Progress 09/01/09 to 08/31/14
Outputs
Target Audience: Target Audiences: People afflicted with diabetes and metabolic syndrome, Researchers studying diabetes and metabolic syndrome, as well as researchers and producers of dietary supplements and food. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provided training for T-32 postdoctoral fellows to understand the role of botanicals on the complex interactions between genetic, molecular and physiological aspects of metabolic syndrome. Each postdoctoral fellow in this training program is encouraged to develop interdisciplinary research efforts to understand the effect and action of botanicals on components of the metabolic syndrome. The program takes advantage of expertise in botanical research and discovery of novel botanical compounds at the Rutgers Department of Plant Biology and Pathology. How have the results been disseminated to communities of interest? Research was described to groups of visitors including NJ farmers and Rutgers graduate and undergraduate students. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact: The focus of the project is the study of botanicals and metabolic syndrome. Although the etiology for metabolic syndrome is not specifically known, obesity, particularly central obesity, and insulin resistance are generally present. It is now recognized that metabolic syndrome develops when individuals are not able to resist metabolic dysfunction at the tissue level (insulin resistance, ectopic fat accumulation, attenuated adipocyte function, etc.) when exposed to an obesogenic environment. Once insulin resistance develops secondary to metabolic dysfunction, insulin secretion is increased (i.e., hyperinsulinemia) in order to compensate for the insulin resistance. Given the compensation, glucose levels remain at or near the normal level. However, when insulin secretory function begins to decline, full compensation of insulin resistance is no longer observed, and hyperglycemia becomes apparent. With development of metabolic dysfunction, there is an increase in cardiovascular disease risk factors (hypertension, dyslipidemia) and accelerated atherosclerosis. Thus, the presence of metabolic syndrome has, and will continue to have, considerable public health significance based on the increased prevalence in the population, the fact it is considered a pre-diabetic state, the observation of increased cardiovascular mortality, and the total health-care costs attributable to these conditions. Thus, the study of botanicals that focus on health maintenance, rather than disease interdiction offers a targeted approach for improving the metabolic syndrome epidemic. The characterization of Artemisia botanicals for improving insulin sensitivity is a good example. Our recent studies examined how the active compounds from PMI-5011 can be formulated with a food matrix such as soy protein to significantly improve the parameters related to the overall effectiveness of the botanical in future clinical work. This research provides significant benefits to the biotechnology and pharmaceutical industries in the State and contribute to value added agriculture of New Jersey. Accomplishments: The scientific goal of the project is to provide a thorough evaluation of specific and compelling hypotheses about the molecular, cellular, and physiological mechanisms by which botanicals promote resistance to the adverse changes at the tissue level resulting from exposure to an obesogenic environment, and hence facilitate maintenance of normal metabolic and functional status at the whole body level. The most highly studied botanical of the Botanical Research Center is an extract of Artemisia dracunculus L. called PMI-5011. A large commercial batch of Artemisia dracunculus (280 kg) was grown in greenhouse conditions and shipped to LSU (frozen) for processing into clinical materials. The polyphenols from Artemisia dracunculus were formulated with soy protein isolate (5011 Nutrasorb) and administered as a food for clinical testing (confirmed by experts at the FDA). Using the TIM model of the upper gastrointestinal tract of humans, the bioaccessibility of the active compounds of Artemisia, specifically the chalcone, 2′,4′-dihydroxy-4-methoxydihydrochalcone (DMC-2), from the soy formulation were determined to be similar to the bioaccessibility of the extract formulated with a commercial excipient and better than the extract alone. The 5011 Nutrasorb had higher relative bioavailability than the extract alone. This formulation was provided to Project 1 investigators and is currently in clinical testing. Artemisias dracunculus, santolinifolia and scoparia were continuously cultivated at Rutgers. The plants were processed into extracts and incorporated into diets for animal studies to determine their effect of insulin signaling pathways. Each of the plants produced a comparable chemical fingerprint. Artemisia santolinifolia and scoparia were fractionated using FCPC and subfractionated using HPLC in the same manner as was performed for A. dracunculus and individual compounds from the active fractions were preliminarily identified using LC-MS as described in previous project reports. A validation study was also initiated for the PMI-5011 project to quantify the DMC-2 in samples of PMI-5011, the quantification of DMC-2 in the plasma of mice treated with PMI-5011 and the synthesis of DMC-2 for use as a chemical standard. Ten grams of DMC-2 were synthesized to greater than 99% purity and the validation analyses of extracts and plasma are ongoing. Separate studies were conducted with extracts of St John's Wort (Hypericum perforatum) produced from plants grown at Rutgers. Seeds from both the USDA seed bank and a commercial source were grown in the greenhouse and compared biochemically to the original extract produced in central Asia that was used in the screening program. Animal studies conducted at the Pennington Biomedical Research Center at Louisiana State University with the extract showed that it did not induce insulin resistance in vivo as it did in vivo using adipocyte cell cultures. In addition, Asclepias incarnata plants were propagated for root mass production. Extract was produced periodically and pure Ikemagenin glycoside (34.0 mg, 0.0057%) was purified from it using a combination of FCPC & HPLC. The pregnane glycoside was hydrolyzed for studies to compare the relative activity of the pregnane with its conjugated glycoside structure. The screening program initiated between Rutgers, PBRC, University of Louisiana at Lafayette and the USGS to expand the botanical research related to metabolic syndrome began during the grant cycle to identify novel botanicals and will continue.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Vance KM, Ribnicky DM, Rogers RC, Hermann GE. Artemisia santolinifolia enhances glutamatergic neurotransmission in the nucleus of the solitary tract. Neurosci Lett. 2014 Oct 17;582:115-9. doi: 10.1016/j.neulet.2014.08.048. Epub 2014 Sep 9. PMID: 25220699 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Cefalu WT, Floyd ZE, Stephens JM, Ribnicky DM. Botanicals and translational medicine: a paradigm shift in research approach. Nutrition. 2014 Jul-Aug;30(7-8 Suppl):S1-3. doi: 10.1016/j.nut.2014.02.009. Epub 2014 Feb 27. No abstract available. PMID: 24985098 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Ribnicky D.M., Roopchand D.E., Poulev A., Kuhn P., Oren A., Cefalu W.T., Raskin I. Artemisia dracunculus L. polyphenols complexed to soy protein show enhanced bioavailability and hypoglycemic activity in C57BL/6 mice. Nutrition. 2014 Jul-Aug; 30 (7-8 Suppl):S4-10. doi: 10.1016/j.nut.2014.03.009. Epub 2014 Mar 28. PMID: 24985105 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Wicks S, Taylor CM, Luo M, Blanchard E 4th, Ribnicky DM, Cefalu WT, Mynatt RL, Welsh DA. Artemisia supplementation differentially affects the mucosal and luminal ileal microbiota of diet-induced obese mice. Nutrition. 2014 Jul-Aug;30(7-8 Suppl):S26-30. doi: 10.1016/j.nut.2014.02.007. Epub 2014 Feb 26. PMID: 24985102 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Boudreau A., Cheng D.M., Ruiz C., Ribnicky D., Allain L., Brassieur C.R., Turnipseed D.P., Cefalu1 W.T., Floyd Z.E. Screening Native Botanicals for Bioactivity: An Interdisciplinary Approach. Nutrition. 2014 Jul-Aug; 30(7-8 Suppl):S11-6. doi: 10.1016/j.nut.2014.02.028. Epub 2014 Mar 15.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Richard AJ, Burris TP, Sanchez-Infantes D, Wang Y, Ribnicky DM, Stephens JM. Artemisia extracts activate PPAR?, promote adipogenesis, and enhance insulin sensitivity in adipose tissue of obese mice. Nutrition. 2014 Jul-Aug;30(7-8 Suppl):S31-6. doi: 10.1016/j.nut.2014.02.013. Epub 2014 Mar 12. PMID: 24985103 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Obanda D.N., Ribnicky D., Raskin I., Cefalu W.T. Bioactives of Artemisia dracunculus L. enhance insulin sensitivity by modulation of ceramide metabolism in rat skeletal muscle cells. Nutrition. 2014 Jul-Aug;30 (7-8 Suppl):S59-66. doi: 10.1016/j.nut.2014.03.006. Epub 2014 Mar 19. PMID: 24985108 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Richard AJ, Fuller S, Fedorcenco V, Beyl R, Burris TP, Mynatt R, Ribnicky DM, Stephens JM. Artemisia scoparia enhances adipocyte development and endocrine function in vitro and enhances insulin action in vivo. PLoS One. 2014 Jun 10;9(6):e98897. doi: 10.1371/journal.pone.0098897. eCollection 2014. PMID: 24915004 [PubMed - in process]
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Vance K.M., Ribnicky D.M., Hermann G.E., and Rogers R.C. St. Johns Wort enhances the synaptic activity of the nucleus of the solitary tract. Neurosci Lett. 2014 Oct 17;582:115-9. doi: 10.1016/j.neulet.2014.08.048. Epub 2014 Sep 9. PMID: 25220699 [PubMed - in process].
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yu, Y., Zhang, X. H., Ebersole, B., Ribnicky, D., and Wang, Z. Q. Bitter melon extract attenuating hepatic steatosis may be mediated by FGF21 and AMPK/Sirt1 signaling in mice. Scientific Reports, Vol. 3, Article No.: 3142, 2013
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Richard, A.J., Amini-Vaughan, Z., Ribnicky, D. M., Stephens, J. M. Naringenin Inhibits Adipogenesis and Reduces Insulin Sensitivity and Adiponectin Expression in Adipocytes. Evidence-Based Complementary and Alternative Medicine, Vol. 2013, Article ID 549750, 10 pages, 2013.
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Target Audiences: People afflicted with diabetes and metabolic syndrome, Researchers studying diabetes and metabolic syndrome, as well as researchers and producers of dietary supplements and food. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project provides training for T-32 postdotroal fellows to understand the role of botanicals in the complex interactions between genetic, molecular, and physiological aspects of metabolic syndrome. Two postdoctoral fellows received training at Rutgers University and five at Pennington Botanical Research Center during this reporting period. Each postdoctoral fellow in this training program is encouraged to develop interdisciplinary research efforts to understand the effect and action of botanicals on components of the metabolic syndrome. The programs takes advantage of expertise in botanical research and discovery of novel botanical compounds through research conducted at the Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University. How have the results been disseminated to communities of interest? Research was described to groups of visitors including NJ farmers and students What do you plan to do during the next reporting period to accomplish the goals? A new screening program was initiated between Rutgers, PBRC, University of Louisiana at Lafayette and the USGS to expand the botanical research related to metabolic syndrome. Plants with folkloric history of traditional use were collected and vouchered in Louisiana and shipped to Rutgers to be made into extracts for testing at PBRC and Rutgers. The first set of extracts were produced at Rutgers and tested for anti-inflammatory activity in RAW macrophages using 4 inflammatory related markers. Inhibitory activity of IL-1β, COX-2 and iNOS was observed for select extracts. Bioavailability of active compounds will be assesed using the plasma of C57B1I6J mice after treatment by gavage, while the relative bioaccessibility will be measured using TIM model.
Impacts
What was accomplished under these goals?
Our research focuses on botanicals that can be used for the prevention/treatment of metabolic syndrome, defined as a condition whose major features consist of obesity, development of Type 2 diabetes and accelerated cardiovascular disease, and how the constituents of botanicals affect insulin signaling pathways and improve insulin resistance, the underlying metabolic dysregulation associated with metabolic syndrome. The extract of Artemisia dracunculus L. (PMI-5011) is the most characterized of the Artemisia species under study both in terms of its biological activity and its composition.PMI 5011 improves insulin action in vitro and in vivo, and its cellular mechanisms are being elucidated. Specific accomplishments include: 1) In preparation for clinical testing, a large commercial crop of Artemisia dracunculus was grown and flash frozen in NJ and further processed at the Louisiana State University food processing pilot plant. The polyphenols from Artemisia dracunculus were formulated with soy protein isolate to be administered as a food for future clinical testing. The food status of the material was confirmed by experts at the FDA. 2) The active polyphenols from Artemisia dracunculus L. were shown to bind effectively to soy protein isolate (Nutrasorb-5011) and have a similar profile to the ethanolic extract (PMI-5011). The relative bioaccessibility of the preparations were compared using the TIM-1 model of the upper gastrointestinal tract in humans. 3) Bioavailability of the active compounds was measured from the plasma of C57Bl6J mice after gavage with the formulations. Bioactivity of the preparations was evaluated using an acute assay of hyperglycemia reduction in C57Bl6J mice after treatment by gavage. Relative to PMI-5011, Nutrasorb-5011 exhibits greater bioaccessibility as measured using the TIM-1 unit, greater relative bioavailability as measured from analysis of plasma from treated mice and greater bioactivity as determined from blood glucose concentrations of treated mice. Our research findings indicate that specific food formulations of Artemisia polyphenols exhibit improved bioaaccessibility, bioavailability and bioactivity. More specifically, these studies examined how the active compounds from plants can be formulated with a food component such as soy protein to significantly improve the parameters related to the overall effectiveness of the botanicals in future clinical work. These efforts provide significant benefits to the biotechnology and pharmaceutical industries in the State and contribute to value added agriculture of New Jersey.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Obanda DN, Cefalu WT. Modulation of cellular insulin signaling and PTP1B effects by lipid metabolites in skeletal muscle cells. J Nutr Biochem. 2013 Aug;24(8):1529-37. doi: 10.1016/j.jnutbio.2012.12.014. Epub 2013 Mar 6. PubMed PMID: 23481236
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Wang ZQ, Zhang XH, Yu Y, Tipton RC, Raskin I, Ribnicky D, Johnson W, Cefalu WT. Artemisia scoparia extract attenuates non-alcoholic fatty liver disease in diet-induced obesity mice by enhancing hepatic insulin and AMPK signaling independently of FGF21 pathway. Metabolism. 2013 Sep;62(9):1239-49. doi: 10.1016/j.metabol.2013.03.004. Epub 2013 May 21. PubMed PMID: 23702383.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ribnicky DM, Roopchand DE, Oren A, Grace M, Poulev A, Lila MA, Havenaar R,
Raskin I. Effects of a high fat meal matrix and protein complexation on the
bioaccessibility of blueberry anthocyanins using the TNO gastrointestinal model
(TIM-1). Food Chem. 2014 Jan 1;142:349-57. doi: 10.1016/j.foodchem.2013.07.073.
Epub 2013 Jul 25. PubMed PMID: 24001852.
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Botanicals have been used for thousands of years as therapeutic agents for many diseases and human conditions. "Metabolic syndrome" is currently defined as a condition whose major features consist of obesity, insulin resistance, development of Type 2 diabetes and accelerated cardiovascular disease and continues to grow at epidemic proportions. Our BRC (Botanical Research Center) focuses on botanicals that can be used for the prevention/treatment of metabolic syndrome. One of the primary research objectives of the BRC as a whole focuses on how the constituents of botanicals affect insulin signaling pathways and improve insulin resistance, the underlying metabolic dysregulation associated with metabolic syndrome. While select species of Artemisia are under current evaluation by the BRC, the extract of Artemisia dracunculus L. (PMI-5011) is the most characterized both in terms of its biological activity and its composition of 6 compounds identified as having antidiabetic activity. PMI 5011, has been shown to improve insulin action in vitro and in vivo, but the cellular mechanisms remain elusive. Using differential proteomics, we have studied mechanisms by which PMI 5011 enhances insulin action in primary human skeletal muscle culture. Bioinformatics analyses determined that several metabolic pathways related to glycolysis, glucose transport and cell signaling were highly represented and differentially regulated in the presence of PMI 5011 indicating that this extract affects several pathways modulating carbohydrate metabolism, including translocation of GLUT4 to the plasma membrane. These findings provide a molecular mechanism by which a botanical extract improves insulin stimulated glucose uptake, transport and metabolism at the cellular level resulting in enhanced whole body insulin sensitivity. In addition to the mechanism work, the bioavailability of active compounds from Artemisia were measured and related to the formulation of the vehicle in C57Bl6J mice. Comparable bioaccessibility (i.e. potential to be bioavailable) of the active compounds was measured using the TNO intestinal model (TIM), a dynamic multi-compartmental, computer-controlled apparatus that closely simulates all in vivo conditions of the upper GI tract of humans. A comparison of test preparations showed significant improvements in the bioaccessibility, bioavailability and bioactivity for specific formulations relative to the extract alone. These effects will have a profound influence on the effective human dose of Artemisia and must be considered when planning clinical studies. PARTICIPANTS: Ilya Raskin - Principal Investigator David Ribnicky - Coordinator Andrew Oren - Laboratory Technician Alexander Poulev - Analytical Support Ruth Dorn - Laboratory Management and ordering supplies TARGET AUDIENCES: People afflicted with diabetes and metabolic syndrome, Researchers studying diabetes and metabolic syndrome, as well as Researchers and producers of dietary supplements and food PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Plants have served as a source of medicinal compounds for thousands of years and the research that we conduct helps to determine how the compounds from medicinal plants are able to provide a biological activity in animals or people. This biological would be considered the medical benefit of the medicinal plant. The research conducted with the gastrointestinal model is providing a way for us to determine the proper dose that should be used for upcoming clinical studies and the formulation that will deliver the maximum amount of the medicinal compounds needed for the medicinal benefit. This research will lead to new alternative strategies for the prevention and treatment of diabetes and should save in health-care expenditures for the State. In addition, the identification of novel plant based preparations for the prevention and treatment of diabetes and metabolic syndrome will be essential for the battle against this growing epidemic. A better understanding of how they work will provide consumers and researchers with information for their most effective use. These efforts contribute to value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- Scherp P, Putluri N, LeBlanc GJ, Wang, ZQ, Zhang XH, Yu, Y, Ribnicky D, Cefalu WT, Kheterpal I. (2012). Proteomic Analysis Reveals Cellular Pathways Regulating Carbohydrate Metabolism that are Modulated in Primary Human Skeletal Muscle Culture due to Treatment with Bioactives from Artemisia Dracunculus L. Journal of Proteomics, Jun 18;75(11):3199-210.
- Eisenman SW, Poulev A, Struwe L, Raskin I, Ribnicky DM. (2011). Qualitative variation of anti-diabetic compounds in different tarragon (Artemisia dracunculus L.) cytotypes, Fitoterapia, Oct;82(7):1062-74.
- Obanda DN, Hernandez A, Ribnicky D, Yu Y, Zhang XH, Wang ZQ, Cefalu WT. (2012). Bioactives of Artemisia dracunculus L. mitigate the role of ceramides in attenuating insulin signaling in rat skeletal muscle cells, Diabetes, Mar;61(3):597-605.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Despite the widespread use of botanicals for treatment and prevention of disease, including metabolic syndrome, the bioavailability of the active compounds from plant sources is generally very low and seldom considered as a significant factor related to bioactivity. However, the fate of these compounds in the GI tract prior to absorption is an important factor for the effective use of botanicals for the treatment and/or prevention of disease, either as dietary supplements or components of food. We have shown that an extract of Artemisia dracunculus (PMI-5011) decreased hyperglycemia in animal models for type 2 diabetes and improved insulin sensitivity in treated clinical subjects. We have also shown that anthocyanin-enriched extracts from low-bush blueberries were hypoglycemic in diabetic C57Bl6J mice while a clinical study recently conducted in patients treated with blueberry puree showed improvement in insulin resistance in the test group. Therefore, we evaluated the bioaccessibility of the bioactive compounds from Artemisia and blueberry using the TNO intestinal model (TIM) of the upper GI tract of humans. The standardized extracts were introduced to TIM in either the fasted or fed state with defined meal matrices. Absorption samples were collected from the jejunal and ileal compartments over a 4 hour run. Using HPLC-MS or HPLC-PDA, the 6 bioactive Artemisia compounds were quantified as highly bioaccessible with a meal matrix while the 20 different anthocyanins from blueberry were determined to be only 15% of the initial amount fed to TIM (ranging from 4% to 23%). The effects of carbohydrate and other meal matrices are being evaluated relative to the bioaccessibility of different types of bioactive compounds. In addition to Artemisia dracunculus, seeds from A. scoparia, A. rutifolia and A. santolinifolia were grown in the greenhouse for bulk plant cultivation to be used for the production of future batches of extract and for the collection of additional seed. Fractions from each of the study species were made using FCPC (fast centrifugal partitioning chromatography) based on separated peaks in the UV chromatogram taken at 254 nm. Ten fractions were collected from each of A. santolinifolia, A. scoparia and A. rutifolia. The extracts and fractions were tested using insulin sensitivity related assays and specific fractions identified as active. A new set of 15 fractions was also made from PMI-5011 using the same FCPC gradient that was used for the other Artemisia species. A recent result revealed a role of ceramide in association with insulin sensitivity in PMI-5011 treated skeletal muscle cultures. New fractions of PMI-5011 showed significant activity by bioactivity guided fractionation and new bioactive compounds are currently being purified and identified. Saint John's Wort extract (SJW) was also discovered to have an effect on adipogenesis. Seeds were also obtained from a commercial source and grown in the greenhouse. Extract and extract fractions were made from the plants and tested in adipogenesis assays. The extract was shown to have a negative effect on insulin sensitivity. PARTICIPANTS: Ilya Raskin - Principal Investigator, David Ribnicky - Coordinator, Andrew Oren - Laboratory Technician, Alexander Poulev - Analytical Support, Ruth Dorn - Laboratory Management and ordering supplies TARGET AUDIENCES: People afflicted with diabetes and metabolic syndrome, Researchers studying diabetes and metabolic syndrome, as well as Researchers and producers of dietary supplements and food. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies are used to help us understand how some of the compounds that are contained in medicinal plants work in animals and people and which compounds are the most biologically active. Multiple Artemisia species are used for this work in order to determine which species will provide the greatest benefit. The studies using Saint John's Wort demonstrate a potential negative effect of the extract on metabolic syndrome which may be significant for public health considerations, and has not been previously reported. This research contributes to the value added agriculture of New Jersey and provides significant benefits to the biotechnology and pharmaceutical industries in the State by suggesting new strategies for the prevention and treatment of diabetes. In doing so, this could save in health-care expenditures for the State. In addition, the identification of novel plant based preparations for the prevention and treatment of diabetes and metabolic syndrome will be essential for the battle against this growing epidemic. A better understanding of how they work will provide consumers and researchers with information for their most effective use. These efforts contribute to value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- Lila, M.A., Ribnicky, D.M., Rojo, L.E., Rojas-Silva, P., Oren, A., Havenaar, R., Janle, E.M. Raskin, I., Yousef, G.G., Grace, M.H. Complementary Approaches to Gauge the Bioavailability and Distribution of Ingested Berry Polyphenolics. J. of Agric. Food Chem. PMID:22111523. 2011.
- Buehrer, B.M., Duffin, D.J., Lea-Currie R., Ribnicky, D.M., Raskin I., Stephens J.M., Cefalu, W.T., and Gimble J. Tools for the identification of bioactives impacting the metabolic syndrome: Screening of a botanical extract library using subcutaneous and visceral human adipose-derived stem cell based assays. Journal of Nutritional Biochemistry. In Press 2011.
- Richard, A.J., Amini, Z., Ribnicky, D.M., and Stephens, J.M. St. John's Wort inhibits insulin signaling in murine and human adipocytes. Biochimica et Biophysica Acta. In Press 2011.
- Zhong Q.W., Yongmei Y., Zhang X.H., Ribnicky D.M., Cefalu W.T. Ecdysterone Enhances Muscle Insulin Signaling by Modulating Acylcarnitine Profile and Mitochondrial Oxidative Phosphorylation Complexes In Mice Fed A High-fat Diet. Diabetes. Accepted 2011.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: PMI-5011 is a botanical extract prepared from Artemisia dracunculus L. (Russian tarragon), a culinary herb with anti diabetic properties. PMI-5011 treatment decreases blood glucose concentrations in STZ-induced diabetic mice and genetically diabetic KK-Ay mice but does not effect blood glucose concentrations in non-diabetic mice or rats. Studies recently completed show that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling by modulating muscle protein degradation and phosphatase activity as a possible mode of action. We have also expanded the research topic area to include anthocyanin bioactivity assessment with berry extracts. Blueberry and maqui berry are rich sources of anthocyanins thought to convey the health benefits of the berries. A major dilemma has persisted with the use of anthocyanins which convey a variety of biological activities but are noted to be highly unstable and poorly bioavailable. Using the TNO intestinal model (TIM) for the human upper gastrointestinal tract, the bioaccessibility of bluberry anthocyanins was evaluated in the fed and fasted state over a time course of 4 h after ingestion. The total bioaccessibility of the individual anthocyanins ranged from 4.3% to 36.9% in the fasted state and did not seem to correlate to the absolute amount of anthocyanin in the extract (i.e. the most bioaccessible anthocyanin was present at about one third the concentration of the most abundant anthocyanin in the extract, but was twice as accessible). The total bioaccessibility of the anthocyanins in the fed state was 17%, only slightly higher than the bioaccessibility in the fasted state (14%) and the bioaccessibility of each anthocyanin was similar overall. For the fed studies, a high fat diet was used to examine the effects of hydrophobic food components on bioaccessibility of polar phytochemicals, but diets of different compositions may exhibit different effects. Certainly, bioaccessibility does not ensure bioavailability, as evidenced in low bioavailability reports for anthocyanins. However, low bioaccessiblity is predictive of low bioavailability. Bioaccessibility determination using the dynamic conditions of TIM is also a unique way to determine the stability of anthocyanins in the biological conditions of the upper gastrointestinal tract. A greater understanding of the metabolism and absorption of important phytonutients like anthocyanins will enable the development of food products with greater potential to provide the maximum therapeutic effects. Determination of bioaccessibility using TIM provides a unique opportunity to explore the fate of important phytochemicals between the act of ingestion and the bioconversion in the colon or modification during absorption in vivo, a process which is otherwise quite difficult to address. PARTICIPANTS: Individuals: Dr. Ilya Raskin (PI) is the laboratory leader and contributes to the design of the experiments and the interpretation of data associated with the project (Effort: 20%). Dr. David Ribnicky (PD) contributes to the design of the experiments and the interpretation of data and conducts the experiments comprising the project. He also assembles the data into reports and manuscripts. DR is also the site manager of the TIM laboratory where the bioaccessibility experiments are performed. Partner Organization: Pennington Biomedical Research Center, Louisiana State University, 6400 Perkins Road, Baton Rouge, LA, 70808 NC State University, Plants for Human Health Institute, 600 Laureate Way, Kannapolis, NC 28081 Netherlands Organization for Applied Scientific Research (TNO), Schoemakerstraat 97 (Building A), 2628 VK Delft, Holland Collaborators and contacts: Dr. Judy Storch, SEBS - Nutritional Sciences, 96 Lipman Drive, New Brunswick, NJ 08901-8525 Dr. Paul Takhistov, SEBS - Food Science, 65 Dudley Road, New Brunswick, NJ 08901-8520 Dr. James Simon, SEBS - Plant Biology & Pathology, 59 Dudley Road, New Brunswick, NJ 08901-8520 Training: Training in techniques used in botanical research, including extraction, analysis, and samples processing, are continuously provided to undergraduate and graduate students, as well as to post-doctoral fellows. TARGET AUDIENCES: The results of our research will target individuals affected by diabetes and metabolic syndrome, medical doctors, and nutritionists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies are used to help us understand how some of the compounds that are contained in medicinal plants work in animals and people with a new emphasis on the compounds from berries and which compounds are the most biologically active. These findings can also help to predict how to properly formulate the botanical medicines to make them more effective at improving medical conditions, such as diabetes. This research contributes to the value added agriculture of New Jersey and provides significant benefits to the biotechnology and pharmaceutical industries in the State by suggesting new strategies for the prevention and treatment of diabetes. In doing so, this could save in health-care expenditures for the State. Properly formulated botanicals can provide significantly greater health benefits for the battle against metabolic syndrome and diabetes. By understanding the mechanisms regulating digestion of botanical bioactives , more effective formulations that maximize the beneficial activities associated with specific plant derived compounds can be produced. These efforts contribute to value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- Ilnytska O., Stuetz A.M., Park-York M., York A.D., Ribnicky D.M., Zuberi A., Cefalu W.T., Argyropoulos G. (2011). Molecular mechanisms for activation of the Agouti-related protein and stimulation of appetite. Diabetes, Jan 60(1): 97-106. Epub 2010 Oct 27.
- Wang Z.Q., Ribnicky D.M., Zhang X.H., Zuberi A., Raskin I., Yu U., Cefalu W.T. (2011). An extract of Artemisia dracunculus L. enhances insulin receptor signaling and modulates gene expression in skeletal muscle in KKay mice. J. Nutr. Biochem. , Jan 22(1): 71-8. Epub 2010 May 5. PMC Journal (In Process)
- Watcho P., Stavniichuk R., Ribnicky D.M., Raskin I., Obrosova I.G. (2010). High fat diet-induced neuropathy of prediabetes and obesity: effect of PMI-5011, an ethanolic extract of Artemisia dracunculus L. Mediators of Inflammation, Volume 2010, 1-10 Article ID 268547
- Kheterpal I., Coleman L., Ku G., Wang Z.Q., Ribnicky D., Cefalu W.T. (2010). Regulation of insulin action by an extract of Artemisia dracunculus L. in primary human skeletal muscle culture, a proteomics approach. Phytother. Res., Sep 24(9): 1278-84. PMC Journal (In Process)
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: PMI-5011 is a botanical extract prepared from Artemisia dracunculus L. (Russian tarragon), a culinary herb with anti diabetic properties. PMI-5011 treatment decreases blood glucose concentrations in STZ-induced diabetic mice and genetically diabetic KK-Ay mice but does not effect blood glucose concentrations in non-diabetic mice or rats. Studies recently completed show that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling by modulating muscle protein degradation and phosphatase activity as a possible mode of action. We have also expanded the research topic area to include anthocyanin bioactivity assessment with berry extracts. Blueberry and maqui berry are rich sources of anthocyanins thought to convey the health benefits of the berries. Treatment by gavage (500 mg/kg body wt) with a phenolic-rich extract and an anthocyanin-enriched blueberry extract formulated with Labrasol lowered elevated blood glucose levels by 33 and 51%, respectively, comparable to the anti-diabetic drug metformin. The greater hypoglycemic activity of the anthocyanin-enriched fraction compared to the initial phenolic-rich extract suggested that the activity was due to the anthocyanin components. Treatment by gavage (300 mg/kg) with the pure anthocyanins, formulated with Labrasol, showed that malvidin-3-O- glucoside was significantly hypoglycemic while delphinidin-3-O-glucoside was not. Anthocyanin enriched and crude extracts of maqui berry were also significantly hypoglycemic in the acute model and most active when provided in a formulation of water only. Additional studies were completed looking at the antidiabetic effects of anthocyanin extracts of berries used in the diets of Native North American Indians (central U.S.) as well as extracts of different berries used by Natives in Alaska. Preliminary studies using the TIM system suggest significant differences in the bioaccessibility of each of the anthocyanins from berry extracts. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies are used to help us understand how some of the compounds that are contained in medicinal plants work in animals and people with a new emphasis on the compounds from berries and which compounds are the most biologically active. These findings can also help to predict how to properly formulate the botanical medicines to make them more effective at improving medical conditions, such as diabetes. This research contributes to the value added agriculture of New Jersey and provides significant benefits to the biotechnology and pharmaceutical industries in the State by suggesting new strategies for the prevention and treatment of diabetes. In doing so, this could save in health-care expenditures for the State.
Publications
- Wang ZQ, Ribnicky DM, Zhang XH, Zuberi A, Raskin I, Yu U, Cefalu WT. An extract of Artemisia dracunculus L. enhances insulin receptor signaling and modulates gene expression in skeletal muscle in KKay mice. J Nutr Biochem 2010 (In Press).
- Kellogg J, Ribnicky D, Wang J, Flint C, Kuhn P, Raskin I, Lila MA. Alaskan wild berry resources and human health under the cloud of climate change. 2010 J Agric Food Chem (In Press). NIHMS# 161682
- Cefalu WT, Ribnicky DM. Modulation of Insulin Action by Botanical Therapeutics. Obes Weight Manag 2009 5(6) 277-281.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: PMI-5011 is a botanical extract prepared from Artemisia dracunculus L. (Russian tarragon), a culinary herb with anti diabetic properties. PMI-5011 treatment decreases blood glucose concentrations in STZ-induced diabetic mice and genetically diabetic KK-Ay mice but does not effect blood glucose concentrations in non-diabetic mice or rats. Bioactivity guided fractionation of the extract using in vitro assays resulted in the purification of 6 compounds that may be responsible for the activities observed in vivo. The presence of these compounds in hydrolyzed plasma was confirmed using ESI-LC-MS. The chalcone, 2′, 4′-dihydroxy-4-methoxydihydrochalcone, was observed to be the most active and abundant compound identified. The chalcone was chemically synthesized for further testing. A non-hypoglycemic pharmaceutical excipient, Labrasol, was used to solubilize the chalcone for testing. C57BL/6J mice maintained on a high fat diet to induce diabetes were gavaged with the chalcone (50, 150 and 300 mg per kg body weight) or metformin (300 mg per kg body weight) as a positive control, after 4h of food restriction. The chalcone had comparable hypoglycemic activity (30+5%) as metformin (34+6%) when administered at 300 mg/kg to the mice and was significantly active at the low dose of 50 mg/kg (14+7%). Blood plasma samples (200 microL) were obtained at 4h post gavage. Native chalcone was detected at high concentrations (1-2 microgram/ml) in hydrolyzed plasma of treated animals but was not detected in unhydrolyzed plasma. The unhydrolyzed plasma contained high concentrations of a modified form of the chalcone with mass spectral properties consistent with a glucuronic acid ester. This glucuronide appears to be the transported form of the chalcone and may also be the active form in vivo. The evaluation of additional formulations is ongoing to identify an appropriate excipient for the next clinical study. The sera samples from the patients that completed a successful clinical study looking at the effect of PMI-5011 were analyzed for the presence of the active compounds. The initial analysis suggests a correlation of the presence of the compounds in the plasma with the patients that were good responders as measured by improved insulin sensitivity. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The elucidation of mechanisms regulating post-ingestive modification of botanical bioactives may lead to more effective formulations that maximize the beneficial activities associated with specific plant derived compounds. These efforts contribute to value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- David M Ribnicky, Peter Kuhn, Alexander Poulev, William T Cefalu; Ilya Raskin 2008. Bioavailability assessment and enhancement of an extract of Artemisia dracunculus for alleviating hyperglycemia in C57. International Journal of Pharmaceutics. Accepted
- Mary H. Grace, David M. Ribnicky, Peter Kuhn, Alexander Poulev, Sithes Logendra Gad G. Yousef; Ilya Raskin, Mary Ann Lila 2008. Hypoglycemic Activity of a Novel Anthocyanin-Rich Formulation from Lowbush Blueberry, Vaccinium angustifolium Aiton Phytochemistry. Accepted
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The botanical extract of Artemisia dracunculus, called PMI-5011, was shown to decrease hyperglycemia and improve insulin sensitivity in animal models for Type 2 diabetes and in vitro studies employing bioactivity guided fractionation of the extract resulted in the purification of 6 compounds suggested to be responsible for the hypoglycemic activity observed in vivo as previously reported. The bioavailability of the compounds was examined because while efforts were made to make a more potent dosage form, extract preparations containing higher concentrations of active compounds were not more active in vivo. To evaluate the bioavailability of active compounds, C57BL/6J mice were gavaged with PMI-5011 at 500 mg/kg body weight, after 4 h of food restriction. Blood plasma samples (200 uL) were obtained at specified times (2, 4, 6, 8 and 24 h) after ingestion, treated with β-glucuronidase and sulfatase to release chemically bound forms of flavonoids and related compounds. The
concentrations of the active compound in the blood were based on the abundance of the masses of the compounds in the sera as measured by electrospray LC-MS and determined to be highest at 4-6 h after gavage. Formulations of the extract with bioenhancers/solubilizers, such as DMSO and mixtures of glyceride esters of polyethylene glycol with fatty acids that are used for self emulsifying drug delivery systems (SEDDS), e.g. Labrasol, were evaluated in vivo for hypoglycemic activity. In addition, the effectiveness of the formulations was evaluated based on the abundance of active compounds in blood sera. At doses of 50-500 mg/kg/day, the hypoglycemic activity of the extract was enhanced 3-5 fold using Labrasol, equal to or greater than the activity for the same dose of the drug metformin used as a positive control in these studies. The active compound, 2′, 4′-dihydroxy-4-methoxydihydrochalcone, was also tested with Labrasol and had equal or greater hypoglycemic activity than
metformin at equal doses of 200-300 mg/kg/day, thus validating the compound as active. Therefore, bioenhancing agents such as Labrasol can be effectively used with multi-component botanical therapeutics such as PMI-5011 to provide greater activity with lower dose. An understanding of the bioactive components and methods to evaluate their bioavailability, however, is essential to effectively test improved formulations. A clinical study looking at the effect of PMI-5011 treatment to improve insulin sensitivity in resistant patients was recently completed with positive results. The sera samples from the patients are currently being analyzed for the presence of the active compounds to see if the plasma concentration of the active compounds is related to the degree of response.
PARTICIPANTS: Our research group have had several successful scientific collaborations with partnering institutions in the area of botanical research. Pennington Biomedical Research Center, Baton Rouge, LA collaborates with our group in research related to Metabolic Syndrome. The University of Illinois, Urbana-Champaign, Il. works with our research group in research related to botanical extracts including those extracts that contain anthocyanins. Training is continuously provided to undergraduate students. Techniques in botanical research including extraction, analysis, and sample processing were taught to students.
Impacts
This research validates that the compounds identified in vitro may be responsible for the in vivo activity of the extract and provides the necessary information to evaluate bioavailability of the botanical. A new formulation was then used to increase the effectiveness of the botanical. These efforts contribute to value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- Bioactives of Artemisia dracunculus L. enhance cellular insulin signaling in primary human skeletal muscle culture. Wang ZQ, Zhang X, Ribnicky DM, Raskin I, Cefalu WT. Metabolism 2008 in press
- Phytochemical composition and metabolic performance enhancing activity of dietary berries traditionally used by Native North Americans.Burns Kraft, T.F., M. Dey, R.B. Rogers, D.M. Ribnicky, D.M. Gipp, W.T. Cefalu, I. Raskin, M.A. Lila. 2008. J. Food Chem. in press
- Botanicals and metabolic syndrome. Cefalu, W.T., J. Ye, A. Zuberi, D.M. Ribnicky, I. Raskin, Z. Liu, Z.Q. Wang, P.J Brantley, L. Howard, M. Lefevre. (2008) Am. J. Clinical Nutrition . in press
- Evaluation of botanicals for improving human health. Ribnicky, D.M., A. Poulev, B. Schmidt, W.T. Cefalu, I. Raskin (2008) Am. J. Clinical Nutrition in press
- A Natural History of Botanical Therapeutics. Barbara Schmidt, David M Ribnicky, Alexander Poulev, Sithes Logendra, William T Cefalu and Ilya Raskin (2008). Metabolism in press
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Progress 01/01/06 to 12/31/06
Outputs
PROGRESS: 2006/01 TO 2006/12 An extract of Artemisia dracunculus, was shown to decrease hyperglycemia and improve insulin sensitivity in animal models for type 2 diabetes. The research on this project was expanded to include studies addressing bioavailability. Previously, in vitro studies, employing bioactivity guided fractionation of the extract, resulted in the purification of 6 compounds that were suggested to be responsible for the activities observed in vivo. The activities included the inhibition of several factors relating to diabetes including aldose reductase activity, PEPCK gene expression (stimulated) and PTP-1B activity and gene expression. One aspect of demonstrating that the compounds have activity in vivo involves the measurement of the compounds in the plasma of treated animals. Methods of plasma analysis were evaluated directly since different treatments were described in the literature. Flavonoids and related compounds, such as those identified as
the actives in the extract, are usually modified in the plasma making their direct measurement difficult and requiring some form of hydrolysis. In order to evaluate the plasma appearance of active metabolites, C57BL/6J mice were gavaged with 500 mg extract per kg body weight, after 4 h of food restriction. Blood plasma samples (200 uL) were obtained at specified times (2, 4, 6, 8 and 24 h) after ingestion. The samples were treated with β glucuronidase and sulfatase, which releases chemically bound forms of flavonoids and related compounds for purposes of detection and partitioned with hexane to selectively remove fatty acids and lipids that interfere with the analysis. A sensitive LC-MS/MS assay was developed to detect and quantitatively estimate the compounds in the plasma. The masses of the active compounds in the blood of treated mice (i.e. sakuranetin, MW 286) were detected by electrospray LC-MS but were not detectable by electron impact LC-MS. The abundance of the ions was
then used to determine the time course for appearance of the components of the extract in the blood plasma. The maximal concentration for most of the compounds occurred at 4-6 h after treatment. In addition, measurements of the compounds in the blood were used to evaluate the effectiveness of Self Emulsifying Drug Delivery Systems (SEDDS) that are known to enhance the solubility and availability of poorly soluble drug compounds. The SEDDS did increase the concentrations of the active compounds in the blood relative to the control treatments.
Impacts
This research will provide credibility to the study of botanicals and, in turn, benefit the value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- David Ribnicky, Sithes Logendra, Alexander Poulev, William T Cefalu & Ilya Raskin Compounds from an extract of Artemisia and methods for treating disorders. Patent filed in March 2006.
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Progress 01/01/05 to 12/31/05
Outputs
Work has continued on the development of an extract from the shoots of Artemisia dracunculus for the treatment and prevention of diabetes and its complications. Efforts have been focused on the identification of specific compounds involved with each of the distinct modes of action that were previously discovered. The Artemisia extract was shown to be a potent aldose reductase (ALR2) inhibitor. Aldose reductase is the first enzyme involved in the polyol pathway that is centrally associated with the etiology of major diabetic complications such as diabetic neuropathy, nephropathy, retinopathy and cardiovascular diseases. Bioactivity guided fractionation and isolation of the compounds that inhibit ALR2 activity was performed with the extract, yielding four identified compounds. The extract was also shown to decrease PEPCK overexpression in diabetic mice. Because of the large amounts of material needed for screening for activity using animals an alternative novel, a
Real-Time PCR-based assay was developed to guide the fractionation and isolation of the PEPCK inhibitors. Two compounds were purified by preparatory HPLC and chemically identified that were able to reduce PEPCK mRNA levels by 68% at 10 g/ml and more than 50% at 25 g/ml, respectively. The effect of these compounds on PEPCK gene expression has not been previously reported. Since the Artemisia extract decreases insulin resistance in skeletal muscle cultures, protein tyrosine phosphatase-1B (PTP-1B) activity was used as a screen to identify active compounds. PTP-1B is a key regulatory element in the insulin-signaling pathway. PTP1B activity and mRNA assays indicated fraction 7 of 10 fractions was the most active and sub-fractions were prepared. Two distinct compounds with an inhibitory effect on PTP1B activity were purified and identified. They decreased PTP-1B activity by 26 % and 36%, respectively. The latter compound significantly reduced PTP 1B mRNA level by 29% suggesting that
suppressed gene expression is involved in the inhibitory effect of this compound on PTP1B activity. The identification of these active compounds substantiates the efficacy of the extract and will provide the information needed to standardize the product and for growers to optimize production.
Impacts
This research will benefit the value added agriculture of New Jersey, as well as provide significant benefits to the biotechnology and pharmaceutical industries in the State.
Publications
- Sithes Logendra, David M Ribnicky, Hui Yang, Alexander Poulev, Jun Ma, Edward J Kennelly, Ilya Raskin (2005. Bioassay-guided isolation of aldose reductase inhibitors from Artemisia dracunculus. Submitted to Journal of Natural Products. David M. Ribnicky, Alexander Poulev, Malcolm Watford, William T. Cefalu, Ilya Raskin (2005) Antihyperglycemic Activity of TARRALIN (trademark), an Ethanolic Extract of Artemisia dracunculus L. Phytomedicine. Accepted.
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Progress 01/01/04 to 12/31/04
Outputs
While the elicitation studies performed with acetate to induce secondary biochemical pathways showed promising results as a model system for the production of bioactive compounds in roots, more recent studies have shifted our focus to shoot production of Artemisia dracunculus for the prevention and treatment of diabetes. The ethanolic extract of A. dracunculus, a plant with a rich history of medicinal properties decreases hyperglycemia in chemically induced and genetically diabetic mice. The extract decreases PEPCK expression in vivo and increases insulin stimulated glucose uptake in muscle cell cultures as potential modes of action. Preliminary evaluation of the initial results of muscle culture assays suggest that the extract lowers insulin resistance in mildly diabetic people. The extract also inhibits aldose reductase activity in vitro suggesting it may be useful for treating the complications of diabetes as well as the alterations in blood glucose metabolism. The
in vitro assays were used for the activity-guided fractionation of the extract necessary for defining a preferred biochemical profile to be used for the standardization and optimization of the extract. The preliminary identification of the active components of the extract suggests that they consist of flavonoids, coumarins and related derivatives. The content of these compounds within the plant was optimized through specific growth and elicitation conditions that provide a consistently active extract. Current efforts are now focused on alternative production methods to further enhance the content of active components thus producing a more efficacious preparation.
Impacts
The proposed study is intended to benefit the value added agriculture of New Jersey as well as provide tangible benefits to the biotechnology and pharmaceutical industry in the State.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
This study is designed to explore the biochemical and molecular mechanisms of acetate-induced elicitation of secondary metabolism in hydroponically grown plants to determine whether elicitation occurs indirectly by inducing signal transduction mechanisms leading to the activation of biosynthetic pathways, or directly by providing carbon building blocks for the formation of secondary metabolites that increase or appear following acetate treatment. The first set of experiments performed was to observe whether the effects of elicitation could be enhanced by in vitro elicitation as opposed to eliciting the entire plant. The roots of Anchusa officinalis and Malva moschata grown in hydroponics were harvested. The fresh roots were placed in elicitors at 1 g fresh weight/10 ml elicitor. The roots were elicited for 24 hours on a shaker. The roots were rinsed prior to lyophilization. Dried roots were ground and extracted in 80% methanol for 48 hours. Extracts were analyzed with
LC-MS. In parallel roots were elicited in vivo. The elicitors used were water, 0.01%, 0.1% acetic acid. There was a decrease in yield of extract from the in vitro elicited roots compared to the whole plants. The quality of the peaks obtained (intensity and number) was similar to the extracts obtained when the whole plant was elicited. There was no benefit found for the in vitro elicitation.
Impacts
The proposed study is intended to benefit the value added agriculture of New Jersey as well as provide tangible benefits to the biotechnology and pharmaceutical industry in the State.
Publications
- There are no publications at this time, since the project just recently started in September 2003.
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Progress 01/01/02 to 12/31/02
Outputs
In the last year we were able to demonstrate and summarize the dramatic effect of elicitation on the production of biologically active phytochemicals in the roots of hydroponically grown plants. To select the most potent and diverse elicitors, we screened 25 known bioactive compounds for their ability to elicit quantitative and qualitative changes in the biochemical composition of roots of several hydroponically grown plant species. Acetate (0.1%), methyl jasmonate (0.1 mM), methyl salicylate (0.8 mM) and chitosan (0.1%) were found to be the most effective. Some of these compounds were previously reported to have elicitor function in roots or cell cultures. These four elicitors were used to induce secondary metabolite synthesis in 588 plant species chosen from a broad taxonomical background. Root extracts produced from elicited and non-elicited plants were screened in anti-cancer assays performed by the National Cancer Institute, Division of Cancer Treatment and
Diagnosis, Developmental Therapeutics Program. As a result, 119 species had at least one anti-cancer activity in either elicited root extracts, non-elicited extracts or both, producing an overall 20% hit rate. Out of 119 active species, 39 were active against one cancer cell line, 25 against two and 55 against three. While all species were tested against the breast cancer cell line, fewer species were tested against cell lines representing the other four forms of cancer. Seventy-six elicited species had unique activity against at least one cancer cell line without detectible activity in the corresponding non-elicited samples. Samples from an additional 17 species were active against one cancer cell line only after elicitation, whereas non-elicited samples from the same species were inactive against this cell line. Data indicate that 64% of plant species (76 out of 119) would have been missed during the more conventional bioprospecting activity, leaving only 43 species active in the
non-elicited state as potential sources of anti-cancer leads. The percentage of missed leads would be even greater if only one cancer cell line was used for screening (79% for breast cancer, 72% for melanoma, and 77% for lung cancer). Only 11 non-elicited species had activity against at least one cancer cell line, while none of the elicited samples from the same species were active. Non-elicited samples of two additional species were active against a particular cancer cell line, while the elicitation of the same species produced activities against different cell lines. The analysis of the effects of different elicitors on plant species clearly demonstrates that for most targets acetate was by far more effective than other elicitors. The quantitative and qualitative effects of acetate on the biochemical profile of the hydroponically grown roots could be easily demonstrated chromatographically in tested species subjected to the biochemical fingerprinting with the HPLC.
Impacts
The above results further demonstrate that root elicitation technology dramatically increases the chances of finding and anti-cancer compounds in plants. This technology was used successfully to specifically identify a number of potential anti-cancer products that can be developed as a nutraceutical, pharmaceutical (botanical drug) or both.
Publications
- Poulev, A., J.M. O'Neal, S. Logendra, R.B. Pouleva, V. Timeva, A.S. Garvey, D. Gleba, I.S. Jenkins, B.T. Halpern, R. Kneer, G.M. Cragg, and I. Raskin. 2002. Elicitation - a new window into plant chemodiversity and phytochemical drug discovery. J. Med. Chem, accepted.
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Progress 01/01/01 to 12/31/01
Outputs
We have successfully carried out the continuation of the project by achieving the following goals with a portion of the financial support obtained from the NJ39101 project. A library of close to 8,000 unique extracts from elicited and non-elicited roots representing 1,400 species from 155 families has been produced. Over 200 in-house anti-microbial leads confirmed, 12 advanced in further development. Over 200 NCI oncology leads confirmed in collaboration with U.S. National Cancer Institute. Nine advanced in further development. Over 20 lead compounds isolated, 5 structures identified. Ability to re-supply the active ingredient confirmed. We have concentrated on the anti-fungal compounds produced from the elicited roots. We have further optimized plant cultivation, elicitation, harvesting and extraction protocols to enhance the ability to identify bioactive compounds in plant roots. Acetic acid was found to be the best elicitor for the stimulation of the production of
anti-fungal and anti-cancer compounds in hydroponically grown plant roots. Eight of the most potent anti-fungal compounds were isolated, partially characterized and tested against three human pathogens, i.e., Candida albicans, Epidermophyton floccosum and Trichophyton mentagrophytes. The Minimum Inhibitory Concentrations (MIC) for these anti-fungal leads ranged from 1 to 100 ug/ml, putting these leads in the potency range of the current anti-fungal pharmaceuticals. In addition, 8 samples were identified by NCI as having strong and selective anti-cancer activity that did not match the selectivity profiles of any known plant product.
Impacts
The above results further demonstrate that root elicitation technology dramatically increases the chances of finding anti-microbial and anti-cancer compounds in plants. This technology was used successfully to specifically identify a number of potential anti-cancer, anti-fungal and anti-microbial products. Each of these products can be developed as a nutraceutical, pharmaceutical (botanical drug) or both. Based on the produced results, a joint development agreement was signed with Eisai Co. (one of the largest Japanese pharmaceutical company) on the discovery and development of novel anti-cancer leads produced in our laboratory partially as a result of the NJ39101 funding. Also a U.S. patent covering aspects of the elicitation technology was filed.
Publications
- Raskin, I.2001. Last year the results of this work were presented at three international meeting and at 8 pharmaceutical companies. One patent was filed on the elicitation technology. A manuscript summarizing the latest results is in preparation and will be submitted shortly.
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Progress 01/01/00 to 12/31/00
Outputs
In previous years we were very successful in using rhizosecretion to optimize the production of various valuable compounds secreted from plant roots and to discover new activities associated with these compounds. We have used genistein as the model compound for this research. Currently, we have expanded our research to eliciting compounds inside the roots of various plants, in addition to looking for them in the hydroponic medium. From the genistine example we believe that most if not all compounds secreted form the roots can be successfully recovered in root extracts. We have specifically concentrated on anti bacterial and anti fungal compounds, such as genistein present in the extracts of hydroponically grown and elicited roots. We have subjected the hydroponically grown roots of 720 different plant species to three to four different abiotic elicitors developed and optimized for the rhizosecretion of genestein. We have developed and implemented the following
procedure for preparing the extracts of the hydroponically grown roots of various plant species and for testing these extracts for anti microbial activity. Bioassay of activity Five different organisms are used for antibacterial and antifungal screening. The standard method used to determine in vitro antibacterial and antifungal activity of plant root extracts consists of utilizing suspensions of microorganisms and monitoring for growth inhibition. Antibacterial and antifungal activity is indicated when a 30 percent or more reduction in growth of cells or spores is visible in the presence of the plant extracts. All extract samples are plated in 3 replicates, the lone well existing as the control. After 24 hours of incubation at 30 degrees C, plates screened for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Saccharomyces cerevisiae are examined for the presence or absence of antibacterial activity. Plates screened for Aspergillus niger are examined for antifungal
activity after 48 hours of incubation at 30 degrees C. Activity is rated from 1 to 5, 1 being the lowest, very little activity, and 5 being the highest, a complete kill. All extracts that show favorable antibacterial or antifungal results are cataloged into a specific database as well as in the daily notebook of the laboratory technician. Using these assays on 720 plant species, approximately 3000 samples we have obtained the following results. 156 samples with antibacterial activity. 77 samples with antifungal activity.
Impacts
The above results demonstrate that root elicitation technology dramatically increases the chances of finding antimicrobial compounds in plants, with an antimicrobial, hit rate, approaching 15 percent. Therefore, the technology we have developed has produced a formidable pipeline of botanical antimicrobial leads for future development. Some of these leads are currently being tested in the advanced antimicrobial screens. Early results are very encouraging. Each of these products can be developed as a nutraceutical, pharmaceutical i.e., botanical drug or both. Industrial partners are being recruited for each of the late development candidates identified as the group's strategic priority.
Publications
- Kneer, R., A.A. Poulev, A. Olesinski, and I. Raskin. 1999. Characterization of the elicitor-induced biosynthesis and secretion of genistein from roots of Lupinus luteus L. J. Experimental Bot. 339: 1553-1559.
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Progress 01/01/99 to 12/31/99
Outputs
Genistein is a multi-functional isoflavonoid naturally secreted from roots of hydroponically grown legume plants. Roots of hydroponically cultivated yellow lupine, Lupinus luteus L., plants, transferred into water secreted minor amounts of genistein (about 5 microgram/g fwt). Secretion of genistein from L. luteus roots (rhizosecretion) was stimulated dramatically to over 100 microgram/g root fresh weight by soluble chitosan, salicylic acid (SA) and potassium cyanide (KCN) supplied at 0.12% (w/v), 800 microM, and 400 microM respectively. Other identified elicitors caused smaller induction of genistein rhizosecretion. Increased levels of genistein in root exudates corresponded to greater amounts of genistein in root tissue. Elicitor-induced rhizosecretion of genistein was based on de-novo synthesis and was inhibited by glyphosate and other less specific metabolic inhibitors. Except for NaF:AlCl3, all tested elicitors of genistein rhizosecretion produced a distinct
bell-shaped dose-response curve. Most of the elicitor-induced rhizosecretion of genistein occurred during the first day, followed by a gradual decline. Additional elicitor treatments had little effect on genistein rhizosecretion, indicating that the induction of genistein rhizosecretion by the identified elicitors is a one-time event. There is a growing interest in the mechanisms of compound exudation from roots and an increasing demand for genistein and other biologically active root-secreted compounds. We propose that many of these compounds can be commercially produced by continuous secretion (exudation) from the roots of hydroponically grown plants. We termed this process "rhizosecretion" and chose genistein as a model compound for characterizing biological mechanism of root exudation and for elucidating conditions that can increase the yield of compounds exuded from plant roots.
Impacts
Genistein also has several desirable pharmacological properties. It has anti-cancer, cancer-preventive and phytoestrogenic activities amongst several others So far most of the commercially available genistein is extracted from the soybean flour, where it is present in low levels. Root exudates may provide a more concentrated source of this valuable compound, if conditions to maximize its rhizosecretion can be optimized in a large-scale commercial hydroponic system.
Publications
- Ralf Kneer, Alexander A. Poulev, Amnon Olesinski, and Ilya Raskin. 1999. Characterization of the elicitor-induced biosynthesis and secretion of genistein from roots of Lupinus luteus L.J. Experiment.Bot.Vol.50, No. 339, pp.1553-1559.
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Progress 01/01/98 to 12/31/98
Outputs
Genistein is a multi-functional isoflavonoid naturally secreted from roots of hydroponically grown legume plants. Roots of hydroponically cultivated yellow lupine, lupinus luteus L., plants, transferred into water secreted minor amounts of genistein 9about 5 ug/g fwt). Secretion of genistein from L. luteus roots (rhizosecretion) was simulated dramatically to over 100ug/g root fresh weight by soluble chitosan, salicylic acid (SA) and potassium cyanide (KCN) supplied at 0.12% (w/w), 800uM, and 400 uM respectively. Other identified elicitors caused smaller induction of genistein rhizosecretion. Increased levels of genistein in root exudates corresponded to greater amounts of genistein in root tissue. Elicitor-inducedrhizosecretion of genistein was based on de-novo synthesis and was inhibited by glyphosate and other less specific metabolic inhibitors. Except for NaF:AICI3, all tested elicitors of genistein rhizosecretion produced a distinct bell-shaped dose-response
curve. most of the elicitor-induced rhizosecretion of genistein occured during the first day, followed by a gradual decline. Additional elicitor treatments had little effect on genistein rhizosecretion, indicating that the induction of genistein rhizosecretion by the identified elicitor is a one-time event.
Impacts
(N/A)
Publications
- No publications reported this period
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