WO2015198346A1 - Composition comprenant un extrait d'alangium salvifolium à activité anti-adipogène ou anti-obésité - Google Patents

Composition comprenant un extrait d'alangium salvifolium à activité anti-adipogène ou anti-obésité Download PDF

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WO2015198346A1
WO2015198346A1 PCT/IN2015/000261 IN2015000261W WO2015198346A1 WO 2015198346 A1 WO2015198346 A1 WO 2015198346A1 IN 2015000261 W IN2015000261 W IN 2015000261W WO 2015198346 A1 WO2015198346 A1 WO 2015198346A1
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extract
alangium
salvifolium
derived
fraction
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Ganga Raju Gokaraju
Rama Raju Gokaraju
Venkata Kanaka Ranga Raju Gokaraju
Trimurtulu Golakoti
Kiran Bhupathiraju
Krishanu Sengupta
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Laila Nutraceuticals
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/40—Cornaceae (Dogwood family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/06—Preparations for care of the skin for countering cellulitis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to herbal ingredients (extracts or purified fractions or compounds) derived from Alangium salvifolium having anti-adipogenic and pro- lipolytic activities for the purpose of control, treatment or prevention of over weight, obesity, lipid storage disease, hyperlipidemia, metabolic syndrome and other metabolic disorders.
  • the present invention further relates to the compositions comprising biologically effective amount of the extracts or purified fractions or compounds derived from Alangium salvifolium optionally containing atleast one compound selected from a bio- enhancing agent, a bioprotecting agent and biologically acceptable carriers or diluents.
  • the invention further relates to a method for treating, controlling or preventing adipogenesis mediated diseases in mammals using herbal ingredients selected from the extracts, purified fractions and the pure compounds derived from Alangium salvifolium or their compositions.
  • the invention further relates to herbal ingredients derived from Alangium salvifolium and their compositions for alleviating at least one condition selected from obesity, overweight, diabetes, atherosclerosis, arteriosclerosis, hypertension, hypercholesterolemia, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases, cardiovascular diseases, endothelial dysfunction, mitochondrial dysfunction, t metabolic syndrome and other metabolic disorders or conditions.
  • Obesity is excess body weight for a particular age, sex and height as a consequence of imbalance between energy intake and energy expenditure.
  • the primary causes of obesity are one or more of the following disorders, which include overeating and inadequate exercise, some genetic disorders, underlying illness (e.g. Hypothyroidism), certain medications, sedentary lifestyle, a high glycemic diet (i.e., a diet that consists of meals that give high post prandial blood sugar), weight cycling (caused by repeated attempts to lose weight by dieting, eating disorders), stress and insufficient sleep.
  • the obesity was not limited to developed countries, but it was rapidly becoming a problem in developing countries as well. The number of those affected, particularly children, are continuing to increase at an alarming rate. Obesity is already responsible for 2-8% of health care costs and 10-13% of deaths in different parts of Europe. Recent studies have shown that approximately a third of variance in adult body weights result from genetic influences. Leptin, an adipocyte and placenta-derived circulating protein, regulates the magnitude of fat stores in the body leading to obesity. Gastrointestinal peptides, neurotransmitters and adipose tissue may also have an etiologic role in obesity.
  • Obesity and adipose tissue expansion increase the risk of hypertension, type 2 diabetes, arthritis, elevated cholesterol, cancer and serious hormonal imbalances in women, leading to sterility.
  • Low caloric diets with or without exercise can help with temporary weight loss; however, diet and exercise alone have not proven successful for long-term solutions in weight management (H. G. Preuss, et al, Nutrition Research, 2004, 24, 45-48).
  • Obesity is the culmination of many underlying mechanisms. Obesity is characterized as uncontrolled adipose tissue mass in the body. An increase in adipose tissue can be the result of the production of new fat cells through the process of adipogenesis and/or the deposition of increased amounts of cytoplasmic triglyceride or lipid droplets per cell. In the adipogenesis process, proliferation of preadipocytes or precursor fat cells needs to be followed by the differentiation of these cells to the mature adipocyte phenotype. Increased lipid accumulation in the mature adipocyte cells is the most important feature of obesity disorder. Peroxisome Proliferator-Activator Receptor gamma (PPAR- ⁇ ) is predominantly expressed in adipocytes and is a key determination factor for adipogenesis.
  • PPAR- ⁇ Peroxisome Proliferator-Activator Receptor gamma
  • Fat is stored as triglycerides form in adipose tissue.
  • the breakdown of this fat in fat cells into glycerol and fatty acids is known as lipolysis.
  • free fatty acids are released into the bloodstream and circulate throughout the body.
  • the hormones called epinephrine, norepinephrine, glucagon and adrenocorticotropic hormone induce lipolysis.
  • Inhibition of the differentiation of pre-adipocytes into mature adipocytes leads to the reduction of new adipose tissue and reduction in the formation of fat reserves. Modulation of adipogenesis and lipolysis in humans may thus lead to reduction in the burden of obesity or overweight (excess body weight).
  • Alangium salvifolium (Alangeaceae) also called as 'Ankola' is extensively cultivated in India. It is a popular folk medicine and has been studied for its antiinflammatory, . antimicrobial, antifertility and cardiotonic activities. Traditionally, Alangium salvifolium seeds have been reported to exhibit a variety of biological activities, including anti-diabetic, anti-cancer, diuretic, anti-inflammatory, antimicrobial, laxative, and anti-epileptic activity.
  • Alangium salvifolium The phytochemical analysis of Alangium salvifolium revealed the presence of alkaloids, glycosides, terpenoids, steroids, tannins and the ethanol extracts of Alangium salvifolium seeds exhibited antidiabetic, anti-epileptic, analgesic and anti-inflammatory activities (Sharma et al., Acta Pol Pharm. 68(6), 897, 2011). Anti-nociceptive and anti-inflammatory activities of Alangium salvifolium flower extract were proven in carrageenan and formalin induced paw edema models in mice (Zahan et al., Pak J Biol Sci. 16(19), 1040, 2013).
  • the main object of the present invention is to provide pharmaceutical, nutraceutical and dietary ingredients comprising the extracts or fractions or compounds derived from Alangium salvifolium or their composition(s) for the prevention, control and treatment of obesity, overweight, metabolic syndrome or other metabolic disorders mediated by adipogenesis, lipolysis, fat or glucose metabolism.
  • Another object of the present invention is to provide an anti-adipogenic and pro- lipolytic extracts or fractions or compounds derived from Alangium salvifolium and their compositions capable of reducing body weight in overweight people, total serum cholesterol level, phospholipids, triglycerides and for treating diabetes, atherosclerosis, arteriosclerosis, hypertension, hypercholesterolemia, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases, cardiovascular diseases, endothelial dysfunction, mitochondrial dysfunction, obesity, metabolic syndrome and other metabolic disorders or conditions.
  • Yet another object of the present invention is to provide extracts or fractions or compounds derived from Alangium salvifolium and their compositions for the amelioration of biomarker proteins or molecules, whose expression/production or molecular interactions are altered in obesity, metabolic syndrome and other metabolic disorders and conditions.
  • the present invention discloses herbal anti-adipogenic and pro-lipolytic supplement comprising a biologically effective amount of an extracts or fractions or pure compounds derived from Alangium salvifolium as a stand-alone active ingredient or compositions thereof.
  • the compositions disclosed by the invention comprises at least one ingredient selected from the extracts or fractions or compounds derived from Alangium salvifolium and optionally in combination with one or more known anti- obese extracts or fractions or agents and powders, along with biologically acceptable carrier or diluents.
  • the invention discloses herbal ingredients selected from the extracts, fractions, enriched fractions or pure compounds derived from Alangium salvifolium or their compositions for the prevention, treatment and control of metabolic syndrome, obesity, overweight, diabetes, atherosclerosis, endothelial dysfunction and other metabolic disorders or conditions; and for amelioration of the production/expression of biological marker proteins associated with obesity, metabolic syndrome and other metabolic disorders which include but not limited to Peroxisome proliferator-activated receptor gamma (PPARy), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBP ), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (P3AR), Perilipin, Adiponectin, Protein
  • the anti-adipogenic and pro-lipolytic ingredients comprising extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions of the present invention is effective for inhibition, amelioration or prevention of various diseases caused by uncontrolled adipogenesis and lipolysis thereof, for example, obesity, overweight, lipid storage disease, hyperlipidemia, atherosclerosis, thrombosis, hypercholesterolemia, hypertension, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases, metabolic syndrome and other metabolic disorders or conditions and also for prevention, control and treatment of inflammatory diseases.
  • diseases caused by uncontrolled adipogenesis and lipolysis thereof for example, obesity, overweight, lipid storage disease, hyperlipidemia, atherosclerosis, thrombosis, hypercholesterolemia, hypertension, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases, metabolic syndrome and other metabolic disorders or conditions and also for prevention, control and treatment of inflammatory diseases.
  • the present invention provides the extracts or fractions or pure compounds derived from dried plant parts of Alangium salvifolium, wherein the plant part can be selected from fruits, rind, bark, tender twigs, flower, stems, leaves, seeds, trunk, aerial parts, roots and mixtures thereof.
  • the dried plant parts of Alangium salvifolium are repeatedly extracted with water or with polar or non-polar organic solvents, alone or in combination. The extracts are combined, filtered, concentrated and then subjected to purification.
  • extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions comprising the active ingredient is formulated into a solid, semi-solid or liquid dosage form suitable for oral and parenteral administration alone or in combination with one or more anti-adipogenic or anti-obesic agents.
  • extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions are formulated into pharmaceuticals, nutraceuticals and dietary supplements including food and beverages.
  • Figure I Selected phytochemicals isolated from Alangium salvifolium
  • ASE03 down regulates the marker proteins of Adipogenesis differentiation processes in 3T3-L1 adipocytes.
  • Representative immunoblots indicate down- regulation of various marker proteins such as PPARY, ADRP, CEBPa, CD36 and perilipin.
  • 3T3-L1 mouse pre-adipocytes were allowed to differentiate in absence or presence of various concentrations of ASE03 as indicated. Vehicle control cultures received only similar concentrations of DMSO. Expression of actin protein was evaluated in each blot as the internal control.
  • Figure III ASE03 down regulates expression of key enzymes responsible for lipogenesis in adipocytes. Representative immunoblots indicate that ASE03 dose- dependently down-regulates the expression of Fatty Acid Synthase and ATP Citrate Lyase in 3T3-L1 adipocytes. Vehicle control cultures received only similar concentrations of DMSO. Expression of actin protein was evaluated in each blot as the internal control.
  • Figure IV Ethanol extract (ASE03) of Alangium salvifolium up-regulates AMPKa phosphorylation in HepG2 human hepatocytes. HepG2 cells were treated with ASE03 at different concentrations as indicated in the figure for 2h.
  • Figure V ASE03 positively modulates AMPK activity in HepG2 human hepatocytes.
  • a SEO 3 treatment also demonstrated hyper phosphorylation of Acetyl CoA Carboxylase at Ser79. Expression of actin in cell lysate samples was considered as the loading control.
  • FIG. VII ASE03 up-regulates phosphorylation of HMG CoA Reductase (HMGCR) in hepatocytes.
  • Representative immunoblot depicts ASE03 increases HMGCR phosphorylation at Ser872 in a bi-phasic manner, with the earliest peak at 15 min and the next at 120 min of exposure. Expression of actin in cell lysate samples was considered as the loading control.
  • Figure VIII Bar diagrammatic representation of mean body weight gain (A) and % reduction in body weight gain over control (B) in diet induced obese model of Sprague Dawley rats following 8 weeks treatment period.
  • the bars Gl to G4 represent weight gain (A) or % reductions in body weight gain (B) in groups supplemented with placebo, ASE03F1 (200 mg/kg), ASE03F1 (400 mg/kg) and sibutramine (10 mg/kg) respectively.
  • Figure IX Bar diagrammatic representation of the levels of different biochemical parameters like Cholesterol (A), LDL (B), Triglycerides (C), Atherogenic index (D) and Coronary artery index (E) in diet induced obese model of Sprague Dawley rats following eight weeks treatment period.
  • the bars Gl to G4 represent the groups supplemented with placebo, ASE03F1 (200 mg/kg), ASE03F1 (400 mg/kg) and sibutramine (10 mg/kg) respectively.
  • adipogenesis proliferation of pre-adipocytes or precursor fat cells is followed by the' differentiation of these cells into mature adipocyte phenotype.
  • An increase in adipose tissue mass can be the result of the production of new fat cells through the process of adipogenesis and the deposition of increased amounts of cytoplasmic triglyceride or lipid droplets in the mature adipocyte cells per cell. Increased lipid accumulation is the most important feature of the adipogenesis process.
  • PPAR- ⁇ Peroxisome Proliferator-Activated Receptor-gamma
  • ADRP adipose differentiation related protein
  • CD36 CD36
  • CEBPa CEBPP
  • Perilipin are other important markers of adipogenesis process.
  • Lipolysis is a catabolic process in adipose tissue leading to the breakdown of triglycerides stored in adipocyte cells and release of fatty acids and glycerol.
  • the 3-adrenergic receptor ( 3AR) is the principal receptor in body's adrenergic system in the regulation of lipolysis and energy expenditure. Tightly regulated balance between lipid synthesis (adipogenesis or lipogenesis) and lipid mobilization (lipolysis) adjusts the fat storage level within cells.
  • the inventors also found that the administration of one or more of the components selected from the extracts, fractions, active compounds derived from the herb Alangium salvifolium in a therapeutically effective amount in cell based studies potently ameliorated the levels of certain biomarker molecules or biological proteins that are altered during metabolic syndrome, obesity, diabetes, atherosclerosis, endothelial dysfunction, hypertension, hypercholesterolemia, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases and other disease conditions associated with metabolic syndrome.
  • adipocyte differentiation markers such as Peroxisome proliferator- activated receptor gamma (PPARy), ADRP, CEBPa, CD36 and intracellular lipid droplet surface associated protein (perilipin) in a dose dependent manner in cellular studies performed using immunoblot assay as summarized and depicted in Figure II.
  • PPAR ⁇ Peroxisome proliferator-activated receptor ⁇
  • CD36 which is a class B scavenger receptor known to function as a fatty acid transporter (FAT) and it facilitates the uptake of long-chain fatty acids (LCFAs) in adipocytes
  • ADRP adipose differentiation related protein
  • ASE03 also down regulates perilipin protein in adipocytes strongly indicating reduced fat store in the cytoplasm.
  • Perilipin forms a protective coating around the lipid droplets in the fat-storing cells in adipose tissue and protects the stored lipids against body's natural lipases, such as hormone-sensitive lipase (HSL).
  • HSL hormone-sensitive lipase
  • the reduced perilipin coat exposes and increases susceptibility of the intravescicular lipids to HSL, which breaks down triglycerides into glycerol and free fatty acids by a process called lipolysis.
  • ASE03 strongly and dose dependently down regulated the expressions of the key enzymes of fatty acid biosynthesis pathway i.e, FAS and ATP citrate Lyase in adipocytes.
  • the results are summarized in Figure III.
  • ASE03 potentially inhibit lipogenesis process in the fat tissue.
  • Fatty acid Synthase (FAS) catalyzes fatty acid synthesis. It catalyzes the synthesis of palmitate from Acetyl-CoA and Malonyl-CoA in presence of NADPH.
  • ATP citrate lyase is the primary enzyme responsible for the synthesis of cytosolic Acetyl-CoA.
  • Acetyl-CoA is the precursor for fatty acid synthesis.
  • the inventors also observed that the extracts of Alangium salvifolium can potentially modulate the phosphorylation of AMPK at Thrl72 in HepG2 human hepatocytes.
  • the treatment of HepG2 human hepatocytes with Alangium salvifolium ethanol extract (ASE03) potently and dose dependently up regulated the AMPK phosphorylation ( Figure IV). This indicates that ASE03 can induce AMPK activation in HepG2 human hepatocytes.
  • AMPK has been considered as the master regulator of metabolic/energy homeostasis in the body via controlling glucose and fat metabolism. Upregulation of AMPK activity by the extracts of Alangium salvifolium indicates the following therapeutic potential for them against:
  • Type 2 or insulin resistant diabetes increases cellular glucose uptake via. increasing glucose uptake).
  • Atherosclerosis, Hypertension and Hypercholesterolemia reduces cholesterol synthesis by inhibiting HMG-Co-A Reductase activity.
  • Fibromyalgia or chronic pain syndrome (as AMPK via improving mitochondrial function through increased expression of Peroxisome Proliferator- Activated Receptor gamma coactivator-1 alpha or PGC-la).
  • Ageing and neurodegenerative diseases (as AMPK increases mitochondrial biogenesis and ROS detoxification by inducing PGC-la synthesis).
  • AMPK activators are also useful for the supplements for sports nutrition as it up-regulates mitochondrial biogenesis, thus increasing the capacity of tissues for aerobic production of ATP.
  • ASE03 positively modulates AMPK activity in HepG2 human hepatocytes.
  • ASE03 up regulates AMPKa phosphorylation at Thrl72 in HepG2 human hepatocytes.
  • ASE03 treatment also demonstrated hyper phosphorylation of Acetyl CoA Carboxylase at Ser79.
  • Hyperphosphorylation at Thrl72 activates AMPK, which in turn phosphorylates Ser79 of Acetyl CoA Carboxylase (ACC), the downstream effector molecule of AMPK.
  • ACC Acetyl CoA Carboxylase
  • Hyper- phosphorylation at the active site switches off ACC activity. Deactivation of ACC is considered as an indicator for AMPK activation.
  • CPT-la Carnitine palmitoyltransferase la
  • CPT-la is the first component and rate-limiting step of the carnitine palmitoyltransferase system, catalyzing the transfer of the acyl group from coenzyme A to carnitine to form palmitoylcarnitine.
  • a translocase then shuttles the acyl carnitine across the inner mitochondrial membrane where it is converted back into palmitoyl- CoA and proceeds for beta-oxidation.
  • ASE03 can be helpful in decreasing adiposity via increasing mitochondrial fat burning, and reducing fatty liver (hepatic steatosis).
  • HMGCR HMG CoA Reductase
  • AMPK phosphorylates and inactivates acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid biosynthesis; and inactivate HMGCR through phosphorylation at Ser872.
  • ASE03 can help in reducing hypercholesterolemia/hypertension via limiting the cholesterol biosynthesis.
  • A. salvifolium extracts can thus be useful for the prevention, treatment and control of overweight, obesity, metabolic syndrome and other metabolic disorders through the modulation of one or more metabolic biomarkers.
  • biomarkers include Peroxisome proliferator-activated receptor gamma (PPARy), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPP), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty- acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor ( 3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase- IB (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT- ⁇ ) and HMG CoA Reductase (HMGCR).
  • the non- limiting metabolic processes include
  • ASE03F1 exhibited 22.29% % and 68.70% reduction in body weight gain in the treatment groups supplemented with 200 mg/kg body weight and 400 mg/kg body weight of ASE03F1 respectively.
  • Sibutramine as a positive control showed 91.84% reduction in body weight gain.
  • the results of body weight gain for the treatment groups and control group are summarized in Figure VIII.
  • ASE03F1 treatment also significantly reduced serum LDL, triglycerides (TG) and cholesterol levels. It also reduced Atherogenic index and Coronary artery index.
  • the efficacy data for Clinical Biochemistry is summarized in Figure IX.
  • Atherogenic Index is the ratio of Total cholesterol and High density lipoprotein
  • Coronary artery index is the ratio of Low density lipoprotein and High density lipoprotein. Both these indices give prediction about cardiovascular disease.
  • the treatment with ASE03F1 is without risks as no atypical signs were observed in animals throughout treatment phase.
  • ASEQ3F1 can be very potent for controlling and treating obesity, overweight, metabolic syndrome and other disease conditions associated metabolic syndrome.
  • the ethanol extract of Alangium salvifolium was subjected to purification over silica gel using solvents of increasing polarity starting from ethylacetate/hexane mixtures through ethylaceate and methanol/ethylacetate mixtures to methanol to obtain eight fraction ASE03/01 to ASE03/08. Some of the fraction were subjected to repeated purification on silica gel or reversed phase silica gel to obtain pure compounds.
  • Some of the compounds are characterized as betulinic acid (1), demethylalangiside (2), psychotrine (3), deoxytubulosine (4) and alangiside (5), loganic acid (6), tubulosine (7), cephaeline (8), salsoline (9) and deacetylipicosidic acid (10), 6-O-methyl-N- deacetyl-6"-0-a-glucopyranosyl-isoipecosidic acid (11).
  • the structures are depicted in Figure I.
  • the some of the fractions and compounds exhibited potent anti- adipogenesis (inhibition of lipid accumulation) and pro-adipolysis (acceleration of lipolysis) activities as summarized in example 14 and Table 4.
  • compositions were prepared by combining ethanol extract of Alangium salvifolium and extracts derived from selected paint extracts.
  • the compositions ASE03F2 to ASE03F7 disclosed in the experimental (example 9) were evaluated for their efficacy to inhibit the lipid accumulation in 3T3-L1 mouse adipocyte cells. All the compositions strongly inhibited the lipid accumulation in adipocyte cells as summarized in example 15 and Table 5, suggesting their potent anti-adipogenesis activity and potential use for treating overweight and obesity, and other metabolic disorders.
  • the herbal extract(s) or fraction(s) or compound(s) or mixtures thereof derived from Alangium salvifolium or their compositions can be used as pharmaceutical/dietary supplement/food ingredient/herbal medicine for the prevention, control and/or treatment of at least one metabolic disorder or condition selected from obesity, overweight, diabetes, atherosclerosis, arteriosclerosis, hypertension, hypercholesterolemia, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases, cardiovascular diseases, endothelial dysfunction, metabolic syndrome and other metabolic disorders or conditions.
  • the phrase/word "ingredient”, "herbal ingredient(s)", 'components' or 'agents' widely used in the specification and claims of the present invention in conjunction with Alangium salvifolium refer to at least one selected from the herbal extract(s) fraction's) and compound(s) or mixtures thereof derived from Alangium salvifolium and the same may be appreciated as such by the person skilled in the art.
  • biologically active components refers to extract(s) or fraction(s) or compound(s) derived from plants, animals and microorganisms.
  • the extract(s) or fraction(s) or mixtures thereof as described in the present invention may optionally be in the form of anhydrous or concentrated or reconstituted extract(s) or fraction(s).
  • the part of the plant to be extracted from Alangium salvifolium is not specifically limited, but it can be selected from fruits, rind, bark, flower, tender twigs, stems, leaves, seed, trunk, aerial parts, heartwood, roots, whole plant and mixture thereof.
  • the plant parts to be extracted also referred to as raw material, are subjected to drying, such as sun drying, shade drying, freeze drying and the like.
  • the present invention discloses a herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredient comprising at least one phytochemical ingredient selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium and their compositions optionally in combination with atleast one component selected from pharmaceutically or dietetically acceptable, vehicle, diluent and carrier for the control, prevention and treatment of at least one condition or disease selected from overweight, obesity, metabolic syndrome and other metabolic disorders.
  • the present invention discloses herbal anti- adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredient composition
  • herbal anti- adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredient composition comprising at least one phytochemical ingredient selected from the extract(s), fraction(s) and pure compound(s) derived from Alangium salvifolium and their compositions and atleast one component selected from phytochemical actives, vehicle, diluent and carrier for the control, prevention and treatment of at least one condition or disease selected from overweight, obesity, metabolic syndrome and other metabolic disorders.
  • the present invention discloses the process for the preparation of anti-adipogenic and pro-lipolytic herbal .
  • supplements containing Alangium salvifolium extract or fraction comprises the steps of:
  • step (a) filtering the extract of step (a) through fine filters
  • step (b) evaporating the filtrate of step (b) to remove solvent and to obtain the concentrated extract;
  • step (c) optionally purifying the concentrate of step (c) to obtain active fraction
  • step (d) optionally purifying the active fraction of step (d) to obtain pure compound or f) optionally mixing the extract of step (c) or purified fraction of step (d) with a known excipient/diluent, anti-obesic agent or antioxidant or bio-enhancer in a mixer followed by sieving and blending to obtain composition(s).
  • the solvent used for extraction is selected from water or hydroalcohol or organic solvents including polar organic solvents and non-polar organic solvents or the mixture thereof.
  • polar organic solvents include, but not limited to, lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propanol, isopropanol, n- butanol, isobutanol, tert-butanol, hydroalchohol, chlorinated solvents and mixtures thereof, and ketones such as dimethyl ketone (acetone), methyl ethyl ketone, methyl isobutyl ketone and the like.
  • non-polar organic solvents include, but not limited to, hexane, methyl acetate, ethyl acetate, butyl acetate, diethyl ether and the like.
  • chlorinated solvents examples include, but not limited to, dichloromethane, chloroform and dichloroethane and the like.
  • the present invention provides the preparation of extracts from fruits of Alangium salvifolium using solvent selected from Water (ASE01), Hydro- alcohol (ASE02) and Ethanol (ASE03).
  • solvent selected from Water (ASE01), Hydro- alcohol (ASE02) and Ethanol (ASE03).
  • the present invention provides the preparation of extracts from tender twigs of Alangium salvifolium using solvent selected from Ethanol (ASE04), Hydro-alcohol (ASE05) and Water (ASE06).
  • solvent selected from Ethanol (ASE04), Hydro-alcohol (ASE05) and Water (ASE06).
  • the extraction method employed in the present invention is selected from immersion extraction, cold homogenizing extraction, hot extraction, continuous extraction, super critical extraction and the like.
  • the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions thus obtained, according to the present invention, is useful as a adipogenesis inhibitor and lipolysis accelerator as these products has exhibited strong anti-adipogenic and pro-lipolytic activities in cell based assays.
  • extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions are used as it is, or the active ingredient is formulated into a solid, semisolid or liquid dosage form by adding a conventional biologically acceptable carrier or diluent.
  • the invention provides therapeutically effective amount of extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions of the present invention which can be administered in a specific dosage form such as oral, topical, transdermal, parenteral or in the form of a kit to a subject or patient in need thereof.
  • Specific dosage form for formulation of the compositions of the present invention includes, but not limited to oral agents such as tablets, soft capsules, hard capsules, pills, granules, powders, emulsions, suspensions, syrups, pellets; topical agents such as cream, gel, emulsions, ointment; enema, medicinal pack, food supplement, inhalers, mouth sprays and the like; and parenteral agents such as injections, drops, infusion solution, suppositories and the like.
  • the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions may be optionally combined with one or more of known anti-adipogenic or anti-obese extracts or powders, including but not limited to Garcinia cambogia extract, green tea extract, green coffee bean extract, Eucalyptus plant extract, double salt of (-)-hydroxycitric acid from Garcinia species, Garcinia mangostana extract, Gymnema sylvestre extract, Lagerstromia speciosa (Banaba) extract, carnitine, Phaseolus vulgaris extract, Citrus aurantium (bitter orange) extract, Chitosan, Sphaeranthus indicus, Conjugated linoleic acid, Glucomannan (Konjac plant extract), Caralluma extract, Sea weed extract, Hoodia gordonii extract, Commiphora mukul gum resin extract, Murraya koenigii extract, Zingiber officinalis extract, Allium
  • the anti-adipogenic and/or pro-lipolytic compositiohs of Alangium salvifolium extracts or fractions further comprise effective amounts of pharmaceutically or nutritionally or dietetically acceptable antioxidant(s), adaptogen(s), anti-inflammatory agents, anti-diabetic agent, bio-protectants, bio-availability enhancers and trace metals or mixtures thereof to form a formulation.
  • the extract(s), fraction(s) or pure compound(s), herein after referred as phytochemical component(s) or ingredient(s) or agents derived from Alangium salvifolium can be used directly or as a composition in combination with an excipient or excipients or other extracts or phytochemicals or mixtures thereof.
  • the inventive composition comprises at least one component selected from plant powder(s), the extract(s), active fraction(s), active compound(s) and pure compounds isolated from Alangium salvifolium and optionally comprising pharmaceutically or dietically acceptable vehicle(s) or carrier(s) for the control and prevention and treatment of overweight, obesity metabolic syndrome and disease indications associated with metabolic syndrome or other metabolic disorders.
  • the invention is unique and the phytochemical ingredients or agents derived from Alangium salvifolium or their composition(s) are effective in alleviating at least one disease or condition selected from Metabolic Syndrome, overweight, obesity, diabetes, insulin resistance/hyperinsulinemia, increased insulin sensitivity, dyslipidemia, hypertriglyceridemia, chylomicronemia and low HDL-cholesterol, lipoprotein aberrations, decreased triglycerides, elevated uric acid levels, fatty liver, polycystic ovarian syndrome, hemochromatosis (iron overload), acanthosis nigricans (dark patches on the skin), impaired glucose tolerance (IGT), including impaired fasting glucose (IFG), and Type 2 diabetes, hypertension, cardiovascular diseases, endothelial dysfunction, atherosclerosis, mitochondrial dysfunction, fibromyalgia or chronic pain syndrome, ageing, neurodegenerative disorders and atherosclerosis.
  • Metabolic Syndrome e.g., Metabolic Syndrome, overweight, obesity, diabetes, insulin resistance/hyperinsulinemia
  • the phytochemical ingredient(s) derived from Alangium salvifolium or its composition(s) are also effective in the amelioration of metabolic marker proteins including but not limited Peroxisome proliferator-activated receptor gamma (PPARy), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPp), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty- acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (p3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase- IB (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransfer
  • compositions may further comprise effective amounts of pharmaceutical or nutraceutical or dietically acceptable antioxidant(s), adaptogen(s), anti-inflammatory agents, anti-obese agents, anti-diabetic agents, bio-protectants and/or bio-availability enhancer(s) and trace metals or an excipient(s) or mixtures thereof to form a formulation administered using any of the method described above.
  • compositions comprising phytochemical agents derived from Alangium salvifolium and the extract(s), fraction(s), active compound(s) or mixtures thereof derived from but not limited to Withania somnifera, Salacia reticulata, Terminalia chebula, Tinospora cordifolia, Citrullus vulgaris, Dolichos biflorus, Sphaeranthus indicus, Garcinia mangostana, Cassia tora, Cassia auriculata, Azadirachta indica, Tephrosia purpurea, Ginkgo biloba, Lagerstroemia speciosa, Ocimum sanctum, Ficus racemosa, Aegle marmelos, Cinnamon extract, Albizia amara, Amorphophallus campanulatus, Murraya koenigii, Cissus quadrangularis, Gendarussa vulgaris, Piper nigrum, Ra
  • the anti-adipogenic and pro-lipolytic formulations of the present invention is prepared by formulating the extracts or purified fractions of Alangium salvifolium, or compositions thereof along with the biologically acceptable carrier or diluents.
  • the examples of the biologically acceptable carrier or diluents employed in the present invention includes, but are not limited to, surfactants, excipients, binders, disintegrants, lubricants, preservatives, stabilizers, buffers, suspensions and drug delivery systems.
  • solid carriers include, but not limited to, glucose, fructose, sucrose, maltose, rice floor, sorbitol, stevioside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-a-tocopherol, glycerin, propylene glycol, glycerin fatty ester, polyglycerin fatty ester, sucrose fatty ester, sorbitan fatty ester, propylene glycol fatty ester, acacia, carrageenan, casein, gelatin, pectin, agar, vitamin B group, nicotinamide, calcium pantothenate, microcrystalline cellulose powder, magnesium stearate, microcel C, aerosol, syloid, amino acids, calcium salts, pigments, flavors, and preservatives.
  • liquid carriers include, distilled water, saline, aqueous glucose solution, alcohol (e.g. ethanol), propylene glycol, and polyethylene glycol; and oily carriers such as various animal and vegetable oils, white soft paraffin, paraffin, and wax.
  • composition of the present invention is delivered in the form of controlled release tablets, using controlled release polymer-based coatings by the techniques known in the art.
  • the said formulation is designed for once a daily administration.
  • the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions can be formulated into any food and beverage forms such as solid food like chocolate or nutritional bars, semisolid food like cream or jam, or gel. Contemplation was also done to formulate the composition of the invention into a beverage and the like, such as refreshing beverage, coffee, tea, milk-contained beverage, lactic acid bacteria beverage, drop, candy, chewing gum, chocolate, gummy candy, yoghurt, ice cream, pudding, soft adzuki-bean jelly, jelly, cookie and the like. These various preparations or foods and drinks are useful as a healthy food for the treatment and/or prevention of obesity.
  • the present invention provides methods of treatment wherein the effective amount of the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions are to be administered or ingested to mammals in the form of above-mentioned nutraceutical and dietary compositions, wherein the dose may not be uniform and varies depending on the nature of the formulation and suggested human or animal dosage of the extract or the fractions, but preferably within a range from 0.01 to 300 mg/kg body weight/day.
  • the quantity of the extract or the purified fraction in the above-mentioned various foods and beverage compositions may not be uniform and varies depending on the nature of the formulation and suggested human or animal dosage of the extract or the fractions, for example, about 0.001 to 50 wt %, preferably about 0.01 to 20 wt %, more preferably about 0.1 to 10 wt %.
  • the percentage of the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium in the compositions of the present invention varies in the range of 0.1% to 99.9% by weight of the above extract based on the total weight of the composition.
  • the animal feed in the present invention is prepared by mixing the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions with various components used in the animal feed for the purpose of inhibition, amelioration or prevention of overweight, obesity, lipid storage disease, hyperlipidemia, cardiovascular disease, atherosclerosis and thrombosis, or for the purpose of inhibition or reduction of an amount of triglyceride or an amount of cholesterol in blood, inhibiting or preventing obesity.
  • the form of the food additive for animal feed is not specifically limited and the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions may be added to food products as it is, or as a composition, to various cooked and processed food products.
  • the quantity may be the same as that used in case of food products.
  • the ingredients may also be added during or after preparation of the animal feeds.
  • the present invention discloses a method of treating, controlling and preventing at least one metabolic disorder selected from obesity, overweight, diabetes, atherosclerosis, arteriosclerosis, hypertension, high blood pressure levels, high cholesterol levels (LDL, HDL, VLDL), abnormal triglyceride levels, hypercholesterolemia, fibromyalgia/chronic pain syndrome, ageing, neurodegenerative diseases, cardiovascular diseases, endothelial dysfunction, metabolic syndrome, atherosclerosis and other metabolic disorders or conditions and also for prevention, control and treatment of inflammatory diseases by administering to a subject in need, a therapeutically effective amount of the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium or their compositions thereof.
  • at least one metabolic disorder selected from obesity, overweight, diabetes, atherosclerosis, arteriosclerosis, hypertension, high blood pressure levels, high cholesterol levels (LDL, HDL, VLDL), abnormal triglyceride levels, hypercholesterolemia, fibromyalgia/chronic pain syndrome, age
  • the present invention discloses a method of controlling metabolic processes selected from acceleration of lipolysis and inhibition of adipogenesis comprising administering to a subject in need thereof a therapeutically effective amount of the extracts, fractions, enriched fraction or pure compounds derived from Alangium salvifolium and their compositions thereof.
  • the present invention discloses a method of ameliorating the biological markers selected from Peroxisome proliferator-activated receptor gamma (PPARy), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta ( ⁇ ), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox- LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (P3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase- IB (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT- ⁇ ) and HMG-CoA Reductase
  • the subject mentioned in above embodiments is mammal or warm blooded animal.
  • the present invention also discloses a method of using the pharmaceutical or dietary supplement or food ingredient selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium, and their compositions, wherein the composition is in comminuted form and/or in unmodified form at a daily dosage and may be administered in any of the forms like powder, capsules, tablets, granules, precipitate, extract, dried extract, liquid, syrup, shots and/or exudates and the like.
  • the present invention discloses anti-adipogenic activity of Alangium salvifolium dried fruit extracts, where the percentage inhibitions of lipid accumulation caused by water extract (ASE01), 50% ethanol extract (ASE02) and ethanol extract (ASE03) of Alangium salvifolium were demonstrated.
  • the results exhibit that, the ethanol extract (ASE03) of Alangium salvifolium potently inhibits lipid accumulation in adipocytes in a dose dependent manner over water extract (ASEO 1 ), 50% ethanol extract (ASE02) as depicted in Table 1.
  • the present invention discloses anti-adipogenic activity of Alangium salvifolium extracts of dried tender twigs, where the percentage inhibitions of lipid accumulation caused by water extract (ASE06), 50% ethanol extract (ASE05) and ethanol extract (ASE04) of Alangium salvifolium were demonstrated.
  • the results exhibit that, the ethanol extract (ASE04) of Alangium salvifolium potently inhibits lipid accumulation in adipocytes in a dose dependent manner over water extract (ASE06), 50%» ethanol extract (ASE05) as depicted in Table 2.
  • the present invention discloses pro-lipolytic activity of ethanol extract (ASE03) of Alangium salvifolium, wherein said extract (ASE03) is effective in increasing lipolysis and % acceleration of lipolysis occur in a dose dependent manner as reported in Table 3.
  • Dried fruit plant material Alangium salvifolium (0.1 Kg) was pulverized into coarse powder and extracted with water (700 mL) for 1 hr. at room temperature. Extraction process was repeated thrice using water in the ratio 1 :4 w/v with respect to the plant material. All the extracts were combined, the combined water extract was fine filtered, and the clear extract was evaporated at 40°C under vacuum to get thick solution (to about 50 ml). The Freeze drying of the above concentrated solution for 35hrs yielded the extract (ASE01) as a dark residue (32g).
  • Dried fruit plant material Alangium salvifolium (0.1 Kg) was pulverized into coarse powder and extracted with 1 : 1 ethanol/water mixture (700 mL) for Jackpot. at room temperature. Extraction process was repeated thrice using 400 mL of 1 : 1 ethanol/water mixture. All the extracts were combined, the combined hydro-alcohol extract was fine filtered, and the clear extract was evaporated at 40°C under vacuum to get thick solution (about 50 ml). The above concentrated solution was freeze dried for 32hrs to give hydro-alcohol extract (ASE02) as a dark residue (32.1 g).
  • Dried fruit plant material Alangium salvifolium (O.lKg) was pulverized into coarse powder and extracted with ethanol (700- mL) for Jackpot at room temperature. Extraction process repeated thrice using 500 mL ethanol each time. All the extracts were combined and the combined ethanol extracts was fine filtered. The clear extract was evaporated at 40°C under vacuum on rotary evaporator to give ethanol extract (ASE03) as a thick paste (1 1.5 g).
  • Dried tender twigs plant material of Alangium salvifolium (0.1 Kg) was pulverized into coarse powder and extracted with 1 : 1 ethanol/water mixture (700 mL) for lhr. at room temperature. Extraction process was repeated thrice using 400 mL of 1 : 1 ethanol/water mixture. All the extracts were combined, the combined hydro-alcohol extract was fine filtered, and the clear extract was evaporated at 40°C under vacuum to get thick solution ( ⁇ 50 ml). The above concentrated solution was freeze dried for 32hrs to give hydro-alcohol extract (ASE05) as dark residue (16.5 g).
  • Dried tender twigs plant material of Alangium salvifolium (0.1 Kg) was pulverized into coarse powder and extracted with water (700 mL) for lhr at room temperature. Extraction process was repeated thrice using water in the ratio 1 :4 w/v with respect to the plant material. All the extracts were combined, the combined water extract was fine filtered, and the clear extract was evaporated at 40°C under vacuum to get thick solution ( ⁇ 50 ml). The Freeze drying of the above concentrated solution for 35hrs yielded the extract (ASE06) as a dark residue (16 g).
  • the ethanol extract of Alangium salvifolium was subjected to purification over silica gel.
  • a sample of lOOg of the extract was adsorbed on 270g of silica and loaded into a column containing 700g of silica.
  • the column was eluted with solvents of increasing polarity starting from 20% ethylacetate/hexane through ethylaceate and methanol/ethylacetate mixtures to methanol to obtain eight fractions ASE03/01 to ASE03/08.
  • the fraction ASE03/04 obtained on elution of the column with 10% methanol/ethylaceate was subjected to repeated purification to obtain betulinic acid (1), demethylalangiside (2), psychotrine (3), deoxytubulosine (4) and alangiside (5).
  • the fraction (ASE03/05) obtained on elution of the column with 20% methanol/ethylaceate yielded loganic acid (6) on further purification.
  • Example 9 Compositions containing the extracts of Alangium salvifolium:
  • Composition ASE03F1 was prepared by combining and blending uniformly 54g of Alangium salvifolium ethanol extract, 44g of microcrystalline cellulose and 2g of Syloid (aerosol).
  • Composition ASE03F2 was prepared by combining and blending uniformly 33.3g of Alangium salvifolium ethanol extract and 66.6g of Amorphophallus campanulatus water extract.
  • Composition ASE03F3 Composition ASE03F3 was prepared by combining and blending uniformly 66.6g of Alangium salvifolium ethanol extract and 33.3g of Commiphora mukul methanol extract standardized to 2.5% guggle sterones.
  • Composition ASE03F4 Composition ASE03F4 was prepared by combining and blending uniformly 75g of Alangium salvifolium ethanol extract and 25g of Garcinia mangostana methanol extract.
  • Composition ASE03F5 was prepared by combining and blending uniformly 66.6g of Alangium salvifolium ethanol extract and 33.3g of Zingiber officianale ethanol extract.
  • Composition ASE03F6 was prepared by combining and blending uniformly 75g of Alangium salvifolium ethanol extract and 25g of Piper nigrum methanol extract standardized to 5% piperine.
  • Composition ASE03F7 was prepared by combining and blending uniformly 50g of Alangium salvifolium ethanol extract and 50g of Sphaeranthus indicus methanol extract.
  • Composition ASE03F8 was prepared by combining and blending uniformly 40g of Alangium salvifolium ethanol extract, 58g of microcrystalline cellulose and 2g of Sylloid (aerosol).
  • Example 10 Assessment of inhibition of lipid accumulation in differentiated adipocytes by Alangium salvifolium extracts
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • the cells incubated only with 0.2% DMSO were considered as the vehicle control. Thereafter, the differentiation medium was replaced by DMEM containing 100 nM insulin and cells were further grown in presence or absence of different concentrations of the extracts derived from Alangium salvifolium fruit for 6 days. After the treatment period, the cells were fixed with 10% buffered formalin for 4 h at room temperature. The fixed cells were stained with Oil Red O solution (0.5 g in 100 ml isopropanol) for 10 min to measure the cellular neutral lipid accumulation.
  • Oil Red O solution 0.5 g in 100 ml isopropanol
  • the anti-adipogenic activity of the Alangium salvifolium derived fruit extracts is represented by percentage inhibition of lipid accumulation (Table 1). *
  • the anti-adipogenic activity of the Alangium salvifolium derived tender twigs extracts is represented by percentage inhibition of lipid accumulation (Table 2).
  • Table 2 Anti-adipogenic activity of Alangium salvifolium dried tender twigs
  • Example 11 Assessment of Pro-Lipolytic Activity of Alangium salvifolium Extracts in Differentiated Adipocytes
  • Equal number of 3T3-L1 mouse pre-adipocyte cells suspended in Dulbecco's Modified Eagle's Medium (DMEM) containing 10%) Fetal Bovine Serum (FBS) was seeded into each well of 48-well plates and incubated for 48 h at 37°C and in 5%> C02.
  • the differentiation of pre-adipocytes was initiated in a differentiation medium, DMEM containing 500 nM insulin, 1.0 ⁇ dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX). After 48h, the differentiation medium was replaced by DMEM containing 100 nM insulin and incubated further for 6 days and then the culture medium was removed.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • the differentiated cell monolayer was washed twice ith phenol red free DMEM. Thereafter, 250 ⁇ ⁇ of incubation solution (phenol red free DMEM containing 2% bovine serum albumin) was added to the wells in triplicate in presence or absence of ethanol extract (ASE03) derived from Alangium salvifolium fruits and the cells were further incubated for 4 h.
  • the vehicle control wells received 0.2% DMSO in the incubation medium.
  • Cell culture supernatants were collected and clarified at 10,000 g for 5 min at 4°C. Released glycerol content in the culture supernatants was measured with glycerol reagent according to the protocol provided in Adipolysis Assay Kit (Millipore, Billerica, MA).
  • the percentage increase in glycerol concentration in the sample solutions compared to the control containing the known concentrations of glycerol corresponds to the percentage acceleration of lipolysis by Alangium salvifolium extract.
  • the percentage increase in lipolysis accelerated by ethanol extract (ASE03) of Alangium salvifolium were determined using the above protocol and data is summarized Table 3.
  • Ethanol Extract (ASE03) of Alangium salvifolium activates AMPKa in HepG2 human hepatocytes
  • the cells Following treatment with ethanol extract (ASE03) of Alangium salvifolium, the cells were placed on ice and washed thrice with chilled phosphate buffered saline. Thereafter, the cells were lysed in cell lysis buffer; and the cell lysates were clarified at T4000g for 10 min at 4°C. The protein content in cell lysates was estimated using BCA protein assay kit (Thermo Scientific, USA). .
  • the expression of phospho and un-phospho AMPKa in the cell lysates was evaluated by immunoblot technique. Briefly, equal amount of cell lysate protein was run in SDS- PAGE and the resolved proteins were electro-blotted onto nitrocellulose membranes. The specifically immuno-reactive proteins were detected by using phospho (Thrl72) - specific and wild type AMPK antibody (Cell Signaling Technology, USA). The immuno-blots were developed by using chemiluminescent West-Pico substrate (Thermo Scientific, USA). The blotted membranes were also reacted with actin specific antibody (Sigma Chemicals, USA) to ensure equal protein loading. The expressions of the immuno reactive protein bands were analyzed by Image Lab Software, version 2.0.1 (BioRad, USA).
  • Mouse pre-adipocyte 3T3-L1 cells are maintained in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 2 mM glutamine, 4.5g/L glucose and 10% fetal bovine serum. Equal number of cells was plated in each well of 24-well culture plates. Cells were pre-treated with multiple concentrations of ASE03 for 2h and followed by addition of differentiation medium containing 500nM insulin, ⁇ . ⁇ dexamethasone, and 0.5mM isobutylmethylxanthine (IBMX) for 48h. Thereafter, cell were further incubated with post differentiation medium (DMEM containing ⁇ insulin) in presence or absence of ASE03 for three days. Vehicle control cultures received only 0.2% DMSO.
  • DMEM Dulbecco's Modified Eagles Medium
  • AMPK/p-AMPK Thrl72
  • ACC/p-ACC Ser79
  • CPTla HMGCR: Equal number of HepG2 human hepatocytes (ATCC, Manassas, VA) suspended in DMEM containing 10% FBS was plated in each cell culture dish. After attachment, the cells were serum starved in DMEM containing 1% FBS for 24hrs. Then the cells were treated with 1 ⁇ g/ml of ASE03 for various time periods.
  • the cells were placed on ice and washed thrice with chilled phosphate buffered saline. Thereafter, the cells were lysed in cell lysis buffer; and the cell lysates were clarified at 14000g for 10 min at 4°C.
  • the protein content in cell lysates was estimated using BCA protein assay kit (Thermo Scientific, USA). Modulations in various protein expressions in the cell lysates were analyzed using immunoblot assays.
  • Immunoblot assay Briefly, equal amount of cell lysate proteins was run in SDS- PAGE and the resolved proteins were electro-blotted onto nitrocellulose membranes. The specific immuno-reactive proteins were detected by using appropriate antibodies.
  • the antibodies against PPARy, ADRP, CEBPa, CD36, Perillipin, FAS, ATP Citrate Lyase, AMPK/p-AMPK (Thrl72), ACC/p-ACC (Ser79), CPTla, HMGCR were purchased from Cell Signaling Technology (Danvers, MA, USA).
  • Antibodies specific to Phospho-HMGCR (Ser872) and Actin were purchased from Merck Millipore (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO).
  • the immuno-blots were developed by using chemiluminescent West-Pico substrate (Thermo Scientific, USA). The blotted membranes were also reacted with actin specific antibody (Sigma Chemicals, USA) to ensure equal protein loading. The expressions of the immuno reactive protein bands were analyzed with help of Image Lab Software, version 2.0.1 (BioRad, USA).
  • ASE03 down regulates adipocyte differentiation marker proteins in 3T3 adipocytes
  • ASE03 potently down regulates PPARY, ADRP, CEBPa, CD36 and perilipin, the marker proteins of Adipogenesis differentiation processes in 3T3-L1 adipocytes.
  • the data is summarized in Figure II. Transformation of preadipocytes to mature adipocytes is tightly controlled by differentiation process and is modulated by members of two families of transcription factors, the CCAAT/enhancer binding proteins (CEBPa) and peroxisome proliferator-activated receptors (PPARs). The formation of white adipose tissue is completely dependent on PPARr and CEBPa.
  • CCAAT/enhancer binding proteins CEBPa
  • PPARs peroxisome proliferator-activated receptors
  • ADRP Adipocyte differentiation related protein
  • CD36 perillipin
  • ASE03 strongly inhibits the adipocyte differentiation process by down regulating the key transcription factors and their target proteins.
  • ASE03 down regulated the expression of key enzymes responsible for lipogenesis in adipocytes.
  • Representative immunoblots summarized in Figure III indicate that ASE03 dose-dependently down-regulates the expression of Fatty Acid Synthase (FAS) and ATP Citrate Lyase in 3T3-L1 adipocytes.
  • Fatty acid Synthase (FAS) catalyzes fatty acid synthesis. It catalyzes the synthesis of palmitate from Acetyl-CoA and Malonyl-CoA in presence of NADPH.
  • ATP citrate lyase is the primary enzyme responsible for the synthesis of cytosolic Acetyl-CoA.
  • Acetyl-CoA is the precursor for fatty acid synthesis.
  • ASE03 by strongly down regulate the expressions of the key enzymes of fatty acid biosynthesis pathway i.e, FAS and ATP citrate Lyase in adipocytes, ASE03 potentially inhibits lipogenesis process in the fat tissue.
  • ASE03 positively modulated AMPK activity in HepG2 human hepatocytes Representative immunoblots depicted in Figure V indicate that ASE03 up regulates AMPKa phosphorylation at Thrl72 in HepG2 human hepatocytes.
  • ASE03 treatment also demonstrated hyper phosphorylation of Acetyl Co A Carboxylase at Ser79.
  • AMPK has been considered as the master regulator of metabolic/energy homeostasis.
  • Hyperphosphorylation at Thr 172 activates AMPK, which in turn phosphorylates Ser79 of Acetyl Co Carboxylase (ACC), the downstream effector molecule of AMPK. Hyper-phosphorylation at the active site switches off ACC activity.
  • ACC Acetyl Co Carboxylase
  • Deactivation of ACC is considered as an indicator for AMPK activation.
  • Activation of AMPK turns on several metabolic pathways, such as stimulates hepatic fatty acid oxidation and ketogenesis, muscle glucose uptake, insulin secretion; on the other hand it inhibits cholesterol synthesis, lipogenesis, and triglyceride synthesis etc.
  • this data indicates that ASE03 activate AMPK pathway and thus might help in reducing hyperglycemia via increasing insulin sensitivity, decreasing obesity via reducing lipogenesis, reducing hypercholesterolemia, triglyceride synthesis etc.
  • CPT-la carnitine palmitoyltransferase-la
  • Carnitine palmitoyltransferase I is the first component and rate-limiting step of the carnitine palmitoyltransferase system, catalyzing the transfer of the acyl group from coenzyme A to carnitine to form palmitoylcarnitine.
  • a translocase then shuttles the acyl carnitine across the inner mitochondrial membrane where it is converted back into palmitoyl- CoA and proceeds for beta-oxidation.
  • CPT-la is the key enzyme in beta- oxidation of long chain fatty acids.
  • ASE03 up-regulates 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) phosphorylation in hepatocytes
  • HMGCR HMG CoA Reductase
  • AMPK phosphorylates and inactivates acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid biosynthesis; and inactivate HMGCR through phosphorylation at Ser872.
  • ASE03 might help in reducing hypercholesterolemia/hypertension via limiting the cholesterol biosynthesis.
  • Example 14 Assessment of inhibition of lipid accumulation and acceleration of adipolysis of fractions (ASE03/01 to ASE03/08) and a few pure compounds [psychotriene (3) and deoxytubulosine (4)] in differentiated adipocytes.
  • the % inhibition of lipid accumulation and % increase of adipolysis in differentiated 3T3-L1 mouse adipocyte cells were evaluated for the fractions ASE03/01 to ASE03/08, and also for the pure compounds psychotriene (3) and deoxytubulosine (4) using the procedures described in example 10 and example 1 1.
  • the compounds psychotriene (3) and deoxytubulosine (4) showed 12.7% and 24.7% inhibition respectively at 0.05 ⁇ g/mL concentration.
  • psychotriene (3) showed 125.7% increase of adipolysis at 10 ⁇ g mL
  • deoxytubulosine (4) showed 144.2% increase at 2.5 g/mL concentration.
  • Example 15 Assessment of inhibition of lipid accumulation of the compositions (ASE03F2 to ASE03F7) in differentiated adipocytes.
  • the % inhibition of lipid accumulation in differentiated 3T3-L1 mouse adipocyte cells were evaluated for the individual extracts and compositions ASE03F2 to ASE03F7 using the experimental procedure described in example 10.
  • the data for % inhibition of lipid accumulation for the individual extracts and compositions ASE03F2 to ASE03F7 are summarized in Table 5.
  • Example 16 _Anti-obese activity of the composition ASE03F1:
  • Treatment Following 6 weeks of induction phase, the animals were treated orally (using oral feeding gavage) with allocated test substances or vehicle daily for 8 weeks.
  • the control group (Gl) rats were administered vehicle (10 mL of 0.5 % CMC-Na) and treatment groups were supplemented with 200 mg of ASE03F1 (G2), 400 mg of ASE03F1 (G3) or 10 mg of positive control, sibutramine (G4) all in 10 mL of 0.5% CMC-Na on daily basis for 8 weeks.
  • Body weights Body weight of individual animal was recorded weekly during the entire duration of the study. Mean body weights for the treatment groups and control group were determined. The body weight gain was calculated at the end of 1 st week, 4 th week and 8 th week after initiation of treatment in comparison to initial body weights. The food intake, clinical biochemistry and Oral Glucose Tolerance Test (OGTT test) were also evaluated for all the animals. In clinical biochemistry total cholesterol (TC), low density lipoproteins (LDL), high density lipoproteins (HDL), triglycerides (TG), alanine aminotransferase and aspartate aminotransferase levels were analyzed with conventionally available kit methods using ILab Aries automatic biochemistry analyzer (Milano, Italy).
  • TC total cholesterol
  • LDL low density lipoproteins
  • HDL high density lipoproteins
  • TG triglycerides
  • alanine aminotransferase and aspartate aminotransferase levels were analyzed with conventionally
  • ASE03F1 Supplementation of ASE03F1 for 8 weeks to obese rats resulted in significant reduction in percentage body weight gain.
  • Sibutramine as a positive control showed 91.84% reduction in body weight gain.
  • the results of body weight gain for the treatment groups and control group are summarized in Figure VIII.
  • ASE03F1 treatment also reduced serum LDL, triglycerides (TG) and cholesterol levels. It also reduced Atherogenic index, Coronary artery index.
  • Atherogenic Index is the ratio of Total cholesterol and High density lipoprotein
  • Coronary artery index is the ratio of Low density lipoprotein and High density lipoprotein. Both these indices give prediction about cardiovascular disease.
  • TC/HDL Atherogenic Index
  • LDL/HDL Coronary artery index
  • ASE03F1 can be very potent for controlling and treating obesity, overweight and other disease conditions associated metabolic syndrome.

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Abstract

L'invention concerne une ou des compositions végétales ou un ou des ingrédients phytochimiques anti-adipogènes et pro-lipolytiques comprenant au moins un composant choisi parmi un ou des extraits, fractions et composés actifs issus d'Alangium salvifolium soit seul, soit en combinaison avec au moins un composant choisi parmi des agents actifs phytochimiques, des véhicules, diluants et supports pharmaceutiquement ou diététiquement acceptables. Les nouveaux ingrédients ou compositions selon la présente invention peuvent être utilisés pour modérer ou traiter le surpoids, l'obésité, le syndrome métabolique ou d'autres troubles métaboliques, et également pour réguler la dépense énergétique, pour prévenir les plaques d'athérosclérose dans les artères coronaires et l'aorte abdominale, pour augmenter la sensibilité à l'insuline, pour améliorer la tolérance au glucose, pour abaisser le taux de triglycérides et équilibrer la glycémie chez les mammifères.
PCT/IN2015/000261 2014-06-24 2015-06-24 Composition comprenant un extrait d'alangium salvifolium à activité anti-adipogène ou anti-obésité WO2015198346A1 (fr)

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WO2018093065A1 (fr) * 2016-11-18 2018-05-24 주식회사 싸이터스에이치앤비 Composition pharmaceutique comprenant de la tubulosine, destinée à la prévention et au traitement du cancer
CN108143972A (zh) * 2018-03-08 2018-06-12 开封红枫叶生物科技有限公司 一种具有燃烧脂肪功能的减肥胶囊及其制备方法
KR101877443B1 (ko) * 2017-11-08 2018-07-11 아주대학교산학협력단 로가닉산 또는 이의 유도체를 유효성분으로 함유하는 비만 예방, 개선 또는 치료용 조성물
IT202000018970A1 (it) * 2020-08-03 2022-02-03 Asoltech Srl Composizione a base di mirra
CN114209681A (zh) * 2021-12-02 2022-03-22 中国海洋大学 海参长链碱及其衍生物在制备调节PPAR-γ制品中的应用
WO2022192413A1 (fr) * 2021-03-09 2022-09-15 Natreon, Inc. Méthodes d'atténuation des maux de dos faisant appel à des compositions de terminalia chebula
CN115053963A (zh) * 2022-06-28 2022-09-16 仙乐健康科技股份有限公司 一种提升体温和促进体表微循环的组合物
CN115417857A (zh) * 2022-08-31 2022-12-02 贵州中医药大学 一种中药八角枫中的哌啶类生物碱及其提取纯化、半合成方法和应用

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TANWER, BS ET AL.: "Phytochemical Evaluation and Quantification of Primary Metabolites of Alangium Salviifolium.", INTERNATIONAL JOUMAL OF PHARMA AND BIO SCIENCES., vol. 1, no. 3, July 2010 (2010-07-01), XP055247060 *
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018093065A1 (fr) * 2016-11-18 2018-05-24 주식회사 싸이터스에이치앤비 Composition pharmaceutique comprenant de la tubulosine, destinée à la prévention et au traitement du cancer
KR20180056366A (ko) * 2016-11-18 2018-05-28 주식회사 싸이터스에이치앤비 튜불로신을 포함하는 암 예방 및 치료용 약제학적 조성물
KR102100826B1 (ko) 2016-11-18 2020-04-27 주식회사 싸이터스에이치앤비 튜불로신을 포함하는 암 예방 및 치료용 약제학적 조성물
KR101877443B1 (ko) * 2017-11-08 2018-07-11 아주대학교산학협력단 로가닉산 또는 이의 유도체를 유효성분으로 함유하는 비만 예방, 개선 또는 치료용 조성물
CN108143972A (zh) * 2018-03-08 2018-06-12 开封红枫叶生物科技有限公司 一种具有燃烧脂肪功能的减肥胶囊及其制备方法
IT202000018970A1 (it) * 2020-08-03 2022-02-03 Asoltech Srl Composizione a base di mirra
WO2022029067A1 (fr) * 2020-08-03 2022-02-10 Asoltech S.R.L. Composition à base de myrrhe
WO2022192413A1 (fr) * 2021-03-09 2022-09-15 Natreon, Inc. Méthodes d'atténuation des maux de dos faisant appel à des compositions de terminalia chebula
CN114209681A (zh) * 2021-12-02 2022-03-22 中国海洋大学 海参长链碱及其衍生物在制备调节PPAR-γ制品中的应用
CN115053963A (zh) * 2022-06-28 2022-09-16 仙乐健康科技股份有限公司 一种提升体温和促进体表微循环的组合物
CN115417857A (zh) * 2022-08-31 2022-12-02 贵州中医药大学 一种中药八角枫中的哌啶类生物碱及其提取纯化、半合成方法和应用
CN115417857B (zh) * 2022-08-31 2023-06-06 贵州中医药大学 一种中药八角枫中的哌啶类生物碱及其提取纯化、半合成方法和应用

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