Claims:We Claim:
1. A method of normalizing a metabolic disorder in a subject comprising the administration of a therapeutically effective amount of a combination consisting of a supercritical CO2, essential oil extract of N. sativa ground seeds and a phytoceutical or therapeutic in amounts that provide a combined AMP-kinase activating activity that is greater than an AMP-kinase activating activity provided by an equivalent amount of either component or the sum of the components.
2. The method according to claim 1, wherein the phytoceutical comprises at least one member of the group consisting essentially of Cayenne pepper, Coleus forskohlii, Cumin, Garcinia spp. (60% hydroxycitric acid), green coffee bean extract, Gymnema sylvestre, Momordica charantia (Bitter Melon), Oregano (carvacrol), Ribes nignum L. (Black current), Trachyspermum ammi L (Arq ajwain), Trigonella foenum-graecum, Vaccinium (blueberry leaf – 20% chlorogenic acid), wheat germ oil and Zingiber officinale (6-gingerol).
3. The method according to claim 1, wherein the phytoceutical is Garcinia spp. standardized to 60% hydroxycitric acid.
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, and cardiovascular disease.
5. A composition for normalizing a metabolic disorder in a subject comprising the administration of a therapeutically effective amount of a combination consisting of a supercritical CO2, essential oil extract of N. sativa ground seeds and a phytoceutical or therapeutic in amounts that provide a combined AMP-kinase activating activity that is greater than an AMP-kinase activating activity provided by an equivalent amount of either component or the sum of the components.
6. The composition according to claim 6, wherein the phytoceutical comprises at least one member of the group consisting essentially of Cayenne pepper, Coleus forskohlii, Cumin, Garcinia spp. (60% hydroxycitric acid), green coffee bean extract, Gymnema sylvestre, Momordica charantia (Bitter Melon), Oregano (carvacrol), Ribes nignum L. (Black current), Trachyspermum ammi L (Arq ajwain), Trigonella foenum-graecum, Vaccinium (blueberry leaf – 20% chlorogenic acid), wheat germ oil and Zingiber officinale (6-gingerol).
7. The composition according to claim 6, wherein the phytoceutical is Garcinia spp. standardized to 60% hydroxycitric acid.
8. The composition according to claim 6, wherein the metabolic disorder comprises at least one member of the group comprising obesity, metabolic syndrome, diabetes, hepatic steatosis, hyperlipidemia, 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 novel, supercritical CO2 essential oil extracts of Nigella sativa seeds, exhibiting positive entourage effects that function alone or synergistically in combination with other phytoceuticals or drugs to improve averse metabolic functioning health. These compositions would be useful in the prevention, treatment or co-treatment of metabolic disorders such as obesity, hyperlipidemia, metabolic syndrome, diabetes, hepatic steatosis, and cardiovascular diseases.
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 now believed to be due to the thymoquinone (TQ) content of the essential oil content, which is also present in lesser amount in the fixed oil along with a variety of other terpenoids (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, riboflavin, 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] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] Garcinia extract, (-)-hydroxycitric acid (HCA) - HCA is a derivative of citric acid and can be found in plant species such as Garcinia cambogia, Garcinia indica, and Garcinia atroviridis, which are native to South Asia [Onakpoya I, Hung SK, Perry R, Wider B, Ernst E: The Use of Garcinia Extract (Hydroxycitric Acid) as a Weight loss Supplement: A Systematic Review and Meta-Analysis of Randomised Clinical Trials. J Obes. 2011;2011:509038]. HCA, is usually marketed as a weight loss supplement [C. Roongpisuthipong, R. Kantawan, and W. Roongpisuth- ipong, “Reduction of adipose tissue and body weight: effect of water soluble calcium hydroxycitrate in Garcinia atroviridis on the short term treatment of obese women in Thailand,” Asia Pacific Journal of Clinical Nutrition, vol. 16, no. 1, pp. 25–29, 2007] (Figure 2A). Some authors have suggested that HCA causes weight loss by competitively inhibiting the enzyme adenosine triphosphatase-citrate-lyase (Figure 2B) [Onakpoya I, Hung SK, Perry R, Wider B, Ernst E: The Use of Garcinia Extract (Hydroxycitric Acid) as a Weight loss Supplement: A Systematic Review and Meta-Analysis of Randomised Clinical Trials. J Obes. 2011;2011:509038]. Weight losses resulting from HCA from Garcinia spp. are small, inconsistent, and require gram quantity doses that frequently cause stomach discomfort [K. Hayamizu, Y. Ishii, I. Kaneko et al., “Effects of Garcinia cambogia (Hydroxycitric Acid) on visceral fat accumulation: a double-blind, randomized, placebo-controlled trial,” Current Therapeutic Research, vol. 64, no. 8, pp. 551–567, 2003]. In a systematic review and meta-analysis of 23 randomised, clinical trials, revealed only a small, statistically significant difference in weight loss favouring HCA over placebo (MD: −0.88kg; 95% CI: −1.75, −0.00) [Onakpoya I, Hung SK, Perry R, Wider B, Ernst E: The Use of Garcinia Extract (Hydroxycitric Acid) as a Weight loss Supplement: A Systematic Review and Meta-Analysis of Randomised Clinical Trials. J Obes. 2011;2011:509038]. Further, no consistent favorable effects have been reported for reducing elevated total cholesterol, LDL cholesterol, HDL cholesterol, or triglycerides.
[0009] 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, peridontal disease, 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 implies that it is beginning to replace undernutrition and infectious diseases as the most significant contributor to ill health in the developed world.
[00010] 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.
[00011] In obese individuals, excessive production of acylation-stimulating protein (ASP), TNFα, 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.
[00012] 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].
[00013] Mitochondrial uncoupling – Increasing thermogenesis appears 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 (Figure 3A). 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.
[00014] Controlling adiposity by targeted modulation of adipocyte mitochondrial membrane potential could offer an attractive alternative to current dietary approaches. It has 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.
[00015] Thermogenesis - Thermogenesis or uncoupling of mitochondrial membrane potential may be activated both directly and indirectly. Direct thermogenesis can result through uncoupling of mitochondrial membrane potential as previously described by DNP (Figure 3A) or forced expression of UCP-1. Indirect activation also occurs through beta-3-adrenergic receptors (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 obesity and type 2 diabetes [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].
[00016] 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.
[00017] Oxidative stress - Current consensus is that obesity results in the production of reactive oxygen (oxidative stress) and nitrogen species, which leads to oxidative myocardial injury. Alterations in myocardial structure and function frequently occur in the late stage of obesity. Oxidative stress, induced by reactive oxygen and nitrogen species derived from obesity, 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 comorbidities associated with obesity.
[00018] Cardiovascular disease is a leading cause of death in obese patients. Cardiomyopathy (CM) is a complicated and chronic process that is secondary to acute cardiac responses in obesity. One of these acute responses is cardiac cell death, which plays a critical role in the initiation and development of CM. Inflammatory response in the obese heart is also a major cause for cardiac cell death. Obesity often causes systemic and cardiac increases in tumor necrosis factor-alpha (TNF), interleukin-18 and PAI-1. How these cytokines induce cardiomyopathies remains unclear, but it has been speculated to relate to oxidative and/or nitrosative stress. Cardiac cell death it is reasoned, is induced by the inflammatory cytokines that are increased in response to obesity. Inflammatory cytokine-induced cardiac cell death is mediated by oxidative stress and is also the major initiator for CM 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].
[00019] 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. AMP-activated protein kinase (AMPK) plays a key role as a master regulator of cellular energy homeostasis (Figure 4A). The kinase is activated in response to cellular stresses that deplete cellular ATP supplies such as low glucose, hypoxia, ischemia, and heat shock (Figure 4B). Due to its role as a central regulator of both lipid and glucose metabolism, AMPK is considered to be a potential therapeutic target for the treatment of obesity, diabetes mellitus, and cancer. AMPK has also been implicated in a number of species as a critical modulator of aging through its interactions with mTOR and sirtuins. 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 (Figure 4B). 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.
[00020] 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 (Figure 4B).
[00021] 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.
[00022] 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.
[00023] Novel, stepwise, supercritical CO2 extractions of ground N. sativa seeds involving changes in temperature, pressure and time, which produce essential oil (EO) and oleoresin (OR) fractions exhibiting an entourage effect among the extracted terpenoids, are described. These EO and OR extracts contain terpenoid fractions in ratios and amounts unlike those of whole N. sativa seeds and unexpectedly exhibit chemical and biological activities in vitro and clinically that differ both qualitatively and quantitatively from whole seeds and the putative, singularly active terpenoid TQ (entourage effect).
[00024] To date no process for supercritical CO2 extraction of N. sativa has been described that is useful for commercial quantities of ground seed and can produce extracts exhibiting the entourage effect. 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 resulting in extracts exhibiting profound examples of the entourage effect.
SUMMARY OF THE INVENTION
[00025] 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
[00026] The present invention relates to the unexpected discovery that supercritical CO2 extracted essential oil from N. sativa seeds exhibit an entourage effect in decreasing mitochondrial membrane potential in adipocytes implying decreased ATP synthesis and increased thermogenesis in excess of its TQ content. The invention provides methods for modifying adipocyte physiology in a subject, comprising administering to the subject a pharmaceutical composition supercritical CO2 extracted essential oil from N. sativa seeds or mixtures thereof. Preferred embodiments provide compositions and methods for enhancing adipocyte thermogenesis or decreasing oxidative stress utilizing supercritical CO2 extracted essential oil from N. sativa seeds.
[00027] The present invention further provides novel compositions of supercritical CO2 EO extracts of N. sativa seeds produced on a commercial scale and exhibiting an entourage effect, in a synergistic combination with HCA or other phytoceuticals, to reduce body weights, waist circumference, adiposity, hyperglycemia, hyperlipidemia, hepatic steatosis, and cardiovascular risk in a subject in need thereof.
[00028] The present invention also 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 CO2 extracted essential oil from N. sativa seeds, exhibiting an entourage effect, in a synergistic, AMPK-activating combination with HCA or other phytoceuticals and continuing the administration of the composition until the signs, signalments or symptoms are reduced.
[00029] 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 CO2 extracted essential oil from N. sativa seeds, which exhibit an entourage effect, in a synergistic AMPK-activating combination with HCA or other phytoceuticals and continuing the administration of the composition until the signs, signalments or symptoms are reduced.
[00030] Additionally, the present invention relates to the unexpected discovery that supercritical CO2 extracted essential oil from N. sativa seeds synergistically activates AMPK with non-thermogenic, AMPK-activating compounds implying superior 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.
[00031] Moreover, the present invention relates to the unexpected discovery that novel, supercritical CO2 extracted essential oil from N. sativa seeds in combination with HCA consumed as two capsules per day can safely decrease body weights, body mass index, waist circumference, adiposity, hepatic steatosis, hyperlipidemia, and cardiovascular risk within 4 weeks and continue these effects through 12 weeks.
BRIEF DESCRIPTION OF THE DRAWINGS
[00032] FIG. 1 depicts structures and relative abundance of terpenoids commonly found in the essential oil fractions EO203 and EO205 of Nigella sativa seeds.
[00033] FIG. 2 representation of the putative anti-obesity effects of Garcinia cambogia/HCA. [A] Possible mechanisms that contribute to anti-obesity effects of HCA. [B] Highlights two reactions that may be perturbed to simulate the anti-obesity effect of HCA.
[00034] FIG. 3 depicts a schematic modeling of mitochondrial membrane uncoupling by [A] dinitrophenol (DNP) and [B] thymoquinone (TQ).
[00035] FIG. 4 depicts [A] tissue-specific functioning of AMPK activation and [B]multiple pathway AMPK activation with downstream pathways.
[00036] FIG. 5 is a schematic of the sequential steps involved in supercritical CO2 extraction of essential oil fractions EO203 and EO205, and the oleoresin fraction OR207 from whole, ground N. sativa seeds.
[00037] FIG. 6 depicts a schematic representation subject disposition in the clinical trial of N. sativa EO1.0% and HCA capsule
[00038] FIG. 7 depicts the synergist action of EO and HCA operating on separate AMPK activation pathways.
[00039] FIG. 8 depicts a schematic of single versus multiple targets in the mechanisms underlying additive or agonistic effects and supra-additive or synergistic effects, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[00040] The compositions of the invention include unique compositions comprising supercritical CO2 extracted EO from whole, ground N. sativa seeds, which exhibit an entourage effect, and a chemical, phytochemical or plant extract capable of activating AMPK independently of uncoupling mitochondrial membrane potential. 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 for their antioxidant or anti-inflammatory properties. The resulting compositions can be consumed as a capsule, 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, exercise endurance or other inflammatory-based pathologies.
[00041] 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.
[00042] 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).
[00043] As used herein, the following abbreviations are defined as: BMI: Body Mass Index; DBP: Diastolic blood pressure; eAG: estimated average glucose; FBG: Fasting blood glucose; HCA: hydroxycitric acid; HDL: High Density Lipoprotein; ICO: Index of central obesity; LDL: Low Density Lipoprotein; MAP: Mean arterial pressure; ND: Not determined; PP: Pulse pressure; PPG: Postprandial glucose; SBP: Systolic blood pressure; TC: Total cholesterol; TG: Triglycerides; TQ: thymoquinone.
[00044] 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.”
[00045] 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%.
[00046] 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.
[00047] 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.
[00048] 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.
[00049] 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.
[00050] The term “phytoceutical” or its equivalent “nutraceutical” as well as verbal variants refer to any plant, combinations of plants, or microbiological material that is consumed for beneficial effects on metabolic functioning in a subject.
[00051] As used herein, "synergistic" means more than the additive effect of the individual components against a mechanism of action. For example, if F1 produces response X, F2 produces response Y, then the combination of F1+F2>X+Y. In some situations, F2 produces no response and the value for Y is equal to zero.
[00052] 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.
[00053] 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.
[00054] As used herein “thermogenesis” represents the process of heat production in organisms. It occurs in all warm-blooded animals. Depending on whether or not they are initiated through locomotion and intentional movement of the muscles, thermogenic processes can be classified as one of the following: (1) exercise-associated thermogenesis (EAT); (2) Non-exercise activity thermogenesis (NEAT), energy expended for everything that is not sleeping, eating or sports-like exercise; and (3) diet- or drug-induced thermogenesis (DIT).
[00055] 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.
[00056] As used herein, "AMPK-related diseases" includes pathologic or pathognomic conditions in which the activation of AMPK provides a salutary effect. Examples of such diseases or conditions include obesity, diabetes, metabolic syndrome, hepatic steatosis, hyperlipidemia, acute inflammatory lung injury, heart disease, reperfusion ischemia, cancer, aging, retinal degeneration, cardiac hypertrophy, non-alcoholic fatty liver disease, hypertension, albuminuria, sporadic Alzheimer's disease, muscular dystrophy, and osteoarthritis.
[00057] 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.
[00058] 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.
[00059] 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.
[00060] 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.
[00061] 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, supercritical CO2, liquid N2, propane, or any combinations of such materials.
[00062] 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. N. sativa essential oil extraction is executed at two steps (Figure 5). At step 203, the essential oil fraction (EO203) is extracted after about 30 min of subjecting the whole, ground N. sativa seeds to about 140 bar pressure and about 50 degree Celsius. Further, at step 205 a second essential oil fraction is extracted (EO205) after about another 120 min of subjecting the whole, ground N. sativa seeds to about 140 bar pressure and about 50 degree Celsius. Hence, the essential oil extraction is executed for a total of about 150 min at about 140 bar pressure and about 50 degree Celsius. At stage two, the whole, ground N. sativa seeds resulting after the extraction of the essential oil in stage one are subjected to about 300 bar pressure and about 60 degree Celsius for about another 180 minutes. Therefore, at step 207 the oleoresin fraction may be obtained at the end of about 230 min (OR207). Once, the essential oil fractions and the oleoresin factions are extracted, defatted N. sativa seeds are obtained at step 209.
[00063] 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.
[00064] 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.
[00065] 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.
[00066] 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.
[00067] 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.
[00068] 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.
[00069] 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.
[00070] As used herein, “entourage effect” is used to describe the combined effect of the terpenoids found in N. sativa essential oil (Figure 1) working together as a whole to produce a greater effect, both qualitatively and quantitatively, than if working separately. Thus, N. sativa essential oils exhibiting qualitatively different terpenoid ratios will produce unique, individualized biological effects. The instant application enables this entourage effect through supercritical CO2 separation of the essential oil of N. sativa into two novel components, EO203 and EO205, which in several examples demonstrate biological activity in excess of its TQ content alone (entourage effect).
[00071] 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.
[00072] 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.
[00073] 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.
[00074] 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.
[00075] 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.
[00076] 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.
[00077] 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.
[00078] 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.
[00079] 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.
[00080] N. sativa seeds contain 36-38% fixed oils, 5% 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 terpenoid components (Figure 1) of the essential oil include thymoquinone (0-57%), p-cymene (7.1-15.5%), carvacrol (5.8-12%), trans-anethole (0.25-38%) 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.
[00081] 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 obesity, metabolic syndrome, diabetes, hepatic steatosis, antioxidant, anti-inflammatory and analgesic actions, anticarcinogenic activity, hypotensive, 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].
[00082] 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, time, and reformulation as described herein. Further, biological activities of extracts in these and other studies with organic solvent extraction were roughly equal to their TQ content with no entourage effect; 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]. As demonstrated in examples, EO extracts obtained as described in Example 1 exhibit potency substantially greater than their TQ content (entourage effect).
[00083] 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 CO2 extraction herein refers to extraction of commercial-scale quantities of N. sativa powdered seed.
[00084] 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 N. sativa EO extracts produced through a sequential series of two lipid extractions (Figure 5) exhibit an entourage effect wherein unexpected in vitro and clinical potency greater than their TQ content is observed. Additionally, the present application teaches that when commercial volumes of N. sativa seeds are processed through the sequential extraction steps described (Figure 5), the resulting essential oils thus produced are capable of synergistically enhancing the effects of other phytoceuticals relating to obesity, metabolic syndrome, T2D, and hepatic steatosis.
[00085] In some example embodiments, N. sativa essential oil extraction is executed at two steps. At step 203, the essential oil fraction (EO203) is extracted after about 30 min of subjecting the whole, ground N. sativa seeds to about 140 bar pressure and about 50 degree Celsius. Further, at step 205 a second essential oil fraction is extracted (EO205) after about another 120 min of subjecting the whole, ground N. sativa seeds to about 140 bar pressure and about 50 degree Celsius. Hence, the essential oil extractions are executed for a total of about 150 min at about 140 bar pressure and about 50 degree Celsius.
[00086] At stage two (207), the whole, ground N. sativa seeds resulting after the extraction of all essential oil in stage one are subjected to about 300 bar pressure and about 60 degree Celsius for about another 180 minutes. Thus, at step 207 the oleoresin fraction may be obtained at the end of about 230 min (OR207). Once, the essential oil fractions and the oleoresin factions are extracted defatted N. sativa seeds are obtained at step 209. Lipid extraction may also be explained in later part of the disclosure.
[00087] 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.
[00088] 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.
[00089] 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.
[00090] 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.
[00091] 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.
[00092] 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.
[00093] Manufacture of the Preferred Embodiments
[00094] 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, and 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.
[00095] In a particularly preferred embodiment, the medical food or nutritional supplement will be formulated into a capsule, 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 a 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.
[00096] 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 mixture 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., N. sativa EO and HCA) 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
[00097] The preferred embodiments contemplate treatment of disorders related to obesity, metabolic syndrome, all forms of diabetes, hepatic steatosis, 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.
[00098] 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 supercritical CO2-extracted N. sativa EO and HCA). 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.
[00099] The preferred embodiments provide compositions and methods to promote normal, adipocyte, myocyte or hepatic functioning relating to subjects exhibiting obesity, T2D, and hepatic steatosis or their co-morbidities. All preferred embodiments provide varying amounts of supercritical CO2, extracted essential oil of N. sativa seeds containing about 1.0% TQ and a second component capable of activating AMPK independently of mitochondrial uncoupling (e.g. HCA).
[000100] Generally, the formulations comprise supercritical CO2 extracted EO from N. sativa seeds and another herb or phytochemical capable of activating AMPK independently of mitochondrial uncoupling. In one embodiment, the composition comprises a supercritical CO2 extracted EO of N. sativa seeds produced through sequential extractions at about 140 bar, about 50°C (EO203) and about 30 minutes followed by a second EO extraction at about 300 bar, about 60°C for about 180 minutes (EO205) combined to produce an TQ content of about 1% and another herb or phytochemical capable of activating AMPK independently of mitochondrial uncoupling.
[000101] In another embodiment, the composition comprises a supercritical CO2 extracted EO of N. sativa seeds produced through sequential extractions at about 140 bar, about 50°C and about 30 minutes (EO203) followed by a second EO extraction at about 300 bar, about 60°C for about 180 minutes (EO205) combined to produce an TQ content of about 1% and HCA capable of activating AMPK independently of mitochondrial uncoupling.
EXAMPLES
Example 1
Supercritical CO2 Extraction of Thymoquinone Fractions of Nigella sativa
[000102] Objective – The objective of this experiment was to produce three supercritical CO2 extracts of N. sativa obtained under various conditions of pressure, temperature and time for future studies of biological activity. As TQ is widely considered the putative active compound in N. sativa essential oil, the TQ content of each of the fractions was determined to compare the relative activity of the supercritical CO2 fractions produced in this example with each other and with prior art.
[000103] 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 through-out the year. The cost generally fluctuates between $2.5/kg to $5.0/kg. The absolute TQ content of whole seeds may vary with region, yearly weather conditions, time of harvest, and length of storage. Therefore, all TQ concentrations in this example for each fraction should be considered relative rather than absolute.
[000104] Grinding and sieving – The N. sativa seeds were ground to a fine powder between 20 – 30 mesh. A magnetic screening system was then used to ensure removal of metallic impurities, particularly iron.
[000105] Supercritical fluid extraction – In this example, the process conditions during the extraction of N. saliva seeds with supercritical CO2 extraction were varied with respect to pressure, temperature and time (Table 2). The extraction was performed in a polyvalent pilot plant extraction set-up as known in the art. Liquid CO2 entering the apparatus was cooled in a condenser before it was pressurized and passed into the system. The flow rate was adjusted manually before the process. During the extraction, the temperatures of the extractor, CO2, and separators were automatically regulated through the recirculation of thermostatic water from three individually regulated water baths.
[000106] The ground N. sativa seeds were loaded into a cylindrical container that was equipped with steel mesh filters on both ends, thus enabling CO2 to pass the cylinder without transport of solids to the exterior. After prepressurization of the total system and the regulation of the CO2 flow rate, the extractor was depressurized and the cylinder was subsequently placed inside the extractor, after which the complete CO2 flow was redirected toward the extractor. The temperature/pressure combinations of the separator vessels were controlled individually. The extraction was stopped by redirecting the CO2 flow again to recirculation over the condenser. The solid residue was removed from the extractor after stepwise depressurization of the entire system. Subsequently, separator vessels were rinsed with hexane, and extracts were collected in UV-opaque bottles to prevent UV-activated degradation of the extract. Pressure/temperature combinations and extraction times for the extracts obtained (EO203, EO205, and OR207,) are presented in Table 2.
[000107] Thymoquinone concentration – TQ content of the various fractions obtained were 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 – Four fractions EO203, EO205, OR207, and Defatted Seeds209 were collected during the three-hour extraction process. Fraction EO203, which was collected over about 30 minutes at about 140 bar and about 50°C in Sep 1, had a TQ content of 2.24%. EO205, which was collected over about the next 120 minutes at about 140 bar and about 50°C, contained 39.3% TQ. Increasing the pressure to about 300 bar and temperature to about 60°C over about the next 180 minutes produced fraction OR207 with a TQ content of 0.24%. The resulting TQ concentration of Defatted seeds209 was 0.01% (Table 2). The extraction protocol is represented schematically in Figure 5.
Table 2
Thymoquinone Content of Supercritical Carbon Dioxide Extracts of N. sativa Seed Powder Under Various Extraction Conditions of Pressure, Temperature and Time

Sample Process conditions
Thymoquinone
(%w/w)
Pressure
(Bar) Temperature
(°C) Time
(min)
EO203 140 50 30 2.24
EO205 140 50 120 39.3
OR207 300 60 180 0.24
Defatted seeds209 - - - 0.01
[000109] As commercial-scaled, supercritical CO2 extractions of N. sativa under the conditions described, represent novel compositions of multiple, bioactive constituents, an in vitro and clinical screening program was developed to assess their potential health benefits and safety in various ratios and in combination with other phytochemicals. Results of the screening of these novel, extracts and combinations are presented in the following examples.
Example 2
Select Supercritical Fluid Extracts of Nigella sativa are Potent Uncouplers of Mitochondrial Membrane Potential in 3T3-L1 Adipocytes
[000110] Objective - The objective of this experiment was to determine the extent supercritical CO2 fluid extracts of N. sativa directly reduce mitochondria membrane potential (m) in 3T3-L1 adipocytes compared to pure TQ or DNP.
[000111] The model- The 3T3-L1 murine fibroblast is 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.
[000112] Chemicals – 2,4-Dinitrophenol and all other chemicals 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 samples used in this study were those described in Example 1.
[000113] 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.
[000114] 3T3-T1 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. 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.
[000115] Treatment with 2,4-Dinitrophenol and Test Material - On Day 6 post differentiation, DNP or test materials TQS (Sigma), EO203, EO205, and OR207 were dissolved in DMSO and added to the culture medium to achieve concentrations of 100 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 was 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 were run concurrently with each experiment. A Packard Fluorocount spectrofluorometer (Model#BF10000, Meridan, CT) set at 560 nm excitation and 590 nm emission was used for quantification of aggregate fluorescence and at 485 nm excitation/530 emission for monomer fluorescence.
[000116] 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.
[000117] Calculation of relative decrease in mitochondrial membrane potential – Aggregate and monomer fluorescence was computed for the 100 µM DNP positive control as well as the test materials TQS, EO203, EO205, and OR207 relative to JC-1 negative controls. The ratio of the monomer to aggregate relative fluorescence was then determined as a measure of relative decrease in m. For statistical comparisons, differences between treatments greater than or equal to 20% were considered statistically different.
Table 3
Mitochondrial Membrane Uncoupling in 3T3-L1 Adipocytes
Test Material [µg/mL] Relative Monomer/Aggregate Ratio
JC-1 DMSO Control -- 1.0a
DNP (100 µM) 18 1.3b
TQS (100% TQ) 25 1.5b
EO203 (2.24% TQ) 25 3.7c
EO205 (39.3% TQ) 25 3.9c
OR207 (0.24% TQ) 25 4.1c
(a) Uncommon superscripts indicate significantly different treatments with difference in relative monomer/aggregate ratio ≥ 20%.
[000118] Results – All test materials exhibited mitochondrial membrane uncoupling potential relative to the DMSO control. Pure TQ at 25 µg/mL decreased m in 3T3-L1 adipocytes to a similar extent as the DNP positive control at 18 µg/mL. The N. sativa extracts EO203, EO205, and OR207, however, decreased mitochondrial membrane potential 3.7- to 4.1-fold relative to solvent controls. Moreover, this mitochondrial membrane uncoupling potential was in excess of the TQ content of each fraction, exhibiting a strong entourage effect among the constitute components. This example demonstrates that the supercritical CO2 extracts of N. sativa possess biological activity unexplained by TQ content alone, rather this activity exemplifies an entourage effect of the specific amounts and ratios of the component terpenoids (Figure 1) in each novel fraction.
Example 3
Hydroxycitric Acid Does Not Uncouple Mitochondrial Membrane Potential in 3T3-L1 Adipocytes
[000119] Objective - The objective of this experiment is to assess the ability of hydroxycitric acid (HCA) to exhibit mitochondrial membrane uncoupling potential.
[000120] Chemicals – HCA, 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.
[000121] Methodology - Methods for assessing the ability of test materials to uncouple mitochondrial membrane potential in 3T3-L1 adipocytes are performed as described in Example 2.
[000122] Results – HCA does not exhibit any increase in uncoupling mitochondrial potential over the DMSO control.
[000123] Conclusion – Unlike supercritical CO2 extracts of N. sativa described in Example 2, HCA is not capable of uncoupling mitochondrial oxidative phosphorylation in 3T3-L1 adipocytes.
Example 4
Supercritical CO2 Extracts of N. sativa Activate AMPK in Myocytes
[000124] Objective - The objective of this experiment was to compare the effect of the AMP mimetic AICAR on AMPK activation to in C2C12 myocytes with the commercial-scale supercritical CO2 N. sativa extracts produced in Example 1.
[000125] The Model- The C2C12 myocyte model was used in this example.
[000126] Chemicals – Penicillin, streptomycin, Dulbecco's modified Eagle's medium (DMEM) was from Mediatech (Herndon, VA) and 10% FBS-HI (fetal bovine serum-heat inactivated) was obtained from Mediatech and Hyclone (Logan, UT). The commercial supercritical CO2 extracts of N. sativa produced in Example 1 were used as the test materials. Unless noted, all other standard reagents were purchased from Sigma (St. Louis, MO).
[000127] Cell culture - Mouse C2C12 myoblasts were obtained from American Type Culture Collection (Manassas, VA), and were 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.
[000128] C2C12 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 myocytes by culturing in DMEM supplemented with 2% horse serum for seven days.
[000129] Treatment - On Day 8 to 10 post differentiation, C2C12 myocytes were incubated in serum-free DMEM plus 0.5% BSA (bovine serum albumin) for three hours. Next, AICAR (Cell Signal, Danvers, MA) was dissolved in phosphate buffered saline (PBS) and added to the culture medium to achieve concentrations of 1 mM. Test materials EO203, EO205 and EO207 were 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 were prepared for determination of activated AMPK (pT172-AMPK).
[000130] Measuring activated AMPKa - pT172-AMPK was quantified using the Biosource AMPKa Immunoassay Kit (Camarillo, CA) without modification. Protein content of the cell lysates was 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) was used for protein determination and a MEL312e BIO-KINETICS READER (Bio-Tek Instruments, Winooski, VT) was used for quantification of pT172-AMPK.
[000131] Calculation of relative activation of AMPK - pT172-AMPK was computed per mg lysate protein and then normalized to the dimethyl sulfoxide (DMSO) negative controls. For statistical comparisons, 95% confidence intervals were computed (Excel, Microsoft, Redman, WA).
Table 4
Activation of AMPK in 3T3-L1 Adipocytes
Test Material [µg/mL] Relative pT172-AMPK
DMSO -- 1.00 (0.92 – 1.08)
AICAR (1 mM) 338 1.45 (1.37 – 1.53)
EO203 (2.24% TQ) 25 1.27 (1.19 – 1.35)
EO205 (39.3% TQ) 25 1.66 (1.58 – 1.74)
OR207 (0.24% TQ) 25 1.05 (0.97 – 1.13)

[000132] Results – Over ten independent assays, 1 mM AICAR increased 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, EO203 and EO205 fractions activated myocyte AMPK, while OR207 had no effect (Table 4). The AICAR positive control induced a 1.45-fold activation of AMPK in assays run with N. sativa supercritical CO2 extracts. AMPK activation by EO203 was similar to AICAR, while EO205 activation exceeded AICAR activation. Based on TQ content, AMPK activation was disproportionately greater in EO203 than EO205, thereby supporting the putative entourage effect seen in essential oils and oleoresins produced through the novel, supercritical CO2 extraction process described in Example 1.
Example 5
Hydroxycitric Acid Activates AMPK in C2C12 Myocytes
[000133] Objective – The objective of this Example is to follow-up the results of Example 4 and assess the effect of HCA on AMPK activation in C2C12 myocytes.
[000134] The Model- The C2C12 murine myocyte model as described in Example 4 is used in this example.
[000135] Chemicals, Cell Culture and Treatment – Chemicals, cell culture procedures, methods and statistical procedures used are as noted in Example 4.
[000136] Test Materials – Hydroxycitric acid (Sigma, St. Louis, MO) is used as the test material and dosed at 25 µg/mL. The concentration for the positive control AICAR was 1.0 mM (338 µg/mL).
[000137] Results – HCA at 25 µg/mL increases pT172-AMPK 1.54-fold relative the DMSO solvent control and greater than the AICAR positive control. The lack of mitochondrial membrane uncoupling by HCA in conjunction with AMPK activation implies a mechanism of action of HCA through CaMKKb (Figure 4B).
Example 6
Synergistic Activation of Myocyte AMPK by Hydroxycitric Acid and Supercritical CO2 Extracts of N. sativa Combinations
[000138] Objective – The objectives of this Example are to follow-up the results of Examples 4 and 5 to test the hypothesis that substances activating AMPK through differing pathways could act synergistically and assess the effect of HCA on AMPK activation in C2C12 myocytes. As shown in Example 2, the likely pathway for EO203, EO205 and OR207 AMPK activation is through decreasing ATP production and increasing the ATP/AMP ratio (Figure 3B). Moreover, HCA is not capable of decreasing mitochondrial membrane potential and would necessarily activate AMPK through a different pathway, most likely the CaMKKb pathway (Figure 4B).
[000139] Methodology - AMPK activation by HCA, and various combinations of OE203, OE205, and OR207 and C2C12 myocytes is performed as described in Examples 4 and 5. Combinations are tested in the following ratios: OE203:HCA [10:1], OE203:HCA [5:1], OE203:HCA[1:5], OE203:HCA [1:10], OE205:HCA [10:1], OE205:HCA [5:1], OE205:HCA[1:5], OE205:HCA [1:10], OR207:HCA [10:1], OR207:HCA [5:1], OR207:HCA[1:5], and OR207:HCA [1:10]
[000140] This skeletal muscle cell line represents a model target cell for favorable metabolic changes in obesity with AMPK activation including increased fatty acid oxidation, glucose uptake, expression of GLUT4, and indirect mitochondrial uncoupling (Figure 5).
[000141] Synergy determination - 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, can be 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.
[000142] Expected relative activation (RA) of AMPK of any multi-component combination is estimated using the relationship:
[000143] [1/Expected RA] = [Fa/RAA] + [Fb/RAB] + … + [Fn/RAN] = 1.00, where Fa = fraction of component A in the combination, Fb = fraction of component B in the combination, Fn = the fraction of the nth component combination, RAA = the observed RA of the component A, RAB = the observed RA of the component B, and RAN = the observed RA of the Nth component.
[000144] The CI is then calculated thusly, CI = Expected [RA]/Observed [RA]. 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. Significance of synergy is inferred when the CI is ≥ 1.20.
[000145] Results - Relative activation of AMPK by OE203, OE205, OR207, and HCA independently are, respectively, 1.27, 1.66, 1.05, and 1.54. Combinations of supercritical CO2 extracts of N. sativa and HCS consisting of 10:1, 5:1, 1:5, and 1:10 ratios, respectively, exhibited synergy in the activation of AMPK when tested at a combined total of 25 µg/mL (Table 5). This conclusion is based on the ability of the mixtures to activate AMPK more than 20% of the expected result estimated from their relative weight in the combination.
Table 5
Expected and Observed Relative Activation of AMPK in C2C12 myocytes by Combinations of Supercritical CO2 fractions of N. sativa and Hydroxycitric Acid.

Test Material† Relative pT172-AMPK
Expected Observed Obs/Exp
HCA (100% Sigma) 1.54 1.54 1.00
OE203 1.27 1.27 1.00
OE203:HCA - [10:1] 1.29 1.67 1.29*
OE203:HCA - [5:1] 1.31 1.70 1.30*
OE203:HCA - [1:5] 1.48 2.11 1.43*
OE203:HCA - [1:10] 1.51 2.08 1.38*
OE205 1.66 1.66 1.00
OE205:HCA - [10:1] 1.65 2.37 1.44*
OE205:HCA - [5:1] 1.64 2.22 1.35*
OE205:HCA - [1:5] 1.55 1.95 1.26*
OE205:HCA - [1:10] 1.55 2.39 1.54*
OR207 1.05 1.05 1.00
OR207:HCA - [10:1] 1.08 1.88 1.74*
OR207:HCA - [5:1] 1.11 1.46 1.32*
OR207:HCA - [1:5] 1.42 1.98 1.39*
OR207:HCA - [1:10] 1.48 1.77 1.20*
† All test materials and combinations are tested at 25 µg/mL.
* Indicates synergy of combination with Obs/Ex = CI ≥ 1.20
[000146] These synergy results provide support for the hypothesis that compounds activating AMPK through differing pathways, (e.g. mitochondrial membrane uncoupling and the CaMKKb pathway) act synergistically (Figure 7). The generalized form of this result can be found in Figure 8, which provides a schematic illustration of single versus multiple targets in the mechanisms underlying additive or agonistic effects and supra-additive or synergistic effects, respectively.
Example 7
Supercritical CO2 Fluid Extracts of Nigella sativa Increase Lypolysis in 3T3-L1 Adipocytes
[000147] Objective – The objective of this experiment was to determine whether the supercritical fluid extracts of N. sativa obtained in Example 1 induce lipolysis in adipocytes.
[000148] 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. The N. sativa samples used in this study were those described in Example 1.
[000149] Cell culture and treatment – Culture and treatment of 3T3-L1 adipocytes was as described in Example 2. Concentrations of the forskolin positive control and test materials are listed in Table 6.
[000150] Glycerol assay - Free fatty acid release (lipolysis) from 3T3-L1 adipocytes was quantified by measuring glycerol secretion into the medium. Glycerol was measured spectrophotometrically using the Free Glycerol Determination Kit (F6428, Sigma) and an EL 312e Microplate BIO-KINETICS spectrophotometer (BioTek, Winooski, VT).
[000151] Data analysis – Glycerol release from adipocytes and myocytes was expressed as relative glycerol content (glycerol index) ± 95% confidence intervals of the DMSO controls of eight observations.
Table 6
Induction of Lipid Release from 3T3-L1 Adipocytes by Supercritical CO2 Essential Oil fractions of N. sativa
Test Material [µg/mL] Glycerol Index (95% CI N=8))
DMSO -- 1.00a (97 – 103)
Forskolin 82 3.34c
Thymoquinone (100%) 5 1.02a
EO203 (2.24% TQ) 10 1.50b
EO205 (39.3% TQ) 10 1.51b
abcUncommon super scripts indicate significant differences among treatments.
[000152] Results – The forskolin positive control and both supercritical CO2 essential oil extracts induced free fatty acid release in adipocytes. Specifically, EO203 and EO205 at 10 µg/mL induced similar free fatty acid secretion from 3T3-L1 adipocytes, while pure TQ at 5 µg/mL failed to significantly induce lipolysis in 3T3-L1 adipocytes. As seen in previous examples, EO203 and EO205 lipolydic activity was out of proportion to their TQ content, a further example of the entourage effect of these preparations.
Example 8
[000153] A Formulation of Supercritical CO2-Extracted Nigella sativa Essential Oil and Aqueous Garcinia Extract Safely Improved Body Weights, Waist Circumference, Blood Lipids and Fatty Liver in Obese Subjects: A Single-Arm, Open Label, Twelve-week Safety and Efficacy Study
[000154] OBJECTIVE – The primary end point of this study was to evaluate the effects of a 12-week administration of capsules containing a supercritical CO2 essential oil extract of N. sativa containing 2 mg TQ and an aqueous extract of Garcinia spp containing 120 mg hydroxycitric acid (Table XX) on body weight, body mass index (BMI), waist and hip circumference, and waist-hip ratio and lipid profile: total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) and triglyceride (TG) in obese subjects. .
[000155] STUDY DESIGN - The study was a single-arm, open label, twelve-week safety and efficacy study of capsules containing a supercritical CO2 essential oil extract of N. sativa containing 2 mg TQ and an aqueous extract of Garcinia spp containing 120 mg hydroxycitric acid in obese subjects for 12 weeks.
Table 7
Composition of N. sativa/Garcicumin Test Material.
Ingredients g/100 mg/Capsule Mg/Day
Nigella sativa extract *(TQ oil, CO2 extracted) 40 200 400
Thymoquinone 0.40 2.0 4.0
Garcinia extract** (Supreem code:8006) 40 200 400
Hydroxycitric acid 24 120 240
Colloidal silicon dioxide (Aerosil/Cabot) 15.2 76 152
Sodium lauryl sulfate 0.96 4.8 96
Talcum 1.84 9.2 18.4
Magnesium stearate 1.0 5.0 10
Polyvinylpyrrolidone 1.0 5.0 10
Isopropyl alcohol Q.S. Q.S. -
Q.S. = as much as is sufficient.
* Supercritical CO2 extracted, mixed EO fractions were used to adjust TQ content to about 1% minimum.
**Garcinia extract: potassium, calcium salt, water soluble form with HCA assay minimum about 60%. Potassium content: Not less than 9%; Calcium content: Not less than 5%
[000156] Study plan - Patients were initially screened to determine suitability. Following the screening visit, patients, who meet all of the inclusion criteria and none of the exclusion criteria, were assigned to receive 12 weeks of 1 capsule bid treatment with the test material. Visits were scheduled for Screening, Day-0, end of week 4, end of week 8, and end of week 12 after enrolment visit to assess safety, efficacy and tolerability.
[000157] Treatment period – Eligible patients were asked to return to the clinic for follow up on Day - 0 (Visit 1), week – 4 (Visit 2), week – 8 (Visit 3) and week – 12 (Visit 4) for clinical assessment. Eligible patients were given test material at Baseline Visit 1. At all these visits, subjects were assessed for treatment efficacy and adverse events. The following information will be collected during this visit: physical measurements including weight, height, waist and hip circumference, blood lipids, any adverse events, and compliance.
[000158] SUBJECTS – Goal of 25 subjects completing the study was set.
Patient Selection Criteria
Inclusion criteria
Subjects meeting the following criteria were recruited for the trial: (1) Adult patients between the age of 20 and 65 years with BMI > 30 kg/m2 ; (2) Patients with waist circumference for males > 102 cms and females > 88 cm; (3) Serum triglyceride > 150 mg %, Serum high density lipoprotein (HDL) < 50 mg%; (4) Patients ready to gibe written informed consent for study participation.
Exclusion criteria
Subjects not meeting the following criteria will be included in the trial: (1) Patients with known allergy to study medications; (2) Patients with impaired liver function test i.e. Elevated Aspartate-aminotransferase (AST) or Alanine-aminotransferase (ALT) > 3x Upper Limit of Normal (ULN) or known liver disease expected to have an impact o survival; (3) Patients with renal impairment (Sr. creatinine > 2.0 mg/dl); (4) Pregnancy or lactation; (5) Patients with Type I diabetes mellitus; (6) Patients considered unsuitable for inclusion
[000159] STUDY ASSESSMENT
Evaluation variables
Primary Efficacy Criteria:
1. Body Weight and Body Mass Index
a. Body weight will be measure in Kg and Body Mass Index (BMI) will be calculated as Kg/m2 where weight is in Kg and height in meters.
2. Waist circumference in cms/Hip circumference in cms
a. Waist & Hip circumference measurement technique:
i. Participant is asked to remove clothing, except for light underwear. Tight clothing, including belts, should be loosened and the pockets emptied.
ii. Participant should stand with feet fairly close together (about 12-15 cm apart) with weight equally distributed on each leg
iii. Participant is asked to breathe normally; the reading of the measurement should be taken at the end of gentle exhaling.
b. Waist circumference measurement
i. Place measuring tape, holding parallel to floor, around abdomen at the level of iliac crest. Hold tape snug but do not compress the skin.
c. Hip circumference measurement
i. Tape should be paced around the point with the maximum circumference over the buttocks. The measuring tape is held firmly, ensuring its horizontal position. When recording, you need to make sure the tape is not too tight or too loose, is lying flat and is horizontal. It may help to have the subject stand on a box to make the measurement easier.
d. Waist-hip ratio
i. Calculated from above measurements
3. Lipid profile: total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) and triglyceride (TG).
[000160] Safety Parameters - All adverse events regardless of treat suspected causal relationship to study drug will be recorded on the adverse event form. Serious adverse events must be reported to sponsor within 24 hours.
[000161] STUDY CONDUCT: Drug administration: At Visit 1, subjects received test material capsules as described in Table XX to be taken orally twice daily for 12 weeks. Subjects were asked to return to the study centre for follow up on weeks 4 (Visit 2), 8 (Visit 3), and 12 (Visit 8) for clinical assessment.
[000162] Drug storage: Test material capsules were Stored at a temperature not exceeding 30°C and Protected from light and moisture.
[000163] Duration of treatment: Test material capsules were taken for 12 weeks.
[000164] Study type: This was an open label, single-arm, clinical study therefore investigator as well as subjects were aware of the investigational product allocated to individual subjects.
[000165] Anthropometric variables: The following anthropometric variables were recorded at the screening visit (V1): age, sex, weight, height, body mass index, waist and hip circumference, and waist-hip ratio.
[000166] Medical history: A relevant medical was recorded at the screening visit (V1). This included all current conditions by diagnosis (where possible) and any concomitant medication, together with significant past conditions, operation and therapeutic or diagnostic procedures. Daily food intake and type of food, addictive habits (like alcohol consumption), sleep, occupation, daily physical exercise etc. were also recorded.
[000167] Physical examination: A physical examination was performed at the screening visit (V1) and visit (V8) as follows: general appearance, respiratory, skin, eyes ears, nose, throat, heart, abdomen, reflexes, lymph nodes and extremities, body temperature, hear rate, respiratory rate, blood pressure, and 12-lead electrocardiogram.
[000168] Laboratory examination: The safety variables included hematology, biochemistry, urinalysis, complete blood count, kidney function, aminotransferases, alkaline phosphatase, and lipid profiles were performed by a central laboratory at the time of screening and at the completion of the study.
[000169] Early discontinuation of individual patients: Patients were informed that they have the right to withdraw from the study at any time without giving a reason. In addition, the investigator had the right remove a patient from the study if, in the investigator’s opinion, it was not in the best medical interest of the patients to continue in the study.
[000170] Patients were considered for removal from the study for the following reasons: (1) Protocol violation by the patient, which may result in improper conclusion; (2) At the discretion of the investigator; (3) Lack of efficacy; and (4) Adverse Events.
[000171] For discontinuation of a patient, the visit V8 assessments were performed as far as possible, at the earliest opportunity. Alternative therapy was to be provided to the subject upon discontinuation from the study. The date of discontinuation from the study and reason for discontinuation were recorded.
[000172] Criteria for removal from study: Following were the criteria that was utilized by the subject, attending physician, or Principal Investigator to remove a subject from study: (1) Disease progression/recurrence; (2) Intercurrent illness that prevents further administration of treatment; (3) Unacceptable adverse event(s); (4) Patient’s decision to withdraw from study; (5) Change in the patient’s condition rendering the patient unacceptable for further treatment in the judgement of the investigator.
[000173] Protocol amendments: (1) Any amendment had to be agreed upon between the investigator and sponsor in the form of a written amendment. Any changes were signed and dated by the Principal Investigator and the sponsor and attached to the original protocol; and (2) All amendments were notified to local ethical committee.
[000174] Ethics - The clinical protocol was approved by the Ethical Committee of the E Ashirrwad Hospital and Research Centre, Section 32, Maratha Section, Near Jijamata Udyan, Udyan, Ulhasnagar, 421004, Maharashta. 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.
RESULTS
[000175] In this single centre, prospective, single arm clinical study, adult obese patients with BMI > 30 kg/m2, waist circumference for male > 102 cm and females > 88 cm, serum triglyceride > 150 mg/dL and serum high density lipoprotein (HDL) < 50 mg/dL were considered for the study.
[000176] Forty-four patients were screened for study eligibility. A total of 27 adults were enrolled and treated with capsules containing N. sativa seed extract containing about 4.0 mg TQ and about 120 mg hydroxycitric acid as an extract of Garcinia spp. one capsule two times a day (one capsule in morning and one capsule in evening) for 12 weeks (Figure 6). Two subjects were lost to the study for failure to return after the initial visit. Mean patient age was 50.0 ± 9.91 years for 19 males (70.4%) and 8 females (29.6%). Mean body weight at baseline was 86.1 ± 9.86 kg, BMI was 31.7 ± 1.05 kg/m2,
[000177] Body weight, body mass index (BMI), waist and hip circumference, and waist-hip ratio and lipid profile: total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL) and triglyceride (TG) were evaluated at week – 4, week – 8 & week – 12 after enrolment visit to assess safety, efficacy and tolerability.
[000178] Significant improvement (p < 0.001) was observed in all the efficacy parameters of patients at weeks 4, 8, and 12 as compared with baseline with a trend of decreasing weight each visit. At weeks 4, 8, and 12, mean body weight was reduced -1.62 + 0.91 kg, -2.81 + 1.31 kg, and -3.48 + 1.70 kg, respectively. At weeks 4, 8, and 12, BMI was reduced -0.59 + 0.34 kg/m2, -1.02 + 0.46 kg/m2, and -1.28 + 0.59 kg/m2, respectively. At week 4, mean waist circumference was reduced by -0.57 + 0.46 cm, which further reduced to -1.04 + 0.64 cm at week 8, and -1.63 + 0.81 at week 12 as compared to baseline. At weeks 4, 8, and 12, hip circumference was reduced -0.14 + 0.25 cm, -0.33 + 0.26 cm, and -0.60 + 0.35 cm, respectively. Waist-hip ratio was reduced by -0.004 ± 0.005, which further reduced to -0.007 ± 0.007 at week 8, and -0.01 ± 0.006 at week 12 relative to baseline.
[000179] All lipidic variables also demonstrated significant improvements (p < 0.001) in all subjects at each week as compared with baseline with a trend of decreasing each visit. At weeks 4, 8, and 12, TC was reduced -3.50 + 2.41 mg/dL, -7.25 + 3.96 mg/dL, and -11.82 + 5.57 mg/dL, respectively. At weeks 4, 8, and 12, LDL was reduced -3.60 + 2.22 mg/dL, -7.61 + 3.46 mg/dL, and -12.34 + 5.14 mg/dL, respectively. At weeks 4, 8, and 12, HDL was increased 1.13 + 0.94 mg/dL, 2.64 + 1.41 mg/dL, and 4.63 + 1.95 mg/dL, respectively. At weeks 4, 8, and 12, TG was decreased -5.15 + 4.72 mg/dL, -11.36 + 7.30 mg/dL, and -19.19 + 11.92 mg/dL, respectively.
[000180] Tolerability – A total of 4 (14.8%) subjects reported adverse reactions during the study. These included eosinophilia, diarrhoea, urinary tract infection, and cough with fever. All were mild in nature and resolved with no consequence with subjects remaining in the study.
CONCLUSIONS
[000181] The dramatic decreases in body weight and serum lipids are consistent with the AMPK activation (Examples 4,5 and 6) and in vitro lipolytic activity (Example 7) of the supercritical CO2, essential oils of Example 1 in combination with HCA. Moreover, to our knowledge, there exists no published, clinical study of N. sativa or HCA capable of reducing body weight or serum lipids at the doses of this example. In a meta-analysis of 23 RCT, only a small, statistically significant difference in weight loss favouring HCA over placebo (MD: −0.88kg; 95% CI: −1.75, −0.00) was noted [Onakpoya I, Hung SK, Perry R, Wider B, Ernst E: The Use of Garcinia Extract (Hydroxycitric Acid) as a Weight loss Supplement: A Systematic Review and Meta-Analysis of Randomised Clinical Trials. J Obes. 2011;2011:509038]. The N. sativa and HCA combination of this example produced nearly 4-tmes the weight loss in the same time period at a dose of the combination one-fourth the daily amount of HCA capable of demonstrating weight loss in the meta-analysis. Thus, with such a dramatic enhancement of the metabolic effects of these two active components, this example represents a clinical example of the entourage and synergy effect of this preparation.
Example 9
Proposed Formulations of Supercritical CO2-Extracted Nigella sativa Essential Oil and Phytoceuticals Capable of Clinically Improving Body Weights, Waist Circumference, Blood Lipids and Fatty Liver in Obese Subjects
[000182] While Example 8 only presents the combination of supercritical CO2-extracted N. sativa essential oil with HCA, it would be obvious for one skilled in the art to assume to expect the same results with combinations of supercritical CO2-extracted N. sativa essential oil and other plant materials or phytochemicals that also exhibit AMPK activation through differing targets (Figure 8). Examples of these combinations resulting from examples presented herein are presented in Table 8.
Table 8
Combinations of plants and phytochemicals with CO2-extracted N. sativa essential oil CO2-extracted N. sativa essential oil for Improving Body Weights, Waist Circumference, Blood Lipids and Fatty Liver in Obese Subjects

Ingredients Mitochondrial AMPK Lipolysis
Active Ingredient #1†
CO2-extracted N. sativa essential oil Y Y Y
Active Ingredient #2†
Cayenne Pepper - Y Y
Coleus forskohlii - - Y
Cumin - Y Y
Green coffee bean extract Y Y Y
Gymnema sylvestre - Y Y
Momordica charantia (Bitter Melon) - Y Y
Oregano (carvacrol) Y Y Y
Ribes nignum L. (Black current) - Y Y
Trachyspermum ammi L (Arq ajwain) - Y Y
Trigonella foenum-graecum (M/AMPK) Y Y Y
Vaccinium (blueberry leaf – 20% chlorogenic acid) - Y Y
Wheat germ oil - Y Y
Zingiber officinale (6-gingerol) - Y Y
Inactive Ingredients††
Colloidal silicon dioxide (Aerosil/Cabot)
Sodium lauryl sulfate
Talcum
Magnesium stearate
Polyvinylpyrrolidone
Isopropyl alcohol
†Active ingredients #1 and #2 can be formulated in ratios of 10:1 to 1:10
††Amounts in similar, final proportions to Table 7
[000183] A formulation for improving body weights, waist circumference, blood lipids and fatty liver in obese subjects is presented in Table 8. Active ingredients #1 and #2 can be formulated in ratios of 10:1 to 1:10 as a combination with the inactive ingredients added in amounts known to one skilled in the art as necessary to allow for formulating capsules, bars, medical foods, etc.
[000184] Thus, there has been disclosed synergistic combinations of novel supercritical CO2 essential oil and oleoresin extracts of N. sativa with a secondary AMK-activating phytochemical. 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.