DESC:FORM 2
THE PATENTS ACT 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. TITLE OF THE INVENTION:

“A COMPOSITION COMPRISING EXTRACT OF ALANGIUM SALVIFOLIUM HAVING ANTI-ADIPOGENIC OR ANTI-OBESIC ACTIVITY”

2. APPLICANT:

(a) NAME: LAILA NUTRACEUTICALS

(b) NATIONALITY: Indian Partnership Firm register under the
Indian Partnership Act, 1932

(c) ADDRESS: 40-15-14, Brindavan Colony, Labbipet, Vijayawada-520010,
Andhra Pradesh, India

3. PREAMBLE TO THE DESCRIPTION:
The following specification describes the invention and the manner in which it is to be performed.

Technical field of Invention:
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, metabolic syndrome and other metabolic disorders or conditions.

Background of the Invention:
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.

During the past 20 years, obesity among adults has risen significantly in the United States. The latest data from the National Center for Health Statistics show that 30 percent of U.S. adults of 20 years of age and older, i.e. over 60 million people, are obese. The percentage of young people, who are overweight, has more than tripled since 1980. More than 16% of the children and teens aged 6-19 years, that is over 9 million young people, are considered overweight.

As per World Health Organization’s latest projections, approximately 1.4 billion adults (age 20+) were overweight. Of these overweight adults, over 200 million men and nearly 300 million women were obese. Thirty five percentage of adults aged 20 and over were overweight in 2008, and 11% were obese. Sixty five percentage of the world's population live in countries where overweight and obesity kills more people than underweight. In 2012, more than 40 million children under the age of 5 were overweight or obese (WHO's fact sheet No. 311, May 2014). WHO further projects that by 2015, approximately 2.3 billion adults will be overweight and more than 700 million worldwide will be obese. Obesity in Europe was recognized as a serious problem, with up to 27% of men, 38% of women and 3 million children are clinically obese. 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.

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. During this process, 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).

There are a few therapeutic interventions based on pharmaceutical drugs, such as phentermine (Fastin, Adipex P), for weight control but these methods exhibit side effects like high blood pressure, headache, insomnia, irritability and nervousness. The other important drug therapy for weight control is Xenical (Roche Pharm. Co. Ltd., Swiss), Reductil (Abbot Co. Ltd., USA). The most common side effects are gas, cramps and diarrhea, elevated blood pressure. All these therapies are based on active ingredients that are of synthetic origin. Effective anti-obese therapies with satisfactory efficacy and acceptable safety have not been developed so far.

More importantly, anti-obese agents of natural origin with proven safety are of great demand to control this growing menace. It is particularly advantageous for inhibition, amelioration and prevention of obesity if an anti-obesity action can be imparted to food products and beverages, which are ordinarily ingested.

Hence, presently there is great demand for development of natural agents for prevention, maintenance and remedy of obesity, which are safe and effective. The major emphasis now for many organizations around the globe has been to develop new dietary ingredients and compositions especially from natural origin. Herbal and natural products containing Gymnema extract, Garcinia extract, or carnitine are known to prevent fat accumulation through the inhibition of fat absorption, enhancement of fat decomposition, and the enhancement of fat consumption by the body.

The Alangium salvifolium (Alangeaceae) also called as ‘Ankola’ is extensively cultivated in India. It is a popular folk medicine and has been studied for its anti-inflammatory, 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, anti-microbial, laxative, and anti-epileptic activity. The phytochemical analysis of Alangium salvifolium revealed the presence of alkaloids, glycosides, terpenoids, steroids, tannins and the ethanol extracts of Alangium salvifolium seeds exhibited anti-diabetic, 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).

However, none of the prior art reported or disclosed the pharmaceutical, nutraceutical and dietary ingredients comprising extracts or fractions or pure compounds derived from Alangium salvifolium or the compositions developed therefrom for the amelioration/inhibition of adipogenesis and /or induction of lipolysis (fat breakdown) or the prevention or treatment or maintenance or management of altered fat metabolism, adipogenesis and/or lipolysis mediated disorders such as obesity, over weight, metabolic syndrome or other metabolic disorders.

Objects of the Invention:
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.

Summary of the Invention:
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 (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG CoA Reductase (HMGCR); and also for the control of the metabolic processes such as inhibition of adipogenesis and/or acceleration of lipolysis.

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.

In further aspect, 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.

In another aspect, 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.

In yet another aspect, 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.

In still yet another aspect, 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.

Brief Description of the Drawings:
Figure I: Selected phytochemicals isolated from Alangium salvifolium

Figure II: 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 PPAR?, 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. Expressions of phosphorylated AMPKa (Thr172) and AMPKa Proteins were detected using immunoblot assay. Expression of actin in cell lysate samples was considered as the loading control. Bar diagram represents the normalized protein expression of p-AMPK vs. AMPK in respective treatments.

Figure V: ASE03 positively modulates AMPK activity in HepG2 human hepatocytes. Representative immunoblots depict ASE03 up-regulates AMPKa phosphorylation at Thr172 in HepG2 human hepatocytes. ASE03 treatment also demonstrated hyper phosphorylation of Acetyl CoA Carboxylase at Ser79. Expression of actin in cell lysate samples was considered as the loading control.

Figure VI: ASE03 up regulates CPT-1a expression in HepG2 human hepatocytes. Representative immunoblot depicts ASE03 increases CPT-1a expression in a bi-phasic manner, with the earliest peak at 60 min of exposure. Expression of actin in cell lysate samples was considered as the loading control.

Figure 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 G1 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 G1 to G4 represent the groups supplemented with placebo, ASE03F1 (200 mg/kg), ASE03F1 (400 mg/kg) and sibutramine (10 mg/kg) respectively.

Detailed Description of the Invention:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

In the adipogenesis process, 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. The uncontrolled fat accumulation in the body during the metabolic process is predominantly driven by the over expression and increased activity of Peroxisome Proliferator-Activated Receptor-gamma (PPAR-?), a ligand activated nuclear receptor that act as a lipid sensor, integrating the homeostatic control of energy, lipid, and glucose metabolism. In addition, adipose differentiation related protein (ADRP), CD36, CEBPa, CEBPß and Perilipin are other important markers of adipogenesis process.

Lipolysis (adipolysis) 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 ß3-adrenergic receptor (ß3AR) 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.

Based on the above information, inventors of the present invention have undertaken the screening of a large number of herbal extracts to assess their inhibitory potential against the lipid accumulation and potential for acceleration of breakdown of stored fat in 3T3-L1 mouse adipocyte cells. It was found that the extracts and purified fractions of Alangium salvifolium fruit raw material exhibit potent anti-adipogenic and pro-lipolytic activity in 3T3-L1 mouse adipocyte cells. The ethanol extract (ASE03) of A. salvifolium showed 49.86% and 74.01% inhibition of lipid accumulation at 1 ?g/mL and 2.5 ?g/mL concentrations respectively. The other extracts also showed potent anti-adipogenesis activity as summarized in Table 1. Extracts of tender twigs of A. salvifolium also showed potent anti-adipogenic activity as summarized in Table 2. In addition, the ethanol extract also potently and dose dependently accelerated the lipolysis in 3T3-L1 adipocyte cells. It exhibited 24.12%, 44.7% and 64.71% increase in lypolysis at 5 ?g/mL, 25 ?g/mL and 50 ?g/mL concentrations respectively as shown in Table 3.

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.

It was observed that the ethanol extract of A. salvifolium (ASE03) potently modulated the levels of several adipocyte differentiation markers such as Peroxisome proliferator-activated receptor gamma (PPAR?), 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. The down regulation of these marker proteins in ASE03 treated adipocytes suggests that the ethanol extract of Alangium salvifolium fruit exerts multiple beneficial roles in controlling the adipogenic differentiation process; by (1) inhibiting cellular differentiation by down regulating Peroxisome proliferator-activated receptor ? (PPAR ?), which is a nuclear receptor that is expressed predominantly in adipose tissue, wherein it is known to play a critical role in adipocyte differentiation and fat deposition, (2) restricting cholesterol ester uptake by inhibiting 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, (3) inhibiting adipose differentiation related protein (ADRP), which play possible role in the formation or stabilization of lipid droplets in adipocytes and enhances the uptake of long chain fatty acids by adipose tissue, (4) by down regulating other critical transcription factors of adipogenesis process, CEBP? (CCAAT/enhancing binding protein alpha) and CEBP? (CCAAT/enhancing binding protein beta), which along with PPAR? are known to cooperatively orchestrate adipocyte differentiation. These are proteins involved in different cellular responses like the control of cellular proliferation, growth, differentiation and metabolism. Together, these markers influence abdominal obesity and related metabolic abnormalities associated with type 2 diabetes and cardiovascular disease.

In addition to the foregoing adipogenesis markers, 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). In presence of ASE03, 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.

In futher cell based studies in 3T3-L1 adipocytes, 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. Thus, 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.

In continuation studies, the inventors also observed that the extracts of Alangium salvifolium can potentially modulate the phosphorylation of AMPK at Thr172 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:
1) Type 2 or insulin resistant diabetes (as AMPK activation increases cellular glucose uptake via. increasing glucose uptake).
2) Obesity and over weight (as AMPK increases thermogenesis via enhanced fatty acid Oxidation; and decreased fatty acid synthesis).
3) Atherosclerosis, Hypertension and Hypercholesterolemia (as AMPK reduces cholesterol synthesis by inhibiting HMG-Co-A Reductase activity).
4) 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-1a).
5) Ageing and neurodegenerative diseases (as AMPK increases mitochondrial biogenesis and ROS detoxification by inducing PGC-1a synthesis).
6) 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.

In a repeated study, it was confirmed that ASE03 positively modulates AMPK activity in HepG2 human hepatocytes. As depicted by representative immunoblots summarized in Figure V, ASE03 up regulates AMPKa phosphorylation at Thr172 in HepG2 human hepatocytes. ASE03 treatment also demonstrated hyper phosphorylation of Acetyl CoA Carboxylase at Ser79. Hyperphosphorylation at Thr172 activates AMPK, which in turn phosphorylates Ser79 of Acetyl CoA Carboxylase (ACC), the downstream effector molecule of AMPK. Hyper-phosphorylation at the active site switches off ACC activity. Deactivation of ACC is considered as an indicator for AMPK activation. Hence, activation of AMPK turns on several metabolic pathways, such as stimulation of hepatic fatty acid oxidation and ketogenesis, muscle glucose uptake, insulin secretion; on the other hand it inhibits cholesterol synthesis, lipogenesis, and triglyceride synthesis etc. Together, our observation 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.

Unexpectedly, ASE03 was also found to upregulate CPT-1a expression in HepG2 human hepatocytes. The data is summarized in Figure VI. Carnitine palmitoyltransferase I? (CPT-1a) is the key enzyme in beta-oxidation of long chain fatty acids. CPT-1a 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. Hence, by inducing CPT-1? expression in hepatic cells, ASE03 can be helpful in decreasing adiposity via increasing mitochondrial fat burning, and reducing fatty liver (hepatic steatosis).

Surprisingly, the HepG2 hepatocyte cells treated with ASE03 also showed up-regulated phosphorylation of HMG CoA Reductase (HMGCR). The data is summarized in Figure VII. HMG CoA Reductase (HMGCR) is a rate limiting enzyme in the mevalonate pathway leading to cholesterol biosynthesis. This enzyme is in activated state when it is dephosphorylated; conversely, inhibition of HMGCR is achieved by its phosphorylation at Ser872. In the metabolic pathway, AMPK plays a crucial role in regulation of HMGCR activity. Upon activation, AMPK phosphorylates and inactivates acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid biosynthesis; and inactivate HMGCR through phosphorylation at Ser872. Hence, our observation strongly suggests that 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. These non-limiting biomarkers include Peroxisome proliferator-activated receptor gamma (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG CoA Reductase (HMGCR). The non-limiting metabolic processes include adipogenesis, adipolysis, fat synthesis and cholesterol synthesis.

The efficacy shown by the extracts of Alangium salvifolium in in vitro models against obesity and other metabolic disorders was further evaluated in an in vivo model of obesity. Obesity was induced in male Sprague Daley rats by supplementing the rats with High Fat Diet (HFD) for six weeks and the obese rats were then treated with one of the test substances for eight weeks of treatment period. The composition ASE03F1 containing the ethanol extract (ASE03) derived from A. salvifolium fruit dose dependently inhibited the body weight gain in high fat diet induced obese rats. 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. In addition, 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 (TC/HDL) is the ratio of Total cholesterol and High density lipoprotein, while Coronary artery index (LDL/HDL) 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. In conclusion, these results suggested that ASE03F1 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??-O-?-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.

Several compositions were prepared by combining ethanol extract of Alangium salvifolium and extracts derived from selected palnt 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.

From the foregoing, it is evident that 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.

For the purpose of this invention, 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.

The phrase “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.

Accordingly, in one preferred embodiment, 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.

In another preferred embodiment, the present invention discloses 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.

In yet another preferred embodiment, 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:
a) extracting the dried Alangium salvifolium plant parts with the water or organic solvent or mixtures thereof;
b) filtering the extract of step (a) through fine filters;
c) evaporating the filtrate of step (b) to remove solvent and to obtain the concentrated extract;
d) optionally purifying the concentrate of step (c) to obtain active fraction;
e) 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.

Examples of the 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.

Examples of the non-polar organic solvents include, but not limited to, hexane, methyl acetate, ethyl acetate, butyl acetate, diethyl ether and the like.

Examples of the chlorinated solvents include, but not limited to, dichloromethane, chloroform and dichloroethane and the like.

In typical embodiment, 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).

In typical embodiment, 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).

Other plant parts of A. salvifolium can also be used for obtaining the extracts. 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.

To obtain full benefit, it is preferable that the above-mentioned 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, semi-solid or liquid dosage form by adding a conventional biologically acceptable carrier or diluent.

In a further embodiment, 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 sativa extract, Cissus quadrangularis, chromium (III) complexes, DHEA, 7-Keto DHEA, and the composition obtained thereof is administered using a method described above.

The anti-adipogenic and/or pro-lipolytic compositions 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.

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 (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG CoA Reductase (HMGCR), which are related to metabolic syndrome and play a role in the prevention, treatment and control of obesity, overweight, diabetes and other metabolic disorders in mammals or subjects or patients in need thereof.

The phytochemical ingredients derived from Alangium salvifolium and their compositions are also effective for amelioration of metabolic processes including but limited to adipogenesis, adipolysis, fat synthesis and cholesterol synthesis

Various exemplary embodiments of the present invention further provides that the 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.

Various exemplary embodiments of the invention further provides, additional dietary supplement agents that can be used for preparing the 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, Raphanus sativus and Rubia cordifolia.

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.

Preferred examples of 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.

Preferred examples of liquid carriers (diluents) 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.

In alternative embodiment, the 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.

In accordance to the present invention, 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.

In another embodiment, 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. Similarly, the ingredients may also be added during or after preparation of the animal feeds.

In another embodiment, 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.

In another embodiment, 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.

In another embodiment, the present invention discloses a method of ameliorating the biological markers selected from Peroxisome proliferator-activated receptor gamma (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG-CoA Reductase (HMGCR) 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 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.

In another embodiment, 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 (ASE01), 50% ethanol extract (ASE02) as depicted in Table 1.

In another embodiment, 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. In further embodiment, 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.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention and they are not to limit the scope of the invention.

Examples:
Preparation of Extracts from Alangium salvifolium Fruits
Example 1: Preparation of Water Extract (ASE01) of Alangium salvifolium
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 40oC 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).

Example 2: Preparation of Hydro-alcohol Extract (ASE02) of Alangium salvifolium
Dried fruit plant material Alangium salvifolium (0.1Kg) was pulverized into coarse powder and extracted with 1:1 ethanol/water mixture (700 mL) for 1hr. 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 40oC 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 (32.1g).

Example 3: Preparation of Ethanol Extract (ASE03) of Alangium salvifolium
Dried fruit plant material Alangium salvifolium (0.1Kg) was pulverized into coarse powder and extracted with ethanol (700 mL) for 1hr 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 40oC under vacuum on rotary evaporator to give ethanol extract (ASE03) as a thick paste (11.5 g).

Example 4: Preparation of Ethanol Extract (ASE04) of Alangium salvifolium tender twigs (Stem and leaves)
Dried tender twigs of Alangium salvifolium (0.1Kg) were pulverized into coarse powder and extracted with ethanol (700 mL) for 1hr at room temperature. Extraction process repeated twice 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 40oC under vacuum on rotary evaporator to give ethanol extract (ASE04) as a thick paste (10.5 g).

Example 5: Preparation of Hydro-alcohol Extract (ASE05) of Alangium salvifolium tender twigs (Stem and leaves)
Dried tender twigs plant material of Alangium salvifolium (0.1Kg) was pulverized into coarse powder and extracted with 1:1 ethanol/water mixture (700 mL) for 1hr. 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 40oC 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).

Example 6: Preparation of Water Extract (ASE06) of Alangium salvifolium tender twigs (Stem and leaves)
Dried tender twigs plant material of Alangium salvifolium (0.1 Kg) was pulverized into coarse powder and extracted with water (700 mL) for 1hr 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 40oC 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).

Example 7: Preparation of methanol Extract (ASE07) of Alangium salvifolium
Dried fruit of the plant material Alangium salvifolium (0.1Kg) was pulverized into coarse powder and extracted with methanol (700 mL) for 1hr at room temperature. Extraction process repeated thrice using 500 mL methanol each time. All the extracts were combined and the combined ethanol extracts was fine filtered. The clear extract was evaporated at 40oC under vacuum on rotary evaporator to give methanol extract (ASE07) as a thick paste (12.9 g).

Example 8: Preparation of fractions (ASE03/01 to ASE03/08 and Pure compounds (1 to 11) from A. salvifolium ethanol extract (ASE03) :
The ethanol extract of Alangium salvifolium was subjected to purification over silica gel. A sample of 100g 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. The fractions obtained on elution of the column with 20 to 30% methanol/ethylacetate were combined (ASE03/06) and subjected to repeated purification to obtain tubulosine (7), cephaeline (8), salsoline (9) and deacetylipicosidic acid (10). Finally, the fractions obtained on elution of the column with 70% methanol/ethylacetate to methanol were combined (ASE03/08) and purified on reversed phase silica column to obtain 6-O-methyl-N-deacetyl-6??-O-?-glucopyranosyl-isoipecosidic acid (11). The chemical structures of compounds, 1 to 11 are depicted in Figure 1.

Example 9: Compositions containing the extracts of Alangium salvifolium:
Composition ASE03F1: 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: 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: 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: 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: Composition ASE03F7 was prepared by combining and blending uniformly 50g of Alangium salvifolium ethanol extract and 50g of Sphaeranthus indicus methanol extract.

Composition ASE03F8: 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
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 48hr at 37°C and in 5% CO2. Cells were pre-incubated with different concentrations of Alangium salvifolium extracts dissolved in medium containing 0.2% DMSO for 2h and then allowed for differentiation in a differentiation medium i.e. DMEM containing 500 nM insulin, 1.0 µM dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX) for 48 h. 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. After removing the unbound stain, the dye retained in the cells was eluted with isopropanol and OD was measured at 550 nm using a micro-plate Reader (SpectraMax M5e, Molecular Devices, USA). Percent inhibition of adipogenesis was calculated using following formula:

The anti-adipogenic activity of the Alangium salvifolium derived fruit extracts is represented by percentage inhibition of lipid accumulation (Table 1).

Table 1: Anti-adipogenic activity of Alangium salvifolium Dried fruit Extracts
Sr. No. Name of the tested product Treatment concentration % Inhibition of lipid accumulation
1. Dried fruit Water Extract (ASE01) 5 µg/ml 74.98

2. Dried fruit 50% Ethanol Extract (ASE02) 5 µg/ml 74.86

3. Dried fruit Ethanol Extract (ASE03) 1 µg/ml 49.86
2.5 µg/ml 74.01

The percentage inhibition of lipid accumulation caused by dried fruit water extract (ASE01), 50% ethanolextract (ASE02) and ethanol extract (ASE03) of Alangium salvifolium were determined using the above protocol and data is summarized in the Table 1. The results of this study exhibited that the dried fruit ethanol extract (ASE03) of Alangium salvifolium potently inhibits lipid accumulation in adipocytes in a dose dependent manner (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 Extracts
Sr. No. Name of the tested product Treatment concentration % Inhibition of lipid accumulation
1. Dried tender twigs Ethanol Extract (ASE04) 5 µg/ml 78.90
2. Dried tender twigs 50% Ethanol Extract (ASE05) 5 µg/ml 77.90
3. Dried tender twigs Water Extract (ASE06) 5 µg/ml 74.50

The percentage inhibition of lipid accumulation caused by dried tender twigs water extract (ASE06), 50% ethanol extract (ASE05) and ethanol extract (ASE04) of Alangium salvifolium were determined using the above protocol and data is summarized in the Table 2. The results of this study exhibited that the dried tender twigs extracts (ASE04, ASE05 and ASE06) of Alangium salvifolium potently inhibits lipid accumulation in adipocytes in a dose dependent manner (Table 2).

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% CO2. The differentiation of pre-adipocytes was initiated in a differentiation medium, DMEM containing 500 nM insulin, 1.0 µM 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. The differentiated cell monolayer was washed twice with phenol red free DMEM. Thereafter, 250 µL 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). Briefly, 25 µL of culture supernatants were incubated with 100 µL of glycerol assay reagent in 96-well plate for 15 min at room temperature. Finally, the absorbance was read in a microplate reader (SpectraMax M5e, Molecular Devices, USA) at 540 nm. A standard curve constructed from the glycerol was used to calculate the concentration of free glycerol in the culture supernatants. Percent increase of adipolysis was measured in terms of glycerol concentration present in the culture supernatants using following formula:

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.

Table3: Pro-lipolytic activity of Alangium salvifolium Extracts
S. No. Name of the tested product Treatment concentration % Acceleration of lipolysis
1 Ethanol Extract (ASE03) 5 µg/ml 24.12
25 µg/ml 44.70
50 µg/ml 64.71

The percentage of induction in lipolytic activity accelerated by Alangium salvifolium ethanol (ASE03) extract is 24.12%, 44.7% and 64.71% at 5µg/ml, 25µg/ml and 50µg/ml, respectively. Ethanol extract (ASE03) is effective in increasing lipolysis and % acceleration of lipolysis appears to occur in a dose dependent manner (Table 3).

Example 12: Ethanol Extract (ASE03) of Alangium salvifolium activates AMPKa in HepG2 human hepatocytes
Equal number of HepG2 human hepatocytes 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 multiple doses of ASE03 and incubated for 2hrs. The vehicle control culture received only 0.2% DMSO in DMEM containing 1% FBS.

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 14000g for 10 min at 4oC. 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 (Thr172) - 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).

Immunoblot analysis revealed that the ethanol extract of Alangium salvifolium (ASE03) up regulated phosphorylation at Thr172 of AMPKa in a dose dependent manner (Figure IV). Hyper-phosphorylation at Thr172 of AMPKa is crucial for activation of its protein kinase activity.

Example 13: Modulation of PPAR?, ADRP, CEBP?, CD36, Perillipin FAS and ATP Citrate Lyase in Mouse pre-adipocyte 3T3-L1 cells and AMPK/p-AMPK (Thr172), ACC/p-ACC (Ser79), CPT-1a and HMGCR in HepG2 human hepatocytes

Cell culture and treatments:
For PPAR?, ADRP, CEBPa, CD36, Perillipin, FAS, ATP Citrate Lyase: 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, 1.0µM dexamethasone, and 0.5mM isobutylmethylxanthine (IBMX) for 48h. Thereafter, cell were further incubated with post differentiation medium (DMEM containing 100nM insulin) in presence or absence of ASE03 for three days. Vehicle control cultures received only 0.2% DMSO.

For AMPK/p-AMPK (Thr172), ACC/p-ACC (Ser79), CPT1a, 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 1µg/ml of ASE03 for various time periods.

Following various treatments with ASE03, 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 4oC. 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 PPAR?, ADRP, CEBPa, CD36, Perillipin, FAS, ATP Citrate Lyase, AMPK/p-AMPK (Thr172), ACC/p-ACC (Ser79), CPT1a, 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
Results and Significance: ASE03 potently down regulates PPAR?, 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 PPAR? and CEBPa. Key PPAR? target proteins such as Adipocyte differentiation related protein (ADRP), CD36, perillipin are heavily expressed as adipocyte specific proteins during the differentiation process. Overall, the data indicate that ASE03 strongly inhibits the adipocyte differentiation process by down regulating the key transcription factors and their target proteins.

ASE03 down-regulates lipogenesis in 3T3-L1 adipocytes
Results and Significance: 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. Thus, 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.

Modulation of metabolic marker proteins AMPK and ACC in ASE03 treated HepG2 human hepatocytes
Results and Significance: ASE03 positively modulated AMPK activity in HepG2 human hepatocytes. Representative immunoblots depicted in Figure V indicate that ASE03 up regulates AMPKa phosphorylation at Thr172 in HepG2 human hepatocytes. ASE03 treatment also demonstrated hyper phosphorylation of Acetyl CoA Carboxylase at Ser79. AMPK has been considered as the master regulator of metabolic/energy homeostasis. Hyperphosphorylation at Thr172 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. 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. Together, 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.

ASE03 up-regulates carnitine palmitoyltransferase-1a (CPT-1a) expression in hepatocytes
Results and Significance: ASE03 strongly up regulated CPT-1a expression in HepG2 human hepatocytes. The data is summarized in Figure VI. 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. Thus, CPT-1a is the key enzyme in beta-oxidation of long chain fatty acids. Our observation indicates that ASE03 induced CPT-1a expression in hepatic cells, suggesting that ASE03 might help in decreasing adipocity via increasing mitochondrial fat burning, and also help in reducing fatty liver (hepatic steatosis).
ASE03 up-regulates 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) phosphorylation in hepatocytes
Results and Significance: ASE03 up-regulated phosphorylation of HMG CoA Reductase (HMGCR) in hepatocytes. The data is summarized in Figure VII. HMG CoA Reductase (HMGCR) is a rate limiting enzyme in the mevalonate pathway leads to cholesterol biosynthesis. This enzyme is in activated state when it is dephosphorylated; conversely, inhibition of HMGCR is achieved by its phosphorylation at Ser872. In the metabolic pathway, AMPK has a crucial role in regulation of HMGCR activity. Upon activation, AMPK phosphorylates and inactivates acetyl-CoA carboxylase, the rate-limiting enzyme of fatty acid biosynthesis; and inactivate HMGCR through phosphorylation at Ser872. However, our observation strongly suggests that 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 11. The compounds psychotriene (3) and deoxytubulosine (4) showed 12.7% and 24.7% inhibition respectively at 0.05 µg/mL concentration. Similarly, psychotriene (3) showed 125.7% increase of adipolysis at 10 µg/mL and deoxytubulosine (4) showed 144.2% increase at 2.5 µg/mL concentration. The data for % inhibition of lipid accumulation and % increase of adipolysis for the fractions ASE03/01 to ASE03/08 are summarized in Table 4.

Table 4: Anti-dipogenesis Activity of the fractions (ASE03/01 to ASE03/08) from ASE03
Sr. No. Extract/Fraction/ Pure Compound % inhibition of adipogenesis
@ 0.5 µg/mL % increase of adipolysis
@ 1.0 µg/mL
1. ASE03 <5 42.26
2. ASE03/01 NA 35.2
3. ASE03/02 NA 65.7
4. ASE03/03 6.7 NA
5. ASE03/04 46.5 53.2
6. ASE03/05 10.4 NA
7. ASE03/06 12.3 73.7
8. ASE03/07 7.6 40.1
9. ASE03/08 44.4 51.2
NA = Not active

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.
Table 5: Anti-adipogenesis activity of compositions
Sr. No. Test substance % inhibition of adipogenesis
@ 2.0µg/mL
1. ASE03 58.9%
2. Amorphophallus campanulatus water extract 7.0%
3. ASE03F2 35.7%
4. Commiphora mukul extract 2.5% guggul sterones 1.4%
5. ASE03F3 53.8%
6. Garcinia mangostana methanol extract 4.0%
7. ASE03F4 56.9%
8. Zingiber officinale ethanol extract NA
9. ASE03F5 58.02%
10. Piper nigrum extract 5% piperine 14%
11. ASE03F6 57.00%
12. Sphaeranthus indicus Extract 0.4%
13. ASE03F7 47.8%

Example 16: Anti-obese activity of the composition ASE03F1:
Efficacy of ASE03F1 in comparison with sibutramine was tested in High Fat Diet induced obesity model of Sprague-Dawley rats.

Induction: Selected healthy Sprague-Dawley rats of 7-8 weeks were made obese experimentally through dietary intervention during the six weeks induction period by feeding high fat diet (60% kcal fat diet supplied by Research Diets Inc., USA, cat. No. D12492) ad libitum. Naïve control group containing 9 animals were fed standard died. Body weights were measured on weekly basis. Obese animals, which showed more than 45% weight increase in body weight by the last day of 6th week induction period were selected for the study and they are randomly divided in to four groups (G1 to G4) with 9 animals in each group. Animals were then shifted to Nutrilab-Rodent standard diet throughout treatment phase.

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 (G1) 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 1st week, 4th week and 8th 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). In OGTT test, Blood sampling was performed by tail clipping and blood glucose level was estimated at pre dose (0 minute) and thereafter on 30, 60, 90 and 120 minutes (post dose) by using glucometer (ACCU - CHEK Active). Outcome of the obesity induction and treatment efficacy was assessed by using derived parameters like body weight gain and percentage body weight gain. Body weight gain was calculated by using formula (body weight on day X – body weight on day 0).

Results and Discussion: Supplementation of ASE03F1 for 8 weeks to obese rats resulted in significant reduction in percentage body weight gain. ASE03F1 dose dependently inhibited the body weight gain in high fat diet induced obese rats. It 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. In addition, ASE03F1 treatment also reduced serum LDL, triglycerides (TG) and cholesterol levels. It also reduced Atherogenic index, Coronary artery index. The efficacy data for Clinical Biochemistry is summarized in Figure IX. Atherogenic Index (TC/HDL) is the ratio of Total cholesterol and High density lipoprotein while Coronary artery index (LDL/HDL) 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. In conclusion, these results suggested that ASE03F1 can be very potent for controlling and treating obesity, overweight and other disease conditions associated metabolic syndrome. ,CLAIMS:We Claim,

1. 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.

2. A 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.

3. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2 for the control, prevention and treatment of atleast one disease or condition selected from obesity, overweight, atherosclerosis, arteriosclerosis, hypercholesterolemia, fibromyalgia/chronic pain syndrome, endothelial dysfunction, ageing, cardiovascular diseases, neurodegenerative diseases, neurological disorders, high blood pressure levels, high cholesterol levels (LDL, HDL, VLDL), abnormal triglyceride levels, hypertension, metabolic syndrome, and other metabolic disorders.

4. The herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredient(s) selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from derived from Alangium salvifolium and their compositions according to claim 1, for the amelioration of the expression or production of atleast one biomarker protein selected from Peroxisome proliferator-activated receptor gamma (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG CoA Reductase (HMGCR).

5. The herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredient selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from derived from Alangium salvifolium and their compositions according to claim 2, for the amelioration of the expression or production of atleast one biomarker protein selected from Peroxisome proliferator-activated receptor gamma (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG CoA Reductase (HMGCR).

6. The herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredients selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium, and their compositions according to claims 1 and 2, for controlling metabolic processes selected from acceleration of lipolysis and inhibition of adipogenesis.

7. The herbal anti-adipogenic and pro-lipolytic 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 according to claim 2, wherein the phytochemical active is at least one phytochemical component selected from Garcinia cambogia extract, green tea extract, green coffee bean extract, Eucalyptus plant extract, double salt of (-)-hydroxycitric acid from Garcinia species, Garcinia mangostana extract, Amorphophallus campanulatus, piperine containing extracts, Sphaeranthus indicus extract, Gymnemasylvestre extract, Lagerstroemia speciosa (Banaba) extract, carnitine, Phaseolus vulgaris extract, Citrus aurantium (bitter orange) extract, Chitosan, Sphaeranthus indicus, Conjugated linoleic acid, Glucomannan (Konjac plant extract), Caralluma extract, Hoodia gordonii extract, Cissus quadrangularis extract, Commiphora mukul gum resin extract, Murraya koenigii, Zingiber officinalis extract, Allium sativa extract, chromium (III) complexes, DHEA, 7-Keto DHEA.

8. The herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredients selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium, and their compositions according to claims 1 and 2, wherein the percentage of Alangium salvifolium derived component in the composition varies in the range from 0.1% to 99.9%.

9. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2, wherein the extracts of fractions derived from Alangium salvifolium are optionally standardized to atleast one phytochemical selected from Betulinic acid (1), demethylalangiside (2), psychotrine (3), deoxytubulosine (4), alangiside (5). loganic acid (6), tubulosine (7), cephaeline (8), salsoline (9) and deacetylipicosidic acid (10), 6-O-methyl-N-deacetyl-6??-O-?-glucopyranosyl-isoipecosidic acid (11).

10. The herbal anti-adipogenic and pro-lipolytic 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 composition according to claim 2, wherein the phytochemical active can be atleast one component selected from the list comprising adaptogen(s), anti-inflammatory agent(s), anti-obese agent(s), anti-diabetic agent(s), anti-hyperglycemic agent(s), hypolipidemic agent(s), anti-obesity agent(s), anti-hypertensive agent(s), anti-platelet agent(s), anti-infective agent(s), anti-atherosclerotic agent(s) and anti-inflammatory agent(s), anti-oxidant(s) and bio-protectant(s) and/or bio-enhancing agent(s).

11. The herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredients according to claims 1 and 2, wherein the extract(s) or active fraction(s) or pure compound(s) or mixtures thereof are derived from atleast one plant part selected from the leaves, fruits, rind, seeds, tender twigs, stem, bark, root, aerial parts, whole plant or mixtures there of obtained from Alangium salvifolium.

12. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2, wherein the vehicle, diluent or carrier can be selected from surfactants, excipients, binders, disintegrators, lubricants, preservatives, stabilizers, buffers, suspensions and drug delivery systems.

13. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2, wherein the vehicle, diluent or carrier can be selected from the solid diluent (carriers) comprising glucose, fructose, sucrose, maltose, 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, amino acids, calcium salts, pigments, flavors, and preservatives and liquid diluent (carrier) comprising, 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.

14. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2, wherein the Alangium salvifolium derived supplement or ingredient or composition is administered orally, topically, transdermally or parenterally to a subject or mammal or warm blooded animal in need of the treatment for overweight, obesity, Metabolic Syndrome or other metabolic disorder.

15. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2, wherein the Alangium salvifolium derived supplement or ingredient or compositions can be formulated as oral agents such as tablets, soft capsule, hard capsule, pills, granules, powders, emulsions, suspensions, syrups, pellets and the like; and parenteral agents such as injection solution, drops, suppositories and the like; topical agents such as cream, gel, emulsions, ointment and the like; transdermal agents such as patches, and food or beverages.

16. The herbal anti-adipogenic and pro-lipolytic 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 according to claims 1 and 2 is prepared by a process comprises the steps of
a) extracting dried Alangium salvifolium plant parts with solvent;
b) filtering the extract of step (a) through fine filters;
c) evaporating the filtrate of step (b) to remove solvent and to obtain the concentrated extract;
d) optionally purifying the concentrate of step (c) to obtain active fraction;
e) 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).

17. The process for preparation of herbal anti-adipogenic and pro-lipolytic pharmaceutical or dietary supplement or food ingredient according to claim 16, wherein said solvent is selected from water or hydro-alcohol or organic solvents including polar organic solvents such as lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, hydroalchohol, chlorinated solvents such as dichloromethane, chloroform, dichloroethane and mixtures thereof, ketones such as dimethyl ketone (acetone), methyl ethyl ketone, methyl isobutyl ketone and the like; and non-polar organic solvents hexane, methyl acetate, ethyl acetate, butyl acetate, diethyl ether and the like or mixture thereof.

18. A method of controlling or treating obesity, overweight, metabolic syndrome disorder and/or other metabolic disorders comprising administering to a subject in need thereof a therapeutically effective amount of a phytochemical ingredient selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium, and their compositions according to claims 1 and 2.

19. The 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 according to claims 1 and 2, 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.

20. The method of promoting lipolysis and/or inhibiting adipogenesis comprising administering a therapeutically effective dose to a subject in need there of a phytochemical ingredient selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium, and their compositions according to claims 1 and 2.

21. The method of ameliorating the biological markers selected from Peroxisome proliferator-activated receptor gamma (PPAR?), Adipose Differentiation Related Protein (ADRP), CCAAT/enhancer-binding protein alpha (CEBPa), CCAAT/enhancer-binding protein beta (CEBPß), adipocyte CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocyte fatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 Adrenergic Receptor (ß3AR), Perilipin, Adiponectin, Protein tyrosine phosphatase-1B (PTP-1B), AMPK, Fatty Acid Synthase, ATP citrate Lyase, Acetyl CoA Carboxylase (ACC), Carnitine palmitoyltransferase I (CPT-1?) and HMG-CoA Reductase (HMGCR) comprising administering a therapeutically effective dose to a subject in need there of a phytochemical ingredient selected from the extract(s) or fraction(s) or pure compound(s) or mixtures thereof derived from Alangium salvifolium, and their compositions according to claims 1 and 2.