Evaluation and Characterization of Novel Botanical Extracts for the Prevention and Treatment of Metabolic Syndrome and Diabetes.

<p>NON-TECHNICAL SUMMARY:<br/> The occurrence of type 2 diabetes continues to soar to epidemic proportions reaching almost 8% (23.6 million) of the population in the U.S. alone. Another 57 million Americans have prediabetes, defined by an impaired fasting glucose values as a result of insulin resistance. Insulin resistance is a key pathophysiologic feature of the "metabolic syndrome" and is strongly associated with co-existing cardiovascular risk factors and accelerated atherosclerosis. Due to the clinical consequences associated with insulin resistance in subjects with metabolic syndrome and type 2 diabetes, clinical regimens directed at increasing insulin sensitivity in vivo remain one of the most desirable goals of treatment. Although it is well established that lifestyle modification can improve insulin resistance and effectively improve many of the risk factors
associated with metabolic syndrome, the success of maintaining lifestyle changes in humans over a chronic period is poor. Therefore, strategies to improve insulin resistance by pharmacological means have represented the traditional approach for clinical medicine. However, because of the widespread use of dietary supplements by the general public, nutritional supplementation with the use of botanicals that effectively increase insulin sensitivity represent a very attractive and novel approach for future studies designed to intervene in the development of metabolic syndrome. The proposed study will benefit the value added agriculture of the State of NJ and provide tangible benefits to the biotechnology and pharmaceutical industries in the State. NJ farmers will benefit from the technologies because they will be provided with the opportunity to grow new crops with a greater value since the
crops will contain bioactive compounds useful for the treatment and prevention of diabetes and metabolic syndrome. Pharmaceutical and nutraceutical companies will use the processed plants as the source of novel ingredients for drugs, foods and or dietary supplements. Therefore, the proposed research will enable agriculture to generate tremendous value through pharmaceutical and nutraceutical manufacturing of products that will promote a healthier population and greener planet. Products that may prevent the onset of metabolic syndrome and diabetes will also save the health care system and State of NJ tremendous amounts of money.
<p>APPROACH:<br/> Methods. Isolation and characterization of the anti-diabetic compounds from Artemisia and Rubus will be guided by in vitro assays, in which the extract was found to be particularly active: 1) non-insulin mediated induction of glycogen accumulation in muscle cells and 2) adiponectin gene expression in adipocytes. Step 1. Fractions of the extracts will be separated and analyzed by preparatory HPLC on a C-8 reverse phase column. Further optimization and fine-tuning of the chromatography conditions for the specific fractions being investigated will be performed. Chemical information, such as spectral data and molecular ions and MS/MS fragmentation fingerprint will be obtained in real time by directing part of the HPLC effluent toward a diode array and mass spectrometer. Compounds will be ionized by different ionization methods such as electrospray, electron
impact, chemical ionization, photoionization to obtain maximum structural information. Mass analysis will be performed using quadrupole ion trap and time-of-flight (TOF) analyzer. Data will be collected and analyzed with the HPLC and MS OEM supplied software, linked with commercially available spectral libraries and the in-house library of plant natural products. This on-line structural analysis will help with the dereplication of the fractions avoiding isolation of well known compounds. Step 2. Structural characterization (deconvolution). High power nuclear magnetic resonance (NMR) instruments (up to 950 MHz available at NCSU) are capable of unequivocal structure determination using sub-milligram quantities of an isolated compound(s). These instruments can also produce structural information from samples of lesser purity. NMR facility at NCSU will be used to structurally characterize
anti-diabetic compound(s) from Artemisia sp. and R. caesius. Center for Gastro-Intestinal Research (CGIR), a collaboration between Rutgers and the Dutch company TNO is focused on the use of the TNO gastroIntestinal Model (TIM), a complex, computer-controlled apparatus that closely simulates in vivo conditions of the gastrointestinal tract of humans. TIM will be used as for pre-clinical studies of bioavailability and intestinal fate of pharmacologically active components of R. caesius and their potential biochemical transformations in each compartment of the gastrointestinal system. In additions, the interaction of the extract and its active components with different diets (e.g. high and low fat, high protein, high carbohydrate) will be studied to determine the recommendations for potential human studies. The bioaccessibility of the active compounds in the TIM system will then be related
to the disposition and bioavailability observed in the animal model in order to demonstrate which factors most negatively impact the bioactivity of the bioactive compounds in vivo and may be used to predict appropriate formulations and doses for clinical effects.
<p>PROGRESS: 2012/01 TO 2012/12<br/>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.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>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.
<p>PROGRESS: 2010/01/01 TO 2010/12/31<br/>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.
<p>PROGRESS: 2009/01/01 TO 2009/12/31<br/>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.
<p>PROGRESS: 2008/01/01 TO 2008/12/31<br/>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.
<p>PROGRESS: 2007/01/01 TO 2007/12/31<br/>
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
?-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.
<p>PROGRESS: 2006/01/01 TO 2006/12/31<br/>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 ? 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.
<p>PROGRESS: 2005/01/01 TO 2005/12/31<br/>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.
<p>PROGRESS: 2004/01/01 TO 2004/12/31<br/>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.
<p>PROGRESS: 2003/01/01 TO 2003/12/31<br/>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.
<p>PROGRESS: 2002/01/01 TO 2002/12/31<br/>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.

<p>PROGRESS: 2001/01/01 TO 2001/12/31<br/>
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.

<p>PROGRESS: 2000/01/01 TO 2000/12/31<br/>
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.

<p>PROGRESS: 1999/01/01 TO 1999/12/31<br/>
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.

<p>PROGRESS: 1998/01/01 TO 1998/12/31<br/>
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.</p>