Plant extracts with antidiabetic effects and other beneficial effects

Description

[0001] The present application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 505,494, filed on May 12, 2017.FIELD OF THE INVENTION

[0002] This invention relates to plant extracts as characterized in the claims containing nutritionally beneficial or medicinally active compounds. Some of these extracts, or the purified compounds contained therein, may be used for the nutritional support, prevention, treatment, or possible cure of various metabolic and other diseases and disorders in human beings and animals, including type 1 and type 2 diabetes, by regulating insulin signaling. This regulatory effect may include modulations of the levels and / or activity of the Insulin Receptor (IR), the Insulin-like Growth Factor (IGF) Receptor, and / or the Insulin Receptor Substrate (IRS) proteins in cells and tissues in the body. A primary focus is directed toward the IRS proteins. Two members of the IRS family of proteins, IRS1 and IRS2, are part of the insulin or insulin-like growth factor signaling pathways, but also mediate signals through other growth factors and cytokines, including IFN-γ, IL-2, IL-4, IL-7, IL-9, IL-13 or IL-15, growth hormone, prolactin, or leptin. IRS1 or IRS2 functional activities also integrate signals emanating from proinflammatory cytokines, including TNF-α, IL-6, IL-1β and related factors. In general, proinflammatory cytokines inhibit IRS1 / IRS2 signaling which contributes to insulin resistance syndromes.BACKGROUND OF THE INVENTION

[0003] Diabetes mellitus is a complex and life-threatening disease that has been known for more than 2000 years. It occurs in mammals as diverse as monkeys, dogs, rats, mice and human beings. The discovery and purification of insulin in 1921 by Banting and Best and its subsequent therapeutic use in people was a landmark advance in medical science and provided a partial treatment for diabetes that is still in widespread use today. Insulin levels are ordinarily adjusted by the body on a moment-to-moment basis to keep the blood sugar level within a narrow physiological range. In the diabetic patient, however, periodic insulin injections can only approximate the normal state because the cellular response to insulin in organs and tissues such as liver, muscle and fat is in many cases is also reduced. Consequently, for these and other reasons which will be discussed in detail below, life threatening complications still occur during the lifetime of treated diabetic patients, especially in the case of type 2 (adult-onset) diabetes. (1)

[0004] Diabetes arises from various causes, including autoimmune-mediated β-cell destruction (type 1 diabetes); insufficient β-cell insulin secretory capacity to compensate for peripheral insulin resistance (type 2 diabetes); and impaired glucose sensing or insulin secretion (Maturity Onset Diabetes of Youth; MODY) (1). Type 1 Diabetes is genetically complex and caused by circulating auto-antibodies against a variety of islet antigens. Insulin is thought to be one of the principle autoantigens in the pathogenesis of type 1 diabetes, but other antigens deserve attention (2). Since new β-cell formation occurs slowly while type 1 diabetes progresses, it might be necessary to treat the disease by accelerating the rate of β-cell regeneration while attenuating the autoimmune response (3).

[0005] Type 2 diabetes is the most prevalent form of diabetes. While it typically manifests at middle age, type 2 diabetes in the developed world is becoming more common in children and adolescents. Physiologic stress-the response to trauma, inflammation, or excess nutrients-promotes type 2 diabetes by activating pathways that impair the post-receptor response to insulin in various tissues (1). Genetic variation also modifies the response to environmental and nutritional factors that promote type 2 diabetes. In a few informative cases, mutations in the insulin receptor or AKT2 explain severe forms of insulin resistance (4). However, common forms of type 2 diabetes are associated with multiple gene variants with modest effects upon insulin action-including peroxisome proliferator-activated receptor gamma (PPARG), peroxisome proliferator activated receptor, gamma, coactivator 1 alpha (PPARGC1A), inward rectifying K+-channel Kir6.2 (KCNJ11), calpain-10 (CAPN10), transcription factor 7-like 2 (TCF7L2), adiponectin (ADIPOQ), adiponectin receptor 2 (ADIPOR2), hepatocyte nuclear factor 4 alpha (HNF4A), uncoupling protein-2, (UCP2), sterol regulatory element binding transcription factor 1 (SREBF1), or high plasma interleukin-6 concentrations (5). Although the effect of each gene is small, these discoveries provide important clues to the pathogenesis of type 2 diabetes.

[0006] Regardless of the underlying etiology, dysregulated insulin signaling exacerbated by chronic hyperglycemia and compensatory hyperinsulinemia promotes a cohort of acute and chronic sequela (6). Untreated diabetes progresses to ketoacidosis (most frequent in type 1 diabetes) or hyperglycemic osmotic stress (most frequent in type 2 diabetes), which are immediate causes of morbidity and mortality. In the long term, diabetes is associated with numerous chronic life-threatening complications. Due to the marked increase in cerebrovascular disease that occurs in diabetics, the incidence of stroke is as much as three-fold higher than in the non-diabetic population. Similarly, cardiovascular diseases such as peripheral vascular disease, congestive heart failure, coronary artery disease and myocardial infarction are uniformly increased in diabetics as a result of the synergistic effects of hyperglycemia with other cardiovascular risk factors. Furthermore, the combined effects of reduced cardiovascular function and systemic oxidative stress result in damage to capillary endothelial cells in the retina (leading to blindness), mesangial cells of the renal glomerulus causing renal failure, and peripheral nerves resulting in neuropathies causing pain and numbness in the extremities (7).

[0007] Diabetes is also associated with age-related degeneration in the central nervous system. Humans beyond 85-90 years of age display less insulin resistance than expected-and centenarians are surprisingly insulin sensitive (8). Compounds that promote peripheral insulin sensitivity and reduce the concentration of circulating insulin required to maintain normal glucose homeostasis provide an ideal treatment of glucose intolerance and its progression to life-threatening diabetes.Insulin, Insulin-like Growth Factors, and Receptors

[0008] Mammals produce three insulin-like peptides - insulin, insulin-like growth factor-1 (IGF-1) and insulin like growth factor-2 (IGF-2) - which activate five homologous insulin-like receptor tyrosine kinases encoded by the insulin receptor (IR) gene and the insulin-like growth factor-1 receptor (IGF1R) gene (Fig. 1A / B). Insulin is produced in pancreatic β-cells in response to circulating glucose concentrations, whereas endocrine IGF-1 is largely secreted from hepatocytes stimulated by nutrients and growth hormone; IGF-1 and IGF-2 are also produced locally in many tissues and cells, including the central nervous system (9). IGF1 can work coordinately with insulin to regulate nutrient homeostasis, insulin sensitivity and pancreatic β-cell function (9). The insulin receptor and IGF receptor genes encode homologous precursors that form covalently linked dimers that are cleaved by proteolysis to generate a tetramer with two extracellular α-subunits and two transmembrane β-subunits. The extracellular α-subunits create the ligand-binding domain that regulates the activity of the tyrosine kinase on the intracellular portion of the transmembrane β-subunits (10).

[0009] High affinity ligand binding induces structural transitions in the catalytic domain of the β-subunit that promote phosphorylation of three tyrosine residues in the kinase regulatory loop (IRa)-Tyr1158, Tyr1162 and Tyr1163 (11). Autophosphorylation releases the regulatory loop from its inhibitory position, which opens the catalytic sites to phosphorylate other proteins (12). The phosphorylated regulatory loop also interacts with other signaling proteins that modulate kinase activity, including Grb10, Grb14, APS and SH2B (13). A fourth tyrosine residue within an NPEY-motif located outside the kinase domain and near the plasma membrane (Tyr960 in IRb; Tyr972 in IRa; Tyr950 in IGF1R) is also phosphorylated, which recruits insulin receptor substrates (IRS-proteins) for tyrosine phosphorylation by the activated receptor kinase (14).Insulin Receptor Substrates

[0010] Cell-based and mouse-based experiments show that most if not all insulin signals are produced or modulated through tyrosine phosphorylation of IRS1, IRS2 or its homologs; or other scaffold proteins including SHC, CBL, APS and SH2B, GAB1, GAB2, DOCK1, and DOCK2 (15). Although the role of each of these substrates merits attention, work with transgenic mice suggests that many insulin responses-especially those that are associated with somatic growth and nutrient homeostasis-are mediated through IRS1 or IRS2 (1).

[0011] The first member of the insulin receptor substrate family of proteins was discovered in 1985, and subsequent research efforts revealed the existence of related IRS family members as well as the signaling pathways to which the IRS proteins are linked. After the discovery that the insulin receptor (IR) possessed a tyrosine kinase enzyme activity, many groups searched for insulin receptor substrates that might regulate downstream signaling from the receptor. The first evidence for the existence of an actual target protein for the insulin receptor, subsequently named an Insulin Receptor Substrate, or "IRS" protein, resulted from the use of phosphotyrosine antibody immunoprecipitates which surprisingly revealed a 185-kDa phosphoprotein (pp185) in insulin-stimulated hepatoma cells (16). Purification and molecular cloning of pp185 revealed one of the first signaling scaffolds as well as the first Insulin Receptor Substrate protein (IRS1) (17,18). IRS1 was determined to be biologically important because it was phosphorylated immediately after insulin stimulation, and catalytically active insulin receptor mutants that failed to phosphorylate IRS1 were biologically inactive.

[0012] Several experiments suggested that other related proteins might exist which led to the purification and cloning of Insulin Receptor Substrate 2 (IRS2), a second member of the IRS family (19,20).

[0013] Experiments in transgenic mice revealed involvement of IRS1 and IRS2 in promoting somatic growth and nutrient homeostasis. Without IRS1, mice are 50% smaller than normal from birth until they die at 2 years of age. Mice without IRS1 have less body fat and are glucose intolerant. In mice, IRS2 is important for peripheral insulin action, as mice lacking IRS2 display glucose intolerance and hyperlipidemia.

[0014] Disruption of the IRS2 gene in mice using standard gene knockout approaches results in diabetes that develops between 8-12 weeks of age. Pancreatic β-cells are lost from these mice as they age, and genes that are important for β-cell function are dysregulated in mice lacking IRS2.

[0015] The IRS-proteins are adapter molecules that link the insulin-like receptors to common downstream signaling cascades (Fig. 1A / B). Four IRS-protein genes have been identified in rodents, but only three of these genes (IRS 1, IRS2 and IRS4) are expressed in humans. IRS1 and IRS2 are broadly expressed in mammalian tissues, whereas IRS4 is largely restricted to the hypothalamus and at low levels in a few other tissues. Each of these proteins is targeted to the activated insulin-like receptors through an NH2-terminal pleckstrin homology (PH) domain. The PTB domain binds specifically to the phosphorylated NPEY-motif in the activated receptor kinases (1). The PH domain also promotes the interaction between IRS proteins and the IR, but the mechanism is poorly understood. The PH domain in the IRS-protein plays a specific role as it can be interchanged among the IRS-proteins without noticeable loss of bioactivity, but heterologous PH domains inhibit IRS1 function when substituted for the normal PH domain (21). In addition to the PH and PTB domains, IRS2 also utilizes another mechanism to interact with the activated insulin receptor (22).IRS→PI3K→AKT cascade

[0016] One of the best studied insulin-like signaling cascades involves the production of PI-3,4,5-P3 by the phosphatidylinositol 3-kinase (PI 3-kinase). The type 1 PI 3-kinase is composed of a regulatory subunit that contains 2 src-homology-2 (SH2) domains and a catalytic subunit that is inhibited by the regulatory subunit until its SH2 domains are occupied by phosphorylated tyrosine residues in the IRS-proteins (23). PI-3,4,5-P3 recruits the Ser / Thr-kinases PDK1 and AKT (also known as PKB) to the plasma membrane where AKT is activated by PDK1-mediated phosphorylation (Fig. 1A / B). AKT phosphorylates many proteins that play a central role in cell survival, growth, proliferation, angiogenesis, metabolism, and migration (24). Phosphorylation of several genuine AKT substrates is especially relevant to insulin-like signaling: GSK3α / β (blocks inhibition of glycogen synthase), AS160 (promotes GLUT4 translocation), the BAD•BCL2 heterodimer (inhibits apoptosis), the FOXO transcription factors (regulates gene expression), p21CIP1 and p27KIP1 (blocks cell cycle inhibition), eNOS (stimulates NO synthesis and vasodilatation), and PDE3b (hydrolyzes cAMP) (Fig. 1A / B). AKT also phosphorylates tuberin (TSC2), which inhibits its GAP activity toward the small G-protein RHEB promoting the accumulation of the RHEB•GTP complex that activates mTOR (24): This pathway provides a direct link between insulin signaling and protein synthesis that is needed for cell growth (Fig. 1A / B).

[0017] The role of IRS-proteins in the PI3K→AKT signaling cascade is validated by a wide array of cell-based and mouse-based experiments. Although IRS1 was originally purified and cloned from rat hepatocytes, the principle role of IRS1 and IRS2 during insulin signaling in hepatocytes in vivo was verified only recently (25). The simplest experiments employ an intraperitoneal injection of insulin into ordinary mice, or mice lacking hepatic IRS1 and IRS2. In ordinary mice, insulin rapidly stimulates Akt phosphorylation, and the phosphorylation of its downstream substrates Foxo1 and Gsk3α / β. Both IRS1 and IRS2 must be deleted to uncouple the insulin receptor from the PI3K→AKT cascade (25). These results confirm the shared but absolute requirement for IRS1 or IRS2 for the hepatic insulin signaling.Transcriptional regulation of IRS2

[0018] The regulation of IRS-protein signaling is an important way to coordinate the intensity and duration of the insulin response among various tissues, but failure of these mechanisms can cause insulin resistance. Transcription of the IRS1 gene is generally stable. By contrast, the production of IRS2 is regulated by multiple nutrient-sensitive transcription factors, including cAMP response element binding protein (CREB) and its binding partner CRTC2, forkhead box O1 (FOXO1), transcription factor E3 (TFE3), and sterol regulatory element binding / factor-1c (SREBF-1c) (26,27). Interestingly, the CREB / CRTC2 transcriptional complex-which binds to cAMP response elements (CRE)-has opposite effects upon IRS2 expression in β-cells and liver. After a meal, the production of ATP from glucose oxidation depolarizes β-cells, which promotes both Ca2+ influx and cAMP production that has many important effects, including the activation of CREB / CRTC2 (26). Thus glucose is coupled directly to IRS2 expression in β-cells, which stimulates β-cell growth and compensatory insulin secretion. By contrast, CREB / CRTC2 promotes IRS2 expression in the fasting liver, which can inhibit the gluconeogenic program by augmenting the basal insulin response.

[0019] In addition to cAMP response elements the promoter region of the IRS2 gene includes elements that bind FOXO family members, an E-box that binds TFE3, and a sterol response element (SRE) recognized by SREBF-1c (27). FOXO1 links the PI3K-AKT cascade to the expression of genes important in cell growth, survival, and metabolism. In liver, IRS1 and IRS2 promote the phosphorylation, nuclear export and degradation of FOXO1, which reduces IRS2 expression. Moreover, SREBF-1c concentrations increase during nutrient excess and chronic insulin stimulation, which inhibits FOXO1-mediated IRS2 expression (28). An imbalance in this reciprocal regulation appears to contribute to pathophysiological effects of over-nutrition leading to the development of the metabolic syndrome and diabetes. Thus, compounds that promote IRS2 signaling are expected to have a strong normalizing effect upon hepatic insulin action, especially during nutrient excess.Insulin resistance and the dysregulation of IRS-protein signaling.

[0020] Insulin resistance is a common pathological state that is associated with many health disorders-obesity, hypertension, chronic infection, dysregulated female reproduction, and kidney and cardiovascular diseases (1). Over the past 15 years, mouse-based experiments have revealed how mutations in genes that mediate the insulin signal, modulate the insulin signal, or respond to the insulin signal contribute to insulin resistance and diabetes. Whereas genetic mutations are obvious sources of life-long insulin resistance, they are usually associated with rare metabolic disorders. Environmental, physiological, and immunological stress causes insulin resistance through heterologous signaling cascades coordinated by complex genetic backgrounds (1).

[0021] Obesity is essentially associated with peripheral insulin resistance. Recent studies reveal a variety of factors secreted from adipose tissue that inhibit insulin signaling-FFAs, tumor necrosis factor-alpha (TNFα), and resistin; or factors that promote insulin signaling-adipocyte complement-related protein of 30 kDa (adiponectin) and leptin. Each one of these factors has specific effects upon gene expression patterns that can alter the response of a cell to insulin. However, the effect of these factors upon the expression or function of the IRS-proteins could contribute to the mechanism for insulin resistance (29). Signaling cascades activated during acute trauma or chronic metabolic or inflammatory stress dysregulate IRS-proteins through various mechanisms, including phosphatase-mediated dephosphorylation, proteasome-mediated degradation, and Ser / Thr-phosphorylation. Dysregulation of IRS-protein function also provides a plausible framework to understand the loss of compensatory β-cell function while peripheral insulin resistance emerges (30).

[0022] Experiments with TNFα reveal one of the first mechanisms linking inflammatory cytokines to insulin resistance (31). TNFα activates the NH2-terminal JUN kinase (JNK), which phosphorylates IRS1 on serine residues that inhibit the activation of the PI 3-kinase / Akt pathway in response to insulin. JNK-mediated phosphorylation of IRS1 may also mediate the effects of cellular stress, including endoplasmic reticulum stress. Insulin itself promotes serine phosphorylation of IRS1 through activation of the PI 3-kinase, revealing feedback regulation that might be mediated by many kinases-AKT, PKCζ, IKKβ, JNK, mTOR and S6K1 (29).The central role of IRS2 signaling in pancreatic β-cells and insulin resistance.

[0023] Mice lacking the gene for Irs1 or Irs2 are insulin resistant, with impaired peripheral glucose utilization. Both types of knockout mice display metabolic dysregulation, but only the Irs2- / - mice develop diabetes between 8-12 weeks of age owing to a near complete loss of pancreatic β-cells (32). This result positions the insulin-like signaling cascade through IRS2 at the center of β-cell function.

[0024] Many factors are required for proper β-cell function, including the homeodomain transcription factor Pdx1. Pdx1 regulates downstream genes needed for β-cell growth and function, and mutations in PDX1 cause autosomal forms of early-onset diabetes in people (MODY). Pdx1 is reduced in Irs2- / - islets and Pdx1 haploinsufficiency further diminishes the function of β-cells lacking Irs2. Glucose and glucagon-like peptide-1 have strong effects upon β-cell growth, which depend upon the Irs2 signaling cascade (Fig. 1B). In β-cells, Irs2 is up regulated by cAMP and Ca2+ agonists-including glucose and glucagon-like peptide-1 (GLP1)-which activate cAMP responsive element binding protein (CREB) and the CREB-regulated transcription co-activator 2 (CRTC22) (34). While many cAMP mediated pathways oppose the action of insulin, the up regulation of IRS2 by glucose and GLP1 reveals an unexpected intersection of these important signals (Fig. 1B). Thus, hyperglycemia resulting from the daily consumption of high caloric food promotes β-cell growth, at least in part by increasing IRS2 expression (34). These results suggest that the Irs2-branch of the insulin-like signaling cascade is the "ordinary gatekeeper" for β-cell plasticity and function. Thus, compounds that promote IRS2 signaling might have beneficial effects upon beta cell growth, survival and function.

[0025] Peripheral insulin resistance contributes to type 2 diabetes, but β-cell failure is an essential feature of all types of diabetes. β-cells frequently fail to compensate for insulin resistance, at least in part because the IRS2-branch of the insulin and IGF signaling cascade which mediates insulin signaling in target tissues also is essential for β-cell growth, function, and survival (32).

[0026] Because insulin resistance is a cause of metabolic dysregulation and diabetes, understanding its molecular basis is an important goal. Genetic mutations are obvious sources of life-long insulin resistance, but they are associated with rare metabolic disorders and thus difficult to identify in the general population. Inflammation is associated with insulin resistance and provides a framework to understand how diet, acute or chronic stress, and obesity might cause insulin resistance.

[0027] Ubiquitin-mediated degradation of IRS-proteins also promotes insulin resistance (Fig. 1A / B). IL6 secreted from leukocytes and adipocytes increases expression of SOCS1 and SOCS3, known for the ability to suppress cytokine signaling. Another function of SOCS1 and SOCS3 is to recruit an elongin BC-based ubiquitin ligase into the IRS-protein complex to mediate ubiquitinylation. Thus, ubiquitin-mediated degradation of IRS-proteins might be a general mechanism of cytokine-induced insulin resistance that contributes to diabetes or β-cell failure (35).

[0028] The activity of protein or lipid phosphatases, including PTP1B, SHIP2 or pTEN modulates insulin sensitivity (FIG. 1). Disruption of each of these genes in mice increases insulin sensitivity, suggesting that each might be a target for inhibitor design. PTP1B resides in the endoplasmic reticulum where it dephosphorylates the insulin receptor during internalization and recycling to the plasma membrane (36). This specialized mechanism appears to limit unwanted side effects associated with inhibition of phosphatases, including unregulated cell growth.

[0029] In US 2016 / 0022752 A1, the inventors described a method of treating an IRS mediated disease or condition (such as diabetes) comprising administering an effective amount of a pharmaceutical composition comprising a botanical extract (preferably an aqueous extract), that provides at least 1x10 4< Insulin Equivalent units in 1 milliliter, which is prepared from a variety of plant species, some of which included edible species.SUMMARY OF THE INVENTION

[0030] The present invention relates to the embodiments as characterized in the claims.

[0031] The insulin receptor substrate (IRS) family of proteins that function immediately downstream of the insulin receptor or insulin like growth factor receptors is of central importance in mediating the effects of insulin on responsive cells. In particular, upregulation of the level or functional activity of IRS2 in humans may result in a therapeutically effective chronic treatment as well as a nutritionally beneficial or supportive effect for patients suffering from diabetes, especially the adult onset (type 2) form of the disease, as well as for other disorders in which IRS protein function is insufficient, abnormal or absent altogether. Further, IRS1 and IRS2 are of central importance in mediating signaling through the insulin like growth factor signaling pathway, and signaling by other growth factors and cytokines as well.

[0032] The present invention relates to a botanical extract that provides at least 1 x 10 4< Insulin Equivalent units in 1 milliliter comprising aqueous extracts from Artemisia dracunculus, Cichorium endivia and Lactuca sativa. Such extracts may be identified using 32D cells expressing IRS2. Such cells may be created using standard methodology (20). Applicants have previously created and described a sophisticated target protein specific cell-based assay system capable of identifying IRS2 branch activators of the insulin mediated signal transduction cascade (38). This system is comprised of both Control and Test cells derived from the 32D myeloid progenitor cell line. For the present invention, Applicants designed a cell-based assay system using an IRS2 overproducing histidinol-resistant Test cell line as well as an appropriate his-resistant Control cell line harboring the expression vector only. Under appropriate cultural conditions, the IRS2 overproducing 32D cells become exquisitely sensitive to activation by insulin. Extracts of the present invention will have a more pronounced effect on the Test cells than on the Control cells, and this effect is quantitated and used to determine the ability of a sample to mimic the effects of insulin as expressed in percentage terms relative to the maximum effect observed following insulin treatment.

[0033] Representative assay results (shown in Tables 1-2 and Figure 5) utilize a 96 well plate format and involve plating Control and Test cells at 25,000 cells per well at time zero. The cells are cultured in IL-3-free medium, and treated for 72 hours with and without 50 nM insulin. Whereas the IRS2-overproducing 32D cell lines become IL-3 independent for the 72 hour duration of the assay (See Figure 5), the Control cells remain absolutely IL-3 dependent and exhibit essentially no cell growth (data not shown). As a result, the assay developed is highly sensitive to compounds (such as insulin) that are capable of activating the IRS2 dependent growth control cascade in IRS2 overproducing 32D cells. Furthermore, potential false positive growth stimulating substances, as emulated by the results of treatment of the cells with IL3, will score positively on the Control cell line as well, and are thus easily eliminated from further consideration and work-up.

[0034] This system was utilized to conduct a high throughput screen consisting of more than 100,000 synthetic and natural product derived compounds in search of agents capable of activating the IRS2 signal transduction cascade. A subset of these compounds included extracts derived from a variety of plant species, some of which included edible species. The latter compounds and extracts were screened because Applicants reasoned that compounds derived from edible plants might also contain compounds that are capable of emulating the biological effects of insulin in an IRS2 dependent manner. If such compounds existed within the subset of edible plants contained within the broader plant kingdom, they would provide a basis for understanding at the molecular level why certain diets, such as the Mediterranean diet, have been shown to be associated with a reduced incidence of diabetes, heart disease, and hypertension, leading to corresponding improvements in lifespan and quality of life (40-45, 52).

[0035] Some publications have proposed the hypothesis that the benefits of such diets result from a lack of deleterious components present in them, including high fat foods, processed foods, refined sugar, artificial sweeteners and the like. Others have suggested the possibly that protective components such as general antioxidants, or essential nutritional components such as vitamins, or minerals, are the reason why certain diets are beneficial to health and well-being (40-45, 52). Applicants reasoned to the contrary that diets associated with good health and well-being might actually contain pharmacologically active components that synergize with or are otherwise beneficial to, normal cellular functioning in human beings and other mammals. By pharmacologically active, Applicants mean that such active components bind to specific sites on proteins, nucleic acids, or other discrete cellular binding sites and exert pharmacological effects. Remarkably, this theory is demonstrated herein to be true, insofar as these efforts by the Applicants have led to the finding that plants derived from selected species, including : Cichorium endivia, var. latifolium; Lactuca sativa, var. longifolia; Lactuca sativa, var. crispa (See Tables 1 and 2), and others, contain one or more compounds detectable within extracts derived from said species that are capable of substantially activating the IRS2 branch of the insulin mediated signal transduction cascade as determined by the IRS2 target protein specific cell-based assay system described above and previously (38,39). Such compounds may be extracted from the aforementioned plant using an aqueous solvent system, as will be described in detail below. One of skill in the art may utilize alternative extraction methods including, but not limited to, the use of organic or inorganic solvents, and / or supercritical fluid extraction using, for example, carbon dioxide.BRIEF DESCRIPTION OF THE FIGURES

[0036] Figs. 1A and 1B depict components of the IRS signaling cascade in muscle and liver cells (1A) and pancreatic beta cells (1B), respectively. There are two main limbs that propagate the signal generated through the IRS-proteins: the PI 3-kinase and the Grb2 / Sos→ras cascade. Activation of the receptors for insulin and IGF-1 results in tyrosine phosphorylation of the IRS-proteins, which bind PI 3-kinase and Grb2 / SOS. The GRB2 / SOS complex promotes GDP / GTP exchange on p21ras, which activates the ras→ raf→ MEK→ ERK1 / 2 cascade. The activated ERK stimulates transcriptional activity by direct phosphorylation of elk1 and by phosphorylation of fos through p90rsk. The activation of PI 3-kinase by IRS-protein recruitment produces PI 3,4P2 and PI 3,4,5P3 (antagonized by the action of PTEN or SHIP2), which recruit PDK1 and AKT to the plasma membrane, where AKT is activated by PDK- and mTOR-mediated phosphorylation. The mTOR kinase is activated by RhebGTP, which accumulates upon inhibition of the GAP activity of the TSC1::TSC2 complex by PKB-mediated phosphorylation. The p70s6k is primed through mTOR-mediated phosphorylation for activation by PDK1. AKT phosphorylates many cellular proteins to inactivating PGC1α, p21 kip< , GSK3β, BAD and AS160, or activate PDE3β and eNOS. The AKT-mediated phosphorylation of the forkhead proteins results in their sequestration in the cytoplasm, which inhibits their influence upon transcriptional activity. Insulin stimulates protein synthesis by altering the intrinsic activity or binding properties of key translation initiation and elongation factors (eIFs and eEFs, respectively) as well as critical ribosomal proteins. This occurs via phosphorylation and / or sequestration of repressive factors into inactive complexes. Components of the translational machinery that are targets of insulin regulation include eIF2B, eIF4E, eEF1, eEF2 and the S6 ribosomal protein (4-6). TNFα activates JNK which can phosphorylate IRS1 inhibiting its interaction with the insulin receptor and subsequent tyrosine phosphorylation. IRS2 expression is promoted by nuclear FOXO, which increases IRS2 expression during fasting conditions. CREB: TORC2 complex also promotes IRS2 expression especially in β-cells, placing IRS2 under the control of glucose and GLP1.

[0037] Tables 1 and 2 show the results obtained with a variety of distinct genera and species of edible plants that Applicants' have discovered possess the desired activity. They are ranked according to their ability to stimulate growth of the IRS2-expressing Test cells as normalized to Insulin, with the response elicited by 50 nM Insulin treatment being defined as 100% (Table 1). The activities listed in Table 1 are among the highest that were obtained from numerous experiments. Considerable variations in activity from lot to lot of plant material may be expected depending upon the time of year that the plant was grown and harvested, the degree of freshness of the plant, the soil and climate conditions in which it was propagated, and so on.

[0038] Figures 2, 3 and 4 show the ability of certain extracts to lower fasting blood glucose in normal (non-diabetic) individuals. Either 75 grams of fresh, raw leaf obtained from a local market, or the indicated amount of lyophilized aqueous extract prepared as described herein were consumed as indicated: Figs 2 and 3: CG-105; Fig. 4: CG-132. Blood glucose measurements were determined using a hand-held portable glucose monitor (Abbott Freestyle Freedom Lite). The glucose monitor and the disposable test strips were obtained from a local pharmacy.

[0039] Fig. 5. shows selected extracts enhance the functioning of insulin in the IRS2 overproducing 32D Test cell system such that the amount of insulin necessary to achieve 100% stimulation of growth of the Test cells is lower than it would otherwise need to be in the absence of the selected extract. When CG-105 extract was added to low-dose insulin treatments on the 32D IRS2 Test Cell system, CG-105 extract was found to enhance the activity of insulin at all insulin doses below the maximum insulin stimulating effect (at 50 nM). This activity is variously referred to herein as Insulin Equivalent Activity (IEA) or Insulin Augmenting Activity (IAA), or the additional terms given in paragraphs [51-69] below.DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to the embodiments as characterized in the claims.

[0041] A substantial amount of effort has been made to attempt to identify plant extracts or compounds derived from plants that possess desirable effects for the treatment of human or animal disease. Numerous extracts, drinks, powders, teas, and so forth are marketed with claims relating to providing nutritional support for, or treatment of, many diseases, including diabetes and related metabolic disorders. None of these preparations has been demonstrated to activate the insulin mediated signal transduction cascade in an IRS2-specific manner (46-51, 53,54, 57-70,72, 74,75, 79-81). Zhang et al. conducted a high-throughput screen of more than 50,000 synthetic compounds and natural products and identified a compound that activated the Insulin Receptor (IR). The compound, however, turned out to not be derived from an edible plant source at all. Rather, the compound was derived from a fungal extract (Pseudomassaria) that had been recovered from leaves of an undetermined plant collected near Kinshasa, Democratic Republic of Congo. This work showed, however, that it was at least possible to identify a small molecule capable of having partial activity toward the Insulin Receptor (IR) (71). Prior to their work, it was believed that only a protein hormone such as insulin could activate its cognate receptor.

[0042] Pinent et al. demonstrated that a class of compounds known as procyanidins, which are derived from grape seeds, which can induce glucose lowering in an animal model, are able to bind to the IR and at least partially activate the receptor (60, 79). However, the authors concluded that the effects of the procyanidins result in activation of the insulin signaling cascade in a different manner than insulin does. Even with purified fractions of grape seed procyanodin extracts (GSPE), the authors were able to obtain only 40% of the activation of the IR as compared to insulin. In addition, the authors could not establish an IRS2-dependent effect of the compounds (79).

[0043] Thus, with the exception of insulin and its corresponding analogs and long-acting formulations, no compounds, including proteins, polypeptides or "small molecules" (i.e. molecules having a molecular weight of 2,000 atomic mass units or less) derived from a genus and species known to be edible have been shown to specifically activate the Insulin / Insulin Receptor / IRS2 signal transduction cascade in mammalian cells. In addition, no small molecules have been demonstrated to activate the insulin signaling cascade through an IRS-2 dependent manner. As discussed above in the Background of the Invention, such compounds, extracts, and methods of identifying them from any source would be desirable. This invention provides such extracts derived from selected genera and species of edible plants containing this highly desirable activity.

[0044] The invention provides a botanical extracts that provide at least 1 x 10 4< Insulin Equivalent units in 1 milliliter comprising aqueous extracts from Artemisia dracunculus, Cichorium endivia and Lactuca sativa for use in a method for the treatment, cure, prevention or nutritional support for various metabolic and other disorders including diabetes, pre-diabetes, metabolic syndrome, obesity, cancer, myelodysplastic syndromes, neurologic disorders such as Alzheimer's disease, dementia and cognitive impairment, attention deficit disorders, premature aging, cardiovascular disorders such as peripheral vascular disease, congestive heart failure, coronary artery disease and myocardial infarction, and others. The invention also provides such extracts for the improvement of certain normal resting states such as baseline cognitive status, the cellular aging process, heart rate, stroke volume, blood pressure (systolic and diastolic), blood flow, cardiac output, and the basal metabolic rate of an organism. These beneficial aspects of the invention result, in part, by regulating the level or functional activity of IRS proteins as a result of administering an effective amount of the extracts of the invention to a subject in need or desiring thereof.

[0045] In one embodiment, the use of the invention provides for restoring or enhancing insulin sensitivity in a cell by upregulating IRS2 function. The use of the invention further provides a method of enhancing pancreatic β-cell function by upregulating IRS2 function. According to the invention, a disease or disorder characterized by reduced or insufficient signaling through IRS2 can be treated by upregulating IRS2 function. Such diseases include, but are not limited to, metabolic disease, diabetes, dyslipidemia, obesity, female infertility, central nervous system disorders, Alzheimer's disease, and disorders of angiogenesis.

[0046] According to the invention, upregulation of IRS2 function includes activation of IRS2 or a complex that includes IRS2. In one embodiment of the invention, upregulation of IRS2 function is also accomplished by activation of IRS2 activity, for example by inhibition of phosphorylation of specific serine, threonine or tyrosine residues of IRS2. In another embodiment, upregulation of IRS2 function is accomplished by enhanced expression of IRS2 or by inhibition of degradation of IRS2. In another embodiment, upregulation of IRS2 function is by modulation of a protein or nucleic acid molecule that participates in the mediation of an insulin effect on insulin-responsive cells. Also, modulation of the coupling function of the PH, PTB, or KRLB domains can improve IRS2 function.

[0047] More than one hundred samples of various edible plant genera and species were obtained from several local and international markets. Both aqueous and organic extractions of the fruits, leaves, stems and roots of various selected genera of edible plants and other plants were prepared. The extraction procedure was performed as follows: 500 milligrams of fresh plant tissue were pulverized with a mortar and pestle. The ground tissue was then added to 2 mL of water, and homogenized for 1 minute at a setting of 6 using a microprobe (Cole Palmer, LabGen 700). The mixture was then spun at 14,000 RPM for 10 minutes. The supernatant containing the aqueous layer was removed and assayed; whereas the pellet was retained and subjected to organic extraction procedure. Between procedures, samples were maintained at 4 °C to minimize endogenous enzymatic activities.

[0048] For larger scale extractions, the procedure was performed as follows: 250 g of wet plant tissue was added to 1L of water, and initial tissue disruption was performed in a table top blender (Kitchen Aid). The blended mixture was then homogenized on ice for 5 minutes at a setting of 20 using a Polytron homogenizer and a standard size probe (Polytron PT2100). The mixture was spun at 10,000 RPM for 10 minutes at 4°C using a JA-10 rotor (Beckman Coulter; Avanti J-25I). The supernatant containing the aqueous layer was removed and assayed. Long term storage was either by refrigeration at 4°C for up to three weeks, or portions of the sample were frozen and lyophilized.

[0049] It is preferable to maintain the pH of the extraction solution above 4.3. We have found that pH values of 4.3 and below may cause precipitation of the active factor from crude extracts, leading to a negative result in the 32D IRS2 cell-based assay system. If the solution is brought to pH values higher than 4.3, activity is restored. However, if the active factor is exposed to pH values that are lower (pH approximately 2.0 or less for extended periods), then restoration of activity by subsequently raising the pH is no longer possible, and the active factor becomes essentially irreversibly inhibited.

[0050] Under certain conditions, the active principle ("active factor", or simply "factor") obtained from CG-105 is susceptible to heat inactivation, whereas the factor is stable to freezing and lyophilization. Essentially no activity is extractable by the neat organic solvents tested, which included ethanol, methanol, phenol, chloroform, acetonitrile, and benzene.

[0051] In addition to emulating the effects of insulin on the Test cell line, it was observed that CG-105 and selected other extracts also increased the overall viability of the cells at the end of the assay on day 3, approximately 72 hours later (Fig. 5).

[0052] After the extraction procedure was complete, each extract obtained was assayed either directly using 1 microliter of aqueous extract derived from a 1 liter preparation using 250 grams of fresh plant material, as described above, or after redissolving 5mg of lyophilized powder in 1 ml of distilled water and using 1 microliter per assay well in the 96 well format (approximately 100 microliter total media volume per well). The assay was performed on the 32D Test cell line stably overproducing IRS2 described above and previously (38). The Test cells consisted of 32D cells harboring a histidinol-selectable expression vector and containing a full-length gene encoding murine IRS2 under the transcriptional control of a promoter functional in 32D cells, whereas the Control cells consisted of 32D cells harboring the same histidinol-selectable expression vector lacking the IRS2 coding region.

[0053] Tables 1 and 2 shows the activities of several of the most active aqueous extracts obtained from selected species. Activities are reported as a percentage of the total insulin activity obtained using 50 nM Insulin as the positive control for signaling through the IRS2 branch of the signal transduction cascade. Table 2 shows the increase in growth of the Test cells relative to the Control cells for each extract that was tested. (The values indicated are determined by subtracting the mean values of the Control cells from the Test cells for each extract, respectively, as shown in Table 1. (Mean and standard deviations for each extract value are as given in Table 1). As is evident from results shown in Table 2, which are organized taxonomically, certain aqueous extracts exhibit insulin like biological activity in the 32D IRS2 Test cell system equal to as much as 40% of the response obtained with insulin. Positively scoring activities ranged from a low of 10% to a high of 40% of the amount of the cellular response obtained with insulin. One of the families shown in Table 2, the Asteraceae family, contains genera and species which uniformly scored positively, though to varying degrees. Other families, such as Lamiaceae or Brassicaceae, contained some members which scored positively and others which were negative. Finally, all of the members of the Amaranthaceae family that were tested were essentially negative (ND = no activity detected). Table 1 - Natural Product Activity Relative to Insulin NPControl Cells (32D his< )Test Cells (32D IRS2< )X ≤ 10% CG148-0.1 + 1.13.9 ± 1.3CG1580.1 ± 2.03.9 ± 1.3CG137-0.1 ± 2.96.1 ± 1.2CG1261.6 ± 0.86.9 ± 1.4CG144-1.7 ± 0.97.3 + 2.4CG1180.6 ± 0.17.8 + 0CG1492.1 ± 1.37.8 ± 0CG1282.0 ± 3.08.3 ± 5.6CG1410.2 ± 0.28.5 ± 1.2CG1461.1 ± 1.38.5 ± 1.2CG1542.5 ± 1.39.1 ± 3.9CG1562.5 ± 09.1 ± 1.3CG1421.0 ± 2.99.8 ± 010% < X ≤ 20% CG1532.8 ± 2.410.4 ± 2.6CG1043.2 ± 0.410.5 + 2.6CG1134.3 ± 1.910.5 ± 2.6CG1234.6 ± 1.811.8 ± 3.9CG1360.7 ± 0.412.2 ± 0CG1311.2 ± 2.413.9 ± 0CG1590.8 ± 0.414.3 ± 1.3CG1150 ± 0.715.7 ± 0CG1212.5 ± 1.815.7 ± 7.8CG1074.3 ± 0.815.8 ± 7.9CG1120.4 ± 1.315.8 ± 2.6CG1251.3 ± 1.816.7 ± 5.6CG1551.3 ± 2.516.9 ± 1.3CG1140.4 ± 1.517.6 ± 9.8CG139-1.0 ± 1.218.3 + 1.2CG1400.7 ± 1.119.5 ± 2.4CG1572.0 ± 2.119.5 ± 3.920% < X ≤ 25% NPControl Cells (32D his< )Test Cells (32D IRS2< )CG1380.1 ± 1.620.7 ± 1.2X>25% NPControl Cells (32D his< )Test Cells (32D IRS2< )CG120-0.3 ± 0.125.5 ± 2.0CG143-2.5 ± 0.325.6 ± 3.7CG132-0.6 ± 1.126.4 ± 1.4CG1350 ± 026.8 ± 4.9CG1101.9 ± 2.131.6 ± 7.9CG1502.4 ± 1.033.8 ± 5.2CG1050.6 ± 036.8 ± 7.9 Table 2. Natural Product Activity Relative to Insulin NP Genus_species % Activity above baseline (T A -C A ) Taxonomic Family: Amaranthaceae CG101Beta vulgarisNDCG102Beta vulgarisNDCG103Beta vulgarisNDCG111Spinacia oleraceaNDCG116Beta vulgarisNDCG117Beta vulgarisND Taxonomic Family: Apiaceae CG127Petroselium hortenseNDCG133Apium graveolensNDCG134Apium graveolens var. dulceNDCG126Petroselinum crispum5CG128Coriandrum sativum6CG118Petroselinum crispum7CG146Anethum graveolens7CG107Daucus carota12CG112Petroselinum crispum15 Taxonomic Family: Asteraceae CG104Cichorium endivia7CG123Taraxacum officinale7CG131Cichorium endivia13CG125Lactuca sativa15CG114Lactuca sativa17CG120Lactuca sativa var. crispa (rd leaf)26CG143Cynara scolymus; Cynara cardunculus26CG132Lactuca sativa var. crispa (gr. leaf)26CG135Artemisia dracunculus27CG110Lactuca sativa var. longifolia30CG105Cichorium endivia var. latifolium36 Taxonomic Family: Brassicaceae CG109Brassica oleracea var. italicaNDCG119Brassica oleracea var. viridisNDCG124Brassica oleracea var. capitataNDCG129Brassica oleracea var. viridisNDCG145Brassica oleracea var. gemmiferaNDCG156Nasturtium officinalis7CG153Brassica rapa8CG142Eruca sativa9CG136Brassica rapa12CG121Brassica nigra13CG115Brassica rapa chinensis16 Taxonomic Family: Cucurbitaceae CG152Cucurbita pepo var. pepoNDCG141Cucumis sativus8 Taxonomic Family: Fabaceae CG150Phaseolus vulgaris31 Taxonomic Family: Lamiaceae CG122Mentha piperitaNDCG137Origanum vulgare6CG154Rosmarinus officinalis7CG159Ocimum basilicum14CG155Salvia officinalis16CG140Origanum majorana19CG138Thymus vulgaris21 Taxonomic Family: Liliaceae CG106 Allium cepaNDCG130Asparagus officinalisNDCG113Allium porrum6CG139Allium schoenoprasum18 Taxonomic Family: Poaceae CG158Cymbopogon4CG157Agropyron Gaertn18 Taxonomic Family: Polygonaceae CG148Rheum ribes4 Taxonomic Family: Solanaceae CG151Solanum lycopersicumNDCG149Solanum melongena6CG144Capsicum L.7 Taxonomic Family: Vitaceae CG147Vitis vinifera; Vitis L.ND

[0054] Based upon the direct comparison to insulin's ability to activate the IRS2 overproducing Test cells, Applicants define the measurement of such activity in the following ways: Insulin Sensitizing Units - (IS units) Insulin Sensitizing Activity (ISA units) Insulin Optimizing Activity - (IOA units) Insulin Optimizing Units - (IO units) Insulin Boosting Activity - (IBA units) Insulin Boosting Units - (IB units) Insulin Amplifying Units - (IA units) Insulin Amplifying Activity - (IAA units) Insulin Intensifying Units - (IIn units) Insulin Intensifying Activity - (IInA units) Insulin Augmenting Activity - (IAA units) Insulin Improving Activity - (IImA units) Insulin Improving Units - (IIm units) Insulin Strengthening Units - (ISt units) Insulin Enriching Units - (IEn units) Insulin Equivalent Units - (IEq units) Insulin Equivalent Activity - (IEA units)

[0055] One Unit of Insulin Equivalent Activity (also known as Insulin Augmenting Activity ) is defined as the minimum amount of material (compound or extract) necessary to increase the growth of the IRS2 overproducing TEST cells by 1% of the level of growth achieved by treatment of the cells with an appropriate amount of insulin necessary to achieve a substantial increase in growth of the Test cells that a skilled investigator would classify as a sufficient positive control result. This is measured in terms of percentage relative to the maximum effect achieved with Insulin treatment under said positive control conditions. For these purposes, and as shown in the experiments of Tables 1 & 2 and Figure 5, 50 nM insulin is utilized as the positive control. This amount has been determined empirically based upon each lot of insulin that is purchased. By way of example, if 1 microliter of plant extract increases the growth of the IRS2 Overproducing 32D Cell line by 20% in the 96-well plate format assay described above, relative to a positive control comprising 50 nM Insulin treatment (normalized to 100%), then said extract is considered to contain 20 units of Insulin Equivalent (or Insulin Augmenting) Activity. In our experience, 50 to 100 nM Insulin is a saturating amount of insulin under most conditions.

[0056] Using standard methodologies for extraction, purification, and filtration, including size-exclusion chromatography, normal and reversed-phase high-pressure liquid chromatography (HPLC), hydrophilic interaction chromatography (HILIC), affinity chromatography, non-sterile and sterile filtration methods, and the like, such activities may be concentrated and increased to as much as 100% of the effect of insulin on the IRS2 Overproducing 32D Cell line (82-84, and references therein). Thus, in one embodiment, the invention provides a botanical extract that contains at least 1 x 10 3< Insulin Equivalent (IE) units per milliliter. In another embodiment, the invention provides a botanical extract that contains at least 1 x 10 4< IE units per milliliter. In another embodiment, the invention provides a botanical extract that contains at least 2 x 10 4< IE units per milliliter. In still another embodiment, the invention provides a botanical extract that contains at least 3.6 x 10 4< IE units per milliliter. In yet another embodiment, the invention provides a botanical extract that contains from 1 x 10 4< to 1 x 10 5< IE units per milliliter. In yet another embodiment, the invention provides a botanical extract that contains from 1 x 10 5< to 1 x 10 6< IE units per milliliter. In still another embodiment, the invention provides a botanical extract that contains from 1 x 10 6< to greater than 1 x 10 7< IE units per milliliter.

[0057] Extraction methods include the use of aqueous-based solvents in neutral, basic or weakly acidic pH ranges, or CO 2 -mediated extraction. Purification methods include size-exclusion chromatography using pore-containing silica-based or polymeric beads for separations depending upon the relative molecular size of a bioactive molecule. Additional purification methods may utilize hydrophobic column matrices (C4 or C18-substituted beads), various hydrophilic interaction chromatography (HILIC) media such as hydroxyl, di-hydroxyl, amide, amino, cyano, and related substituted side chains, affinity chromatography media, or ion exchange media employing amino or carboxylic acid moieties cross-linked to a 3 or 5 micron beads. Sterile or non-sterile filtration methods include filter papers such as Whatman 3MM, sterile nylon-membrane-based filters with 0.45 or 0.2 micron exclusion cutoffs, insoluble filtration medias such as glass wool, cellulose, silica, diatomaceous earth (DE), nylon fabric, stainless steel plates containing micron and submicron sized pores, crossflow cartridge filtration systems, centrifugation-based separation, and the like.

[0058] Disclosed herein is a botanical extract that provides at least 1 x 10 4< to 1 x 10 5< Insulin Equivalent units in 1 milliliter where the botanical extract comprises extracts from Artemisia dracunculus, Cichoria endivia and Lactuca sativa and a metal (e.g., chromium, iron, manganese, zinc, or copper). In certain embodiments, the extracts from Artemisia dracunculus, Cichoria endivia and Lactuca sativa are dried aqueous extracts where the aqueous extracts are produced by a process comprising extracting leaves of Artemisia dracunculus, Cichoria endivia and Lactuca sativa.

[0059] Also discosed herein is a pharmaceutical composition or nutritional supplement comprising the extracts described above, optionally in combination with a metal. The pharmaceutical composition or nutritional supplement may be in a conventional dosage form, e.g., a tablet or a capsule such as a hard- or soft-shelled gelatin or hydroxypropylmethylcellulose capsule. The pharmaceutical composition or nutritional supplement may also be in the form of a powder, e.g., a maltodextrin-containing powder, which may be reconstituted into solution by dissolving in water or another suitable liquid before use.

[0060] In one embodiment, the pharmaceutical composition or nutritional supplement in tablet or capsule form comprises: a dried herbal extract of Artemisia dracunculus; a dried herbal extract of Cichoria endivia; a dried herbal extract of Lactuca sativa; and optionally, chromium.

[0061] In certain embodiments, the pharmaceutical composition or nutritional supplement is in tablet form. In certain embodiments, the pharmaceutical composition or nutritional supplement in tablet form comprises: 0.7 mg of a dried herbal extract of Artemisia dracunculus; 962.7 mg of a dried herbal extract of Cichoria endivia; 16.7 mg of a dried herbal extract of Lactuca sativa; and 1.2 mcg chromium; optionally also comprising at least one pharmaceutically acceptable excipient selected from the group consisting of: dicalcium phosphate, microcrystalline cellulose, silicon dioxide, hydroxypropyl cellulose, stearic acid, croscarmellose sodium, and magnesium stearate; and combinations thereof.

[0062] In certain embodiments, the pharmaceutical composition or nutritional supplement is in tablet, capsule, or powder form. In certain embodiments, the pharmaceutical composition or nutritional supplement in tablet, capsule, or powder form comprises: a total of 50 mg to 1,500 mg of: a dried herbal extract of Artemisia dracunculus; a dried herbal extract of Cichoria endivia; a dried herbal extract of Lactuca sativa; and optionally, chromium. optionally also comprising at least one pharmaceutically acceptable excipient selected from the group consisting of: dicalcium phosphate, microcrystalline cellulose, silicon dioxide, hydroxypropyl cellulose, stearic acid, croscarmellose sodium, and magnesium stearate; and combinations thereof.

[0063] In certain embodiments, the at least one pharmaceutically acceptable excipient is present in an amount of 15-25% of the total weight. In certain embodiments, the extracts of Artemisia dracunculus, Cichoria endivia and Lactuca sativa are present in a ratio of about 1:1,375:24 (w / w / w).

[0064] Disclosed herein is the pharmaceutical compositions or nutritional supplements described above for use in a method of treating an IRS mediated disease or condition. In certain embodiments, the IRS mediated disease or condition is diabetes, pre-diabetes, metabolic syndrome, insulin resistance, or dementia. In certain embodiments, the method further comprises administering an antidiabetic agent, insulin, metformin, exenatide, vildagliptin, sitagliptin, a DPP4 inhibitor, meglitinide, exendin-4, liraglutide, or a GLP1 agonist. The pharmaceutical composition or nutritional supplement disclosed above may be administered in a separate pharmaceutical formulation from the antidiabetic agent, insulin, metformin, exenatide, vildagliptin, sitagliptin, a DPP4 inhibitor, meglitinide, exendin-4, liraglutide, or GLP1 agonist. Alternatively, the pharmaceutical composition or nutritional supplement disclosed above may be administered in the same pharmaceutical formulation as the antidiabetic agent, insulin, metformin, exenatide, vildagliptin, sitagliptin, a DPP4 inhibitor, meglitinide, exendin-4, liraglutide, a sodium-glucose transporter type 2 (SGLT-2) inhibitor such as empagliflozin, canagliflozin, or dapagliflozin, or a GLP1 agonist. In certain embodiments, the pharmaceutical composition or nutritional supplement is administered orally twice per day, 30-60 minutes before meals.

[0065] Disclosed herein is the pharmaceutical compositions or nutritional supplements described above for use in a method of stimulating IRS2-dependent signal transduction in a subject in need thereof.

[0066] Disclosed herein is an in vitro method of stimulating IRS2-dependent signal transduction comprising contacting a cell with the pharmaceutical compositions or nutritional supplements described above.

[0067] This application describes methods of providing nutritional support, preventing, inducing durable long-term remissions or curing patients with diabetes, metabolic disorders, central nervous system diseases, obesity, fertility and other human disorders as discussed above. The invention is particularly concerned with the IRS proteins and with modulation of the activity of IRS2-mediated cellular signaling pathways as a mechanism for treating human disease and / or providing beneficial nutritional support.

[0068] The application also provides an assay to select for specific cultivars with even higher activity or selecting progeny from crosses of cultivars or species for selecting individuals again with high levels of activity.

[0069] As shown in Figures 2 and 3, a 75g raw dose of a selected genus and species (Cichorium endivia, var. latifolium; designated CG-105), when administered orally, lowers fasting blood glucose in a human being. The volunteer test subjects are the Applicants. Arrows indicate time of oral administration. Also shown in Figure 2 are analogous human results of fasting blood glucose before and after administration of a 1.9 gram dose of lyophilized aqueous extract prepared from CG-105 and administered in gelatin capsules. These results demonstrate that this species of plant is capable of inducing a modest fasting blood glucose lowering effect in non-diabetic human beings.

[0070] Modulation of IRS function can involve one of the following non-limiting mechanisms. One possible mechanism involves modifying (i.e., promoting or inhibiting) the IRS2 binding interaction with various proteins both upstream and downstream that interact with (bind to) IRS2. These include, for example, the human Insulin Receptor (hIR) which binds to and phosphorylates IRS1 and IRS2, the N-terminal c-jun kinase (JNK), PKC isoforms, ERK1 or ERK2, as well additional upstream or downstream signaling elements such as src homology 2 (SH2) domain-containing proteins that bind to IRS2 and may also phosphorylate, dephosphorylate or otherwise modify IRS.

[0071] Another mechanism involves changing the specific pattern of covalent modifications of IRS such as the phosphorylation state of serine, threonine and tyrosine residues, ubiquitination patterns, acetylation or other covalent modifications that alter the function, intracellular localization, or stability of IRS proteins.

[0072] A third mechanism involves controlling the expression of the IRS genes in specific cells, including beta cells, brain cells, liver cells muscle cells, reproductive cells and tissues involved in reproduction, fat cells, mammary cells, bone cells and immune system cells, essentially any cells of the body where IRS2 might be naturally expressed. IRS2 is regulated by transcription factors such as CREB, CRTC2, Foxo1, TFE3, and SREBP1. Accordingly, increased IRS2 expression can result from increased activity of the transcription factors that stimulate the transcription of the IRS2 genes. IRS2 expression is also modulated in part by cAMP levels.

[0073] IRS is sensitive to proteolytic degradation. Accordingly, compounds that interfere with IRS degradation, for example by interacting with IRS to block degradation or by inhibiting a protease directly, can be used to upregulate IRS signaling activity.

[0074] Methods for assessing the effects of compounds on IRS signaling, in vitro and in vivo, are known in the art. For example, cell based assays can be used to confirm increases in IRS signaling. Further, various experimental strategies are available to confirm IRS function, including measuring glucose uptake in response to insulin stimulation, or determining expression of known downstream genes. To observe regulation of IRS expression, reporter genes linked to IRS expression control sequences may be constructed.

[0075] Compounds that upregulate the expression or cellular activity of IRS are used to promote IRS signaling. Upregulating IRS in specific tissues can target or prevent diseases involving those specific tissues or cells. For example, upregulation of IRS2 in pancreatic β-cells improves glucose stimulated insulin secretion. Drugs that upregulate the IRS2 gene or promote IRS2 signaling in β-cells will promote β-cell function and are useful to treat or prevent diabetes. Further, the level or functional activity of IRS2 can be modulated in human beings and other mammals in order to ameliorate or prevent the failure or destruction of pancreatic β-cells that causes certain forms of diabetes, and reduce the need for insulin by peripheral insulin sensitive tissues.

[0076] IRS genes also functions in peripheral tissues that respond to insulin. Upregulation of the IRS2 gene or upregulation of IRS2 signaling function makes tissues more sensitive to insulin and thus less insulin is needed to elicit an appropriate response.

[0077] Upregulation of IRS expression or an increase of IRS signaling function is also useful to treat other diseases and disorders. Compounds that promote IRS function are useful for reversing catabolism during acute trauma. Insulin resistance is a major problem during acute trauma. Decreased insulin secretion during acute trauma exacerbates autophagy, which increases muscle and tissue wasting that can progress to kidney disease. Insulin resistance and decreased insulin secretion leads to massive catabolism that can threaten survival in the early period of repair. Both processes can be explained in part by the loss of IRS signaling due to inhibition by inflammatory processes and activation of autophagy. Drugs that promote IRS2 function, prevent IRS2 degradation, or promote IRS2 expression reverse these effects.

[0078] A major problem with obesity is that peripheral tissues become insulin resistant; if β-cells fail to make enough insulin to overcome the insulin resistance then diabetes develops. Applicants have previously discussed how insulin resistance and diabetes can be treated with compounds that upregulate IRS2 in β-cells and / or peripheral tissues. Upregulating IRS2 in β-cells promotes glucose sensitivity and insulin secretion, and upregulating IRS2 in peripheral tissues reduces the insulin requirements. Accordingly, the incidence of life threatening complications of obesity can be reduced.

[0079] Approximately half of the growth of a mouse brain depends on the expression of the IRS2 gene. Drugs that promote IRS2 signaling promote neural growth and regeneration in mammals and people. IRS2 signaling also plays a role in dephosphorylation of the Tau protein, a marker of Alzheimer disease. Upregulation of IRS2 in the hippocampus should promote normal function and contribute to the prevention of the neuronal degeneration associated with Alzheimer disease. Accordingly, extracts of the invention will be beneficial for dementia, including Alzheimer's disease.

[0080] IRS2 signaling also plays a role in feeding behavior. Mice lacking IRS2 tend to gain weight as a result of the inability of the brain to properly assess whether insulin has been secreted or not after a meal, so the brain can not determine whether a meal has in fact been consumed. Upregulation of IRS2 in the hypothalamus, and particularly the arcuate nucleus of the hypothalmus, will promote appetite regulation that results in reduced weight gain or even weight loss

[0081] IRS2 signaling plays a role in fertility. Notably, female mice lacking IRS2 are infertile. By upregulating IRS2 signaling or IRS2 gene expression in pituitary gonadotrophs or ovaries, ovulation may be enhanced

[0082] IRS2 promotes retinal growth. Mice lacking IRS2 display increased loss of retinal neurons, especially rod and cones, leading to blindness. Thus, compounds having insulin augmenting activity are useful for reducing or preventing retinal degeneration and promoting retinal growth and regeneration.

[0083] The invention also provides for coadministration of an extract alone or together with a pharmaceutically acceptable salt, ester, amide, prodrug, or solvate, to a subject in combination with a second therapeutic agent or other treatment.

[0084] Second therapeutic agents for treatment of diabetes and related conditions include biguanides (including, but not limited to metformin), which reduce hepatic glucose output and increase uptake of glucose by the periphery, insulin secretagogues (including but not limited to sulfonylureas and meglitinides, such as repaglinide) which trigger or enhance insulin release by pancreatic β-cells, and PPARγ, PPARα, and PPARα / γ modulators (e.g., thiazolidinediones such as pioglitazone and rosiglitazone).

[0085] Additional second therapeutic agents include GLP1 receptor agonists, including but not limited to GLP1 analogs such as exendin-4 and liraglutide and agents that inhibit degradation of GLP1 by dipeptidyl peptidase-4 (DPP-4). Vildagliptin and sitagliptin are non-limiting examples of DPP-4 inhibitors.

[0086] Still other second therapeutic agents include the sodium glucose transporter type 2 (SGLT-2) inhibitors, which reduce the ability of the kidney to reabsorb glucose after it passes through the glomerulus and into the nephron. SLGT-2 inhibitors including, but not limited to empagliflozin, canagliflozin, or dapagliflozin inhibit reabsorption of glucose by the nephron resulting in large amounts of glucose remaining in the urine. This class of compounds has a significant blood glucose lowering effect but also markedly increases the likelihood of bladder infections and pyelonephritis due to the resulting glucosuria.

[0087] In certain embodiments of the invention, extracts are coadministered with insulin replacement therapy.

[0088] According to the invention, extracts are coadministered with statins and / or other lipid lowering drugs such as MTP inhibitors and LDLR upregulators, antihypertensive agents such as angiotensin antagonists, e.g., losartan, irbesartan, olmesartan, candesartan, and telmisartan, calcium channel antagonists, e.g. lacidipine, ACE inhibitors, e.g., enalapril, and β-andrenergic blockers (β-blockers), e.g., atenolol, labetalol, and nebivolol.

[0089] In another embodiment, a subject is prescribed an extract of the invention in combination with instructions to consume foods with a low glycemic index.

[0090] In a combination therapy, the extract is administered before, during, or after another thereapy as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after administering the second therapeutic agent. For example, an extract of the invention can be administered daily while extended release metformin is administered daily (55, 56). Also, therapy with an extract of the invention can be commenced before, during, or after commencing therapy with another agent. For example, therapy with an extract of the invention can be introduced into a patient already receiving therapy with an insulin secretagogue. In addition, extracts of the present invention may be administered once or twice daily in conjuction with other nutritional supplements, vitamins, nutraceuticals, or dietary supplements. Examples include GCE, chlorogenic acid, chicoric acid, cinnamon and various other hydroxycinnamic acids, chromium, chromium picolinate, a multivitamin, and so on.

[0091] In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of the extracts of the present invention, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

[0092] In another aspect, the present invention provides nutritionally beneficial or supportive compositions which comprise a nutritionally beneficial or supportive amount of the extracts of the present invention, formulated together with one or more active or inactive ingredients carriers (additives) and / or diluents. As described in detail below, the nutritional supplement formulations of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drinks, foods, chewable pastes or gums, drenches (aqueous or non-aqueous solutions or suspensions), capsules, tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. The term "composition" whether in singular or plural form, refers both to extracts from plants that show a positive result in the IRS2 Cell-based assay system described above.

[0093] The phrase "pharmaceutical composition" necessarily includes, when appropriate, nutraceutical compositions, nutritional / dietary supplements, and the like.

[0094] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals with toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. By the term "metal" is meant any element of the periodic table, including but not limited to those elements also known as "transition metals", "inner-transition metals", and "post-transition metals" that is generally considered to have metallic chemical properties and is capable of being present within the body of a mammal without resulting in toxicity or death of the mammal. Examples of a metals embraced by this definition include, but are not limited to, lithium, beryllium, sodium, magnesium, aluminum, potassium, calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, and the like. (86)REFERENCES

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Claims

1. A pharmaceutical composition or nutritional supplement in tablet, capsule, or powder form comprising: a dried herbal extract of Artemisia dracunculus; a dried herbal extract of Cichorium endivia; a dried herbal extract of Lactuca sativa; and chromium, wherein the extracts of Artemisia dracunculus, Cichorium endivia and Lactuca sativa are present in a ratio of 1: 1,375:24 (w / w / w).

2. The pharmaceutical composition or nutritional supplement of claim 1 in tablet form comprising: 0.7 mg of a dried herbal extract of Artemisia dracunculus; 962.7 mg of a dried herbal extract of Cichorium endivia; 16.7 mg of a dried herbal extract of Lactuca sativa; and 1.2 mcg chromium.

3. The pharmaceutical composition or nutritional supplement of claim 1 comprising a total of 50 mg to 1,500 mg of: a dried herbal extract of Artemisia dracunculus; a dried herbal extract of Cichorium endivia; a dried herbal extract of Lactuca sativa; and optionally, chromium.

4. The pharmaceutical composition or nutritional supplement in tablet, capsule, or powderform of claim 1 further comprising at least one pharmaceutically acceptable excipient selected from the group consisting of: dicalcium phosphate, microcrystalline cellulose, silicon dioxide, hydroxypropyl cellulose, stearic acid, croscarmellose sodium, and magnesium stearate; and combinations thereof, preferably wherein the at least one pharmaceutically acceptable excipient is present in an amount of 15-25% of the total weight.

5. The pharmaceutical composition or nutritional supplement of claim 1 for use in treating an Insulin Receptor Substrate (IRS) mediated disease or condition, wherein the IRS mediated disease or condition is diabetes, pre-diabetes, metabolic syndrome, insulin resistance, or dementia.

6. The pharmaceutical composition or nutritional supplement for use according to claim 5 which further comprises administering an antidiabetic agent, insulin, metformin, exenatide, a sulfonylurea, vildagliptin, sitagliptin, a DPP4 inhibitor, meglitinide, exendin-4, liraglutide, a thiazolidinedione, empagliflozin, canagliflozin, dapagliflozin or a GLP1 agonist.

7. The pharmaceutical composition or nutritional supplement for use according to claim 5 or 6 wherein the pharmaceutical composition or nutritional supplement is administered orally twice per day, 30-60 minutes before meals.

8. The pharmaceutical composition or nutritional supplement of claim 1 for use in stimulating IRS2-dependent signal transduction in a subject.

9. An in vitro method of stimulating IRS2-dependent signal transduction comprising contacting a cell with the pharmaceutical composition or nutritional supplement of claim 1.