WO2018194444A1 - A composition for regulating appetite and a method thereof - Google Patents

A composition for regulating appetite and a method thereof Download PDF

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Publication number
WO2018194444A1
WO2018194444A1 PCT/MY2018/050017 MY2018050017W WO2018194444A1 WO 2018194444 A1 WO2018194444 A1 WO 2018194444A1 MY 2018050017 W MY2018050017 W MY 2018050017W WO 2018194444 A1 WO2018194444 A1 WO 2018194444A1
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Prior art keywords
tocotrienol
composition
trf
subject
leptin
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PCT/MY2018/050017
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French (fr)
Inventor
Nardev RAMANATHAN
Wei Ney Yap
Jordan Todorov Petkov
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Kl-Kepong Oleomas Sdn Bhd
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Publication of WO2018194444A1 publication Critical patent/WO2018194444A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/889—Arecaceae, Palmae or Palmaceae (Palm family), e.g. date or coconut palm or palmetto
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • the invention relates to a composition for regulating appetite and promoting satiety.
  • the invention relates to a composition for maintaining a steady and balanced appetite containing a naturally-derived active compound, and a method thereof.
  • the composition is useful in promoting satiety and managing body weight.
  • Obesity is one of the largest healthcare problems facing the developed countries, as well as the world today.
  • Nations that were historically grappling with undernutrition and starvation, such as India and China, are now struggling with obesity problems. For instance, China, for the first time, reported that obesity kills more people than undernutrition does.
  • These emerging nations have seen huge rise in a middle class to affluent population.
  • the obesity problem has become more prevalent in recent years owing to lifestyle related issues, including unhealthy diet such as consumption of processed foods and fast foods with massive amounts of saturated fats and oil, as well as sedative lifestyle and lack of regular exercise.
  • a stress related lifestyle can often exacerbate these problems.
  • Obesity has also been identified as a risk factor for the development of type 2 diabetes mellitus, in which more than 85% of individuals with type 2 diabetes mellitus are obese. It is estimated that the exponential growth of obesity will substantially attributable to major changes in the number and average ages of the population of many countries, for instance India and China.
  • Leptin is a hormone that is almost largely secreted by adipocytes, that regulates satiety or appetite by interacting with receptors in the brain.
  • Other tissues in the body have also been identified to contribute to leptin secretion in the body.
  • the stomach is also known as an organ which secrets considerable amount of leptin.
  • Other sources include the placenta and ovaries in females, skeletal muscle and the pituitary gland. Low levels of leptin in the abovementioned weight-reduced state can be interpreted as a state of hunger by the brain and so lead to increase of appetite and hence the resulting hyperphagia or overeating behavior.
  • Another U. S. patent publication no. US 2013022240 A 1 also discloses a method for reducing weight using a composition comprising fucoxanthin extract, tocotrienols, fucoidan or a combination thereof. It is disclosed in this document that the weight reducing effect of the fucoxanthin-comprising composition is based on fat metabolism. It is however not disclosed in this document on how the fucoxanthin extract, tocotrienols and fucoidan inter-reacted among each other to achieve such weight reducing effect. There is also no disclosure on managing or reducing weight through regulating of appetite or leptin level.
  • vitamin E including tocopherols and tocotrienols
  • tocopherols and tocotrienols has been used in the manufacture of medicament or supplement for weight management, either as one of the essential or optional ingredients.
  • most of the prior art documents disclose tocopherol- or tocotrienol-containing composition or method for reducing weight or combating obesity thereof via regulation or alteration of fat metabolism of a subject. Therefore, new compositions or methods specifically developed using naturally-derived compound, such as vitamin E, for use in regulating appetite are therefore desirable.
  • One of the objects of the invention is to provide a composition for naturally regulating satiety or appetite, particularly by promoting satiety, via leptin enhancement in the body of a subject.
  • the invention also aims to provide a method to regulate, for example, to promote satiety by using naturally-derived active compound, in order to prevent uncontrolled hunger pangs that may result by massive weight loss after dieting, so that the weight loss after dieting can be sustainably maintained.
  • At least one of the proceeding objects is met, in whole or in part, by the invention, in which the one of the embodiments of the invention describes a composition for regulating appetite in a subject comprising a plant-derived tocotrienol or derivative thereof selected from a tocotrienol-rich fraction (TRF), alpha-tocotrienol (a- tocotrienol), beta-tocotrienol ( ⁇ -tocotrienol), gamma-tocotrienol ( ⁇ -tocotrienol), delta- tocotrienol ( ⁇ -tocotrienol) or a combination of any two or more thereof.
  • TRF tocotrienol-rich fraction
  • alpha-tocotrienol alpha-tocotrienol
  • beta-tocotrienol beta-tocotrienol
  • gamma-tocotrienol ⁇ -tocotrienol
  • delta- tocotrienol ⁇ -tocotrienol
  • the tocotrienol or derivative thereof is derived from palm, annatto, grape, rice, wheat, barley, rye, oat or a combination of any two or more thereof.
  • the tocotrienol or derivative thereof contains 50% to 99% of total tocotrienol content by weight.
  • the tocotrienol or derivative thereof is a TRF derived from palm, containing 50% to 99% of total tocotrienol content by weight.
  • the composition comprises essentially of a TRF derived from palm, containing 50% to 99% of total tocotrienol content by weight.
  • the TRF contains a-tocopherol, a- tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, phytosterol, squalene, fatty acid or a combination of any two or more thereof.
  • composition in the form of an oral, intravenous or intradermal preparation.
  • the appetite regulation in the subject is achieved by increasing leptin expression, secretion or the combination thereof in adipocytes.
  • a further embodiment of the invention discloses a method for managing, for example, reducing the body weight of a subject comprising a step of administering to a subject an effective amount of a composition according to the foregoing embodiments.
  • the method can be a non-therapeutic method.
  • Another further embodiment of the invention discloses a method for regulating appetite in a subject comprising a step of administering to a subject an effective amount of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a- tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol or a combination of any two or more thereof.
  • the method can be in vivo or in vitro.
  • Yet another further embodiment of the invention discloses use of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a- tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol or a combination of any two or more thereof, in the manufacture of a medicament for regulating appetite in a subject.
  • Figure 1 shows (A) a light microscopy image of TRF-treated adipocytes (20 ⁇ , also the IC 50 concentration for TRF in cells) (8 days short term culture) under high glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per ⁇ g protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), **p ⁇ 0.01; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF-treated adipocytes (right) in comparison to the control (left), *p ⁇ 0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p ⁇ 0.05.
  • Figure 2 shows (A) a light microscopy image of a-tocopherol-treated adipocytes
  • FIG. 1 shows (A) a light microscopy image of TRF-treated adipocytes (20 ⁇ ) (8 days short term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per ⁇ g protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), **p ⁇ 0.01; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF-treated adipocytes (right) in comparison to the control (left), *p ⁇ 0.05, as described in one embodiment of the invention.
  • Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p ⁇ 0.05. shows (A) a light microscopy image of a-tocopherol-treated adipocytes (20 ⁇ ) (8 days short term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per ⁇ g protein secreted into the extracellular environment) by such a-tocopherol- treated adipocytes (right) in comparison to the control (left); (C) a photograph of such ⁇ -tocopherol-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such ⁇ -tocopherol-treated adipocytes (right) in comparison to the control (left), as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p ⁇ 0.05.
  • FIG. 1 shows (A) a light microscopy image of TRF-treated adipocytes (20 ⁇ ) (21 days long term culture) under high glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per ⁇ g protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), ***p ⁇ 0.001; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF- treated adipocytes (right) in comparison to the control (left), *p ⁇ 0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p ⁇ 0.05.
  • Figure 6 shows (A) a light microscopy image of a-tocopherol-treated adipocytes
  • FIG. 1 shows (A) a light microscopy image of TRF-treated adipocytes (20 ⁇ ) (21 days long term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per ⁇ g protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), *p ⁇ 0.05; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF-treated adipocytes (right) in comparison to the control (left), *p ⁇ 0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p ⁇ 0.05.
  • Figure 8 shows (A) a light microscopy image of ⁇ -tocopherol-treated adipocytes (20 ⁇ ) (21 days long term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per ⁇ g protein secreted into the extracellular environment) by such a-tocopherol- treated adipocytes (right) in comparison to the control (left); (C) a photograph of such a-tocopherol-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such a-tocopherol-treated adipocytes (right) in comparison to the control (left), as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p ⁇ 0.05.
  • the invention discloses a composition for regulating appetite in a subject comprising a plant-derived tocotrienol or derivative thereof selected from a TRF, a-tocotrienol, ⁇ - tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol or a combination of any two or more thereof.
  • the composition can also be known as an appetite reducer or an appetite regulator.
  • regulating appetite it refers to regulating or preventing food craving and extreme hunger sensation by promoting satiety or fullness.
  • the appetite regulating or reducing effect of the composition of the invention can be reasonably sustainable, it also assists in maintaining the body weight or sustaining the weight loss in a certain rate. It is to be appreciated that the composition does not reduce the appetite of a subject in a massive way, but it stabilizes and keeps the satiety balance in the subject to prevent the subject from overeating, to compensate for the sudden drop in fat mass during weight loss.
  • the tocotrienol or derivative thereof used in the composition can be a TRF.
  • TRF is a fraction obtained from a purification process of a naturally occurring vitamin E-containing compound extracted from a plant source, for example palm oil.
  • the purification process applied can be a molecular distillation process, which is capable of producing TRF with different concentrations of vitamin E (tocotrienols and tocopherols) (e.g. 30, 50, 70, 95%) by removing impurities (e.g. glyceride, sterols, etc).
  • impurities e.g. glyceride, sterols, etc.
  • TRF contains a relatively higher concentration of tocotrienol as compared to a naturally occurring fraction of its own source which has not gone through the purification process.
  • the TRF used can contain approximately 50% to 99% of total tocotrienol content.
  • This TRF can be obtained from various sources.
  • the TRF can be derived from palm, annatto, grape, rice, wheat, barley, rye, oat or a combination of any two or more thereof.
  • the TRF used in the composition is derived from palm ⁇ Elaeis guineensis jacq.), for example, from palm oil. It is to be appreciated that, the proportion of the contents of vitamin E, i.e. tocotrienols and tocopherols, is naturally varied among different sources.
  • the TRF produced from different sources of plant can contain different proportions of tocotrienols to tocopherols.
  • a 95% purity of TRF produced from palm oil may contain approximately 70% of tocotrienols and 25% of a-tocopherol.
  • a 70% purity of TRF produced from palm oil can contain approximately 50% of tocotrienols and 20% a-tocopherol.
  • a TRF containing 70% to 99% of total tocotrienol content is used in the composition.
  • the TRF can also contain trace amount of plant phytosterols, squalene, fatty acid (e.g.
  • the TRF used in the composition contains ⁇ -tocopherol, a-tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol and ⁇ - tocotrienol, in a specific proportion.
  • the TRF contains approximately 5% to 25% of ⁇ -tocopherol and 50% to 95% of tocotrienols, including a-tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol and ⁇ -tocotrienol.
  • the tocotrienol or derivative thereof is present in an amount of approximately 0.01% to 50%, for example 0.01% to 50% of TRF, by weight of the composition.
  • the composition can contain approximately 50% to 99.99% of other active ingredients, or carrier, excipient or the combination thereof.
  • the composition comprises essentially of palm-derived TRF with 50% to 99% of total tocotrienol content by weight.
  • the composition can contain essentially of 0.01% to 50% of such palm-derived TRF, without other additional active ingredient for regulating appetite, promoting satiety and/or regulating body weight.
  • the composition can contain approximately 50% to 99.99% of other active ingredients, or carrier, excipient or the combination thereof.
  • the composition can be in the form of an oral, intravenous or intradermal preparation.
  • the oral preparation can be a solid, semi-solid or liquid preparation; while the intravenous or intradermal preparation can be of semi-solid or liquid preparation. These preparations can be a water-soluble or an oil-soluble preparation.
  • the oral preparations can include the form of pill, capsule, tablet, powder, lozenges, dragees, granules, pellets, beads, paste, syrup, solution, suspension, shake, cream or emulsion.
  • These oral preparations can also be directly prepared and stored in a solid or semi-solid dosage form for consumption. They can also be prepared and stored in the dosage form of capsule, powder sachet, pellet or effervescent tablet for reconstitution into water or solvent before oral consumption or other administration methods.
  • they can also be prepared or incorporated into foodstuffs or beverage for easy consumption.
  • these oral preparations can be prepared in the form of a dietary supplement, a nutrition snack bar, a slimming tea, a meal replacement drink, or others.
  • the composition can also be formatted into a sustained-release formulation.
  • the composition can be in the form of an intravenous or intradermal preparation, such as solution, suspension, emulsion, or any other dosage forms suitable for injection.
  • the composition as described in the embodiment of the invention is a nutraceutical composition.
  • this nutraceutical composition can contain one or more carrier, excipient or the combination thereof.
  • the carrier, excipient or the combination thereof can be present in an amount of 50% to 99.99% by weight of the composition.
  • the carrier or excipient can be a stabilizing agent, binding agent, filling agent, emulsifying agent, wetting agent, suspending agent, lubricating agent, dispersing agent, diluent, desiccant, solubiliser, flavouring agent, colouring agent, preservative or a combination of any two or more thereof.
  • the composition can further comprise one or more other biologically active agents, for example, biologically active agents with therapeutic effects suitable for regulating appetite, promoting satiety, managing body weight, combating obesity and/or their related metabolic diseases or disorders. These biologically active agents can also be naturally derived from plants or vegetables.
  • They can be an additional satiety or appetite regulating agent, a fat or carbohydrate absorption altering agent, a metabolism altering agent, a calories burning agent, a detoxification agent, an enzyme, an antioxidant, a bioflavonoid, a phytonutrient, an essential oil, a polyphenol, a vitamin, a mineral or a combination of any two or more thereof.
  • the composition can also further comprise a pharmaceutically active agent or drug suitable for regulating appetite, promoting satiety, managing body weight, combating obesity and/or their related metabolic diseases or disorders.
  • the composition for regulating appetite can reduce appetite in the subject by increasing leptin expression, secretion or the combination thereof in adipocytes, according to the embodiments of the invention.
  • increasing leptin expression it refers to increasing of gene and protein expression of the hormone leptin in the adipocytes (also known as fat cells); while “increasing leptin secretion” refers to increasing of the export of intracellularly synthesized leptin into the extracellular environment and ultimately the blood circulatory system.
  • composition of the invention containing the TRF is capable of increasing or upregulating of leptin expression in the adipocytes, and hence resulting in an increase in leptin levels in the adipocytes as well as the endocrine system.
  • a further embodiment of the invention discloses a non-therapeutic method of managing, for example, reducing the body weight of a subject comprising a step of administering to a subject an effective amount of a composition according to the foregoing embodiments.
  • the non-therapeutic method can be a cosmetic method of managing or reducing body weight.
  • another further embodiment of the invention also discloses a method for regulating appetite in a subject comprising a step of administering to a subject an effective amount of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a-tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol or a combination of any two or more thereof.
  • the method can be in vivo or in vitro. In both in vitro and in vivo analysis, the increase of leptin level in adipocytes can be observed.
  • the composition can be administered orally, intravenously or intradermally.
  • the composition can be administered in the forms of oral preparation such as pill, capsule, tablet, powder, lozenges, dragees, granules, pellets, beads, paste, syrup, solution, suspension, shake, cream or emulsion for oral consumption; or in the form of an intravenous or intradermal preparation, such as solution, suspension, emulsion, or any other dosage forms suitable for injection.
  • the composition can also be prepared and stored in the dosage form of capsule, powder sachet, pellet or effervescent tablet for reconstitution into water or solvent before administered via oral consumption or other administration methods.
  • the composition can be administered alone, or to be presented as a nutraceutical composition which contains one or more carrier, excipient or the combination thereof.
  • the composition can contain essentially of 0.01% to 50% of such palm-derived TRF, with or without any other additional active ingredient for regulating appetite, promoting satiety and/or regulating body weight.
  • the composition can be administered to the subject in need once or twice a day, or as necessary.
  • determination of an appropriate dosage for a particular appetite-related disease or disorder, or metabolic condition is within the skill of the art.
  • eating disorder such as hyperphagia, bulimia nervosa or obesity
  • a person having ordinary skill in the art, such as a nutritionist can readily determine and prescribe the effective amount of the composition required for regulating the appetite of the subject, in order to achieve the desired effects.
  • the subject is a human.
  • the subject can be a mammal other than a human, for example a primate, a canine, a feline, an ursine, an equine, a leporine or a rodent.
  • the subject can be an adipocyte or a fat cell.
  • the regulation of appetite of the subject can be achieved by increasing leptin expression, secretion or the combination thereof in adipocytes.
  • the increase of leptin secretion in adipocytes of a subject treated with the composition of this invention can be in a rate of 10% to 50% as compared to that of an untreated subject.
  • Adipocytes or fat cells which have been previously known to be fat storage tissues, are now increasingly becoming appreciated for its endocrine functions, i.e. for secreting many important hormones crucial for metabolic homeostasis, including leptin.
  • Leptin and leptin receptor signaling are most crucial of the factors identified to date that coordinate the control of food intake and energy expenditure in response to an altered energy state. Leptin can act on the hypothalamic arcuate nucleus neurons producing signaling compounds such as anorexigenic proopiomelanocortin (POMC) and orexigenic neuropeptide Y (NPY)/Agouti-related protein (AgRP).
  • POMC anorexigenic proopiomelanocortin
  • NPY orexigenic neuropeptide Y
  • AgRP Agouti-related protein
  • leptin suppresses energy intake and stimulates energy expenditure, leading to a reduction in stored body fat.
  • the increase of levels of leptin in the adipocytes may trigger the signaling pathway of leptin in the central nervous system, particularly, in the hypothalamus, for the system to signal the sensation of satiety in a subject.
  • JAK-STAT signaling is a representative signaling pathway through which leptin regulates food intake and energy homeostasis in the hypothalamus.
  • Leptin can bind to its receptors and initiates downstream signaling through the sequential phosphorylation of the tyrosine kinase Janus kinase (JAK) and the transcription factor signal transducer and activators of transcription (STAT).
  • the phosphorylation of STAT induces its dimerization and translocation to the nucleus, where it binds DNA and modulates the transcription of genes involved in food intake and energy homeostasis.
  • Example 1 shows a method of preparation of adipocyte cell line and culture conditions according to one embodiment of the invention, in which a preadipocytic cell line that undergo a high efficiency of differentiation when induced to undergo adipogenesis can be used, for example, 3T3-L1 murine preadipocytes.
  • a test sample of a-tocopherol can also be included in the study as a comparison to the effect of TRF.
  • the culture conditions prepared can include both the long term and short term conditions, as the long term cultures are crucial in demonstrating that the leptin enhancement from adipocytes is sustainable, given that a key aim for use of TRF-containing composition is to maintain enhanced leptin secretion.
  • High and low glucose conditions can be used, in which low glucose conditions can represent the blood glucose level of a normal health individual, whereas the high glucose media can be relevant in revealing if the leptin enhancement effect can also be observed in diabetic individuals or those with poor glycemic control.
  • Example 2 The leptin secretion and triglyceride accumulation in short term culture of adipocytes under high glucose condition is shown in Example 2.
  • An unpaired t-test analysis can be utilized to assess for statistical significance.
  • the TRF treatment in high glucose condition exhibits greater leptin secretion and a subsequent lower triglyceride accumulation in the adipocytes as compared to the untreated cells, in which the difference shown is significant (p ⁇ 0.01).
  • p ⁇ 0.01 the difference shown in the experiment conducted under low glucose condition as illustrated in Example 3 and Figure 3
  • a similar significant difference of p ⁇ 0.01 can be observed, indicating that the TRF-treated adipocytes also exhibit greater leptin secretion and lower triglyceride accumulation under low glucose condition.
  • Example 6 shows an exemplary method of animal model (rat) studies and protein assays from adipose tissues of the euthanized rats. From the data illustrated in Figure 9, leptin levels can be observed to be significantly increased by approximately 50% (p ⁇ 0.05) within the adipose tissue itself, as compared to unsupplemented control groups.
  • An enhanced delivery system of TRF composition which is a water-soluble TRF formulation suitable for rat consumption, can be used in the animal study.
  • the TRF composition used can contain an emulsifier, an oil carrier or a combination thereof, for effective delivery of the tocotrienols and tocopherols.
  • the method as described in the embodiment of the invention can naturally enhance leptin secretion from adipocytes treated with the TRF-containing composition, under both high and low glucose conditions, indicating that the TRF-containing composition is effective in both diabetic and normal individuals respectively, and such effects are sustainable at a reasonable level.
  • the TRF-containing composition is also capable of decreasing triglyceride accumulation in the samples of adipocytes tested, asides from increasing leptin secretion.
  • the enhancement of leptin secretion by the composition of the invention can result in the promotion of satiety or fullness, lowering of calorie intake, decrease of blood glucose levels, lowering of blood cholesterol level, decrease of body fat, and others.
  • This method is potentially helpful in managing of body weight, thus combating obesity. As obesity is one of the biggest risk factors for diabetes and cardiovascular-related morbidities, this method indirectly helps to reduce the risk of developing these diseases.
  • the regulation of appetite can hence help to maintain the overall health of the gastrointestinal and circulatory system of the subject.
  • the invention also discloses use of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a-tocotrienol, ⁇ -tocotrienol, ⁇ - tocotrienol, ⁇ -tocotrienol or a combination of any two or more thereof, in the manufacture of a medicament for regulating appetite in a subject, according to yet another embodiment.
  • the medicament can be an oral preparation containing TRF, for example, a dietary supplement pill for regulating appetite or promoting satiety.
  • the composition can contain an emulsifier, an oil carrier or a combination thereof; in which the emulsifier can be a non-ionic surfactant (e.g.
  • 3T3-L1 murine preadipocytes were grown in Dulbecco's modified Eagle's medium (DMEM) high glucose (HG) at 4.5g/L containing 10% newborn calf serum (NCS) (heat inactivated at 65°C for 30 minutes). Cells were seeded at 1.0 x 10 4 cells/cm 2. Two days after reaching confluence, cultures were induced to differentiate in DMEM HG at 4.5g/L or low glucose (LG) at l.Og/L containing 10% FBS, supplemented with ⁇ insulin, 0.5mM 3-isobutyl-l-methyl-xanthine (IB MX), and ⁇ dexamethasone for 2 days and then supplemented with ⁇ insulin alone for 2 days.
  • DMEM Dulbecco's modified Eagle's medium
  • HG high glucose
  • NCS newborn calf serum
  • TRF a-tocopherol
  • the standard culture medium containing 4.5g/L glucose is of 4.5 times higher than normal glucose levels, which mimics the condition of a diabetic individual or someone with poor glycemic control.
  • Example 2 Leptin secretion and triglyceride accumulation in short term culture of adipocytes under high glucose condition
  • STC short term culture
  • Example 4 Leptin secretion and triglyceride accumulation in long term culture of adipocytes under high glucose condition
  • Example 2 The experimental method of Example 2 was applied, in which the culture was differentiated for 21 days was used.
  • HG conditions were used to culture the adipocytes under LTC, leptin secretion remained sustainably enhanced.
  • the leptin secretion was increased by 40% (Figure 5(B)) (p ⁇ 0.001) compared to untreated cells with a concomitant decrease of 43% (Figure 5(D)) in the triglyceride levels (p ⁇ 0.05).
  • Figure 5(B) p ⁇ 0.001
  • Figure 5(D) the triglyceride levels
  • Example 5 Leptin secretion and triglyceride accumulation in long term culture of adipocytes under low glucose condition
  • TRF sample is a proprietary self-emulsifying delivery system of TRF containing tocotrienols and tocopherol, with an emulsifier (e.g. Span-20 & Tween 80) and an oil carrier (e.g. glycerol trioleate).
  • an emulsifier e.g. Span-20 & Tween 80
  • an oil carrier e.g. glycerol trioleate
  • Rats were fed 18.75mg/kg/day body weight of the TRF sample (which is a human equivalence dose of 182.43mg/day for a 60kg adult) for a period of 8 weeks. This is also a dose that is well within the recommended doses (no-observed-adverse-effect level or NOAEL dose) for rats.
  • the average rat in this experiment weighed ⁇ 400g, and these rats consume approximately 25ml of water per day for a 400g rat body weight based on our in-house observations.
  • One cage holds two rats and can hold two water bottles. And each water bottle holds 250ml of water. Results are cumulated and reported fortnightly, thus the effective drink volume in 2 weeks for 1 rat would be expected to be 350ml.
  • the effective drink volume in 2 weeks for 1 rat would be expected to be 350ml.
  • TRF 37Sm.g TRF to be added into sack, water bottk the equation above was used to arrive at 375 mg TRF sample that was added to water bottles for the rat consumption.
  • the TRF delivery system was composed of 20% pure TRF, thus this was factored into the equation above. Bottles were checked every alternate day to calculate the drink volume as well as ensure appropriate fluid consumption (to ensure rats were not dehydrated) by the rats. Bottles with TRF sample were also reconstituted fresh each time before being placed into the cages.
  • tissue lysis buffer 50mM pH7.4, 150mM NaCl, 0.1% sodium dodecyl sulphate (SDS), lOmM ethylenediaminetetraacetic acid (EDTA), 1 % NP-40 (v/v) and Complete Protease Inhibitor Cocktail (Roche)
  • the tissues were homogenized using a sonicator.
  • the samples were then spun down on a tabletop centrifuge 13,000 rpm for 10 minutes at 4°C. The supernatant was aspirated carefully and pipetted into a new Eppendorf tube.
  • the protein levels in the supernatant were quantified using a Pierce BCA Protein Assay kit according to the manufacturer's protocol.
  • the purified tissue lysates were then subjected to leptin quantification using a Leptin ELISA kit (Crystal Chem Inc. #90030) according to the manufacturer's protocol.
  • the resultant data were normalised to the protein content in the cells (based on the BCA protein quantitation done earlier).
  • the results were subjected to an unpaired student's t-test to compare significant differences between the 2 groups (treated with TRF and non-treated water control) and p-value taken to be significant ⁇ 0.05.
  • Example 7 An oral formulation of the composition for regulating appetite
  • Table 2 shows an example of the composition in the dosage form of capsule.

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Abstract

The invention relates to a composition for regulating appetite in a subject comprising a plant-derived tocotrienol or derivative thereof selected from a tocotrienol-rich fraction, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol or a combination of any two or more thereof. The invention also relates to a method for regulating appetite in a subject comprising a step of administering to a subject an effective amount of the composition, in which the regulation of appetite can be achieved by increasing leptin expression, secretion or the combination thereof in adipocytes. The composition of the invention may also be used in the manufacture of a medicament for regulating appetite or promoting satiety.

Description

A COMPOSITION FOR REGULATING APPETITE AND A METHOD
THEREOF
FIELD OF INVENTION
The invention relates to a composition for regulating appetite and promoting satiety. In more particular, the invention relates to a composition for maintaining a steady and balanced appetite containing a naturally-derived active compound, and a method thereof. The composition is useful in promoting satiety and managing body weight.
BACKGROUND OF THE INVENTION
Obesity is one of the largest healthcare problems facing the developed countries, as well as the world today. Nations that were historically grappling with undernutrition and starvation, such as India and China, are now struggling with obesity problems. For instance, China, for the first time, reported that obesity kills more people than undernutrition does. These emerging nations have seen huge rise in a middle class to affluent population. The obesity problem has become more prevalent in recent years owing to lifestyle related issues, including unhealthy diet such as consumption of processed foods and fast foods with massive amounts of saturated fats and oil, as well as sedative lifestyle and lack of regular exercise. In addition, a stress related lifestyle can often exacerbate these problems. Obesity has also been identified as a risk factor for the development of type 2 diabetes mellitus, in which more than 85% of individuals with type 2 diabetes mellitus are obese. It is estimated that the exponential growth of obesity will substantially attributable to major changes in the number and average ages of the population of many countries, for instance India and China.
This huge incidence of obesity has prompted a lot of public awareness campaigns to encourage a healthy lifestyle that includes healthy food and regular exercise. Many also attempt to reduce the caloric intake by following diet plans or consuming supplements that encourage weight loss. Some of these diet fads may lead to rapid weight loss; however, the dieters often regain the lost mass within a short period, or sometimes even exceeding the weight gain prior to the beginning of the diet plan. Therefore, whilst there are many weight loss management strategies in the art, the most challenging aspect in managing weight is often not in losing weight, but sustaining the weight loss. This has often been attributed to the sudden drop in fat mass which is the primary producer of leptin. It has been suggested in the art that this weight-reduced state is a state of leptin deficiency.
Leptin is a hormone that is almost largely secreted by adipocytes, that regulates satiety or appetite by interacting with receptors in the brain. Other tissues in the body have also been identified to contribute to leptin secretion in the body. For example, the stomach is also known as an organ which secrets considerable amount of leptin. Other sources include the placenta and ovaries in females, skeletal muscle and the pituitary gland. Low levels of leptin in the abovementioned weight-reduced state can be interpreted as a state of hunger by the brain and so lead to increase of appetite and hence the resulting hyperphagia or overeating behavior. Accordingly, reduced fat mass in the body of a subject would lead to a sudden drop in leptin levels which then trigger the well-known phenomenon of binge eating. Therefore, a quick relapse could occur whereby the subject would regain his/her original obese state, or even a more severe obese state. Therefore, one of the approaches to remedy such issues of leptin deficiency-induced weight gain is through regulation of appetite, in order to prevent the state of hunger and hence eliminate the need to binge eat experienced by the brain. Such regulation of appetite can be achieved by a number of methods, including increase of leptin level in the adipocytes.
There are a number of technologies disclosed in the art relating to composition or method for combating obesity or managing weight, in which a naturally-derived active compound is applied as the constituent of the composition. For example, U. S. patent publication no. US7399784B2 discloses an anti-obesity medicament containing a phytyl substituted chromanol and an obesity-promoting drug, wherein the phytyl substituted chromanol is a tocopherol or a tocotrienol. However, it is disclosed in this patent document that the composition inhibits or reduces triglyceride accumulation in adipocytes and promotes a resistance to excess triglyceride accumulation in adipocytes, to prevent or treat obesity or undesirable weight gain. The phytyl substituted chromanol used in the composition is to counteract the side effect of the obesity-promoting drug. There is no disclosure provided in this document to prevent obesity or manage weight gain by regulating of appetite or leptin level.
Another U. S. patent publication no. US 2013022240 A 1 also discloses a method for reducing weight using a composition comprising fucoxanthin extract, tocotrienols, fucoidan or a combination thereof. It is disclosed in this document that the weight reducing effect of the fucoxanthin-comprising composition is based on fat metabolism. It is however not disclosed in this document on how the fucoxanthin extract, tocotrienols and fucoidan inter-reacted among each other to achieve such weight reducing effect. There is also no disclosure on managing or reducing weight through regulating of appetite or leptin level.
From the prior art documents, it is known that vitamin E, including tocopherols and tocotrienols, has been used in the manufacture of medicament or supplement for weight management, either as one of the essential or optional ingredients. However, most of the prior art documents disclose tocopherol- or tocotrienol-containing composition or method for reducing weight or combating obesity thereof via regulation or alteration of fat metabolism of a subject. Therefore, new compositions or methods specifically developed using naturally-derived compound, such as vitamin E, for use in regulating appetite are therefore desirable.
SUMMARY OF INVENTION One of the objects of the invention is to provide a composition for naturally regulating satiety or appetite, particularly by promoting satiety, via leptin enhancement in the body of a subject. The invention also aims to provide a method to regulate, for example, to promote satiety by using naturally-derived active compound, in order to prevent uncontrolled hunger pangs that may result by massive weight loss after dieting, so that the weight loss after dieting can be sustainably maintained.
At least one of the proceeding objects is met, in whole or in part, by the invention, in which the one of the embodiments of the invention describes a composition for regulating appetite in a subject comprising a plant-derived tocotrienol or derivative thereof selected from a tocotrienol-rich fraction (TRF), alpha-tocotrienol (a- tocotrienol), beta-tocotrienol (β-tocotrienol), gamma-tocotrienol (γ-tocotrienol), delta- tocotrienol (δ-tocotrienol) or a combination of any two or more thereof.
According to one embodiment of the invention, the tocotrienol or derivative thereof is derived from palm, annatto, grape, rice, wheat, barley, rye, oat or a combination of any two or more thereof.
In accordance with another embodiment of the invention, the tocotrienol or derivative thereof contains 50% to 99% of total tocotrienol content by weight.
In accordance with a preferred embodiment of the invention, the tocotrienol or derivative thereof is a TRF derived from palm, containing 50% to 99% of total tocotrienol content by weight. In certain embodiments, the composition comprises essentially of a TRF derived from palm, containing 50% to 99% of total tocotrienol content by weight.
According to another embodiment of the invention, the TRF contains a-tocopherol, a- tocotrienol, β-tocotrienol, γ-tocotrienol, δ-tocotrienol, phytosterol, squalene, fatty acid or a combination of any two or more thereof.
Still another embodiment of the invention discloses that the composition is in the form of an oral, intravenous or intradermal preparation.
In a further embodiment of the invention, the appetite regulation in the subject is achieved by increasing leptin expression, secretion or the combination thereof in adipocytes.
A further embodiment of the invention discloses a method for managing, for example, reducing the body weight of a subject comprising a step of administering to a subject an effective amount of a composition according to the foregoing embodiments. The method can be a non-therapeutic method.
Another further embodiment of the invention discloses a method for regulating appetite in a subject comprising a step of administering to a subject an effective amount of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a- tocotrienol, β -tocotrienol, γ-tocotrienol, δ-tocotrienol or a combination of any two or more thereof. In certain embodiments, the method can be in vivo or in vitro.
Yet another further embodiment of the invention discloses use of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a- tocotrienol, β -tocotrienol, γ-tocotrienol, δ-tocotrienol or a combination of any two or more thereof, in the manufacture of a medicament for regulating appetite in a subject.
The present preferred embodiments of the invention consist of novel features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings and particularly pointed out in the appended claims; it being understood that various changes in the details may be effected by those skilled in the arts but without departing from the scope of the invention or sacrificing any of the advantages of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS For the purposes of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.
Figure 1 shows (A) a light microscopy image of TRF-treated adipocytes (20μΜ, also the IC50 concentration for TRF in cells) (8 days short term culture) under high glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), **p<0.01; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF-treated adipocytes (right) in comparison to the control (left), *p<0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05.
Figure 2 shows (A) a light microscopy image of a-tocopherol-treated adipocytes
(20μΜ) (8 days short term culture) under high glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such a-tocopherol- treated adipocytes (right) in comparison to the control (left); (C) a photograph of such α-tocopherol-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such α-tocopherol-treated adipocytes (right) in comparison to the control (left), as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05. shows (A) a light microscopy image of TRF-treated adipocytes (20μΜ) (8 days short term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), **p<0.01; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF-treated adipocytes (right) in comparison to the control (left), *p<0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05. shows (A) a light microscopy image of a-tocopherol-treated adipocytes (20μΜ) (8 days short term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such a-tocopherol- treated adipocytes (right) in comparison to the control (left); (C) a photograph of such α-tocopherol-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such α-tocopherol-treated adipocytes (right) in comparison to the control (left), as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05.
shows (A) a light microscopy image of TRF-treated adipocytes (20μΜ) (21 days long term culture) under high glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), ***p<0.001; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF- treated adipocytes (right) in comparison to the control (left), *p<0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05.
Figure 6 shows (A) a light microscopy image of a-tocopherol-treated adipocytes
(20μΜ) (21 days long term culture) under high glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such a-tocopherol- treated adipocytes (right) in comparison to the control (left); (C) a photograph of such α-tocopherol-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such α-tocopherol-treated adipocytes (right) in comparison to the control (left), as described in one embodiment of the invention.
Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05.
shows (A) a light microscopy image of TRF-treated adipocytes (20μΜ) (21 days long term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such TRF-treated adipocytes (right) in comparison to the control (left), *p<0.05; (C) a photograph of such TRF-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such TRF-treated adipocytes (right) in comparison to the control (left), *p<0.05, as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05.
Figure 8 shows (A) a light microscopy image of α-tocopherol-treated adipocytes (20μΜ) (21 days long term culture) under low glucose condition; (B) a graph of leptin secretion (expressed as ng of leptin per μg protein secreted into the extracellular environment) by such a-tocopherol- treated adipocytes (right) in comparison to the control (left); (C) a photograph of such a-tocopherol-treated adipocyte culture tested by oil Red O stain for lipids; and (D) a graph of triglyceride accumulation by such a-tocopherol-treated adipocytes (right) in comparison to the control (left), as described in one embodiment of the invention. Statistical significance was determined by Student's unpaired t-test with statistical significance taken at p<0.05.
Figure 9 is a graph showing leptin levels (expressed as ng of leptin per μg protein in adipose tissue) in adipose tissues of 2 groups of rats fed with an enhanced delivery system of TRF sample (n=8) and water only (n=8) as quantified by ELISA, in which the results were statistically significant (*p<0.05) and were obtained by unpaired t-tests, as described in one embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the invention shall be described according to the preferred embodiments of the invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.
The invention discloses a composition for regulating appetite in a subject comprising a plant-derived tocotrienol or derivative thereof selected from a TRF, a-tocotrienol, β- tocotrienol, γ-tocotrienol, δ-tocotrienol or a combination of any two or more thereof. In certain embodiments of the invention, the composition can also be known as an appetite reducer or an appetite regulator.
By the term "regulating appetite", it refers to regulating or preventing food craving and extreme hunger sensation by promoting satiety or fullness. As the appetite regulating or reducing effect of the composition of the invention can be reasonably sustainable, it also assists in maintaining the body weight or sustaining the weight loss in a certain rate. It is to be appreciated that the composition does not reduce the appetite of a subject in a massive way, but it stabilizes and keeps the satiety balance in the subject to prevent the subject from overeating, to compensate for the sudden drop in fat mass during weight loss.
As set forth in the preceding description, the tocotrienol or derivative thereof used in the composition can be a TRF. TRF is a fraction obtained from a purification process of a naturally occurring vitamin E-containing compound extracted from a plant source, for example palm oil. The purification process applied can be a molecular distillation process, which is capable of producing TRF with different concentrations of vitamin E (tocotrienols and tocopherols) (e.g. 30, 50, 70, 95%) by removing impurities (e.g. glyceride, sterols, etc). In other words, the more impurities being removed from the TRF, the higher purity of TRF can be produced. Hence, TRF contains a relatively higher concentration of tocotrienol as compared to a naturally occurring fraction of its own source which has not gone through the purification process.
Preferably, the TRF used can contain approximately 50% to 99% of total tocotrienol content. This TRF can be obtained from various sources. According to one of the embodiments of the invention, the TRF can be derived from palm, annatto, grape, rice, wheat, barley, rye, oat or a combination of any two or more thereof. Preferably, the TRF used in the composition is derived from palm {Elaeis guineensis jacq.), for example, from palm oil. It is to be appreciated that, the proportion of the contents of vitamin E, i.e. tocotrienols and tocopherols, is naturally varied among different sources. Therefore, the TRF produced from different sources of plant can contain different proportions of tocotrienols to tocopherols. For example, a 95% purity of TRF produced from palm oil may contain approximately 70% of tocotrienols and 25% of a-tocopherol. As another example, a 70% purity of TRF produced from palm oil can contain approximately 50% of tocotrienols and 20% a-tocopherol. More preferably, a TRF containing 70% to 99% of total tocotrienol content is used in the composition. Asides from tocotrienols and tocopherols, the TRF can also contain trace amount of plant phytosterols, squalene, fatty acid (e.g. monoglycerides, diglycerides or triglycerides) and others. According to one preferred embodiment of the invention, the TRF used in the composition contains α-tocopherol, a-tocotrienol, β-tocotrienol, γ-tocotrienol and δ- tocotrienol, in a specific proportion. For example, the TRF contains approximately 5% to 25% of α-tocopherol and 50% to 95% of tocotrienols, including a-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol.
As a constituent in the composition of the invention, the tocotrienol or derivative thereof is present in an amount of approximately 0.01% to 50%, for example 0.01% to 50% of TRF, by weight of the composition. In a suitable form of preparation, the composition can contain approximately 50% to 99.99% of other active ingredients, or carrier, excipient or the combination thereof.
In certain embodiments, the composition comprises essentially of palm-derived TRF with 50% to 99% of total tocotrienol content by weight. Preferably, the composition can contain essentially of 0.01% to 50% of such palm-derived TRF, without other additional active ingredient for regulating appetite, promoting satiety and/or regulating body weight. In a suitable form of preparation, the composition can contain approximately 50% to 99.99% of other active ingredients, or carrier, excipient or the combination thereof. In accordance with one embodiment of the invention, the composition can be in the form of an oral, intravenous or intradermal preparation. In certain embodiments, the oral preparation can be a solid, semi-solid or liquid preparation; while the intravenous or intradermal preparation can be of semi-solid or liquid preparation. These preparations can be a water-soluble or an oil-soluble preparation.
According to certain embodiments of the invention, the oral preparations can include the form of pill, capsule, tablet, powder, lozenges, dragees, granules, pellets, beads, paste, syrup, solution, suspension, shake, cream or emulsion. These oral preparations can also be directly prepared and stored in a solid or semi-solid dosage form for consumption. They can also be prepared and stored in the dosage form of capsule, powder sachet, pellet or effervescent tablet for reconstitution into water or solvent before oral consumption or other administration methods. In certain embodiments, they can also be prepared or incorporated into foodstuffs or beverage for easy consumption. For example, these oral preparations can be prepared in the form of a dietary supplement, a nutrition snack bar, a slimming tea, a meal replacement drink, or others. The composition can also be formatted into a sustained-release formulation.
In accordance with another embodiment, the composition can be in the form of an intravenous or intradermal preparation, such as solution, suspension, emulsion, or any other dosage forms suitable for injection. It could be appreciated that the composition as described in the embodiment of the invention is a nutraceutical composition. Accordingly, this nutraceutical composition can contain one or more carrier, excipient or the combination thereof. As set for in the foregoing description, the carrier, excipient or the combination thereof can be present in an amount of 50% to 99.99% by weight of the composition. In particular, the carrier or excipient can be a stabilizing agent, binding agent, filling agent, emulsifying agent, wetting agent, suspending agent, lubricating agent, dispersing agent, diluent, desiccant, solubiliser, flavouring agent, colouring agent, preservative or a combination of any two or more thereof. In certain embodiments, the composition can further comprise one or more other biologically active agents, for example, biologically active agents with therapeutic effects suitable for regulating appetite, promoting satiety, managing body weight, combating obesity and/or their related metabolic diseases or disorders. These biologically active agents can also be naturally derived from plants or vegetables. They can be an additional satiety or appetite regulating agent, a fat or carbohydrate absorption altering agent, a metabolism altering agent, a calories burning agent, a detoxification agent, an enzyme, an antioxidant, a bioflavonoid, a phytonutrient, an essential oil, a polyphenol, a vitamin, a mineral or a combination of any two or more thereof.
In certain other embodiments, the composition can also further comprise a pharmaceutically active agent or drug suitable for regulating appetite, promoting satiety, managing body weight, combating obesity and/or their related metabolic diseases or disorders.
The composition for regulating appetite can reduce appetite in the subject by increasing leptin expression, secretion or the combination thereof in adipocytes, according to the embodiments of the invention. By the terms "increasing leptin expression", it refers to increasing of gene and protein expression of the hormone leptin in the adipocytes (also known as fat cells); while "increasing leptin secretion" refers to increasing of the export of intracellularly synthesized leptin into the extracellular environment and ultimately the blood circulatory system. Without wishing to be bound by the theory, it is believed that the composition of the invention containing the TRF is capable of increasing or upregulating of leptin expression in the adipocytes, and hence resulting in an increase in leptin levels in the adipocytes as well as the endocrine system.
A further embodiment of the invention discloses a non-therapeutic method of managing, for example, reducing the body weight of a subject comprising a step of administering to a subject an effective amount of a composition according to the foregoing embodiments. The non-therapeutic method can be a cosmetic method of managing or reducing body weight.
Apart from the appetite regulating composition described, another further embodiment of the invention also discloses a method for regulating appetite in a subject comprising a step of administering to a subject an effective amount of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a-tocotrienol, β -tocotrienol, γ-tocotrienol, δ-tocotrienol or a combination of any two or more thereof. In certain embodiments, the method can be in vivo or in vitro. In both in vitro and in vivo analysis, the increase of leptin level in adipocytes can be observed.
As set forth in the preceding description, the composition can be administered orally, intravenously or intradermally. Accordingly, the composition can be administered in the forms of oral preparation such as pill, capsule, tablet, powder, lozenges, dragees, granules, pellets, beads, paste, syrup, solution, suspension, shake, cream or emulsion for oral consumption; or in the form of an intravenous or intradermal preparation, such as solution, suspension, emulsion, or any other dosage forms suitable for injection. In certain embodiments, the composition can also be prepared and stored in the dosage form of capsule, powder sachet, pellet or effervescent tablet for reconstitution into water or solvent before administered via oral consumption or other administration methods. As set forth in the foregoing embodiments, the composition can be administered alone, or to be presented as a nutraceutical composition which contains one or more carrier, excipient or the combination thereof. Preferably, the composition can contain essentially of 0.01% to 50% of such palm-derived TRF, with or without any other additional active ingredient for regulating appetite, promoting satiety and/or regulating body weight.
In accordance with the embodiment of the invention, the composition can be administered to the subject in need once or twice a day, or as necessary. In the form of a nutraceutical composition containing the composition as set forth in the foregoing description, determination of an appropriate dosage for a particular appetite-related disease or disorder, or metabolic condition is within the skill of the art. For example, for therapeutic applications for eating disorder such as hyperphagia, bulimia nervosa or obesity, a person having ordinary skill in the art, such as a nutritionist, can readily determine and prescribe the effective amount of the composition required for regulating the appetite of the subject, in order to achieve the desired effects. According to the preferred embodiment of the present invention, the subject is a human. In other embodiments, the subject can be a mammal other than a human, for example a primate, a canine, a feline, an ursine, an equine, a leporine or a rodent. In other embodiments, the subject can be an adipocyte or a fat cell. In accordance with certain embodiments of the invention, the regulation of appetite of the subject can be achieved by increasing leptin expression, secretion or the combination thereof in adipocytes. Without wishing to be bound by theory, it is believed that the addition of the TRF-containing composition as described in the embodiments of the invention can lead to significant enhancement in leptin secretion. Exemplary methods of the leptin enhancement effect of the TRF-containing composition are further detailed in Examples 1 to 5, using adipocytes cultured in both short term and long term conditions, in both high and low glucose conditions. According to one embodiment of the invention, the increase of leptin secretion in adipocytes of a subject treated with the composition of this invention can be in a rate of 10% to 50% as compared to that of an untreated subject.
Adipocytes or fat cells, which have been previously known to be fat storage tissues, are now increasingly becoming appreciated for its endocrine functions, i.e. for secreting many important hormones crucial for metabolic homeostasis, including leptin. Leptin and leptin receptor signaling are most crucial of the factors identified to date that coordinate the control of food intake and energy expenditure in response to an altered energy state. Leptin can act on the hypothalamic arcuate nucleus neurons producing signaling compounds such as anorexigenic proopiomelanocortin (POMC) and orexigenic neuropeptide Y (NPY)/Agouti-related protein (AgRP). Under certain conditions, leptin suppresses energy intake and stimulates energy expenditure, leading to a reduction in stored body fat. Without wishing to be bound by theory, it is believed that the increase of levels of leptin in the adipocytes may trigger the signaling pathway of leptin in the central nervous system, particularly, in the hypothalamus, for the system to signal the sensation of satiety in a subject.
In accordance with certain embodiments, JAK-STAT signaling is a representative signaling pathway through which leptin regulates food intake and energy homeostasis in the hypothalamus. Leptin can bind to its receptors and initiates downstream signaling through the sequential phosphorylation of the tyrosine kinase Janus kinase (JAK) and the transcription factor signal transducer and activators of transcription (STAT). The phosphorylation of STAT induces its dimerization and translocation to the nucleus, where it binds DNA and modulates the transcription of genes involved in food intake and energy homeostasis.
Example 1 shows a method of preparation of adipocyte cell line and culture conditions according to one embodiment of the invention, in which a preadipocytic cell line that undergo a high efficiency of differentiation when induced to undergo adipogenesis can be used, for example, 3T3-L1 murine preadipocytes. In the experiment of studying the leptin secretion enhancement effect of TRF, a test sample of a-tocopherol can also be included in the study as a comparison to the effect of TRF. As set forth in the foregoing description, the culture conditions prepared can include both the long term and short term conditions, as the long term cultures are crucial in demonstrating that the leptin enhancement from adipocytes is sustainable, given that a key aim for use of TRF-containing composition is to maintain enhanced leptin secretion. High and low glucose conditions can be used, in which low glucose conditions can represent the blood glucose level of a normal health individual, whereas the high glucose media can be relevant in revealing if the leptin enhancement effect can also be observed in diabetic individuals or those with poor glycemic control.
The leptin secretion and triglyceride accumulation in short term culture of adipocytes under high glucose condition is shown in Example 2. An unpaired t-test analysis can be utilized to assess for statistical significance. As shown in Figure 1, the TRF treatment in high glucose condition exhibits greater leptin secretion and a subsequent lower triglyceride accumulation in the adipocytes as compared to the untreated cells, in which the difference shown is significant (p<0.01). In the experiment conducted under low glucose condition as illustrated in Example 3 and Figure 3, a similar significant difference of p<0.01 can be observed, indicating that the TRF-treated adipocytes also exhibit greater leptin secretion and lower triglyceride accumulation under low glucose condition. On the contrary, there is no significant difference in leptin secretion and triglyceride accumulation observed between the a-tocopherol- treated adipocytes and the untreated cells, as illustrated in Figures 2 and 4. Exemplary method of the leptin secretion and triglyceride accumulation in long term culture of adipocytes under high glucose condition and low glucose conditions are respectively shown in Examples 4 and 5. It can be illustrated in Figure 5 that leptin secretion remains sustainably enhanced when high glucose condition is used to culture the adipocytes under long term culture condition treated with TRF From Figure 7, the TRF-treated adipocytes also show the enhanced leptin secretion in low glucose conditions. This data suggests that the enhancement in leptin secretion observed in the short term culture condition can also be sustained at comparable levels in a long term culture condition when adipocytes are treated with TRF. Therefore, the enhanced leptin secretion and concomitant decrease in triglycerides in the TRF- treated cells are not a short-lived effect. Such significant leptin secretion and concomitant decrease in triglycerides could however not be seen in the a-tocopherol- treated cells, as illustrated in Figures 6 and 8.
Additionally, the findings of the in vitro experiments can also be corroborated with in vivo animal experiments (for example, rat study) by demonstrating the elevated levels of leptin in adipose tissues. Example 6 shows an exemplary method of animal model (rat) studies and protein assays from adipose tissues of the euthanized rats. From the data illustrated in Figure 9, leptin levels can be observed to be significantly increased by approximately 50% (p<0.05) within the adipose tissue itself, as compared to unsupplemented control groups. An enhanced delivery system of TRF composition, which is a water-soluble TRF formulation suitable for rat consumption, can be used in the animal study. According to a preferred embodiment of the invention, the TRF composition used can contain an emulsifier, an oil carrier or a combination thereof, for effective delivery of the tocotrienols and tocopherols.
As shown in Examples 1 to 6, the method as described in the embodiment of the invention can naturally enhance leptin secretion from adipocytes treated with the TRF-containing composition, under both high and low glucose conditions, indicating that the TRF-containing composition is effective in both diabetic and normal individuals respectively, and such effects are sustainable at a reasonable level. As shown by the experimental data, the TRF-containing composition is also capable of decreasing triglyceride accumulation in the samples of adipocytes tested, asides from increasing leptin secretion.
In certain embodiments, the enhancement of leptin secretion by the composition of the invention can result in the promotion of satiety or fullness, lowering of calorie intake, decrease of blood glucose levels, lowering of blood cholesterol level, decrease of body fat, and others. This method is potentially helpful in managing of body weight, thus combating obesity. As obesity is one of the biggest risk factors for diabetes and cardiovascular-related morbidities, this method indirectly helps to reduce the risk of developing these diseases. The regulation of appetite can hence help to maintain the overall health of the gastrointestinal and circulatory system of the subject.
The invention also discloses use of a composition containing a plant-derived tocotrienol or derivative thereof selected from a TRF, a-tocotrienol, β-tocotrienol, γ- tocotrienol, δ-tocotrienol or a combination of any two or more thereof, in the manufacture of a medicament for regulating appetite in a subject, according to yet another embodiment. Preferably, the medicament can be an oral preparation containing TRF, for example, a dietary supplement pill for regulating appetite or promoting satiety. Preferably, the composition can contain an emulsifier, an oil carrier or a combination thereof; in which the emulsifier can be a non-ionic surfactant (e.g. Polysorbate 80), sorbitan monolaurate (e.g. Span 20), while the oil carrier can be a vegetable oil (e.g. soybean oil, olive oil or glycerol trioleate). An example of the oral formulation is further detailed in Example 7. While the invention has been disclosed in connection with the preferred embodiments shown and described in detail, various modifications and improvements thereon will become readily apparent to those skilled in the art. Accordingly, the scope of the invention is not to be limited by the following non-limiting examples, but is to be understood in the broadest sense allowable by law.
EXAMPLE
Example 1 Preparation of adipocyte cell line and culture conditions
3T3-L1 murine preadipocytes were grown in Dulbecco's modified Eagle's medium (DMEM) high glucose (HG) at 4.5g/L containing 10% newborn calf serum (NCS) (heat inactivated at 65°C for 30 minutes). Cells were seeded at 1.0 x 10 4 cells/cm 2. Two days after reaching confluence, cultures were induced to differentiate in DMEM HG at 4.5g/L or low glucose (LG) at l.Og/L containing 10% FBS, supplemented with ΙμΜ insulin, 0.5mM 3-isobutyl-l-methyl-xanthine (IB MX), and ΙμΜ dexamethasone for 2 days and then supplemented with ΙμΜ insulin alone for 2 days. After 4 days, cells were cultured in DMEM containing 10% FBS and with media change every 2 days subsequently. TRF with purity 95% was used. This TRF contains 21.1% alpha- tocopherol, 22.5% alpha-tocotrienol, 2.7% beta-tocotrienol, 36.7% gamma-tocotrienol and 11.8% delta-tocotrienol. TRF were supplemented every 2 days from the start of differentiation until the end of differentiation. A test sample of a-tocopherol (aTP) was also included in the study as a comparison to the effect of TRF.
Cells were differentiated and prepared under the conditions as listed in Table 1.
Table 1
Test samples Culture conditions
TRF Short term culture (8 days)
High glucose (4.5g/L)
TRF Short term culture (8 days)
Low glucose (l.Og/L) TRF Long term culture (21 days)
High glucose (4.5g/L)
TRF Long term culture (21 days)
Low glucose (l.Og/L)
a-tocopherol Short term culture (8 days)
High glucose (4.5g/L)
a-tocopherol Short term culture (8 days)
Low glucose (l.Og/L)
a-tocopherol Long term culture (21 days)
High glucose (4.5g/L)
a-tocopherol Long term culture (21 days)
Low glucose (l.Og/L)
As normal human blood glucose levels are approximately 5mmol/L, a closer amount of glucose of the l.Og/L was hence used in this experiment as the low glucose medium. Whilst, the standard culture medium containing 4.5g/L glucose is of 4.5 times higher than normal glucose levels, which mimics the condition of a diabetic individual or someone with poor glycemic control.
Example 2 Leptin secretion and triglyceride accumulation in short term culture of adipocytes under high glucose condition
The short term culture (STC) data indicate that there was not only an increased amount of leptin secretion by TRF but also a concomitant reduction in the triglycerides level in the cells (Figure 1). The data representing 3 independent experiments (n=3) was shown in Figures 1 and 2. Student's unpaired t-test analysis was utilized to assess for statistical significance, in which differences were considered statistically significant when p<0.05. By using commercially available ELISA kits from Crystal Chem (Catalogue No: 90030) specific for leptin, a 30% increment of leptin secretion into the cell culture media (Figure 1(B)) (p<0.01) was observed as compared to untreated cells. Oil Red O stain for lipids was applied. Additionally, a 62% reduction of triglycerides was also observed as compared to untreated cells (Figure 1(D)) using a commercially available kit from Abeam (Catalogue No: ab65336) (p<0.05), in the TRF-treated cells, compared to untreated cells. When aTP was used under same conditions, there was no significant change in leptin secretion and triglyceride levels observed as compared to untreated cells (Figure 2). Example 3 Leptin secretion and triglyceride accumulation in short term culture of adipocytes under low glucose condition The experimental method of Example 2 was applied, in which the HG was substituted by LG condition. When LG conditions were used to culture the adipocytes under STC, leptin secretion continued to stay enhanced while triglyceride levels were lowered, similar to what was observed in the STC-HG condition. We observed a 40% increment in leptin secretion (p<0.01) (Figure 3(B)) and a 60% reduction in triglyceride levels (p<0.05). (Figure 3D) compared to untreated cells. The leptin secretion was even higher than when cultured under HG conditions, suggesting that the excessive glucose in the medium may in fact impair leptin secretion. Under the same conditions with aTP treatment, we found no significant changes in leptin and triglyceride levels compared to untreated cells (Figure 4). Although a 70% increase in leptin was observed, this was not found to be statistically significant (p=0.20) given the large variance observed (Figure 4(D)).
Example 4 Leptin secretion and triglyceride accumulation in long term culture of adipocytes under high glucose condition
The experimental method of Example 2 was applied, in which the culture was differentiated for 21 days was used. When HG conditions were used to culture the adipocytes under LTC, leptin secretion remained sustainably enhanced. In fact, the leptin secretion was increased by 40% (Figure 5(B)) (p<0.001) compared to untreated cells with a concomitant decrease of 43% (Figure 5(D)) in the triglyceride levels (p<0.05). This data suggests that what was seen after 8 days of inducing differentiation is also sustained at comparable levels at 21 days of differentiation. Thus, the enhanced leptin and concomitant decrease in triglycerides is not only a short-lived effect. On the other hand, when aTP was used under same conditions, we found no significant changes in leptin secretion nor triglyceride levels compared to untreated cells (Figure 6). The triglyceride levels were in fact 15% higher with aTP treatment compared to untreated cells, although this was not statistically significant (p=0.10) (Figure 6(D)).
Example 5 Leptin secretion and triglyceride accumulation in long term culture of adipocytes under low glucose condition
The experimental method of Example 4 was applied, in which the HG was substituted by LG condition. Under LG conditions, leptin secretion was significantly enhanced with a 20% increase compared to untreated cells although (p<0.05) (Figure 7(B)). There was a concomitant decrease of 40% in triglyceride levels and this was found to be statistically significant (p<0.05). Under the same conditions with aTP treatment, we found no significant changes in leptin secretion and triglyceride levels compared to untreated cells (Figure 8). The triglyceride levels were in fact 15% higher with aTP treatment compared to untreated cells, although this was not statistically significant (p=0.20) (Figure 8(D)).
Example 6 Animal model (rat) studies and protein assays from adipose tissues
Four- month-old rats were primed on a Vitamin E stripped diet (Research Diets Inc., D 13022804) for a period of 4 weeks. This 4-week priming was essential to eliminate all traces of background a-tocopherol normally incorporated in the rodent chow diet. A total of 8 rats were fed a TRF sample mixed in the drinking water and 8 control rats were fed only water as control. The TRF sample is a proprietary self-emulsifying delivery system of TRF containing tocotrienols and tocopherol, with an emulsifier (e.g. Span-20 & Tween 80) and an oil carrier (e.g. glycerol trioleate). Rats were fed 18.75mg/kg/day body weight of the TRF sample (which is a human equivalence dose of 182.43mg/day for a 60kg adult) for a period of 8 weeks. This is also a dose that is well within the recommended doses (no-observed-adverse-effect level or NOAEL dose) for rats. The average rat in this experiment weighed ~400g, and these rats consume approximately 25ml of water per day for a 400g rat body weight based on our in-house observations. One cage holds two rats and can hold two water bottles. And each water bottle holds 250ml of water. Results are cumulated and reported fortnightly, thus the effective drink volume in 2 weeks for 1 rat would be expected to be 350ml. Thus, to achieve a 18.75mg/kg body weight dose of TRF sample daily,
IS.lS-mg TRF τ 100% enhanced deliver-
X QAkg average rat weight X
k.Q (20% pure TRF)
250ml total water in bottle
X
25 ml consumed per rat daily
= 37Sm.g TRF to be added into sack, water bottk the equation above was used to arrive at 375 mg TRF sample that was added to water bottles for the rat consumption. The TRF delivery system was composed of 20% pure TRF, thus this was factored into the equation above. Bottles were checked every alternate day to calculate the drink volume as well as ensure appropriate fluid consumption (to ensure rats were not dehydrated) by the rats. Bottles with TRF sample were also reconstituted fresh each time before being placed into the cages.
At the end of the experiment (8 weeks), all the rats were euthanized and lOOmg of their gonadal (epididymal adipose) tissue collected. The gonadal adipose tissue makes up the largest fat depot in the rodent, comprising 30% of all dissectible fat, and thus makes a good representation of the fat content. The lOOmg of tissue was placed in an Eppendorf tube containing tissue lysis buffer (50mM pH7.4, 150mM NaCl, 0.1% sodium dodecyl sulphate (SDS), lOmM ethylenediaminetetraacetic acid (EDTA), 1 % NP-40 (v/v) and Complete Protease Inhibitor Cocktail (Roche)), pre-chilled on ice. The tissues were homogenized using a sonicator. The samples were then spun down on a tabletop centrifuge 13,000 rpm for 10 minutes at 4°C. The supernatant was aspirated carefully and pipetted into a new Eppendorf tube. The protein levels in the supernatant were quantified using a Pierce BCA Protein Assay kit according to the manufacturer's protocol. The purified tissue lysates were then subjected to leptin quantification using a Leptin ELISA kit (Crystal Chem Inc. #90030) according to the manufacturer's protocol. The resultant data were normalised to the protein content in the cells (based on the BCA protein quantitation done earlier). The results were subjected to an unpaired student's t-test to compare significant differences between the 2 groups (treated with TRF and non-treated water control) and p-value taken to be significant <0.05.
Leptin levels in the adipose tissue were significantly increased by 50% (p<0.05), compared to unsupplemented control groups (Figure 9). as quantified by ELISA. Two groups of rats fed with either TRF(n=8), or water only (n=8) were analyzed for leptin protein levels within adipose tissues. The results were subjected to an unpaired student's t-test and p-value taken to be significant <0.05.
Example 7 An oral formulation of the composition for regulating appetite
Table 2 shows an example of the composition in the dosage form of capsule.
Table 2
Ingredient Amount (% wt)
TRF 0.01-50%
surfactant/emulsifier 10-60%
oil carrier 0-70%

Claims

1. A composition for regulating appetite in a subject comprising a plant-derived tocotrienol or derivative thereof selected from a tocotrienol-rich fraction, alpha- tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol or a combination of any two or more thereof.
2. A composition according to claim 1, wherein the tocotrienol or derivative thereof is derived from palm, annatto, grape, rice, wheat, barley, rye, oat or a combination of any two or more thereof.
3. A composition according to claims 1 or 2, wherein the tocotrienol or derivative thereof contains 50% to 99% of total tocotrienol content by weight.
4. A composition according to any of claims 1 to 3, wherein the tocotrienol or derivative thereof is a tocotrienol-rich fraction derived from palm, containing 50% to 99% of total tocotrienol content by weight.
5. A composition according to any of claims 1 to 4, wherein the composition comprises essentially of a tocotrienol-rich fraction derived from palm, containing
50% to 99% of total tocotrienol content by weight.
6. A composition according to any of claims 1 to 5, wherein the tocotrienol-rich fraction contains alpha-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma- tocotrienol, delta-tocotrienol, phytosterol, squalene, fatty acid or a combination of any two or more thereof.
7. A composition according to any of claims 1 to 6, wherein the composition is in the form of an oral, intravenous or intradermal preparation.
8. A composition according to any of claims 1 to 7, wherein the appetite regulation in the subject is achieved by increasing leptin expression, secretion or the combination thereof in adipocytes.
9. A method for managing body weight of a subject comprising a step of administering to a subject an effective amount of a composition according to claims 1 to 8.
10. A method for regulating appetite in a subject comprising a step of administering to a subject an effective amount of a composition containing a plant-derived tocotrienol or derivative thereof selected from a tocotrienol-rich fraction, alpha- tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta- tocotrienol or a combination of any two or more thereof.
11. A method according to claim 10, wherein the tocotrienol or derivative thereof is derived from palm, annatto, grape, rice, wheat, barley, rye, oat or a combination of any two or more thereof.
12. A method according to claims 10 or 11, wherein the tocotrienol or derivative thereof contains 50% to 99% of total tocotrienol content by weight.
13. A method according to any of claims 10 to 12, wherein the tocotrienol or derivative thereof is a tocotrienol-rich fraction derived from palm, containing 50% to 99% of total tocotrienol content by weight.
14. A method according to any of claims 10 to 13, wherein the composition comprises essentially of a tocotrienol-rich fraction derived from palm, containing 50% to
99% of total tocotrienol content by weight.
15. A method according to any of claims 10 to 14, wherein the tocotrienol-rich fraction contains alpha-tocopherol, alpha-tocotrienol, beta-tocotrienol, gamma- tocotrienol, delta-tocotrienol, phytosterol, squalene, fatty acid or a combination of any two or more thereof.
16. A method according to any of claims 10 to 15, wherein the composition is administered orally, intravenously or intradermally.
17. A method according to any of claims 10 to 16, wherein the appetite regulation in the subject is achieved by increasing leptin expression, secretion or the combination thereof in adipocytes.
Use of a composition containing a plant-derived tocotrienol or derivative thereof selected from a tocotrienol-rich fraction, alpha-tocotrienol, beta-tocotrienol, gamma-tocotrienol, delta-tocotrienol or a combination of any two or more thereof, in the manufacture of a medicament for regulating appetite in a subject.
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