activity of crude leaf extracts and essential oils of six Ocimum species against Human Microbial Pathogens. STEM 2018. April 25, 2018. Alabama A&M University.


    Progress 09/01/16 to 08/31/17

    Outputs
    Target Audience:A poster based on results of research on antimicrobial activity of basil accessions was presented at the 2017 Annual Conference of the American Council for Medicinally Active Plants at Clemson University, SC. The target audience was students, scientists, professionals and community members interested in medicinal plants. This is in accordance with dissemination of research results generated in this project. Several undergraduate and graduate students, post-doctiral Fellows, scientists and other professionals visited the poster and graduate student presenting the poster could dialogue with the audience and gain inputs on research on basil. Changes/Problems:Field experiments to develop organic production practices planned for crop growing season in 2017 could not be planted due to unusually heavy and frequent rains during the planting season (late may thru July). These experiments will be planted in 2018 along with experiments planned for 2018. A one-year no-cost extension will be sought to properly accomplish project goals and objectives. What opportunities for training and professional development has the project provided?One high school andone undergraduate students were trained in greenhouse and field research on basilin this project. They learnt research methodology, data collection, tabulation and poster preparation. One graduate student is being trained in assessing antimicrobial properties of basil as the primary area of research towards her doctiral dissertation. One more graduate student has been recruited inthis project to train in assessment of antidiabetic properties of basil using cell culture and animal models. Both graduate students are being trained in phytochemical analyses using basil leaf mass from plants in this project. How have the results been disseminated to communities of interest?A high school student interned during the summer and presented a poster on basil at high school students presentation, target audience was high school science students, faculty and administrators. A graduate student presented a poster in the grauate student poster competitions at an international conference in Clemson University, SC. A target audience of students, faculty, scientists, professionals and medicinal plant growers viewed and learned about basil and its medicinal values. One full aper has been published in an international peer reviewed journal. One full paper has been submitted to a peer reviewed international journal. What do you plan to do during the next reporting period to accomplish the goals?Continue to assess adapatability, growth, abd yield of a large number of basil genotypes in replicated field trials. Complete field trials to develop organic production system for basil. Determine inter-and intra-specific variation for antidiabetic activity among Ocimum species. Determine the mode of action of antidiabetic activity of basil.

    Impacts
    What was accomplished under these goals? Field experiments plannedduring the cropgrowing season in 2017 could not be planted due to unusually heavy and frequent rains that rendered soils super saturated and prevented field operations such as land preparation. However, about one hundred and one basil accessions belonging to seven species, Ocimum africanum, O. americanum, O. basilicum, O. campechianum, O. gratissimum, O. seloi, and O. tenuiflorum were planted in replicated field plots. Plant biomass and partictioning to stems and leaves were determined at harvest. Fresh leaves from 17 accessions representing the seven species were harvested at peak vegetative phase to enable antimicrobial studies and essential oil and phtochemical profiling. The experiments planned for 2017 will be carried out in 2018. Leaf extracts of seventeen accessions belonging to the seven species was accomplished by the Co-PI at the USDA-ARS, Oxford, MS and fractions were sent to the Co-PI at Samford University to conduct assays for determining antidiabetic activity using cell cultures.

    Publications

    • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Manjula Bomma, Florence Okafor, S.R. Mentreddy, Leopold Nyochembeng, William Setzer. 2017. The Antimicrobial Activity of crude Leaf Extracts of Ocimum spp (O.basilicum and O.tenuiflorum) accessions on Bacterial Pathogens
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Monday Mbila, Sampson Hopkinson, and Srinivasa Mentreddy. 2017. Effects of Alabama Soil Types on the Plant Biomass and Elements in Holy Basil. International Journal of Agriculture, Environment and Bioresearch, 2 ( 03): 413-433. www.ijaeb.org
    • Type: Other Status: Other Year Published: 2017 Citation: Kaylyn Steger, Manjula Bomma, & S.R. Mentreddy. 2017. Effects of Water Stress on the Growth of Basil. High School student presentation. Summer Research, Department of Biological and Environmental Sciences, Alabama A&M University.
    • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Lokesh K Mishra1, Dipayan Sarkar, Rao Mentreddy, and Kalidas Shetty. 2017. Phenolic Antioxidant-Linked Anti-Hyperglycemic and Anti-Microbial Properties of Selected Species of Asian Basil (Ocimum spp.).Journal of Herbs, Spices and Medicinal Plants.


    Progress 09/01/15 to 08/31/16

    Outputs
    Target Audience:Medicinal plant growers, organic famers, scientists, faculty, extension service professionals, and students. Changes/Problems:The project started off as planned and we could accomplish tasks set for year 1 activities. Originally we proposed to screen 100 genotypes belonging to a large number of Ocimum species for their antihyperglycemic activity. However, due to government restrictions in countries from where germplasm lines of Ocimum species were sought, we could not access any germplasm lines other than what we could obtain from USDA GRIN source. Also, due to the lack of a full time graduate student and enough undergraduate students' help, all one hundred and four lines could not be sampled for extraction purposes. We changed this to sixteen accessions belonging to six species. Remedy: We will continue to seek new Ocimum species from sources where available. Next year, we will strive for employing a full time graduate student and acouple more undergraduatestudents to harvest 104 genotypes and extract them using the most appropriate method for quick screening for antihyperglycemic activity. What opportunities for training and professional development has the project provided?One graduate student and one undergraduate student were trained in raising greenhouse seedlings, field preparation, agronomic research, planting and caring of Ocimum genotypes. They learneddata collection including recording flowering dates, biomass production, and tabulation. The graduate student was trained in extraction methods of basil leaves. How have the results been disseminated to communities of interest?A field day was organized to show a wide range of basil genotypes to medicinal plant growers, organic farmers, extension service professionals, faculty, and students. A group of high school students was given a tour and knowledge of the culinary and medicinal value of Ocimum genotypes. One high school student presented a poster on water stress effects on growth and elemental content of leaves of seven Ocimum species as part of her summer apprenticeship program, during summer of 2016. What do you plan to do during the next reporting period to accomplish the goals?Chemical profiling of sixteen genotypes belonging to six Ocimum species; Screen 104 genotypes, if possible or sixteen selected genotypes representative of six speciesfor their antihyperglycemic activity, using a quick put through cell culture assay.

    Impacts
    What was accomplished under these goals? Objective 1. This year, one hundred and four Ocimum genotypes belonging to O. africanum,O. americanum, O. balsilicum, O. campechianum, O. gratissimum, O. selloi, and O. tenuiflorum were planted in a randomized block design with four replications. All of the genotypes were scored for insect and disease, fresh and dry biomass production was determined, and leaf samples were collected for extraction and determining antihyperglycemicactivity. A graduate studentcompleted a comparison of extraction methods using hydrodistillation chloroform and methanol to determine the best extraction methods for profiling phytochemicals in seventeen genotypes belonging to seven species. This work is in progress. Following extraction of leaves, cell culture research to determine the inter-specific variation for antihyperglycemic activity will be determined.

    Publications

    • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Lokesh K Mishra,Dipayan Sarkar, Rao Mentreddy, and Kalidas Shetty. 2018. Phenolic Antioxidant-Linked Anti-Hyperglycemic and Anti-Microbial Properties of Selected Species of Asian Basil (Ocimum spp.). Journal of Herbal Medicine.