Claims:We Claim:
1. A method of enhancing the normalization of a metabolic disorder in a subject comprising the administration of a therapeutically effective amount of supercritical CO2, defatted N. sativa ground seeds and at least one member of the group comprising a phytoceutical, and a pharmaceutical.
2. The method according to claim 1, wherein the phytoceutical comprises at least one member of the group comprising whole, ground N. sativa seeds. whole, ground Trigonella foenum-graecum seeds, and Momordica charantia.
3. The method according to claim 1, wherein the therapeutic comprises at least one member of the group comprising metformin, glimepiride, insulin, sitagliptin, and teneligliptin.
4. The method according to claim 1, wherein the metabolic disorder comprises at least one member of the group comprising obesity, metabolic syndrome, diabetes, hepatic steatosis, hyperlipidemia, leaky gut, irritable bowel disease, and cardiovascular disease.
5. A composition for enhancing the normalization of a metabolic disorder in a subject comprising a therapeutically effective amount of at least one member of the group comprising a supercritical CO2 defatted N. sativa ground seeds, a phytoceutical, and a pharmaceutical.
6. The composition according to claim 5, wherein the phytoceutical comprises at least one member of the group comprising whole, ground N. sativa seeds. whole, ground Trigonella foenum-graecum seeds, and Momordica charantia.
7. The composition according to claim 5, the therapeutic comprises at least one member of the group comprising metformin, glimepiride, insulin, sitagliptin, and teneligliptin.
8. The composition according to claim 5, wherein the metabolic disorder comprises at least one member of the group comprising obesity, metabolic syndrome, diabetes, hepatic steatosis, hyperlipidemia, leaky gut, irritable bowel disease, and cardiovascular disease.
, Description:PREAMBLE
The following specification particularly describes the invention and the manner in which it is performed:-

Field of the Invention
[0001] The present invention relates to a novel, defatted product of Nigella sativa seeds that functions alone or in combination with other phytoceuticals or drugs to improve averse metabolic functioning and improved health. These compositions would be useful in the prevention, treatment or co-treatment of metabolic disorders such as obesity, diabetes, and metabolic syndrome as well ––as hyperlipidemia, hepatic steatosis. hypertension and exercise recovery.
BACKGROUND OF THE INVENTION
Description of the Related Art
[0002] Nigella sativa belongs to family Ranunculaceae and is commonly known as black seed or black curcumin. In the Indian subcontinent, Arabian countries, and Europe, N. sativa seeds, alone or in combination with honey, have been used for centuries to treat various human conditions, including obesity, hyperglycemia. elevated plasma lipids, cardiovascular disorders systemic inflammation, infectious diseases, inflammation, cough, bronchitis, headache, eczema, fever, dizziness influenza. and wounds [Yimer EM, Tuem KB, Karim A, Ur-Rehman N, Anwar F: Nigella sativa L. (Black Cumin): A Promising Natural Remedy for Wide Range of Illnesses. Evid Based Complement Alternat Med. 2019:1528635]. Through recent decades of modern research, much of the biological activity of the seeds is believed to be due to the essential oil content, especially thymoquinone (TQ), which is also present in the fixed oil. Little research has been published on the non-lipid components (e.g. alkaloids and melanin) of N. sativa that are found primarily in the pericarp (Table 1 and Figure 1).
[0003] The seeds of N. sativa are characterized by a very low degree of toxicity. Administration of either the seed, its extract or its oil has been shown not to induce significant toxicity or adverse effects on liver or kidney functions even at extremely high doses [Ali BH, Blunden G. 2003. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 17: 299-305]. Thus, N. sativa seed possess the necessary safety factor for commercialization in the dietary supplement or pharmaceutical market.
Table 1
Chemical Content of Various Fractions of N. sativa Seeds†

SEED FRACTION
COMPONENT CONTENT % [W/W]
Fixed Oils 36
Essential fatty acids in fixed oil Myristic acid (C14) 0.50
Palmitic acid (C16) 13.7
Palmitoleic acid (C16 ?-9) 0.1
Stearic acid (C18) 2.6
Linoleic acid (C18 ?-6) 57.9
Linolenic acid (C18 ??? 0.2
Arachidic acid (C20) 1.3
Essential oil components a-Pinene, camphene, b-pinene, sabinene, ß-myrcene, a-terpinene, limonene, b-phellandrene, 1,8-cineole, g-terpinene, p-cymene (7.1-15.5%), a-terpinolene, 2-heptanal, thujone, trans-sabinenehydrate, longipinene, camphor, linalool, cis-Sabinenehydrate, longifoline (1.0-8.0%), bornylacetate, 2-undecanone, 4-terpineol (2.0-6.6%), borneol, carvone, thymoquinone (27-57%), 2-tridecanone, t-anethole (0.25-2.3%), p-cymene-8-ol, p-anisaldehyde, thymol and carvacrol (5.8-11.6%) 0.5 – 1.5
Polar and Non-fat components Melanin, alkaloids, protein, thiamin, ribovflavin, pyridoxine, niacin, folic acid, and calcium. 58
†Burits M, Bucar F: Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000;14(5):323-328.
As seen in Table 1, the lipid content of N. sativa seeds represents approximately 36 % of the dry weight.
[0004] N. sativa is one of only two Nigella species reported among the natural Nigella species to contain pyrozol-type alkaloids, nigellidine and nigellicine (Figure 1A). These compounds, as well as the isoquinoline alkaloids, nigellicimine and nigellicimine-N-oxide (Figure 1A), are almost exclusively accumulated in the seed coat [Botnick I, Xue W, Bar E, et al.: Distribution of primary and specialized metabolites in Nigella sativa seeds, a spice with vast traditional and historical uses. Molecules. 2012;17(9):10159-10177].
[0005] Melanin refers to a group of high molecular weight, black, and brown pigments formed through the oxidation and polymerization of phenolic compounds (Figure 1B). This pigment is present in all kingdoms of living organisms, but it remains the most enigmatic pigment in plants. The poor solubility of melanin in particular solvents and its complex polymeric nature significantly constrain its study [Glagoleva AY, Shoeva OY, Khlestkina EK: Melanin Pigment in Plants: Current Knowledge and Future Perspectives. Front Plant Sci. 2020;11:770]. The characteristic black color of N. sativa is due to the melanin content (approximately 2%) of the seed coat. The plant melanin has been associated with many protective roles such as photo-protection [Novikov, D.A., V.P. Kurchenko and I.I. Azarko, 2000. [Photoprotective properties of melanins from grape (Vitis vinifera) and black tea (Thea sinensis)]. Radiatsionnaia Biol. Radioecol., 41: 664-670], hepato-protection [Hung, Y.C., V.M. Sava, S.Y. Makan, M.Y. Hong and G.S. Huang, 2004. Preventive effect of Thea sinensis melanin against acetaminophen-induced hepatic injury in mice. J. Agric. Food Chem., 52: 5284-5289], nephron-protection [Hung, Y.C., G.S. Huang, L.W. Lin, M.Y. Hong and P.S. Se, 2007. Thea sinensis melanin prevents cisplatin-induced nephrotoxicity in mice. Food Chem. Toxicol., 45: 1123-1130], anti-inflammatory effect and antioxidant effect [Avramidis, N., A. Kourounakis, L. Hadjipetrou and V. Senchuk, 1998. Anti-inflammatory and immunomodulating properties of grape melanin: Inhibitory effects on paw edema and adjuvant induced disease. Arzneimittel-Forschung, 48: 764-771. Plant melanin from N. sativa or other sources, however, have not been clinically studied. Further, supercritical CO2 extraction has never been used to isolate melanin from lipids.
[0006] Extraction processes - Traditional solvent extraction is time-consuming, requires multiple steps, and consumes large amounts of organic solvents. The amount and the price of organic solvent directly influences the total cost of producing an acceptable product. Moreover, when the final product is used as a food ingredient, it is absolutely necessary to remove all potentially toxic solvents.
[0007] Supercritical fluid extraction (SFE) has already proven itself as an attractive technique for selectively removing compounds from complex food matrices. Extraction with liquid or supercritical CO2 is essentially a simple concept, although specialized equipment and technically skilled operators are needed to bring concept to reality. CO2 can exist in solid, liquid or gaseous phase, in common with all chemical substances. Furthermore, if the liquid phase is taken beyond the so-called critical points of temperature and pressure, a supercritical fluid is formed, which in simple terms can be considered as a dense gas. Both liquid and supercritical CO2 act effectively as solvents. While liquid CO2 is excellent for dissolving relatively non-polar, small molecules (liquid CO2 can be compared to hexane in this regard), supercritical CO2 allows the extraction of larger and more polar compounds. Through the judicious manipulation of pressure and temperature, it is theoretically possible to target an infinite range of polarities for extraction. Thus, supercritical extraction has the potential for creating novel extracts of commonly used herbs.
[0008] Supercritical CO2 is pumped through the plant material in the extraction columns, where extraction of the desired plant components takes place. After passing through the expansion valve, the extract-laden CO2 is depressurized and the extract precipitates out of solution in the separator. The gaseous CO2 can be recycled for further extractions.
[0009] What sets liquid and supercritical CO2 apart from other solvents such as hexane and ethanol are two key properties. Firstly, once the extraction has been affected, the CO2 solvent is released as a gas and recycled in the process, so that a solvent-free extract is produce. This has two immediate benefits – the extract is free of all solvent residues, and importantly so is the extracted material, which can then be further used for processing if required. Secondly, the solvating power of CO2 can be manipulated readily by altering temperature and pressure. This means that extraction can be highly selective and novel through the production of literally an infinite range of extraction polarities.
[00010] Trigonella foenum-graecum (fenugreek) has been cited in ancient texts as well as Ayurveda and traditional Chinese medicine. Clinically reported uses of fenugreek, which largely overlap with N. sativa, include weight loss, hyperglycemia, elevated plasma lipids, systemic inflammation, microbial infections, increasing postpartum milk secretion, and general health improvement [Nagulapalli Venkata KC, Swaroop A, Bagchi D, Bishayee A: A small plant with big benefits: Fenugreek (Trigonella foenum-graecum Linn.) for disease prevention and health promotion. Mol Nutr Food Res. 2017;61(6)]. Fenugreek is also used to enhance the sensory quality of foods as a stabilizer, emulsifier and adhesive [Wani SA, Kumar P: Fenugreek: A review on its nutraceutical properties and utilization in various food products. J Saudi Soc Agric Sci. 2016].
[00011] It has been estimated that in 2019 approximately 463 million people globally were diabetic (~90% type 2 diabetic), with China (25.1%), India (16.6%), and the United States of America (6.69%) representing nearly 50% of that total. Additionally, increases in the worldwide prevalence of diabetes of 25 and 51% are projected over the next 10 and 25 years, respectively [Saeedi P, Petersohn I, Salpea P, et al.: Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9^th edition. Diabetes Research and Clinical Practice. 2019;157]. In India alone, the prevalence of type 2 diabetes (T2D) as well as prediabetes has been determined to be approximately 12.1 and 14.0% respectively, with no differences between genders [Ramachandran A, Snehalatha C, Kapur A, et al.: High prevalence of diabetes and impaired glucose tolerance in India: National Urban Diabetes Survey. Diabetologia. 2001;44(9):1094-1101]. In India patients with T2D exhibited a two-fold risk of coronary artery disease-related deaths compared with white, T2D Europeans [Forouhi NG, Sattar N, Tillin T, McKeigue PM, Chaturvedi N: Do known risk factors explain the higher coronary heart disease mortality in South Asian compared with European men? Prospective follow-up of the Southall and Brent studies, UK. Diabetologia. 2006;49(11):2580-2588].
[00012] Prediabetes is generally asymptomatic and studies suggest it goes largely undiagnosed until the development of T2D or its complications [Ramachandran A, Snehalatha C, Kapur A, et al.: High prevalence of diabetes and impaired glucose tolerance in India: National Urban Diabetes Survey. Diabetologia. 2001;44(9):1094-1101]. Further, meta-analyses have revealed that T2D and its precursor prediabetes are a likely consequence of the "NAFLD pathway", in which nonalcoholic fatty liver disease (NAFLD) establishes multiple sites of metabolic dysregulation (multiple-hit pathogenesis) that become strong determinants for progression to prediabetes, metabolic syndrome (MetS), and T2D[Lonardo A, Ballestri S, Marchesini G, Angulo P, Loria P: Nonalcoholic fatty liver disease: a precursor of the metabolic syndrome. Dig Liver Dis. 2015;47(3):181-190]. The defining characteristic of NAFLD is excess fat stored in liver cells adversely modifying hepatic functioning.
[00013] When therapeutic intervention for T2D or associated liver disease is warranted, however, metformin remains the recommended drug of choice [Qaseem A, Barry MJ, Humphrey LL, Forciea MA: Oral Pharmacologic Treatment of Type 2 Diabetes Mellitus: A Clinical Practice Guideline Update From the American College of Physicians. Annals of Internal Medicine. 2017;166(4):279-290]. Moreover, drugs alone are neither economically nor clinically sustainable. In addition to the obvious financial burden of a pharmaceutical solution, factors such as a monotherapy failure rate approaching 50% over 5 years [Jeon JY, Lee SJ, Lee S, et al.: Failure of monotherapy in clinical practice in patients with type 2 diabetes: The Korean National Diabetes Program. J Diabetes Investig. 2018;9(5):1144-1152], and low patient compliance due to the complexity of treatment for developing comorbidities [Marcum ZA, Gellad WF: Medication Adherence to Multidrug Regimens. Clinics in Geriatric Medicine. 2012;28(2):287-300], impose significant impediments to the management of T2D through current therapeutic guidelines. In support of a multiple-hit pathophysiology, studies have now indicated that early antidiabetic drug failure is related to increased hepatic transaminase levels within the normal range [Irace C, Rossetti M, Carallo C, et al.: Transaminase levels in the upper normal range are associated with oral hypoglycemic drug therapy failure in patients with type 2 diabetes. Acta Diabetol. 2012;49(3):193-197]. Such small increases in AST and ALT may be early indicators of the NAFLD pathway activation toward the development of T2D and reflective of a multitude of simultaneous, macro- and micro-metabolic alterations occurring within the patient. Moreover, its asymptomatic nature precludes therapeutic intervention.
[00014] Obesity is a disease resulting from a prolonged positive imbalance between energy intake and energy expenditure. Excess body weight is one of the most important risk factors for all-cause morbidity and mortality. The likelihood of developing conditions such as type 2 diabetes, heart disease, cancer and osteoporosis of weight-bearing joints increases with body weight. The rapidly increasing world-wide incidence of obesity and its association with serious comorbid diseases means it is beginning to replace undernutrition and infectious diseases as the most significant contributor to ill health in the developed world.
[00015] It is now generally accepted that adipose tissue acts as an endocrine organ producing a number of biologically active peptides with an important role in the regulation of food intake, energy expenditure and a series of metabolic processes. Adipose tissue secretes a number of bioactive peptides collectively termed adipokines. Through their secretory function, adipocytes lie at the heart of a complex network capable of influencing several physiological processes. Dysregulation of adipokine production with alteration of adipocyte mass has been implicated in metabolic and cardiovascular complications of obesity. In obese individuals, excessive production of acylation-stimulating protein (ASP), TNFa, IL-6 or resistin deteriorates insulin action in muscles and liver, while increased angiotensinogen and PAI-1 secretion favors hypertension and impaired fibrinolysis. Leptin regulates energy balance and exerts an insulin-sensitizing effect. These beneficial effects are reduced in obesity due to leptin resistance. Adiponectin increases insulin action in muscles and liver and exerts an anti-atherogenic effect. Further, adiponectin is the only known adipokine whose circulating levels are decreased in the obese state. The thiazolidinedione anti-diabetic drugs increase plasma adiponectin, supporting the idea that adipokine-targeted pharmacology represents a promising therapeutic approach to control type 2 diabetes and cardiovascular diseases in obesity.
[00016] Metabolism of white adipose tissue is involved in the control of body fat content, especially visceral adipose tissue. Adipose tissue plays a central role in the control of energy homeostasis through the storage and turnover of triglycerides and through the secretion of factors that affect satiety and fuel utilization. Mitochondrial remodeling and increased energy expenditure in white fat may affect whole-body energy homeostasis and insulin sensitivity [Wilson-Fritch L, Nicoloro S, Chouinard M, Lazar MA, Chui PC, et al. 2004. Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone. J Clin Invest 114: 1281-9].
[00017] Oxidative stress - Current consensus is that hyperglycemia results in the production of reactive oxygen (oxidative stress) and nitrogen species, which leads to oxidative myocardial injury. Alterations in myocardial structure and function occur in the late stage of diabetes. These chronic alterations are believed to result from acute cardiac responses to suddenly increased glucose levels at the early stage of diabetes. Oxidative stress, induced by reactive oxygen and nitrogen species derived from hyperglycemia, causes abnormal gene expression, altered signal transduction, and the activation of pathways leading to programmed myocardial cell deaths. The resulting myocardial cell loss thus plays a critical role in the development of diabetic cardiomyopathy.
[00018] Mitochondrial uncoupling – Controlling adiposity by targeted modulation of adipocyte mitochondrial membrane potential could offer an attractive alternative to current dietary approaches. It has recently been reported that forced uncoupling protein 1 (UCP1) expression in white adipocytes derived from a murine (3T3-L1) preadipocyte cell line reduced the total lipid accumulation by approximately 30% without affecting other adipocyte markers, such as cytosolic glycerol-3-phosphate dehydrogenase activity and leptin production. The expression of UCP1 also decreased glycerol output and increased glucose uptake, lactate output, and the sensitivity of cellular ATP content to nutrient removal [Si Y, Palani S, Jayaraman A, Lee K. 2007. Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism. J Lipid Res 48: 826-36]. These results suggest that the targeting reduction in intracellular lipid of adipocytes by uncoupling mitochondrial membrane potential represents a feasible mechanism for identification of anti-obesity molecules. Nevertheless, the putative role of various mitochondrial protonophores in white fat cells in the control of adiposity remains to be clarified.
[00019] Thermogenesis - Thermogenesis or uncoupling of mitochondrial membrane potential may be activated both indirectly and directly. Indirect activation occurs through ß3AR and ß3 agonists (ß3AA). In the early 1980s, an "atypical" beta-adrenergic receptor was discovered and subsequently called ß3AR. Further clinical testing will be necessary, using compounds with improved oral bioavailability and potency, to help assess the physiology of the ß3AR in humans and its attractiveness as a potential therapeutic for the treatment of type 2 diabetes and obesity [de Souza CJ, Burkey BF. 2001. Beta 3-adrenoceptor agonists as anti-diabetic and anti-obesity drugs in humans. Curr Pharm Des 7: 1433-49].
[00020] Adaptive thermogenesis - Adaptive thermogenesis represents the decrease in energy expenditure (EE) beyond what could be predicted from the changes in fat mass or fat-free mass under conditions of standardized physical activity in response to a decrease in energy intake. Thus, there exists the potential of adaptive thermogenesis to impede obesity treatment on a short- and long-term basis, at least in some individuals. In some cases, the adaptive decrease in thermogenesis was shown to be significantly related to a single cycle of body weight loss and regain, an increase in plasma organochlorine concentration following weight loss. This suggests that energy metabolism might be sensitive to stimuli of different physiological nature and that adaptive thermogenesis could be quantitatively more important than what is generally perceived by health professionals and nutrition specialists. However, from a clinical point of view, several issues remain to be investigated in order to more clearly identify adaptive thermogenesis determining factors and to develop strategies to cope with them. Along these lines, it is concluded that unsuccessful weight loss interventions and reduced body weight maintenance could be partly due, in some vulnerable individuals, to the adaptive thermogenesis, which is multicausal, quantitatively significant, and has the capacity to compensate for a given prescribed energy deficit, possibly going beyond any good compliance of some patients.
[00021] Additional approaches to increasing thermogenesis appear necessary to affect sustained weight loss in obese subjects. One of these approaches with demonstrated proof-of-concept in humans is direct, chemical stimulation of thermogenesis through chemical uncoupling of mitochondrial membrane potential using 2,4-dinitrophenol (DNP). Doubling metabolic rate by selectively and modestly uncoupling adipocyte thermogenesis should produce few adverse side-effects as this level of increase would only be equivalent to mild exercise. DNP is a lipid –soluble, weak acid that acts as a protonophore because it can cross membranes protonated, lose its proton and return as the anion, then reprotonate and repeat the cycle. In this way, it increases the basal proton conductance of mitochondria and uncouples oxidative phosphorylation. The overall result is a decrease in ATP formation for an equivalent amount of oxidation.
[00022] Improvements in the treatment of noncardiac complications from diabetes have resulted in heart disease becoming a leading cause of death in diabetic patients. Pathogenesis of diabetic cardiomyopathy (DCM) is a complicated and chronic process that is secondary to acute cardiac responses to diabetes. One of the acute responses is cardiac cell death that plays a critical role in the initiation and development of DCM. Besides hyperglycemia, inflammatory response in the diabetic heart is also a major cause for cardiac cell death. Diabetes or obesity often causes systemic and cardiac increases in tumor necrosis factor-alpha (TNF?), interleukin-18 and PAI-1. However, how these cytokines cause cardiac cell death remains unclear. It has been considered to relate to oxidative and/or nitrosative stress. Cardiac cell death is induced by the inflammatory cytokines that are increased in response to diabetes. Inflammatory cytokine-induced cardiac cell death is mediated by oxidative stress and is also the major initiator for DCM development [Wang, Y. H., and Cai, L. Diabetes/obesity-related inflammation, cardiac cell death and cardiomyopathy. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2006, 31, 814-8].
[00023] AMP-activated protein kinase – The 5'-AMP-activated protein kinase (AMPK) functions as an intracellular fuel sensor that affects metabolism and gene expression in humans and rodents. AMPK has been described as an integrator of regulatory signals monitoring systemic and cellular energy status. Recently, it has been proposed that AMPK could provide a link in metabolic defects underlying progression to the metabolic syndrome. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit alpha and two regulatory subunits beta and gamma. Rising AMP and falling ATP activate AMPK. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKKbeta (calmodulin-dependent protein kinase kinase). The AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression.
[00024] As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole-body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischemia playing a role in glucose transport and fatty acid oxidation. In liver, activation of AMPK results in enhanced fatty acid oxidation as well as decreased glucose production [Viollet, B., Mounier, R., Leclerc, J., Yazigi, A., Foretz, M., and Andreelli, F. Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders. Diabetes Metab 2007, 33, 395-402]. The net effect of AMPK activation is stimulation of hepatic fatty acid oxidation and ketogenesis, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipolysis and lipogenesis, stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulation of insulin secretion by pancreatic beta-cells.
[00025] 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) represents a useful tool for identifying new target pathways and processes regulated by the AMPK protein kinase cascade. Incubation of rat hepatocytes with AICAR results in accumulation of the monophosphorylated derivative (5-aminoimidaz-ole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells.
[00026] AMPK also regulates food intake and energy expenditure at the whole-body level, in particular by mediating the effects of insulin sensitizing adipokines leptin and adiponectin. AMPK is robustly activated during skeletal muscle contraction and myocardial ischemia playing a role in glucose transport and fatty acid oxidation.
[00027] Additional approaches to affect sustained weight loss in obese subjects represent a critical need. Further, compounds or formulations that safely and effectively activate AMPK may function to stimulate hepatic fatty acid oxidation and ketogenesis, inhibit cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibit adipocyte lipolysis and lipogenesis, stimulate of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulate insulin secretion by pancreatic beta-cells.
[00028] Inflammatory bowel disease - Each year inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, afflict more than one million people in the United States [Baumgart DC, Bernstein CN, Abbas Z, et al. IBD Around the world: Comparing the epidemiology, diagnosis, and treatment: Proceedings of the World Digestive Health Day 2010 - Inflammatory bowel disease task force meeting. Inflamm Bowel Dis 2010]. The healthy gastrointestinal tract absorbs only the small molecules like those that are product of complete digestion. These molecules are the amino acids, simple sugars, fatty acids, vitamins, and minerals that the body requires for all the processes of life to function properly. The intestines, small intestine in particular, only allow these substances to enter the body due to the fact that the cells that make up the intestinal wall are tightly packed together. The intestines also contain special proteins called 'carrier proteins' that are responsible for binding to certain nutrients and transporting them through the intestinal wall and into the bloodstream.
[00029] Leaky Gut Syndrome (LGS) is common parlance for the disruption of the intestinal membrane integrity as a result of oxidative stressors or pro-inflammatory mediators, which compromise the ability of the intestinal wall to keep out large and undesirable molecules. Hence the name, as substances that are normally kept outside the body and within the intestines, are "leaking" across the intestinal wall and into the body as a whole. This happens when the spaces between the cells of the intestinal wall become enlarged for various reasons and allow larger, less digested particles and toxins to pass through—causing LGS. The body then recognizes these particles as foreign “invaders,” and the immune system attempts to fight them off—which can set the stage for various autoimmune disorders.
[00030] This disruption of intestinal membrane integrity is applicable to the pathognomic impacts of asthma, arthritis, food allergies, ulcers, Crohn’s disease, ulcerative colitis, celiac disease, autoimmune diseases, alcoholism, chronic fatigue, joint pain, migraines, diarrhea, parasitic infections, dysbiosis, candidiasis, multiple sclerosis, diabetes, multiple sclerosis, vasculitis, Addison’s disease, lupus, thyroiditis, and fibromyalgia.
[00031] A novel, defatted, product of ground N. sativa seeds produced by sequential steps of supercritical CO2 extraction involving changes in temperature, pressure and time is described. This SDNS product contains about 20 to 35% more alkaloids and melanin that whole N. sativa seeds and unexpectedly exhibits chemical and biological activities in vitro and clinically that differ both qualitatively and quantitatively from whole seeds and TQ, the putative active component of N. sativa seed extracts.
[00032] To date no process for supercritical lipid removal of N. sativa has been described that is useful for commercial quantities of ground seed. It is well known in the art, that changes in scale will profoundly affect the quality and quantity of the extract produced. The procedure described herein can be used in the extraction of commercial quantities of N. sativa seed in amounts greater than about 10 kg.
SUMMARY OF THE INVENTION
[00033] Before invention embodiments are disclosed and described, it is to be understood that no limitation to the particular structures, process steps, or materials disclosed herein is intended, but also includes equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used to describe particular examples only and is not intended to be limiting. The same reference numerals in different drawings represent the same element. Numbers provided in flow charts and processes are provided for clarity in illustrating steps and operations and do not necessarily indicate a particular order or sequence. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise.
Objects
[00034] The present invention further provides compositions of supercritical CO2 defatted N. sativa seeds produced on a commercial scale, alone or in combination with other phytoceuticals, to reduce body weights, waist circumference, adiposity, hyperglycemia, hyperlipidemia, hepatic steatosis, and cardiovascular risk in a subject in need thereof.
[00035] The present invention provides a composition for treating diseases or pathologies related to obesity, type 1 or type 2 diabetes, metabolic syndrome, hepatic steatosis, inflammation, and oxidative stress, in an animal comprising administering to an animal exhibiting signs, signalments, or symptoms of the pathology or disease an effective amount of a formulation comprising supercritical, defatted N. sativa seeds and continuing the administration of the composition until the signs, signalments or symptoms are reduced.
[00036] The present invention further provides a method of treating diseases or pathologies related to obesity, type 1 or type 2 diabetes, metabolic syndrome, hepatic steatosis, inflammation, and oxidative stress, in an animal comprising administering to an animal exhibiting signs, signalments, or symptoms of the pathology or disease an effective amount of a formulation comprising supercritical, defatted N. sativa seeds and continuing the administration of the composition until the signs, signalments or symptoms are reduced.
[00037] The present invention relates to the unexpected discovery that defatted N. sativa seeds decrease mitochondrial membrane potential in adipocytes implying decreased ATP synthesis and increased thermogenesis. The invention provides methods for modifying adipocyte physiology in a subject, comprising administering to the subject a pharmaceutical composition of a supercritical CO2 defatted N. sativa seeds or mixtures thereof. Preferred embodiments provide compositions and methods for enhancing adipocyte thermogenesis or decreasing oxidative stress utilizing supercritical fluid defatted N. sativa seeds.
[00038] Further, the present invention relates to the unexpected discovery that defatted N. sativa seeds inhibit iNOS-mediated NO biosynthesis as a result of the action of multiple external stimuli to adipocytes implying inhibition of protein nitrosylation.
[00039] Additionally, the present invention relates to the unexpected discovery that defatted N. sativa seeds dramatically activate AMPK implying stimulation of hepatic fatty acid oxidation and ketogenesis, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipolysis and lipogenesis, stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake, and modulation of insulin secretion by pancreatic beta-cells.
[00040] Moreover, the present invention relates to the unexpected discovery that defatted N. sativa seeds in combination with whole, ground N. sativa and fenugreek in a flat bread, consumed as a normal part of an unmodified diet, can safely decrease body weights, waist circumference, adiposity, hyperglycemia, hyperlipidemia, hepatic steatosis, and cardiovascular risk.
BRIEF DESCRIPTION OF THE DRAWINGS
[00041] FIG 1 depicts is the structures of [A] alkaloids and [B] melanin found in supercritical defatted Nigella sativa seeds.
[00042] FIG 2 is a schematic of the sequential steps involved in the lipid removal from whole, ground N. sativa seeds using supercritical CO2 extraction.
[00043] FIG 3 depicts a schematic representation of the clinical trial.
[00044] FIG 4 depicts absolute changes in total body weight and total fat mass for all subjects at 6 and 12 weeks.
[00045] FIG 5 depicts (A) Linear regression of absolute HbA1c changes over 12 weeks; (B) Absolute HbA1c changes (± SEM) over 12 weeks for the HbA1c = 7.0 and HbA1c < 7.0 post hoc subgroups at baseline and 12 weeks; (C) Fasting blood glucose (FBG) mg/dL (±SEM) over 12 weeks for the HbA1c = 7.0 and HbA1c < 7.0 post hoc subgroups, respectively, at baseline, 6 weeks, and 12 weeks; (D) Post postprandial blood glucose (PPBG) mg/dL (± SEM) for the HbA1c = 7.0 and HbA1c < 7.0 post hoc subgroups at baseline, 6 weeks, and 12 weeks.
[00046] FIG 6 depicts individual changes in (A) fatty liver index; (B) lipid accumulation product, and (C) hepatic stenosis index,
DETAILED DESCRIPTION OF THE INVENTION
[00047] The compositions of the invention include a unique composition comprising supercritical fluid defatted N. sativa seeds. These compositions may be used for reducing body weight, adiposity, hyperglycemia, metabolic syndrome, type 1 or type 2 diabetes, hyperlipidemia, hepatic steatosis, and cardiovascular risk as well as their antioxidant or anti-inflammatory properties. The resulting compositions can be consumed as a medial food or dietary supplement to address obesity, adiposity, hyperglycemia, metabolic syndrome, type 1 or type 2 diabetes, hyperlipidemia, hepatic steatosis, and cardiovascular risk, oxidative stress, benign prostate hyperplasia, leaky gut syndrome, irritable bowel syndrome, increasing exercise endurance or other inflammatory-based pathologies.
[00048] The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
[00049] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufman et al., Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth general definitions of medical terms and the general principles of pharmacology, respectively, include Stedman’s Medical Dictionary [26th edition] and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).
[00050] As used herein, the following abbreviations are defined as: AKP: Alkaline phosphatase; ALT: Alanine transaminase; AST: Aspartate aminotransferase; BMI: Body Mass Index; BUN: Blood urea nitrogen; DBP: Diastolic blood pressure; DM: Diabetic; DM/OW: Diabetic and overweight; eAG: estimated average glucose; FBG: Fasting blood glucose; GGT: Gamma-glutamyl transferase; HbA1c: Glycated Hemoglobin; HDL: High Density Lipoprotein; ICO: Index of central obesity; LDL: Low Density Lipoprotein; MAP: Mean arterial pressure; ND: Not determined; OW: Over weight; PP: Pulse pressure; PPG: Postprandial glucose; SBP: Systolic blood pressure; T3: Triiodothyronine; T4: Thyroxine; TC: Total cholesterol; TG: Triglycerides; TSH: Thyroid stimulating hormone; VLDL: Very low density lipoprotein.
[00051] In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. As used in this specification, the singular forms "a," "an" and "the" specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. Additionally, as used herein, unless specifically indicated otherwise, the word "or" is used in the "inclusive" sense of "and/or" and not the "exclusive" sense of "either/or.” The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.
[00052] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for a variable that is inherently discrete, the variable can be equal to any integer value of the numerical range, including the end-points of the range. Similarly, for a variable that is inherently continuous, the variable can be equal to any real value of the numerical range, including the end-points of the range. As an example, a variable that is described as having values between 0 and 2 can be 0, 1 or 2 for variables that are inherently discrete, and can be 0.0, 0.1, 0.01, 0.001, or any other real value for variables that are inherently continuous.
[00053] As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term "comprising" means that the compound or composition includes at least the recited features or compounds, but may also include additional features or compounds.
[00054] Reference is made hereinafter in detail to specific embodiments of the invention. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well known process operations have not been described in detail, in order not to unnecessarily obscure the present invention.
[00055] Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted.
[00056] The term “phytoceutical” and its verbal variants refer to any plant, combinations of plants, or microbiological material that is consumed for beneficial effects on metabolic functioning in a subject.
[00057] The term “treat” and its verbal variants refer to palliation or amelioration of an undesirable physiological state. Thus, for example, where the physiological state is poor glucose tolerance, “treatment” refers to improving the glucose tolerance of a treated subject. As another example, where the physiological state is obesity, the term “treatment” refers to reducing the body fat mass, improving the body mass or improving the body fat ratio of a subject. Treatment of diabetes means improvement of blood glucose control. Treatment of inflammatory diseases means reducing the inflammatory response either systemically or locally within the body. Treatment of osteoporosis means an increase in the density of bone mineralization or a favorable change in metabolic or systemic markers of bone mineralization. The person skilled in the art will recognize that treatment may, but need not always, include remission or cure.
[00058] The term “prevent” and its variants refer to prophylaxis against a particular undesirable physiological condition. The prophylaxis may be partial or complete. Partial prophylaxis may result in the delayed onset of a physiological condition. The person skilled in the art will recognize the desirability of delaying onset of a physiological condition, and will know to administer the compositions of the invention to subjects who are at risk for certain physiological conditions in order to delay the onset of those conditions. For example, the person skilled in the art will recognize that obese subjects are at elevated risk for coronary artery disease. Thus, the person skilled in the art will administer compositions of the invention in order to increase insulin sensitivity in an obese, whereby the onset of diabetes mellitus or dyslipemia may be prevented entirely or delayed.
As used herein “adaptive thermogenesis” represents the decrease in energy expenditure beyond what could be predicted from the changes in fat mass or fat-free mass under conditions of standardized physical activity in response to a decrease in energy intake.
[00059] As used herein the term “oxidative stress” is used to describe the effect of oxidation in which an abnormal level of reactive oxygen species (ROS), such as the free radicals (e.g. hydroxyl, nitric acid, superoxide) or the non-radicals (e.g. hydrogen peroxide, lipid peroxide) lead to damage (called oxidative damage) to specific molecules with consequential injury to cells or tissue. Increased production of ROS occurs as a result of fungal or viral infection, inflammation, ageing, UV radiation, pollution, excessive alcohol consumption, cigarette smoking, etc. Removal or neutralization of ROS is achieved with antioxidants, endogenous (e.g. catalase, glutathione, superoxide dismutase) or exogenous (e.g. vitamins A, C, E, bioflavonoids, carotenoids). Oxidative damage to the eye, particularly the retina and the lens, is a contributing factor to age-related macular degeneration and cataract.
[00060] All forms of life maintain a reducing environment within their cells. This reducing environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA.
[00061] In humans, oxidative stress is involved in the etiology of many diseases, such as obesity, atherosclerosis, metabolic syndrome, type 1 and type 2 diabetes, hepatic steatosis, Parkinson’s disease, cardiac arrest, myocardial infarction, Alzheimer’s disease, Fragile X syndrome and chronic fatigue syndrome. Short-term oxidative stress, however, may also be important in prevention of aging by induction of a process named mitohormesis. ROS can be beneficial, as they are used by the immune system as a way to attack and kill invading pathogens. ROS are also used in cell signaling. This is dubbed redox signaling and is a critical component of such pathognomic conditions as obesity, metabolic syndrome, colitis, irritable bowel syndrome and adaptive thermogenesis.
[00062] The methods of the present invention are intended for use with any subject that may experience the benefits of the methods of the invention. Thus, in accordance with the invention, "subjects" include humans as well as non-human subject, particularly domesticated animals. It will be understood that the subject to which a compound of the invention is administered need not suffer from a specific traumatic state. Indeed, the compounds of the invention may be administered prophylactically, prior to any development of symptoms. The term "therapeutic," "therapeutically," and permutations of these terms are used to encompass therapeutic, palliative as well as prophylactic uses.
[00063] As used herein, the term "solvent" refers to a liquid of gaseous, aqueous or organic nature possessing the necessary characteristics to extract solid material from the hop plant product. Examples of solvents would include, but not limited to, water, steam, superheated water, methanol, ethanol, hexane, chloroform, liquid CO2, liquid N2, propane, or any combinations of such materials.
[00064] As used herein, the term "CO2 extract" refers to the solid material resulting from exposing more than 10 kg powdered N. sativa seeds to a liquid or supercritical CO2 preparation followed by subsequent removal of the CO2.
[00065] As used herein, the term “defatted, CO2 extract" refers to the solid material resulting from exposing more than 10 kg powdered N. sativa seeds to a liquid or supercritical CO2 preparation at sufficient temperature and pressure to remove essentially all fat and fat-soluble materials followed by subsequent removal of the CO2.
[00066] As used herein, “decreased secretion or biosynthesis,” means to decrease by at least 3%, the rate of secretion or amount of biosynthesis of the referent compound. The invention further provides a method of decreasing adipocyte or myocyte concentrations of inflammatory mediators in a subject, comprising administering to the subject an amount of the composition sufficient to decrease NO secretion from adipocytes or myocytes in the subject. In general, a decrease in adipocyte or myocyte NO secretion or biosynthesis will result in improvements in such conditions as obesity, metabolic syndrome, colitis, irritable bowel syndrome and adaptive thermogenesis.
[00067] As used herein, “linear inhibitory effect” refers to a linear decrease in secretion or biosynthesis resulting from all concentrations of the inhibiting material over a dose-response curve. For example, inhibition at low concentrations followed by a failure of inhibition or increased secretion at higher concentrations represents a lack of a linear inhibitory effect.
[00068] As used herein, “Leaky Gut Syndrome (LGS)” is an increase in permeability of the intestinal mucosa to luminal macromolecules, antigens and toxins associated with inflammatory degenerative and/or atrophic mucosal damage. LGS can lead to any number of seemingly unrelated symptoms affecting every organ system in the body. LGS has also been linked with having a causative role in a large number of distinct illnesses. Many of these are autoimmune diseases, which means the immune system attacks the body's own cells. LGS plays a role in these types of illness because it increases immune reactions to food particles and then cross reactivity may occur meaning that the immune system attacks body tissues that are chemically similar to the foods to which it has become sensitized. A sampling of the many diseases in which leaky gut syndrome may have a role includes: rheumatoid arthritis, osteoarthritis, asthma, multiple sclerosis, vasculitis, Crohn’s Disease, colitis, Addison’s Disease, lupus, thyroiditis, chronic fatigue syndrome, and fibromyalgia.
[00069] As used herein, the term “CLA isomers” refers to fatty acids with the same 18-carbon, polyunsaturated structure. In the case of CLA, each isomer is derived from the 18-carbon essential polyunsaturated fat linoleic acid (18:2n-6), which has two cis-double bonds at carbons 9 and 12. Cis9-CLA has been shown to regulate adiposity in animals and humans. The trans10-CLA isomer (t10-CLA), however, is associated with hyperglycemia, insulin resistance and dyslipidemia as well as elevated levels of inflammatory prostaglandins and cytokines. These stressors can impair the adipocyte’s ability to synthesize or store fatty acids as triglycerides, causing lipids to accumulate in hepatocytes and myocytes and resulting in steatosis and insulin resistance, respectively. These issues raise concern about the safe and effective use of supplements containing t10-CLA as a dietary strategy for weight loss.
[00070] In some aspects the compositions further comprise a pharmaceutically acceptable excipient where the pharmaceutically acceptable excipient is selected from the group consisting of coatings, isotonic and absorption delaying agents, binders, adhesives, lubricants, disintergrants, coloring agents, flavoring agents, sweetening agents, absorbants, detergents, and emulsifying agents. In yet further aspects, the composition additionally comprises one or more members selected from the group consisting of antioxidants, vitamins, minerals, proteins, fats, and carbohydrates.
[00071] The term "therapeutically effective amount" is used to denote treatments at dosages effective to achieve the therapeutic result sought. Furthermore, one of skill will appreciate that the therapeutically effective amount of the compound of the invention may be lowered or increased by fine-tuning and/or by administering more than one compound of the invention, or by administering a compound of the invention with another compound. See, for example, Meiner, C.L., "Clinical Trials: Design, Conduct, and Analysis," Monographs in Epidemiology and Biostatistics, Vol. 8 Oxford University Press, USA (1986). The invention therefore provides a method to tailor the administration/treatment to the particular exigencies specific to a given mammal. As illustrated in the following examples, therapeutically effective amounts may be easily determined, for example, empirically by starting at relatively low amounts and by step-wise increments with concurrent evaluation of beneficial effect.
[00072] As used herein, “more effectively” is used to describe relative biological responses of compounds or formulations wherein the response elicited by one formulation is greater per unit dose than the other.
[00073] The term "pharmaceutically acceptable" is used in the sense of being compatible with the other ingredients of the compositions and not deleterious to the recipient thereof.
[00074] As used herein, “compounds” may be identified either by their chemical structure, chemical name, or common name. When the chemical structure and chemical or common name conflict, the chemical structure is determinative of the identity of the compound. The compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated or identified compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The compounds may also exist in several tautomeric forms including the enol form, the keto form and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated or identified compounds. The compounds described also encompass isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds of the invention include, but are not limited to, 2H, 3H, 13C, 14C, 15N, 18O, 17O, etc. Compounds may exist in unsolvated forms as well as solvated forms, including hydrated forms and as N-oxides. In general, compounds may be hydrated, solvated or N-oxides. Certain compounds may exist in multiple crystalline or amorphous forms. Also contemplated within the scope of the invention are congeners, analogs, hydrolysis products, metabolites and precursor or prodrugs of the compound. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present invention.
[00075] The compounds according to the invention are optionally formulated in a pharmaceutically acceptable vehicle with any of the well-known pharmaceutically acceptable carriers, including diluents and excipients (see Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, Mack Publishing Co., Easton, PA 1990 and Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type of pharmaceutically acceptable carrier/vehicle employed in generating the compositions of the invention will vary depending upon the mode of administration of the composition to a mammal, generally pharmaceutically acceptable carriers are physiologically inert and non-toxic. Formulations of compositions according to the invention may contain more than one type of compound of the invention), as well any other pharmacologically active ingredient useful for the treatment of the symptom/condition being treated.
[00076] The compounds of the present invention may be provided in a pharmaceutically acceptable vehicle using formulation methods known to those of ordinary skill in the art. The compositions of the invention can be administered by standard routes. The compositions of the invention include those suitable for oral, inhalation, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intratracheal). In addition, polymers may be added according to standard methodologies in the art for sustained release of a given compound.
[00077] It is contemplated within the scope of the invention that compositions used to treat a disease or condition will use a pharmaceutical grade compound and that the composition will further comprise a pharmaceutically acceptable carrier. It is further contemplated that these compositions of the invention may be prepared in unit dosage forms appropriate to both the route of administration and the disease and patient to be treated. The compositions may conveniently be presented in dosage unit form be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the vehicle that constitutes one or more auxiliary constituents. In general, the compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid vehicle or a finely divided solid vehicle or both, and then, if necessary, shaping the product into the desired composition.
[00078] The term "dosage unit" is understood to mean a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active ingredient as such or a mixture of it with solid or liquid pharmaceutical vehicle materials.
[00079] Compositions suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets, soft gels or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid, such as ethanol or glycerol; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. Such oils may be edible oils, such as e.g. cottonseed oil, sesame oil, coconut oil or peanut oil. Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, gum arabic, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose and polyvinylpyrrolidone. The active ingredient may also be administered in the form of a bolus, electuary or paste.
[00080] In addition to the compositions described above, the compositions of the invention may also be formulated as a depot preparation. Such long-acting compositions may be administered by implantation (e.g. subcutaneously, intraabdominally, or intramuscularly) or by intramuscular injection. Thus, for example, the active ingredient may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in a pharmaceutically acceptable oil), or an ion exchange resin.
[00081] The compounds of this invention either alone or in combination with each other or other compounds generally will be administered in a convenient composition. The following representative composition examples are illustrative only and are not intended to limit the scope of the present invention. In the compositions that follow, "active ingredient" means a compound of this invention.
[00082] As used herein, "therapeutically effective time window" means the time interval wherein administration of the compounds of the invention to the subject in need thereof reduces or eliminates the deleterious effects or symptoms. In a preferred embodiment, the compound of the invention is administered proximate to the deleterious effects or symptoms.
[00083] Nigella sativa Linn. (family: Ranunculacease), commonly known as black seed or black curcumin, is an annual plant that has been traditionally used in the Indian subcontinent, Arabian countries, and Europe for culinary and medicinal purposes as a natural remedy for a number of illnesses and conditions that include asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness and influenza. The seeds or its oil are used as a carminative, diuretic, lactoagogue, and vermifuge. They are also used in food as a spice and a condiment.
[00084] N. sativa seeds contain 36-38% fixed oils, melanin, proteins, alkaloids, saponin and 0.4-2.5% essential oil. The fixed oil is composed mainly of unsaturated fatty acids, including the unusual C20:2 arachidic and eiosadienoic acids. Major components of the essential oil include thymoquinone (27-57%), p-cymene (7.1-15.5%), carvacrol (5.8-11.6%), trans-anethole (0.25-2.3%) p-terpineol (2.0-6.6%) and longifoline (1.0-8.0%). TQ readily dimerizes to form dithymoquinone and as used herein, TQ will also refer to the naturally occurring dimmer dithymoquinone.
[00085] Many studies have been conducted, particularly during the past two decades, on the effect of N. sativa seed extracts on various body systems in vivo or in vitro. Included among those physiological variables studied are antioxidant, anti-inflammatory and analgesic actions, anticarcinogenic activity, hypotensive, antidiabetic, antiulcer, antimicrobial and antiparasitic responses [Ali BH, Blunden G. 2003. Pharmacological and toxicological properties of Nigella sativa. Phytother Res 17: 299-305]. This body of research teaches that extraction methodology is a primary determinant of the effectiveness of the resulting N. sativa seed extract [see for example: Kokoska, L., J. Havlik, et al. (2008). "Comparison of chemical composition and antibacterial activity of Nigella sativa seed essential oils obtained by different extraction methods." J Food Prot 71(12): 2475-2480].
[00086] Recently studies have been reported using supercritical liquid extracts of N. sativa seeds containing 2.0 to 2.8 percent TQ, but none with extracts produced under the conditions of pressure, temperature and time as described herein. Further, biological activities of extracts in these and other studies with organic solvent extraction were roughly equal to their TQ content and none of the reported studies were applicable to commercially-scaled quantities of N. sativa extract [Ismail, M., G. Al-Naqeep, et al. (2010). "Nigella sativa thymoquinone-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats." Free Radic Biol Med 48(5): 664-672; Al-Naqeep, G., M. Ismail, et al. (2009). "Regulation of Low-Density Lipoprotein Receptor and 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene Expression by Thymoquinone-Rich Fraction and Thymoquinone in HepG2 Cells." J Nutrigenet Nutrigenomics 2(4-5): 163-172].
[00087] As used herein, “commercial-scale quantities” of N. sativa seed are considered quantities of raw material in excess of about 10 kg. All supercritical fluid extraction herein refers to extraction of commercial-scale quantities of N. sativa powdered seed.
[00088] All prior art, including reports using supercritical extraction of about 100 g or less of N. sativa, teach that TQ is the active component of N. sativa. The present application, however, teaches that supercritical CO2 defatted N. sativa seeds produced through a sequential series of two lipid extractions (Figure 2) exhibits unexpected in vitro and clinical potency greater than its TQ content. Additionally, the present application teaches that when commercial volumes of N. sativa seeds are defatted through the sequential lipid extraction steps described (Figure 2), the resulting material is capable of synergistically enhancing the effects of other phytoceuticals relating to hepatic steatosis, metabolic syndrome, T2D and obesity.
[00089] In some example embodiments, whole, ground N sativa seeds may be obtained at step 201 to perform lipid extraction process. This process may comprise two stages. At stage one, essential oil fraction is extracted. Extraction of essential oil is executed at two steps. At step 203, the essential oil fraction is extracted after 30 min of subjecting the whole, ground N. sativa seeds to 140 bar pressure and 50 degree Celsius. Further, at step 205. the essential oil fraction is extracted after another 120 min of subjecting the whole, ground N. sativa seeds to 140 bar pressure and 50 degree Celsius. Hence, the essential oil extraction is executed for a total of 150 min at 140 bar pressure and 50 degree Celsius.
[00090] At stage two the whole, ground N sativa seeds resulted after stage one is subjected to 300 bar pressure and 60 degree Celsius for another 180 min, Therefore, at step 207 Oleoresin fraction may be obtained at the end of 230 min. Once, the essential oil fractions and the oleoresin factions are extracted to perform lipid extraction defatted N. sativa seeds are obtained at step 209. Lipid extraction may also be explained in later part of the disclosure.
[00091] The present compositions can be provided in any convenient form. It can be provided as dietary supplement in capsule or tablet form. It can be formulated into a food or drink, and provided, for example, as a snack bar, a cereal, a drink, a gum, or in any other easily ingested form. It can also be provided as a cream or lotion for topical application. One trained in the art can readily formulate the present composition into any of these convenient forms for oral or topical administration.
[00092] The amounts of other additives per unit serving are a matter of design and will depend upon the total number of unit servings of the nutritional supplement daily administered to the patient. The total amount of other ingredients will also depend, in part, upon the condition of the patient. Preferably, the amount of other ingredients will be a fraction or multiplier of the RDA or DRI amounts. For example, the nutritional supplement will comprise 50% RDI (Reference Daily Intake) of vitamins and minerals per unit dosage and the patient will consume two units per day.
[00093] Flavors, coloring agents, spices, nuts and the like can be incorporated into the product. Flavorings can be in the form of flavored extracts, volatile oils, chocolate flavorings (e.g., non-caffeinated cocoa or chocolate, chocolate substitutes such as carob), peanut butter flavoring, cookie crumbs, crisp rice, vanilla or any commercially available flavoring. Flavorings can be protected with mixed tocopherols. Examples of useful flavorings include but are not limited to pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or pure vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, walnut oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch or toffee. In a preferred embodiment, the nutritional supplement contains berry or other fruit flavor. The food compositions may further be coated, for example with a yogurt coating if it is as a bar.
[00094] Emulsifiers may be added for stability of the final product. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), or mono- and di-glycerides. Other emulsifiers are readily apparent to the skilled artisan and selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.
[00095] Preservatives may also be added to the nutritional supplement to extend product shelf life. Preferably, preservatives such as potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate or calcium disodium EDTA are used.
[00096] In addition to the carbohydrates described above, the nutritional supplement can contain natural or artificial sweeteners, e.g., glucose, sucrose, fructose, saccharides, cyclamates, aspartamine, sucralose, aspartame, acesulfame K, or sorbitol.
Manufacture of the Preferred Embodiments
[00097] The medical foods or nutritional supplements of the present invention may be formulated using any pharmaceutically acceptable forms of the vitamins, minerals and other nutrients discussed above, including their salts. They may be formulated into capsules, tablets, powders, suspensions, gels or liquids optionally comprising a physiologically acceptable carrier, such as but not limited to water, milk, juice, soda, starch, vegetable oils, salt solutions, hydroxymethyl cellulose, carbohydrate. In a preferred embodiment, the nutritional supplements may be formulated as powders, for example, for mixing with consumable liquids, such as milk, juice, sodas, water or consumable gels or syrups for mixing into other nutritional liquids or foods. The nutritional supplements of this invention may be formulated with other foods or liquids to provide pre-measured supplemental foods, such as single serving chapatis, beverages or bars, for example.
[00098] In a particularly preferred embodiment, the medical food or nutritional supplement will be formulated into a dietary staple, a form that has consumer appeal, is easy to administer and incorporate into one's daily regimen, thus increasing the chances of patient compliance. To manufacture the beverage, the ingredients are dried and made readily soluble in water. For the manufacture of other foods or beverages, the ingredients comprising the nutritional supplement of this invention can be added to traditional formulations or they can be used to replace traditional ingredients. Those skilled in food formulating will be able to design appropriate foods or beverages with the objective of this invention in mind.
[00099] The medical food can be made in a variety of forms, such as flat breads, puddings, confections, (i.e., candy), nutritional beverages, ice cream, frozen confections and novelties, or non-baked, extruded food products such as bars. The preferred form is a flour mixture for various flat breads or mixtures to add to a beverage or a non-baked extruded nutritional bar. In another embodiment, the ingredients can be separately assembled. For example, certain of the ingredients (e.g., the ground defatted N. sativa seeds and whole N. sativa ground seeds) can be assembled into a tablet or capsule using known techniques for their manufacture. The remaining ingredients can be assembled into a powder or nutritional bar. For the manufacture of a food product, the dry ingredients are added with the liquid ingredients in a mixer and mixed until the dough phase is reached; the dough is cooked; and the product is cooled and consumed. The two assembled forms comprise the nutritional supplement and can be packaged together or separately, such as in the form of a kit, as described below. Further, they can be administered together or separately, as desired.
Use of Preferred Embodiments
[000100] The preferred embodiments contemplate treatment of disorders related to hepatic steatosis, metabolic syndrome, all forms of diabetes, obesity, oxidative stress, inflammation, and cardiovascular risk. A pharmaceutically acceptable carrier may also be used in the present compositions and formulations. The recommended daily amounts of each ingredient, as described herein, serve as a guideline for formulating the medical foods and nutritional supplements of this invention. The actual amount of each ingredient per unit dosage will depend upon the number of units administered daily to the individual in need thereof. This is a matter of product design and is well within the skill of the nutritional supplement formulator.
[000101] The ingredients can be administered in a single formulation or they can be separately administered. For example, it may be desirable to administer the compounds in a form that masks their taste (e.g., capsule or pill form) rather than incorporating them into the nutritional composition itself (e.g. chapatis). Thus, the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the nutritional compositions of the invention (e.g., nutritional supplement in the form of a powder and capsules containing defatted N. sativa seeds). Optionally associated with such container(s) can be a notice in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed together in any combination, present in a formulation or tablet.
[000102] The preferred embodiments provide compositions and methods to promote normal hepatic, adipocyte or myocyte functioning relating to obesity, T2D, and hepatic steatosis. All preferred embodiments provide varying amounts of supercritical CO2, defatted N. sativa seeds containing about less than 0.01% TQ. Generally, the formulations comprise supercritical CO2, defatted, ground N. sativa seeds with whole, ground N. sativa and another herb or phytochemical. In one embodiment, the composition comprises a supercritical CO2-defatted ground N. sativa seeds produced through sequential extractions at 140 bar, 50°C and 150 minutes followed by a second extraction at 300 bar, 60°C for180 minutes.
EXAMPLES
Example 1
Supercritical CO2 Defatting of Nigella sativa Powdered Seeds
[000103] Objective – The objective of this experiment was to produce a unique supercritical CO2 defatted N. sativa seed product to assess its potential for metabolic activity and value as a unique medical food. It is well known in the art that scale of the extraction process can greatly affect the components of the extract and that extraction results obtained with small quantities of substrate do not reflect results obtained with larger, commercial quantities.
[000104] Raw material purchase – The black seeds of N. sativa are seasonal, available from April to June of every year. The main source is in northern parts of India particularly in Uttar Pradesh. Stocked material is available throughout the year. The identity of the commercial N. sativa purchased was confirmed at Supreem Pharma (Mysore, India).
[000105] Grinding and sieving - Once the commercial quantity of N. sativa seeds was purchased it was ground to a fine powder between 20 – 30 mesh. A magnetic screening system was used to ensure removal of metallic impurities, particularly iron.
[000106] Supercritical fluid extraction – In this example, the process conditions during the defatting of N. saliva seeds with supercritical CO2 extraction were varied with respect to pressure, temperature and time to optimize the extraction of lipids while increasing the melanin and alkaloid content of the extracted seeds as depicted in Figure 2. In general, all procedures were performed according to one skilled in the art without modification. The commercial-scale extraction was performed in a polyvalent pilot plant extraction set-up. Liquid CO2 entering the apparatus was cooled in condenser C before it was pressurized and passed into the system. The flow rate was adjusted manually before the experiment. During the extraction process, the temperatures of the extractor, CO2, and separations 1 and 2 (S1/S2 Figure 2) were automatically regulated through the recirculation of thermostatic water from three individually regulated water baths.
[000107] Thymoquinone concentration – TQ content of the defatted N. sativa seeds was determined by HPLC analysis as described by Ghosheh (Ghosheh OA, Houdi AA, Crooks PA. High performance liquid chromatographic analysis of the pharmacologically active quinones and related compounds in the oil of the black seed (Nigella sativa L.). J Pharm Biomed Anal. Apr 1999;19(5):757-762) with no modifications.
[000108] Results – Following supercritical CO2 delipidation of ground N. sativa seeds, the TQ content was determined to be less than 0.01%.
[000109] As commercial-scaled supercritical CO2 delipidation of N. sativa has never before been attempted, and considered. An in vitro and clinical screening program was developed to assess its potential health benefits and safety. Results of the screening of this novel, potentia phytoceutical are presented in the following Examples.
Example 2
Free Radical Quenching Activity of Supercritical Carbon Dioxide Defatted Nigella sativa Seeds is Equivalent to Thymoquinone
[000110] Objective - The objective of this experiment is to compare the antioxidant (free radical scavenging) activity of the defatted N. sativa seeds obtained by supercritical CO2 extraction in Example 1 with the pure TQ marker compound.
[000111] Chemicals – TQ, 2,2-diphenyl-1-picrylhydrazyl and all other compounds used in this example are purchased from Sigma (St. Louis, MO) and are of the highest purity commercially available. The defatted N. sativa sample used in this study is the commercial-scale product described in Example 1.
[000112] Methodology - Antioxidant activity is determined utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH), which is a stable radical. The odd electron in the DPPH free radical gives a strong absorption maximum at 550 nm and is purple in color. The color turns from purple to yellow as the molar absorption of the DPPH radical at 550 nm is reduced when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H. The test samples are dissolved in methanol containing 1% dimethyl sulfoxide and added to microtiter wells in 100 µL aliquots to 100 µL of a 100 µM DPPH solution in methanol. Readings are taken at 10, 30 and 60 minutes following the addition of the test material. Percent inhibition of the DPPH radical by the test material is computed relative to the inhibition of the DPPH radical by the vitamin E analog trolox and tabulated as µmol trolox/g test material.
[000113] Results – The SDNS exhibits antioxidant activity in the DPPH assay in the form of free radical savaging activity (Table 2). The antioxidant activity exhibited by SDNS, however, is approximately 20% better than pure TQ. Thus, the antioxidant activity of the SDNS is quantitatively superior to TQ. This example demonstrates that the chemical behavior of defatted N. sativa seeds is largely a function of extraction conditions such as solvent, temperature and time and independent of TQ concentration.
Table 2
Free Radical Savaging Activity of Supercritical Carbon Dioxide Defatted N. sativa Seeds Relative to Thymoquinone

Test Material Thymoquinone Content
[%] DPPH Reducing Activity(1)
[µmol Trolox/g TQ]
Thymoquinone (Sigma) 100 45.2
SDNS 0.010 54.1
(1)Computed on basis of TQ content.
SDNS: supercritical CO2, defatted Nigella sativa seeds
Conclusion –As the prior art teaches that the TQ content of the essential oil fraction of N. sativa solely contributes to the antioxidant activity, unexpectedly, the unique combination of compounds extracted from N. sativa in SDNS behaves synergistically to greatly exceed the antioxidant activity of the putative active component TQ.
Example 3
Synergistic Free Radical Quenching of a Three-component Phytocomplex
[000114] Objective – The objective of this study is to evaluate the potential for synergy of a novel, three-component phytocomplex to scavenge free radicals
[000115] Methodology – The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay as described in Example 2 is used to assess scavenging of free radicals by the test materials.
[000116] Test Materials – The SDNS created in Example 1, commercial, ground N. sativa seeds, and ground fenugreek seeds are used as the test materials in this study.
[000117] Calculations - The median inhibitory concentration (IC50) for free radical quenching activity is calculated using CalcuSyn (BIOSOFT, Ferguson, MO). This statistical package performs multiple drug dose-effect calculations using the median effect methods described by T-C Chou and P. Talaly [(1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22, 27-55.] hereby incorporated by reference.
[000118] Briefly, the analysis correlates the “Dose” and the “Effect” in the simplest possible form: fa/fu = (C/Cm)m, where C is the concentration or dose of the compound and Cm is the median-effective dose signifying the potency. Cm is determined from the x-intercept of the median-effect plot. The fraction affected by the concentration of the test material is fa and the fraction unaffected by the concentration is fu (fu = 1 – fa). The exponent m is the parameter signifying the sigmoidicity or shape of the dose-effect curve; it is estimated by the slope of the median-effect plot.
[000119] The median-effect plot is a graph of x= log(C) vs y = log(fa/fu) and is based on the logarithmic form of Chou’s median-effect equation. The goodness of fit for the data to the median-effect equation is represented by the linear correlation coefficient r of the median-effect plot. Usually, the experimental data from enzyme or receptor systems have an r > 0.96, from tissue culture an r > 0.90 and from animal systems r > 0.85.
[000120] Synergy of test components is quantified using the combination index (CI) parameter. This parameter defines only the additive effect rather than synergism or antagonism. Synergy, however, is defined as a more than expected additive effect (CI >1.00), and antagonism as a less than expected additive effect (CI<1.00) as described below.
[000121] Expected median inhibitory concentrations of any multi-component combination is estimated using the relationship:
[1/Expected IC50] = [Fa/IC50A] + [Fb/IC50B] + … + [Fn/IC50N] = 1.00
where Fa = mole fraction of component A in the combination and Fn = the mole fraction of the nth component combination and IC50A = the observed IC50 of the component A.
[000122] The CI is then calculated thusly, CI = Expected [IC50]/Observed [IC50].
[000123] Using the designation of CI = 1.00 as the additive effect, for mutually exclusive compounds that have the same mode of action or for mutually non-exclusive drugs that have totally independent modes of action the following relationships are defined: CI < 1.00, = 1.00, and > 1.00 indicating antagonism, additivity and synergy, respectively.
Table 3
Determining Combination Index for Free Radical Quenching of a Three-component Phytocomplex

Test Material Observed IC50
[µg/mL] Relative Amount
[F] Fn/[IC50]
[µg/mL]-1
Ground N. sativa seed 0.975 0.429 0.440
Defatted N. sativa seed 0.195 0.429 2.20
Ground Fenugreek 6.35 0.143 0.022
Phytocomplex 0.210 1.00 2.66
Phytocomplex contains relative amounts [F] of each of the three test materials; Expected IC50 for the phytocomplex = 1/[2.66] = 0.376 µg/mL.

[000124] Conclusion – With CI = 1.79, the 3-component phytocomplex containing ground N. seeds, SDNS, and ground fenugreek, unexpectedly produces 1.8-times the free radical quenching capacity than expected from the sum of its components indicating synergy of the combination.
Example 4
Inhibition of Prostate Specific Antigen Secretion from LNCaP Prostate Cells
by Defatted N. sativa Seeds
[000125] Objective - While the effects of TQ and various extracts of N. sativa have shown cytotoxicity to a number of tumor cell lines, no studies have been performed on the effect of defatted N. sativa seeds on the secretion of prostate specific antigen (PSA), a marker for prostate hyperplasia as well as cancer. While PSA is present in small quantities in the serum of normal men, it is often elevated in the presence of nonproliverative as well as neoplastic prostate disorders. Examples of noncancerous or nonproliverative prostate disorders exhibiting elevated PSA are benign prostate hyperplasia and infections of the prostate. The objective of this experiment was to determine whether commercial-scale, supercritical CO2, defatted N. sativa would affect prostate specific antigen secretion from non-proliferating prostate cells in a manner similar to pure TQ alone.
[000126] Chemicals – PSA was quantified from the cell culture supernatant fluid using the Quantikine Human KLK3/PSA Immunoassay kit (R&D Systems, Inc., Minnaepolis, MN). All other materials used in this example were purchased from Sigma (St. Louis, MO) or otherwise noted and were of the highest purity commercially available. The N. sativa sample used in this study was the SDNS described in Example 1.
[000127] Cell culture and treatment – The LNCaP prostate cell line, which produces PSA, was used to study the effects of commercial-scale, SCNS of Example 1 and pure TQ on the secretion of PSA. LNCaP cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and sub-cultured according to instructions supplied by ATCC. Prior to experiments, cells were cultured in DMEM containing 10% FBS-HI added 50 units penicillin/ml and 50 µg streptomycin/ml, and maintained in log phase prior to experimental setup. Cells were grown in a 5% CO2 humidified incubator at 37°C. Components of medium included: (1) 10% FBS/DMEM (Fetal Bovine Serum/Dulbecco’s Modified Eagle’s Medium) containing 4.5 g glucose/L; (2) 50 U/ml penicillin; and (3) 50 µg/ml streptomycin. Growth medium was made by adding 50 ml of heat inactivated FBS and 5 ml of penicillin/streptomycin to 500 ml DMEM. This medium was stored at 4°C. Before use, the medium was warmed to 37°C in a water bath.
[000128] LNCaP cells were seeded at an initial density of 6x104 cells/cm2 in 96-well plates. For two days, the cells were allowed grow to reach confluence. On day three post seeding, cells were treated with TQ, or SDNS at the concentrations listed in Table 4. Twenty-four hours later, the supernatant media were sampled and assayed for PSA.
Table 4
Summary of Effects on Prostate Specific Antigen Secretion by LNCaP Prostate Cells by Supercritical Defatted N. seeds and Pure Thymoquinone
Test Material Effect on PSA Secretion over 24 Hours
Thymoquinone (Sigma) Decreased PSA at 5 and 10 µg/mL, dramatic increase in PSA secretion at 50 and 100 µg/mL.
SDNS Decrease in PSA secretion at both doses tested 250 and 500 µg/mL.
SDNS: supercritical CO2, Defatted Nigella sativa Seeds
[000129] Results –Unexpectedly, TQ did not exhibit a linear dose-response curve and decreased PSA only at the two lowest doses tested while dramatically increasing PSA secretion at the highest doses, 166 and 207%. The response of SDNS differed qualitatively from that of TQ alone, nearly completely decreasing PSA secretion at both of concentrations.
[000130] It is clear from this summary that SDNS produced novel effects on PSA secretion from prostate cells implying differences in composition and biological activity not anticipated in the prior art.
Example 5
Defatted Nigella sativa Seeds are Potent Uncouplers of Mitochondrial Membrane Potential in 3T3-L1 Adipocytes
[000131] Objective - The objective of this experiment is to determine whether commercial-scale, supercritical CO2 defatted N. sativa seeds produced in Example 1 directly reduce mitochondria membrane potential in 3T3-L1 adipocytes compared to pure TQ or DNP, indicating their potential to induce weight loss in a subject.
[000132] The model- 3T3-L1 murine fibroblast are routinely used to study the potential effects of compounds on white adipose tissue in vitro. This cell line allows investigation of stimuli and mechanisms that regulate inflammatory mediators of cytokine secretion of the adipocyte. As preadipocytes, 3T3-L1 cells have a fibroblastic appearance. They replicate in culture until they form a confluent monolayer, after which cell-cell contact triggers Go/G1 growth arrest. Terminal differentiation of 3T3-L1 cells to adipocytes depends on proliferation of both pre- and post-confluent preadipocytes. Subsequent stimulation with 3-isobutyl-1-methylxanthane, dexamethasone, and high doses of insulin (MDI) for two days prompts these cells to undergo post-confluent mitotic clonal expansion, exit the cell cycle, and begin to express adipocyte-specific genes. Approximately five days after induction of differentiation, more than 90% of the cells display the characteristic lipid-filled adipocyte phenotype. At this stage of differentiation, response to mitochondrial uncouplers such as DNP may be assessed.
[000133] Chemicals – 2,4-Dinitrophenol and all other chemicals used in this example are purchased from Sigma (St. Louis, MO) or otherwise noted and are of the highest purity commercially available. The defatted N. sativa commercial-scale extracts used in this study are those described in Example 1.
[000134] Cell culture and Treatment - The murine fibroblast cell line 3T3-L1 is purchased from the American Type Culture Collection (Manassas, VA) and sub-cultured according to instructions from the supplier. Prior to experiments, cells are cultured in DMEM containing 10% FBS-HI added 50 units penicillin/ml and 50 µg streptomycin/ml, and maintained in log phase prior to experimental setup. Cells are grown in a 5% CO2 humidified incubator at 37°C. Components of the pre-confluent medium include: (1) 10% FBS/DMEM (Fetal Bovine Serum/Dulbecco’s Modified Eagle’s Medium) containing 4.5 g glucose/L; (2) 50 U/ml penicillin; and (3) 50 µg/ml streptomycin. Growth medium is made by adding 50 ml of heat inactivated FBS and 5 ml of penicillin/streptomycin to 500 ml DMEM. This medium is stored at 4°C. Before use, the medium is warmed to 37°C in a water bath.
[000135] 3T3-T1 cells are seeded at an initial density of 6x104 cells/cm2 in 96-well plates. For two days, the cells are allowed grow to reach confluence. Following confluence, the cells are forced to differentiate into adipocytes by the addition of differentiation medium; this medium consisted of (1) 10% FBS/DMEM (high glucose); (2) 0.5 mM methylisobutylxanthine; (3) 0.5 µM dexamethasone and (4) 10 µg/ml insulin (MDI medium). After three days, the medium is changed to post-differentiation medium consisting of 10 µg/ml insulin in 10% FBS/DMEM.
[000136] Treatment with 2,4-Dinitrophenol and Test Material - On Day 6 post differentiation, DNP or defatted N. sativa seeds are dissolved in DMSO and added to the culture medium to achieve concentrations of 500 µM for DNP and 25 µg/mL for the test materials each in eight wells of a single column 60 min at 37°C. JC-1 is then added to the test and negative control columns in 10 µL DMSO to achieve a final concentration of 5 µM and allowed to incubate at 37°C for an additional 30 min. A DMSO and solvent plus JC-1 control are run concurrently with each experiment. A Packard Fluorocount spectrofluorometer (Model#BF10000, Meridan, CT) set at 560 nm excitation and 590 nm emission is used for quantification of aggregate fluorescence and at 485 nm excitation/530 emission for monomer fluorescence.
[000137] Measuring mitochondrial membrane potential changes (??m) - JC-1 (Sigma, St. Louis, MO) has advantages over other cationic dyes in that it can selectively enter into mitochondria and reversibly change color from green to red as the membrane potential increases. In healthy cells with high mitochondrial membrane potential (??m), JC-1 spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the other hand, in cells with low ??m, JC-1 remains in the monomeric form exhibiting only green fluorescence. The changes in ??m by different forms of JC-1 as either green or red fluorescence are both quantified by a fluorescence plate reader with appropriate filter sets.
[000138] Calculation of relative decrease in mitochondrial membrane potential – Aggregate and monomer fluorescence are computed for the 500 µM DNP positive control as well as the defatted N. sativa seeds relative to JC-1 negative controls. The ratio of the monomer to aggregate relative fluorescence is then determined as a measure of relative decrease in ??m.
[000139] Results – The positive control DNP, at 500 µM, decreases ??m in 3T3-L1 adipocytes to a similar extent as 25 µg/mL pure TQ, approximately 30 to 40%. The supercritical CO2 defatted N. sativa, however, decreases mitochondrial membrane potential 3.7-fold relative to solvent controls. This example demonstrates that the commercial-scale, supercritical CO2 defatted N. sativa possess biological activity unexplained by TQ content alone.
Example 6
Defatted Nigella sativa Inhibits Inflammation-Stimulated Nitric Oxide
Biosynthesis in Adipocytes
[000140] Objective – The objective of this experiment was to determine whether the commercial-scale, supercritical CO2 defatted N. sativa seeds obtained in Example 1 reduce inflammation-induced NO secretion in adipocytes as effectively as TQ alone.
[000141] The model- The murine 3T3-L1 preadipocyte model was used in this Example. This model was selected to serve as the surrogate for adipocytes that are exposed to a variety of the inflammatory stimuli of invading bacteria, modeled by lipopolysaccharide (LPS), as well as the counter-inflammatory responses of infiltrating macrophage, modeled by interferon gamma (IF?) and tumor necrosis factor alpha (TNF?). Additionally, alterations in NO levels have been demonstrated in pathologic conditions in humans such as obesity, diabetes, hepatic steatosis, hypertension, leaky gut, osteoarthritis, osteoporosis, and interstitial cystitis.
[000142] Chemicals - Heat-inactivated fetal bovine serum (HIFBS), penicillin and streptomycin solution, and Dulbecco’s Modification of Eagle’s Medium (DMEM) were purchased from Mediatech (Herndon, VA). 2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)-amino-2-deoxy-d-glucose (2-NBDG) and N-methyl-4-hydrazino-7-nitrobenzofurazan (NBDM) were obtained from Invitrogen (Carlsbad, CA). TQ, bacterial lipopolysaccharide (LPS), murine TNF? and Interferon-? and all standard chemicals, unless noted, were obtained from Sigma (St Louis, MO) and were of the highest purity commercially available.
[000143] Cell culture and treatment – The murine fibroblast cell line 3T3-L1 was purchased from the American Type Culture Collection (Manassas, VA) and sub-cultured according to instructions from the supplier. Prior to experiments, cells were cultured in DMEM containing 10% FBS-HI added 50 units penicillin/ml and 50 µg streptomycin/ml, and maintained in log phase prior to experimental setup. Cells were grown in a 5% CO2 humidified incubator at 37°C. Components of the pre-confluent medium included: (1) 10% FBS/DMEM (Fetal Bovine Serum/Dulbecco’s Modified Eagle’s Medium) containing 4.5 g glucose/L; (2) 50 U/ml penicillin; and (3) 50 µg/ml streptomycin. Growth medium was made by adding 50 ml of heat inactivated FBS and 5 ml of penicillin/streptomycin to 500 ml DMEM. This medium was stored at 4°C. Before use, the medium was warmed to 37°C in a water bath.
[000144] 3T3-T1 cells were seeded at an initial density of 6x104 cells/cm2 in 24-well plates. For two days, the cells were allowed grow to reach confluence. Following confluence, the cells were forced to differentiate into adipocytes by the addition of differentiation medium; this medium consisted of (1) 10% FBS/DMEM (high glucose); (2) 0.5 mM methylisobutylxanthine; (3) 0.5 µM dexamethasone and (4) 10 µg/ml insulin (MDI medium). After three days, the medium was changed to post-differentiation medium consisting of 10 µg/ml insulin in 10% FBS/DMEM.
[000145] For assessing the effect of defatted N. sativa on cytokine-stimulated NO-production in 3T3-L1 adipoctyes, D6 adipoctyes were treated with 50, 10, 5, or 1 µg TQ or SDNS/mL for 1 hour and then stimulated with a cytokine mixture containing 1 µg LPS/mL, 50 ng TNF?/mL and 100 U IF?/mL (LTI) for 20 hours. L-NG-Nitroarginine methyl ester (L-NAME) at 200 µM (47 µg/mL) was used as the positive control and 0.1% DMSO for the negative control.
[000146] Nitric oxide determination - NBD methylhydrazine (NBDM, N-methyl-4-hydrazino-7-nitrobenzofurazan) was used to detect N-methyl-4-amino-7-nitrobenzofuazan, the fluorescent product of the NBDM reagent with nitrite (Buldt A, Karst U. Determination of nitrite in waters by microplate fluorescence spectroscopy and HPLC with fluorescence detection. Anal Chem. Aug 1 1999;71(15):3003-3007). In a separate, black-walled, 96-well, microtiter plate, a 7 µL aliquot of a 4.8 x 10-4 mol NBDM/L solution was added to 200 µL of the supernatant media followed by the addition of 15 µL of concentrated phosphoric acid. After a reaction time of 30 minutes at ambient temperature, the fluorescence was read with 485 nm excitation filter and a 530 nm emission filter in a Packard Fluorocount spectrofluorometer (Model#BF10000, Meridan, CT). Fluoresence was linear in the range of 3.59 x 10-7 to 1.44 x10-5 mol nitrite/L. The standard deviation was 3.5 percent for 1.44 x 10-6 mol nitrite/L. Experiments were performed a minimum of three times with eight replicates per dose, capturing the median inhibitory concentration (IC50) when possible.
[000147] Calculations - The median inhibitory concentrations (IC50) and 95% confidence interval were calculated using CalcuSyn (BIOSOFT, Ferguson, MO). This statistical package performs multiple drug dose-effect calculations using the Median Effect methods described by Chou and Talalay [Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984;22:27-55].
Table 5.
Median Inhibitory Concentrations of Defatted N. sativa and Thymoquinone for iNOS-mediated NO Biosynthesis in Adipocytes

Test Material IC50*
[µg/mL]
TQS (100% TQ) 1.9 (1.4 - 2.7)
SCNS (<0.0005 µg TQ equivalents/mL) 0.92†
*Value computed on the basis of TQ content.
†Significantly different (p<0.05) from TQS.
SDNS: supercritical CO2, Defatted Nigella sativa Seeds
[000148] Results – TQ alone was a potent inhibitor of iNOS-mediated NO production in adipocytes stimulated with the trivalent cytokine mixture and exhibited a median inhibitory concentrations of 1.9 µg TQ/mL (Table 5). These results are consistent with published results for NO inhibition in the LPS-stimulated macrophages (IC50 = 1.4 – 2.8 µg/mL). [El-Mahmoudy A, Matsuyama H, Borgan MA, et al. TQ suppresses expression of inducible nitric oxide synthase in rat macrophages. Int Immunopharmacol. Oct 2002;2(11):1603-1611]. SDNS was also a potent inhibitor of LTI-stimulated, NO biosynthesis in 3T3-L1 adipocytes, exceeding even the potency of pure TQ
[000149] These unexpected results serve to demonstrate the superior NO inhibitory potency of SDNS over TQ alone. Such a finding has thus far not been reported in the prior art.
Example 7
Supercritical Defatted Nigella sativa Increases Lypolysis in 3T3-L1 Adipocytes
[000150] Objective – The objective of this experiment is to determine whether the supercritical fluid CO2 defatted N. sativa obtained in Example 1 can induce lipolysis in adipocytes.
[000151] Chemicals - All chemicals used in this example were purchased from Sigma (St. Louis, MO) or otherwise noted and were of the highest purity commercially available. Then defatted N. sativa used in this study is described in Example 1.
[000152] Cell culture and treatment – Culture and treatment of 3T3-L1 adipocytes is as described in Example 5. The forskolin positive control is dosed at 82 µg/mL, while TQ, and SDNS as described in Example 1 are dosed at 5 µg/mL.
[000153] Glycerol assay - Free fatty acid release from 3T3-L1 adipocytes is quantified by measuring glycerol secretion into the medium. Glycerol is measured spectrophotometrically using the Free Glycerol Determination Kit (F6428, Sigma) and an EL 312e Microplate BIO-KINETICS spectrophotometer (BioTek, Winooski, VT).
[000154] \Data analysis – Glycerol release from adipocytes is expressed as the percent increase in free fatty acid secretion and relative glycerol content (glycerol index) of eight observations for one of three representative experiments.
[000155] Results – The forskolin positive control, TQ, and SDNS induced free fatty acid release in adipocytes. Specifically, SDNS exhibiting an increase in free fatty acid release of 102% relative to forskolin in adipocytes, and is twice as potent as TQS (Sigma) on a weight basis.
Example 8
Supercritical Defatted Nigella sativa Activates AMPK in Myocytes
[000156] Objective - The objective of this experiment is to compare the effect of the AMP mimetic AICAR on AMPK activation in C2C12 myocytes with the commercial-scale supercritical CO2 defatted N. sativa produced in Example 1.
[000157] The Model- The C2C12 myocyte model is used in this example.
[000158] Chemicals – Penicillin, streptomycin, Dulbecco's modified Eagle's medium (DMEM) is from Mediatech (Herndon, VA) and 10% FBS-HI (fetal bovine serum-heat inactivated) is obtained from Mediatech and Hyclone (Logan, UT). The commercial supercritical CO2 defatted N. sativa produced in Example 1 is used as the test materials. Unless noted, all other standard reagents are purchased from Sigma (St. Louis, MO).
[000159] Cell culture - Mouse C2C12 myoblasts are obtained from American Type Culture Collection (Manassas, VA), and are maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under a humidified atmosphere of 5% CO2.
[000160] C2C12 cells are seeded at an initial density of 6x104 cells/cm2 in 24-well plates. For two days, the cells are allowed grow to reach confluence. Following confluence, the cells are forced to differentiate into myocytes by culturing in DMEM supplemented with 2% horse serum for seven days.
[000161] Treatment - On Day 8 to 10 post differentiation, C2C12 myocytes are incubated in serum-free DMEM plus 0.5% BSA (bovine serum albumin) for three hours. Next, AICAR (Cell Signal, Danvers, MA) is dissolved in phosphate buffered saline (PBS) and added to the culture medium to achieve concentrations of 1 mM. SDNS was added in DMSO to achieve a final concentration of 25 µg test material/mL and one percent DMSO. After 30 minutes at 37°C, cell lysates are prepared for determination of activated AMPK.
[000162] Measuring activated AMPKa - pT172-AMPK is quantified using the Biosource AMPK Immunoassay Kit (Camarillo, CA) without modification. Protein content of the cell lysates is determined with the Active Motif fluorescent protein assay reagent (Carlsbad CA, Hoefelschweiger, B. K., Duerkop, A., and Wolfbeis, O. S. Novel type of general protein assay using a chromogenic and fluorogenic amine-reactive probe. Anal Biochem 2005, 344, 122-9). A Packard Fluorocount spectrofluorometer (Model#BF10000, Meridan, CT) is used for protein determination and a MEL312e BIO-KINETICS READER (Bio-Tek Instruments, Winooski, VT) is used for quantification of pT172-AMPK.
[000163] Calculation of relative activation of AMPK - pT172-AMPK is computed per mg lysate protein and then normalized to the dimethyl sulfoxide (DMSO) negative controls. For statistical comparisons, 95% confidence intervals are computed (Excel, Microsoft, Redman, WA).
[000164] Results – Over ten independent assays, 1 mM AICAR increases pT172-AMPK an average of 1.67-fold (95% CI = 1.26 – 2.21) in C2C12 myocytes relative to the DMSO negative controls. In three independent assays, SDNS also activates myocyte AMPK. In studies of direct comparisons, 25 µg SDNS/mL is 14% more active (p<0.05) than 1 mM AICAR in activating AMPK.
Example 9
Commercial Supercritical Defatted Nigella sativa Seeds Attenuate LPS/Oxidant-mediated Loss of Transepithelial Electrical Resistance in Caco-2 Intestinal Epithelial Cells More Effectively Than Thymoquinone
[000165] Objective - The objective of this experiment was to assess the effect of the commercial-scale SDNS produced in Example 1 on the loss of transepithelial electrical resistance in Caco-2 monolayers induced by a cytokine/prooxidant stimulus.
[000166] Caco-2 Cells - The protocol for growing and differentiation the human Caco-2 colon andenocarcinoma cells was a modification of Protocol 3 as described by Yamashita et al. [Yamashita, S., Konishi, K., Yamazaki, Y., Taki, Y., Sakane, T., Sezaki, H., and Furuyama, Y. (2002) New and better protocols for a short-term Caco-2 cell culture system, J Pharm Sci 91, 669-679]. Caco-2 Human Colon Adenocarcinoma cells were obtained from ATCC (Rockville, MD; catalog #HTB-37) and maintained in a growth media: DMEM (Dulbecco’s Modified Eagles Medium 1x) containing L-glutamine, glucose; Cellgro catalog #35-010CV with the following additions: (1) Penicillin (5,000 IU/mL)/Streptomycin (5,000 µg/mL) Cellgro catalog #30-001C1, (2) 10% FBS – Fetal Bovine Serum, Characterized; Hyclone catalog #SH30071.3, and (3) 1% NEAA – Non Essential Amino Acids; Cellgro catalog #25-025-CI; cells were cultured at 37 C in a humidified air-5% CO2 atmosphere in T175 flasks.
[000167] Differentiation of Caco-2 cells to intestinal epithelial cells - BIOCOATR HTS Caco-2 Assay System kits (Becton Dickinson, NJ; catalog #354801) consisting of 24-well fibrillar collagen coated inserts and feeder trays were used in all experiments and plated as follows. Media was removed from the T175 flasks and cells were washed with 10 mL PBS (phosphate buffered saline 1X without Ca++, without Mg++; Cellgro #25-053-CI). PBS was removed and 5 mL of Trypsin/EDTA (1x, 0.25% Trypsin/2.21mM EDTA in HBSS; Cellgro #25-053-CI) were added to the flask and the flask placed at 37 C until cells were visibly floating, approximately 3-5 minutes. Five mL of growth media were added to the flask to neutralize the trypsin. This solution was then transferred to a sterile 50 mL tube. Eight µL were sampled and placed in a hemocytometer for counting under a microscope. Five-hundred µL of cell solution were placed in the upper chamber of each insert so that the final density per well was at least 6.6x105 cells/cm2. Thirty-five mL of growth media were then added to the feeder tray and plate incubated at 37 C with 5% CO2 and 100% humidity for 20-24 hours. At this time, media were removed from the inserts by decanting and from the feeder tray by aspiration. Media were then added for the cell differentiation phase; Entero-STIMTM Medium was prepared as per assay kit instructions (Becton Dickinson, NJ; catalog #354801), 500 µL to the upper chamber of each insert and 35 mL added to the feeder tray.
[000168] This medium was refreshed after 48 hours. The following day the plates were prepared for treatment. Media were removed from the upper chamber of the inserts so that the final volume was 300 mL. The insert plate was removed from the feeder tray and placed directly on a 24-well plate. One mL of Entero-STIM TM Medium was added to each well.
[000169] Test materials and controls were solubilized or suspended by sonication for 5 minutes in DMSO as a 500X stock and added to top wells in 0.6 µL to achieve the tabulated concentrations. DMSO was added to both negative and positive controls at the same 0.1% concentration as the test wells. Test materials remained on the monolayers for 1 hour, at which time all positive and test cells were treated with 10 uM H2O2 and 50 µg/mL lipopolysaccharide (LPS, Sigma, St.Louis, MO).
[000170] Transepithelial Electrical Resistance Assay - Baseline transepithelial electrical resistance (TEER) measurements of the Caco-2 monolayers were made using a MillicellR-ERS system (Millipore Corporation, Bedford, MA). Measurements of TEER were made 1, 2, and 3 hours post treatment.
Table 6
Effect of supercritical CO2 defatted Nigella sativa seeds on LPS/oxidant-mediated decrease of transepithelial electrical resistance in Caco-2 cells

Treatment Test Concentration
[µg TQ equivalents/mL]
Relative Loss of TEER
LPS/H2O2 - 100 ± 7.41
Thymoquinone (Sigma) 5.0 111
SDNS 0.0005 85.8*
**Significantly less than LPS/H2O2 positive control (p<0.05)
SDNS: supercritical CO2, Defatted Nigella sativa Seeds
[000171] Data Analysis - After TEER values were normalized to their zero-hour control, the percent change from the positive LPS/H2O2 control at three hours was tabulated for each fraction. Ninety-five percent confidence intervals (CI) were computed for the positive control, which was set to 100 (Excel, Microsoft, Redmond, WA). Values were considered significantly different from the positive control if the mean of the four test wells of the samples fell outside this 95% CI (p<0.05).
[000172] Results –TQ had no effect on the LPS/H2O2-mediated loss of TEER in Caco-2 monolayers (Table 6). SCNS, however, reduced the relative LPS/H2O2-stimulated loss of TEER approximately14 percent.
[000173] Conclusions – Unexpectedly, the degree to which SDNS attenuated LPS/H2O2 stimulation loss of TEER in differentiated Caco-2 monolayers was out of proportion to its TQ content.
Example 10
Supercritical, Defatted Nigella sativa Seeds Attenuate trans-10, cis12-Conjugated Linoleic Acid Isomer Loss of Transepithelial Electrical Resistance in Caco-2 Intestinal Epithelial Cells
[000174] Background – Drugs, such as HIV-1 protease inhibitors, and select dietary supplements, such as conjugated linoleic acid (t10-CLA), have been clinically shown to breach the integrity of the mucosal epithelial barrier, and allow translocation of virus and bacteria to impair the gastrointestinal mucosal barrier contributing to diarrhea, loss of intestinal membrane integrity (leaky gut syndrome, LGS), as well as systemic inflammation. In vitro, HIV-1 protease inhibitors and t10-CLA have been shown to negatively affect TEER in Caco-2 cells.
[000175] Objective - The objective of this experiment was to assess the ability of the commercial-scale supercritical CO2 defatted N. sativa produced in Example 1 to attenuate the loss of transepithelial electrical resistance in Caco-2 monolayers induced by the inflammatory conjugated linoleic acid isomer t10-CLA.
[000176] Methods – Experimental methods and data analysis for assessing the loss of TEER in t10-CLA stimulated Caco-2 cells were as described in Example 9 with the exception that 50 µM t10-CLA was used to induce TEER loss in place of the LPS/H2O2 positive control.
Table 7
Effect of supercritical CO2, Defatted Nigella sativa Seeds on t10-CLA-mediated decrease of transepithelial electrical resistance in Caco-2 Cells

Treatment Test Concentration
[µg TQ equivalents/mL]
Relative Loss of TEER
Solvent Only - 0.00*
t10-CLA 50 µM 100 ± 5.81
Thymoquinone (Sigma) 5.0 44.5*
SDNS 0.0005 0.00*
*Significantly less than t10-CLA 50 µM positive control (p<0.05)
SDNS: supercritical CO2, Defatted Nigella sativa Seeds
[000177] Results - SDNS as well as TQ inhibited loss of TEER in t10-CLA-stimulated Caco-2 cells indicating the ability to reduce intestinal monolayer disruption in response to a pro-inflammatory stimulus (Table 7). Unexpectedly, the degree to which SDNS attenuated t10-CLA stimulation in differentiated Caco-2 monolayers was quantitatively and qualitatively more potent than pure TQ.
Example 11
Supercritical CO2, Defatted Nigella sativa Seeds Attenuates trans-10, cis12-Conjugated Linoleic Acid Isomer Mediated Inflammation in 3T3-L1 Adipocytes
[000178] Background - Inflammation plays a role in insulin resistance in adipocytes. One manifestation of this inflammatory response is increased IL-6 and decreased adiponectin secretion by the adipocyte.
[000179] Objective - The objective of this experiment was to assess the effect of the commercial-scale SDNS produced in Example 1 on the increase in IL-6 secretion and decreased adiponectin secretion in 3T3-L1 adipocytes induced by inflammation as induced by the t10-CLA isomer.
[000180] The Model- The 3T3-L1 murine fibroblast model as used in Example 4 was used in this experiments.
[000181] Test Materials – Commercial-scale SDNS produced in Example 1 was used as the test material. Powdered t10-CLA, used to induce inflammation, was provided from Lipid Nutrition (Wormerveer, The Netherlands).
[000182] Treatment –Test materials were added four hours prior to the addition of t10-CLA at a concentration of 50 µM. Following overnight incubation, the supernatant media were sampled for determination of IL-6 and adiponectin.
[000183] Interleukin-6 assay - The IL-6 secreted into the medium in response to TNF??stimulation was quantified using the Quantikine® Mouse IL-6 Immunoassay kit with no modifications (R&D Systems, Minneapolis, MN). Information supplied by the manufacturer indicated that recovery of IL-6 spiked in mouse cell culture media averaged 99% with a 1:2 dilution and the minimum detectable IL-6 concentration ranged from 1.3 to 1.8 pg/mL. All supernatant media samples were diluted 1:30 for quantification.
[000184] Adiponectin assay - The adiponectin secreted into the medium was quantified using the Mouse Adiponectin Quantikine® Immunoassay kit with no modifications (R&D Systems, Minneapolis, MN). Information supplied by the manufacturer indicated that recovery of adiponectin spiked in mouse cell culture media averaged 103% and the minimum detectable adiponectin concentration ranged from 0.001 to 0.007 ng/ml.
[000185] Statistical Calculations and Interpretation – Test materials and were assayed in duplicate, while solvent controls were replicated eight times. IL-6 and adiponectin secretion were represented relative to the IL-6 and adiponectin secretion of the t10-CLA only controls as the IL-6 and adiponectin index and differences among the means were analyzed by the student’s t-test assuming a five percent probability of a type I error (Excel; Microsoft, Redmond, WA).
[000186] Results - Treatment with 50 µM t10-CLA induced a 8-fold increase in IL-6 secretion and 65 percent reduction in adiponectin secretion relative to controls (Table 8). SDNS as well as TQ inhibited IL-6 secretion in t10-CLA-stimulated adipocytes indicating the ability to reduce secretion of inflammatory cytokines in response to a pro-inflammatory stimulus. Both SDNS and TQ attenuated t10-CLA-stimulated decrease of adiponectin secretion. Unexpectedly, the degree to which SDNS affected t10-CLA stimulation in 3T3-L1 adipocytes was out of proportion to the TQ content implying novel inhibitory components.
Table 8
Effect of Supercritical CO2, Defatted Nigella sativa Seeds on t10-CLA-mediated inflammation in 3T3-L1 Adipocytes

Treatment Test Concentration
[µg TQ equivalents/mL]
IL-6 Index
Adiponectin Index
Solvent Only - 12.5 222
t10-CLA 50 µM 100±11 100±14
Thymoquinone (Sigma) 5.0 75* 128*
SDNS <0.0005 54* 143*
*Significantly less than t10-CLA 50 µM positive control (p<0.05)
SDNS: supercritical CO2, Defatted Nigella sativa Seeds
[000187] The attenuation of IL-6 secretion and inhibition of adiponectin secretion of adipocytes as demonstrated in this example underscores the potential of SDNS to overcome the diabetogenic effects of the t10-CLA inflammation induction model. Thus, SDNS would be useful to inhibit inflammation-induced obesity and stimulate weight loss and development of metabolic syndrome or type 2 diabetes.
Example 12
Chapatis, Supplemented with a Novel Phytoceutical Combination, Safely Improved HbA1c, Body Weight, Waist Circumference, Blood Lipids and Fatty Liver in Overweight and Diabetic Subjects: A Twelve-week Safety and Efficacy Study
[000188] Objective - The primary objectives of this exploratory study were to assess the safety and potential efficacy of a unique combination of defatted N. sativa seeds, powdered N. sativa seeds and fenugreek seed supplementation of a staple food in overweight and T2D subjects.
[000189] Study design - This study was an exploratory, interventional, single-arm, 12-week trial conducted at the JSS Ayurveda Medical Hospital in Mysore, India. Wheat flour chapatis, fortified to provide, respectively, 4.7 and 0.75 g powdered N. sativa and fenugreek seed/day with the consumption of four chapatis (Table 9), were supplied six days/week for 12 weeks to overweight (OW), T2D (DM) or DM and overweight subjects (DM/OW). Participants were instructed not to modify their diet, physical activity, lifestyle habits, current prescription drugs, or dietary supplements.
Table 9
Macronutrient and Supplement Content of Wheat Flour, Supplemented Wheat Flour, and Daily Chapatis Consumption.

Macronutrient composition Wheat Flour Supplemented Flour† Chapatis††
Calories [kcal] 323 320 300
[g/100g] [g/100g] [g/day]
Moisture 11.0 10.8 76.4
Carbohydrate 61.0 57.5 53.9
Total ash 1.43 1.76 1.65
Crude protein 14.4 14.7 13.8
Total fat 2.32 3.43 3.22
Saturated fatty acids 0.570 0.770 0.722
Monounsaturated fatty acids 0.380 0.630 0.591
Polyunsaturated fatty acids 1.37 2.03 1.90
Trans fatty acids ND ND ND
Dietary fiber 9.83 10.9 10.26
Insoluble dietary fiber 8.87 10.0 9.41
Soluble dietary fiber 0.960 0.904 0.848
Seed Powder Supplement
Whole Nigella powder 2.50 2.35
Defatted Nigella seed powder 2.50 2.35
Ground fenugreek seeds powder 0.800 0.750
† Commercial whole wheat flour (Pillsbury Chakki Fresh Atta 100% Atta, 0% Maida)
††1 kg of the supplemented wheat flour was mixed with 700 to 750 mL water to form dough; 45 to 50 g of the dough were grilled resulting in finished chapatis weighting 38 to 45g.
ND: None detected with Limit of Quantitation = 0.10%
[000190] Subject selection - Subjects for this study were recruited in Mysore, India beginning September 25, 2018, with completion of the trial on April 26, 2019. Recruitment notices, describing the trial, were posted in relevant departments of the JSS Ayurveda Medical Hospital in Mysore and the local newspaper to advertise the study. A shown in FIG. 3, At step 301, the process of clinical trial includes initial screening of 78 subjects. At step 303, the process of clinical trial includes selection of only 48 of 78 subjects screened based upon entrance, exclusion criteria, and regular consumption of chapatis, as well as willingness to participate six-days/week for 12 weeks, which is explained in detail in later part of the disclosure. Briefly, individuals between the ages of 18 to 75 with a body mass index greater than 25 (OW) or T2D (DM) subjects with one or more of the following: HbA1c = 6.0, fasting blood glucose > 110 mg/dL, postprandial glucose = 150 mg/dL or on medication for T2D over one year without adequate blood sugar control were selected to participate. Those individuals meeting both T2D and overweight criteria were classified as DM/OW. Concurrent medications of two months or longer were not discontinued for the trial. Further, the selected subjects had some inclusion criteria which were classified into body mass and diabetic. Body mass index of subjects equal to or greater than 25 was considered as over-weight. Whereas diabetic subjects exhibited one or more of the following: glycosylated hemoglobin A1c>=6.0, Fasting blood glucose >110mg/dL, postprandial glucose =150 mg/dl, failing to meet therapeutic goals on medication for type 2 diabetes over one year.
[000191] Investigational product – N. sativa and Fenugreek fortified chapatis (NFC) - Commercial whole wheat flour (Pillsbury Chakki Fresh Atta 100% Atta, 0% Maida) was formulated to contain 5% N. sativa (kalonji) and 0.8 % fenugreek (methi) ground seed. The identity of the commercial N. sativa and fenugreek seeds was confirmed at Supreem Pharma (Mysore, India). One-half of the N. sativa seed powder (2.5% of the formulated Atta flour) was a defatted seed powder produced by supercritical carbon dioxide extraction as described in Example 1 and depicted schematically in Figure 2. As a result of flour supplementation, the fat content increased by 49%, representing 66 and 48% increases in MUFA and PUFA, respectively (Table 9). Chapatis were prepared twice daily at Supreem Pharma by adding 700 to 750 mL of water to 1 kg of supplemented wheat flour to form the chapatis dough; balls of 45 to 50 g; the dough were pressed and grilled using Emami Healthy and Tasty refined sunflower oil (Emami Agrotech Ltd., Kolkata, India) resulting in finished chapatis weighting 38 to 45 g (N. sativa/fenugreek/chapatis – NFC). Two, pre-cooked NFC were delivered to each subject twice per day, Monday through Saturday. With each delivery, subjects were questioned concerning compliance including changes in diets or physical activity, taste, and tolerance of the NFC.
[000192] Clinical measurements - Clinical measurements and frequency includes 2 chapatis (bid) morning/afternoon 6 days/week for 12 weeks. Anthropometric variables were measured at all clinical visits by a single physician of the JSS Ayurveda Medical Hospital assigned to monitor the trial. Subjects were requested to remove any outdoor clothes before measurements, breathe normally, and stand with feet fairly close together (about 12-15 cm apart) with weight equally distributed on each leg. For measuring of body weight, a scale with accuracy 0.1 kg was used. Waist circumference was measured at the midpoint between the lowest rib and the top of the hipbone (iliac crest) with a flexible plastic measuring tape. BP was measured using a standard sphygmomanometer after resting for 10 min. The measurement was taken from a bare left arm in a quiet room.
[000193] Mean arterial pressure (MAP) was computed as (SBP+2*DBP)/3 and pulse pressure (PP) = (SBP – DBP). Estimated average glucose over 12 weeks (eAG) was computed using the formula: eAG(mg/dL) = ((28.7 x HbA1c) - 46.7). Analysis of whole blood and serum samples was conducted by the JSS Ayurveda Medical Hospital diagnostic laboratory. Reference ranges for the interpretation of CMP were based on historical hospital records and as published.
[000194] Cardio-metabolic algorithms - In addition to waist, hip and waist/hip measurements, the Index of Central Obesity (ICO) was computed as a parameter of central obesity using the formula: Waist Circumference(cm)/Height(cm). The NCEP ATP III guidelines, were employed to determine the number of metabolic syndrome (MetS) diagnostic criteria exhibited by each subject. The ASCVD algorithm (http://static.heart.org/riskcalc/app/index.html#!/baseline-risk) was used to estimate the risk of stroke or heart disease over ten years (Cardiovascular Risk Score/CRS). The fatty liver index (FLI), was calculated from serum TG, BMI, GGT, and WC according to the equation: FLI = (e 0.953*loge (TG) + 0.139*BMI + 0.718*loge (GGT) + 0.053*WC - 15.745) / (1 + e 0.953*loge (TG) + 0.139*BMI + 0.718*loge (GGT) + 0.053*WC - 15.745) * 100. The Lipid Accumulation Product (LAP) was computed, respectively, for men and women as: LAPM = (WC(cm) - 65) x TG(mmol/L), LAPW = (WC(cm) - 58) x TG(mmol/L). The formula: HSI = 8 x ALT/AST + BMI(+ 2 if type 2 diabetes yes, + 2 if female) was used to compute the Hepatic Steatosis Index.
[000195] Ethics - The clinical protocol was approved by the Ethical Committee of the JSS Ayurveda Medical Hospital, Mysore- 570028. This study was conducted based on good clinical practice International Conference on Harmonisation guidance and the ethical principles of the Declaration of Helsinki. Before enrollment, every participant received complete instructions concerning the protocol and the objectives of the study in nontechnical terms and they then executed a written, informed consent document. A personal copy of the executed, informed consent document was provided to each subject.
[000196] Statistical methods - An earlier, single-arm study was used to estimate sample size [Tripp ML, Dahlberg CJ, Eliason S, et al.: A Low-Glycemic, Mediterranean Diet and Lifestyle Modification Program with Targeted Nutraceuticals Reduces Body Weight, Improves Cardiometabolic Variables and Longevity Biomarkers in Overweight Subjects: A 13-Week Observational Trial. J Med Food. 2019;22(5):479-489]. In this previous trial, a -4.1% difference (P<0.01) from a median baseline value for HbA1c of 5.8% was detected using the paired t-test. From this effect size and standard deviation of the difference, a group size of 35 was estimated for HgA1c and glycemic variables, with the probability of a type I error at 5% and type II error at 20%. A goal of 40 subjects, therefore, was established to account for possible attrition. A per protocol analysis was conducted with subjects completing the trial with compliance set at 100%.
[000197] Clinical response variables and the number of MetS criteria met were normally distributed and analyzed using paired t-test or 1-way, repeated measures ANOVA. The post hoc analysis of the HbA1c = 7.0 subgroup vs HbA1c < 7.0 subgroup analysis was conducted using a 2-way, fixed effects ANOVA model. Cardiometabolic indices including CRS, FLI, LAP, and HSI, and their percent change from baseline were not normally distributed and analyzed using the nonparametric Wilcoxon matched-pairs signed-rank test. Tabulated baseline values are presented as means ± SEM for normally distributed data and medians with parenthetic ranges for nonparametric results. Ninety-five percent confidence intervals of the relative differences were computed for both means and medians. Person's linear correlation coefficient r was used to assess the relationship between HgA1c and other variables. StatMate and GraphPad Software (San Diego, CA) were used, respectively, for power and statistical analysis. Using two-tailed tests, the probability of a type I error was set at the nominal 5% level for all variables.
Results
[000198] Subject engagement and compliance - After initial, in-person screening interviews, which included medical records of 78 subjects, 48 were selected to participate based on inclusion/ exclusion criteria (Figure 3). Exclusion for screens include any one of conditions such as unable to read/write and understand the study protocol, change in prescription of medications over-the-counter medications, medical foods, and nutritional supplements within 30 days prior to Day 1 and the duration of the study, use of an investigational drug or participation in an investigational study within 30 days prior to Day 1 and for the duration of the study, use of anticoagulant medications (heparin compounds, platelet inhibitors or warfarin) within 30 days prior to Day 1 and for the duration of the study. Eight subjects withdrew from the study in the first weeks due to personal reasons unrelated to the trial. Due to the twice-daily contact with the subjects over 12 weeks. As shown in step 305, the clinical trial process includes 40 screens completing the trial with 100% compliance (i.e. no noteworthy changes in diet or physical activity and consumption of NFC two times/day, six days/week).
[000199] Baseline description of the subjects - Overall, 47.5% (19M/21F) of the subjects were male with a mean age of 49.8 years and a range of 32 to 72 years. The mean age and range for females were 40.4 years (23 to 65). Fifteen subjects were classified as OW, 9 as DM and 16 as DM/OW with males comprising 5/15 (33%), 9/9 (100%), and 5/16 (68.7%) of each subgroup, respectively. At 37.0 years, OW males were younger than DM males (54.4 years; P= 0.0049) and OW/DM males (54.2 years; P= 0.0131). Similarly, OW females were younger than DM/OW females 34.4 vs 45.9 and P= 0.0068 (Table 10).
Table 10
Baseline Anthropometric and Metabolic Characteristics of the Three Subgroups

Variable OW
(N=15) DM
(N=9) DM/OW
(N=16)
Anthropometric†
Gender M/F (%M) 5/10 (33.3%) 9/0 (100%) 5/11 (68.7%)
Male Age (Yr/Range) 37.0a (32 to 42) 54.4b (42 to 63) 54.2b (41 to 72)
Female Age (Yr/Range) 34.4a (23 to 45) -- 45.9b (32 to 65)
Height (cm) 162a ± 1.71 173b ± 2.82 158a ± 2.62
Weight (kg) 74.1a ± 1.98 69.4a ± 1.88 74.5a ± 3.22
BMI (kg/m2) 28.3b ± 0.657 23.3a ± 0.439 29.6b ± 0.776
Waist (cm) 92.1a ± 1.92 90.4a ± 1.72 97.3b ± 2.36
Hip (cm) 108b ± 1.60 97.9a ± 0.920 106b 2.55
Waist/Hip 0.857a ± 0.0206 0.924b ± 0.0147 0.917b ± 0.0191
Index of Central Obesity 0.569a ± 0.00995 0.525a ± 0.0116 0.615b ± 0.0129
Glycemic†
HbA1c =7.0 (% of subjects in group) 0/15 (0.0%)a 7/9 (77.8%)b 12/16 (75%)b
HbA1c (%) 5.29a ± 0.0661 7.92b ± 0.398 7.97b ± 0.392
FBG (mg/dL) 92.9a ± 1.99 137b ± 15.1 164b ± 11.3
PPBG (mg/dL) 129a ± 4.30 228b ± 24.7 232b ± 14.9
eAG (mg/dL) 105a ± 1.90 181b ± 11.4 182b ± 11.2
Vascular†
SBP (mm Hg) 120a ± 2.05 123a ± 2.89 126a ± 2.88
DBP (mm Hg) 76.9a ± 1.36 80.7a ± 3.56 80.9a ± 2.26
MAP (mm Hg) 91.3a ± 1.28 94.9a ± 3.08 95.9a ± 2.35
PP (mm Hg) 43.2a ± 2.09 42.7a ± 2.77 45.0a ± 1.70
Lipidic†
TC (mg/dL) 214a ± 7.41 197a ± 8.83 196a ± 11.0
TC-HDL (mg/dL) 174a ± 7.25 157a ± 9.03 157a ± 10.7
LDL (mg/dL) 137 a ± 7.13 127 a ± 8.47 126 a ± 9.45
VLDL (mg/dL) 32.3a ± 1.88 30.9a ± 2.95 27.8a ± 2.12
HDL (mg/dL) 39.9a ± 0.888 39.7a ± 0.289 38.5a ± 0.585
TG (mg/dL) 163a ± 9.39 156a ± 14.7 139a ± 11.1
Lipidic Ratios†
TC/HDL 5.38a ± 0.194 4.98a ± 0.249 5.07a ± 0.255
LDL.HDL 3.43a ± 0.172 3.20a ± 0.227 3.25a ± 0.221
VLDL/HDL 0.815a ± 0.0498 0.782a ± 0.0782 0.719a ± 0.0506
TG/HDL 4.21a ± 0.297 3.69a ± 0.285 3.53a ± 0.323
Hepatic†
GGT (IU/L) 9–48 16.5a ±1.07 23.1b ±1.97 15.7a ± 1.93
AST (IU/L) 10 to 40 6 high 28.8a ± 1.92 41.4b ± 5.83 30.0a ± 2.57
ALT (IU/L) 7–56 U/ L 35.7ab ±2.64 44.3b ± 5.58 31.6a ± 2.47
AST/ALT 0-1 0.836a ± 0.0564 0.925a ± 0.0450 0.976a ± 0.0695
ALP (IU/L) 44 to 147 i 148a ± 8.45 175a ± 17.9 167a ± 13.5
Bilirubin (mg/dL) 0.1 – 1.2 0.653a ± 0.0477 0.611a ± 0.0807 0.644a ± 0.0545
Direct Bilirubin (mg/dL) 0.220a ± 0.0200 0.222a ± 0.0465 0.213a ± 0.0256
Total Protein (mg/dL) 8 6.59a ± 0.108 6.88a ± 0.134 6.76a ± 0.102
Albumin (g/dL) 1 out 4.27a ± 0.114 4.39a ± 0.170 4.36a ± 0.104
Globulin (g/dL) 2.32a ± 0.104 2.43a ± 0.124 2.41a ± 0.0566
A/G 3h 1L 1.93a ± 0.160 1.86a ± 0.163 1.83a ± 0.0676
Renal†
BUN (mg/dL) 20-40 24.5a ± 1.29 23.0a ± 1.74 26.8a ±1.56
Creatinine (mg/dL) 0.8-1.4 0.880a ± 0.0327 0.989a ± 0.0484 0.994a ± 0.0295
BUN/Creatinine 28.6a ± 2.22 23.2a ± 1.17 27.3a ± 1.91
Thyroid†
T3 (ng/mL) 0.8 – 2.0 1.16a ± 0.0475 1.17a ± 0.0512 1.17a ± 0.0616
T4 (mg/dL) 5.13 – 14 7.83a ± 0.483 8.00a ± 0.299 7.90a ± 0.655
TSH (mg/dL) 0.27 – 4.2 3.19a ± 0.439 2.77a ± 0.306 3.48a ± 0.584
Metabolic Syndrome†
Number of diagnostic criteria met 2.20a ± 0.296 2.33a ± 0.167 3.31b ± 0.254
Cardiometabolic Indices††
Cardiovascular Risk (%) 1.20a
(0.700 to 2.90) 11.9b
(4.20 to 23.1) 3.75b
(0.800 to 38.8)
Fatty Liver Index (Score) 42.0b
(25.0 to 80.0) 34.0a
(24.0 to 45.0) 50.0b
{25.0 to 84.0)
Lipid Accumulation Product (Score) 58.1b
(27.8 to 115) 41.2a
(29.2 to 57.6) 52.4ab
(31.7 to 98.2)
Hepatic Steatosis Index (Score) 39.1b
(34.6 to 48.8) 33.5a
(32.2 to 36.8) 38.4b
(31.9 to 46.3)
† Values are means ± SEM; common letter superscripts indicate nonsignificant differences P>0.05 determined by ANOVA
†† Values are medians with parenthetic range; common letter superscripts indicate nonsignificant differences P>0.05 determined by Kruskal-Wallis test
AKP: Alkaline phosphatase; ALT: Alanine transaminase; AST: Aspartate aminotransferase; BMI: Body Mass Index; BUN: Blood urea nitrogen; DBP: Diastolic blood pressure; DM: Diabetic; DM/OW: Diabetic and overweight; eAG: estimated average glucose; FBG: Fasting blood glucose; GGT: Gamma-glutamyl transferase; HbA1c: Glycated Hemoglobin; HDL: High Density Lipoprotein; ICO: Index of central obesity; LDL: Low Density Lipoprotein; MAP: Mean arterial pressure; ND: Not determined; OW: Over weight; PP: Pulse pressure; PPG: Postprandial glucose; SBP: Systolic blood pressure; T3: Triiodothyronine; T4: Thyroxine; TC: Total cholesterol; TG: Triglycerides; TSH: Thyroid stimulating hormone; VLDL: Very low density lipoprotein.
[000200] Eight (88.9%) and 11 (68.8%) of the nine DM and 16 DM/OW subjects, respectively, were taking prescription drugs, ayurvedic medications, or supplements. Median years on medication were 5.0 and 2.8 years for DM and DM/OW, respectively. In the DM subgroup, metformin (1 subject), metformin with glimepiride (4 subjects), and insulin (1 subject) were the prescribed drugs, while combinations of antidiabetic ayurvedic medications and bitter melon (1subject) as well as bitter melon alone (1 subject) were taken by two subjects. Eight (50.0%) DM/OW subjects were receiving oral hyperglycemic agents (OHA) including metformin (1 subject), combinations of metformin with glimepiride, sitagliptin or teneligliptin (5 subjects), glimepiride (1 subject) or glimepiride plus insulin (1 subject). One of these eight was also prescribed insulin in addition to the metformin combinations. One subject was being treated with the antihypertensive agent metoprolol in addition to a metformin/glimepiride combination. Three DM/OW subjects (18.8%) were taking ayurvedic medications, one with a bitter melon supplement. A preliminary statistical analysis of HbA1c, FBG, eAG and PPBG at baseline, weeks 6, and 12 in these subjects versus DM subjects that had not received OHA or taken supplements (N=6) indicated no differences between these subgroups. Therefore, both OHA-medicated and nonmedicated diabetics were grouped for statistical analyses.
[000201] Adherence to protocol eligibility requirements is reflected in the baseline subgrouping descriptions (Table 10). While body weights were similar over subgroups, BMI was lower in DM than OW (P= 0.0003) or DM/OW (P <0.0001) as DM were taller. WC and ICO were highest in the DM/OW subgroup. The percent of subjects with HbA1c = 7.0 and %HbA1c was similar in the DM (7/9; mean HbA1c=7.92%) and DM/OW (12/16; mean HbA1c=7.97%) subgroups and greater than the OW (0/15; mean HbA1c=5.29%). Similarly, FBG, eAG, and PPG were equal in DM and DM/OW and both elevated with respect to OW.
[000202] Blood pressures, lipid profiles, and ratios as well as kidney and thyroid function did not differ among subgroups. At baseline, DM exhibited generally higher GGT, AST, ALT, and ALP than OW or DM/OW and mean AST (41.4 IU/L) and ALP (175 IU/L) exceeded the normal, reference ranges of 40 and 147 IU/L, respectively. All other hepatic biomarkers were in the normal, reference ranges and did not differ among subgroups.
[000203] Overall, being DM/OW put subjects at greatest risk of a diagnosis of MetS as 75% (95% CI = 50.5 to 89.8%) of DM/OW exhibited = 3 diagnostic criteria for MetS, while only 46.7% (24.8 to 69.9%) and 33.3% (12.1 to 64.6%) OW and DM, respectively, were diagnostic for MetS. Similarly, the mean number of diagnostic criteria met were greatest (P<0.05) in the DM/OW subgroup 3.32 versus 2.20 and 2.33 for OW and DM, respectively (Table 2). The risk of a cardiovascular event over the next ten years (CRS) was higher in the DM (11.9%) and DM/OW (3.75%) than in the OW (1.20%) subjects (P<0.05 for both). FLI, LAP, and HSI, indices of hepatic fat accumulation, were elevated in the OW and DM/OW subgroups relative to the DM subjects (P<0.05), possibly related to a lower BMI and WC in the DM subjects and reflecting differences in underlying metabolic dysfunction.
[000204] Tolerability - Volunteers were given 2 chapatis b.i.d., as a regular diet, without restricting their normal food habits and no other special instruction in terms of exercise or workouts. NFC (Nigella-fenugreek chapatis) were well received and their taste was rated “good” to “excellent” overall. Subjects routinely commented on feeling lighter and clothes fitting better during the study. Four subjects reported relief of chronic constipation, while five subjects initially recounted instances of transient gastric upset with four chapatis per day and were switched to three chapatis per day with no further issues.
[000205] Anthropometric measurements - All anthropometric variables were positively modified at the completion of 12 weeks and exhibited a trend to increasing improvement (P<0.0001) with time on study (Table 11). Specifically, mean BW was reduced -1.55 kg (95% CI = -2.29 to -0.808 kg; P= 0.0001), BMI -0.645 kg/m2 (-1.02 to -0.272 kg/m2; P= 0.0012), WC -3.05 cm (-4.00 to -2.10 cm; P <0.0001), HC -1.90 cm (-3.01 to -0.781 cm; P=0.0013) W/H –1.50% (-2.78 to -0.214%; P=0.0234), and ICO -3.25% (-4.21 to -2.29%; P<0.0001). The absolute weight losses of -1.3 and -1.6 kg were essentially all due to fat loss, respectively, -1.2 and -1.6 kg at 6 and 12 weeks (Figure 4). There were no differences in the relative changes in anthropometric measurements among criteria subgroups (data not shown).
Table 11
Mean Anthropometric and Metabolic Variables Over the Study

Variable Baseline
(N=40) Week 6
(N=40)
P-value Week 12
(N=40)
P-value P-value (Trend)
Anthropometric†
Weight (kg) 73.2 ± 1.55 71.9 ± 1.51
(-1.79%) <0.0001
71.7 ± 1.54
(-2.12%) 0.0001 <0.0001
BMI (kg/m2) 27.7 ± 0.560
27.2 ± 0.552
(-1.81%) <0.0001 27.1 ± 0.557
(-2.32%) 0.0012 <0.0001
Waist (cm) 93.8 ± 1.31 91.8 ± 1.28
(-2.21%) <0.0001 90.8 ± 1.29
(-3.25%) <0.0001 <0.0001
Hip (cm) 105.0 ± 1.329 103.5 ± 1.265
(-1.42%) 0.0006 103.1 ± 1.305
(-1.81%) 0.0013 <0.0001
Waist/Hip 0.896 ± 0.0121 0.889±0.0120
(-0.781%) 0.1208 0.883 ± 0.114
(-1.45%) 0.0234 0.0081
Index of Central Obesity 0.577±0.00867 0.565±0.00881
(-2.08%) <0.0001 0.559±0.00838
(-3.12%) <0.0001 <0.0001
Glycemic†
HbA1c (%) 6.95 ± 0.272 --- --- 6.56 ± 0.221
(-5.65%) 0.0002 NC
FBG (mg/dL) 131 ± 7.49 119 ± 6.64
(-9.31%) 0.0405 122 ± 6.03
(-7.46%) 0.0866 0.1034
PPBG (mg/dL) 193 ± 11.3 178 ± 9.83
(-7.46%) 0.1012 177 ± 7.41
(-9.15%) 0.0341 0.0465
eAG (mg/dL) 153 ± 7.82 --- --- 142 ± 6.35
(-7.39%) 0.0002 NC
Vascular†
SBP (mm Hg) 123 ± 1.55 123 ± 1.31
(-0.28%) 0.6149 124 ± 1.99
(0.99%) 0.3911 0.3129
DBP (mm Hg) 79.4 ± 1.31 78.5 ± 0.894
(-1.07%) 0.3285 78.2 ± 1.41
(-1.47%) 0.5131 0.4239
MAP (mm Hg) 94.0 ± 1.27 93.3 ± 0.907
(-0.73%) 0.3085 93.6 ± 1.45
(-0.38%) 0.7969 0.7563
PP (mm Hg) 43.8a ± 1.19 44.3 ± 1.19
(1.14%) 0.7465 46.3 ± 1.56
(5.14%) 0.1275 0.1275
Lipidic†
TC (mg/dL) 202.7 ± 5.61
--- --- 186.7 ± 4.49
(-7.88%) 0.0012 NC
TC-HDL (mg/dL) 163.4 ± 5.49 --- --- 148.2 ± 4.47
(-9.39%) 0.0016 NC
LDL (mg/dL) 130.2 ± 4.96
--- --- 121.5 ± 4.23
(-6.68%) 0.0603
(NS) NC
VLDL (mg/dL) 30.2 ± 1.29 --- --- 26.8 ± 0.989
(-11.1%) 0.0020 NC
HDL (mg/dL) 39.3 ± 0.416 --- --- 38.5 ± 0.410
(-1.91%) 0.0842
(NS) NC
TG (mg/dL) 152 ± 6.61 --- --- 138 ± 5.03
(-9.21%) 0.0022 NC
Lipidic Ratios†
TC/HDL 5.16 ± 0.137 --- --- 4.86 ± 0.123
(-5.85%) 0.0161 NC
LDL/HDL 3.31 ± 0.119 --- --- 3.17 ± 0.117
(-4.23%) 0.2189
(NS) NC
VLDL/HDL 0.769 ± 0.0325 --- --- 0.697 ± 0.0232
(-9.44%) 0.0150 NC
TG/HDL 3.87 ± 0.166 --- --- 3.59 ± 0.124
(-7.44%) 0.0211 NC
Hepatic†
GGT (IU/L) 17.7 ± 0.849 --- --- 15.3 ± 0.809
(-13.5%) 0.0048 NC
AST (mg/dL) 32.1 ± 1.93 --- --- 26.8 ± 1.60
(-16.5%) 0.0003 NC
ALT (mg/dL) 36.0 ± 1.98 --- --- 29.6 ± 1.60
(-17.8%) <0.0001 NC
AST/ALT 0.912 ± 0.0369 --- --- 0.935 ± 0.0438
(2.47%) 0.6564
(NS) NC
ALP (mg/dL) 161.9 ± 7.45 --- --- 141.4 ± 5.75
(-12.7%) <0.0001 NC
Bilirubin (mg/dL) 0.640 ± 0.0354 --- --- 0.538 ± 0.0301
(-16.1%) 0.0093 NC
Direct Bilirubin (mg/dL) 0.218 ±0.0160 --- --- 0.203 ±0.0136
(-6.88%) 0.4602 (NS) NC
Protein (mg/dL) 6.73 ± 0.0659 --- --- 6.63 ± 0.0671
(-1.45%) 0.0781
(NS) NC
Albumin (g/dL) 4.34 ± 0.0693 --- --- 4.20 ± 0.0511
(-3.06%) 0.0044 NC
Globulin (g/dL) 2.38 ± 0.0520 --- --- 2.43 ± 0.0492
(1.89%) 0.4123 (NS) NC
Albumin/Globulin 1.87 ± 0.0737 --- --- 1.76 ±0.0465
(-5.88%) 0.1078 (NS) NC
Renal†
Urea (mg/dL) 25.1 ± 0.892 --- --- 23.6 ± 0.647
(-5.78%) 0.6564
(NS) NC

Creatinine (mg/dL) 0.948 ± 0.0218 --- --- 0.938 ± 0.0122
(-1.05%) 0.6564
(NS) NC
Urea/Creatinine 26.9 ± 1.18 --- --- 25.3 ± 0.707
(-5.95%) 0.6564
(NS) NC
Thyroid†
T3 (ng/mL) 1.17 ± 0.0318 --- --- 1.16 ± 0.0370
(-0.21%) 0.9389
(NS) NC
T4 (mg/dL) 7.89 ± 0.319 --- --- 7.99 ± 0.237
(1.16%) 0.5812 (NS) NC
TSH (mg/dL) 3.21 ± 0.291 --- --- 3.15 ± 0.339
(-2.07%) 0.7803 (NS) NC
Metabolic Syndrome†† Baseline 12 Weeks % Change
# of criteria met 2.68 ± 0.174 2.38 ± 0.174 0.0503
(NS) -11.2
% with = 3 criteria
(95% CI) 55.0
(39.6 to 70.4) --- --- 45.0
(29.6 to 60.4) 0.374
(NS) -18.2
Cardiometabolic Indices††
Cardiovascular Risk Score (%) 2.80
(0.700 to 38.8) 2.50
(0.500 to 34.0) 0.00210 -10.7
Fatty Liver Index 40.5
(24.0 to 84.0) 31.0
(10 to 77.0) <0.0001 -23.5
Lipid Accumulation Product 52.4
(27.8 to115) 42.6
(14.8 to 92.1) <0.0001 -18.7
Hepatic Steatosis Index 37.7
(31.9 to 48.8) 32.5
(7.06 to 48.3) <0.0001 -13.8
† Values are means ± SEM; differences from baseline determined by paired-t or repeated measures ANOVA; trend analysis for nonzero slope over 12 weeks.
†† Values are medians with parenthetic range. P-values determined by Wilcoxon matched-pairs signed rank test.
AKP: Alkaline phosphatase; ALT: Alanine transaminase; AST: Aspartate aminotransferase; BMI: Body Mass Index; BUN: Blood urea nitrogen; DBP: Diastolic blood pressure; DM: Diabetic; DM/OW: Diabetic and overweight; eAG: estimated average glucose; FBG: Fasting blood glucose; GGT: Gamma-glutamyl transferase; HbA1c: Glycated Hemoglobin; HDL: High Density Lipoprotein; ICO: Index of central obesity; LDL: Low Density Lipoprotein; MAP: Mean arterial pressure; ND: Not determined; OW: Over weight; PP: Pulse pressure; PPG: Postprandial glucose; SBP: Systolic blood pressure; T3: Triiodothyronine; T4: Thyroxine; TC: Total cholesterol; TG: Triglycerides; TSH: Thyroid stimulating hormone; VLDL: Very low density lipoprotein.
[000206] Glycemic responses - After 12 weeks of NFC consumption, mean absolute HbA1c change from baseline over all subjects was -0.393% ( -0.587 to -0.198; P=0.0002) representing a relative HbA1c decrease of -5.65% (-8.58 to -2.85%). Concordant relative decreases were seen in FBG and PPBG of -9.31% (-18.1 to -0.420%; P=0.0405), and -7.46% (-16.4 to1.52%; P=0.1012), respectively, at 6 weeks and -6.95% (-15.0 to 1.05%; P=0.0866) and -8.39% (-16.2 to -0.663%; P=0.0341) at 12 weeks. Moreover, PPBG decreases from baseline through 6 and 12 weeks exhibited a linear trend for improvement (P=0.0465). eAG, which reflects the average blood glucose concentration over the previous 12 weeks, decreased -7.39% (-11.1 to -3.71%; P=0.0002) from 153 to 142 mg/dL over all subjects (Table 11).
[000207] Among all variables, glycemic responses exhibited the most pronounced differences between the criteria subgroups. As a post hoc subgroup, diabetic subjects exhibited higher baseline HbA1c, FBG, PPBG, and eAG versus nondiabetics (Table 11). As expected, the relationships between HbA1c with FBG and PPBG were linear with (r = 0.839; P<0.0001; N=80) and (r = 0.870; P<0.0001; N=80) from baseline through week 12, respectively (data not shown). Moreover, the absolute change in HbA1c was linearly correlated to baseline HbA1c with r = -0.658 (P <0.0001; N=40) with a decrease of -0.232 in HbA1c for each unit increase in baseline HbA1c level (Figure 5A). Therefore, post hoc subgroups of baseline HbA1c=7.0 (Range 7.00 to 10.8; N=19) and baseline HbA1c<7.0 (Range 4.90 to 6.00; N=21) were created to assess changes in HbA1c, FBG, and PPBG over 12 weeks as a function of diabetic severity (Figures 5B, 5C and 5D, respectively).
[000208] After 12 weeks, absolute HbA1c fell -0.689 (-0.942 to -0.436; P <0.0001) relative to baseline (Figure 5B) in the HbA1c =7.0 subgroup. FBG fell -22.4 mg/dL (-3.51 to -41.3; P= 0.0160) and -18.8 mg/dL (-37.7 to 0.118; P= 0.0518) at 6 and 12 weeks respectively (Figure 5C). Over the same time periods, PPBG fell -36.3 mg/dL (-62.8 to -9.82; P= 0.0045) and -38.2 mg/dL (-64.7 to -11.7; P= 0.0026). There were no differences from baseline in the HbA1c<7.0 subgroup for any of the glycemic variables at 6 or 12 weeks (Figure 5B, 5C, and 5D).
[000209] Of the 19 subjects previously non-responsive to diabetic medications over one year, HbA1c decreased from 8.17 to 7.61 (-5.58; 95% CI = -0.897 to -0.219; P <0.0001) in 12 weeks.
[000210] Blood pressure - Consumption of NFC exhibited no treatment effects on any of the four measures of vascular pressure. Variations in SBP by subject during the study did not result in categorical changes as recently defined (Optimal, Normal, High normal, or Grade 1 hypertension) [Williams B, Mancia G, Spiering W, et al.: 2018 ESC/ESH Guidelines for the management of arterial hypertension. Rev Esp Cardiol (Engl Ed). 2019;72(2):160]. Additionally, no treatment effects were noted for MAP or PP and a majority of subjects exhibited normal MAP (38/40) and PP (39/40) from baseline through week 12 (Table 11).
[000211] Lipids - After 12 weeks, total cholesterol, non-HDL cholesterol, VLDL and TG were decreased, respectively, -7.88% (-12.4 to -3.33%; P= 0.0012), -9.39% (-15.0 to -3.77%; P=0.0016), -11.1% (-17.9 to -4.30%; P=0.0020) and -9.21% (-14.9 to -3.51%; P=0.0022) by NFC (Table 11). Neither LDL nor HDL were changed from baseline P= 0.0603 and P= 0.0842, respectively. No differences were seen in the relative changes in lipid biomarkers when adjusted for diabetic severity (data not shown).
[000212] Lipidic ratios - After 12 weeks of consuming NFC, decreases from mean baseline values (N=40) were noted for TC/HDL, VLDL/HDL, and TG/HDL, respectively, -5.85% (-10.5 to -1.15%; P=0.016), -9.44% (-16.9 to -1.94%; P=0.015), and -7.44% (-13.7 to -1.18%; P=0.021). LDL/HDL was not changed from baseline (Table 11). No differences were seen in the responses of the lipidic ratios among criteria subgroups (data not shown).
[000213] Hepatic function – Positive modifying effects were observed in hepatic biomarkers GGT -13.5%; (-22.5 to -4.37%; P=0.0048), AST -16.5% (-24.8 to -8.15%; P=0.0003), ALT -17.8% (-26.1 to -9.42%; P<0.0001), ALP -12.7%; (-18.3 to -6.92%; P<0.0001), and bilirubin -16.1% (-27.8 to -4.17%; P=0.0093). Mean ALP dropped from 162 IU/L, above the normal, reference range of 147 IU, to 141 IU/L at the completion of 12 weeks (Table 11). Relative responses for all hepatic biomarkers were similar among criteria subgroups (data not shown).
[000214] Renal and thyroid assessment - No adverse effects on renal or thyroid function were seen from consuming the NFC over 12 weeks (Table 11). Average BUN, creatinine, and BUN/creatinine were unchanged from baseline values with 95% confidence intervals of percent change respectively, (-12.2 to 0.637%; P=0.0761), (-5.81 to 3.70%; P=0.6564) and (-13.3 to 1.36%; P=0.1080), respectively).
[000215] Similarly, mean T3, T4 and TSH did not change during the study with 95% confidence intervals of percent change respectively, (-5.60 to 5.18%; P=0.939), (-3.05 to 5.35% P=0.581), and (-17.0 yo 12.9%; P=0.780) (Table 11). No differences were seen in relative renal or thyroid changes among the criteria subgroups (data not shown).
[000216] Cardio-metabolic indices - Along with an -11.2% (-22.4 to 0.0135%; P= 0.0503) decrease in mean number of diagnostic criteria per subject, the CRS, whose variables partially overlap with MetS criteria, exhibited a median -10.7% decrease (-14.3 to -3.57%; P=0.0021) over all subjects after 12 weeks (Data not shown). FIGURE 6A, FIGURE 6B and, FIGURE 6C illustrates graphs obtained to track Fatty Liver Index (FLI), Lipid Accumulation Product (LAP) and , Hepatic stenosis respectively over duration of the clinical trial. Further, measures of hepatic fat accumulation, FLI, LAP and HSI, all decreased significantly with median decreases of -19.8 (-27.2 to -14.8%), -13.8% (-24.6 to -10.6%) and -7.53% (-53.1 to -2.76%) with P<0.0001for all, respectively.
[000217] The dramatic decreases in body fat and hepatic fat accumulation are consistent with the in vitro lipolytic (Example 7) and AMPK activation (Example 8) activity of the supercritical carbon dioxide, defatted N. sativa ground seeds of Example 1. Moreover, to our knowledge, there exists no published, clinical study of a N. sativa containing food product capable of reducing body weight or hepatic steatosis with such a high degree of fat reducing specificity.
[000218] Studies of N. sativa and fenugreek administered separately in bread matrices were generally negative. MetS patients consuming 3 g powdered N. sativa daily in 100 g bread for 2 months did not exhibit significant decreases in FBG [Mohtashami A: Effects of Bread with Nigella Sativa on Blood Glucose, Blood Pressure and Anthropometric Indices in Patients with Metabolic Syndrome. Clin Nutr Res. 2019;8(2):138-147]. In the second study, fenugreek administered as a single serving of 56 g of bread containing 2.8 g fenugreek failed to significantly affect PPBG in eight, diet-controlled diabetics [, although a decrease in insulin secretion over 2 hours versus placebo was reported [Losso JN, Holliday DL, Finley JW, et al.: Fenugreek bread: a treatment for diabetes mellitus. J Med Food. 2009;12(5):1046-1049].
[000219] To date, no clinical study of N. sativa, or fenugreek, alone or in combination in a food, has demonstrated the range or degree of positive effects on adiposity, glycemic control, lipidic biomarkers, or hepatic functioning as Example 12.
[000220] Thus, there has been disclosed a novel supercritical CO2 defatted N. sativa composition resulting from sequential lipid removal of commercial scale quantities of powdered seed produced under specific conditions of temperature, pressure and time of lipid removal. Methods for the production of this formulation and uses have been described. It will be readily apparent to those skilled in the art, however, that various changes and modifications of an obvious nature may be made without departing from the spirit of the invention, and all such changes and modifications are considered to fall within the scope of the invention as defined by the appended claims. Such changes and modifications would include, but not be limited to, the incipient ingredients added to affect the food, capsule, tablet, powder, lotion, or bar manufacturing process as well as additional herbs, phytoceuticals, vitamins, flavorings and carriers. Other such changes or modifications would include the use of herbs or other botanical products containing the combinations of the preferred embodiments disclosed above. Many additional modifications and variations of the embodiments described herein may be made without departing from the scope, as is apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only.