WO2009064082A1 - Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism - Google Patents

Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism Download PDF

Info

Publication number
WO2009064082A1
WO2009064082A1 PCT/KR2008/006294 KR2008006294W WO2009064082A1 WO 2009064082 A1 WO2009064082 A1 WO 2009064082A1 KR 2008006294 W KR2008006294 W KR 2008006294W WO 2009064082 A1 WO2009064082 A1 WO 2009064082A1
Authority
WO
WIPO (PCT)
Prior art keywords
galla rhois
obesity
extract
fraction
prevention
Prior art date
Application number
PCT/KR2008/006294
Other languages
French (fr)
Inventor
Se Chan Kang
Han Choi
Young Jun Cheong
Young Hwan Jung
Hee Jeong Kim
Eun Hwa Son
Original Assignee
Igsbio
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020080104394A external-priority patent/KR101078002B1/en
Application filed by Igsbio filed Critical Igsbio
Publication of WO2009064082A1 publication Critical patent/WO2009064082A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • 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
    • A61K31/351—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 not condensed with another ring
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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 present invention relates to a composition comprising a Galla Rhois extract for the prevention and treatment of obesity based on the mechanism of autophage inducing system.
  • Obesity is a body condition referring to a state when an excess body fat relative to body weight has accumulated in the body due to the imbalance between energy supply and consumption by inappropriate control by the adipocytes, which are present about 20 billion or more in the body. If the number of adipocytes is increased and energy and triglycerides are excessively accumulated therein, the size of adipocytes is increased, leading to obesity.
  • Primary function of adipocytes is to store and release energy in the body. When energy supply exceeds energy consumption, most of the excess energy is converted into triglycerides and accumulated in the adipocytes.
  • the accumulated triglycerides are degraded into free fatty acids and glycerol to be used as an energy source.
  • the accumulation of triglycerides in the adipocytes due to the energy surplus is a major cause of obesity.
  • Korean Patent No. 588469 discloses a method of inhibiting the differentiation of adipocytes and accumulation of fats in the differentiated adipocytes by using shogaol isolated from ginger. The patent merely discloses that shogaol inhibits the accumulation of fats in adipocytes, but there was no teaching regarding the effect of preventing obesity or reducing body weight in an obesity- induced animal model, thus limiting its applicability.
  • autophagocytosis Different from natural death or necrosis which involves in the death of a whole cell, autophagocytosis, a new type of cell death, relates to the death and regeneration of intracellular organelles such as mitochondria, endoplasmic reticulum and the like. It was believed that these intracellular organelles are usually maintained under static conditions, but recent study has discovered that they undergo a continuous process of apoptosis and regeneration. The old intracellular organelle undergone a degenerative change becomes extinct through such a process and is provided as a nutrient source for the regeneration of a new intracellular organelle. Autophage inducing system (autophagocytosis) can prevent the death of a whole cell or induce the same via the apoptosis and regeneration process of intracellular organelles.
  • autophage inducing system autophagocytosis
  • Galla Rhois is a nest of a parasitic bug, Mellaphis chinensis Bell, formed in the leaves of Rhus javanica L. or other Anacardiaceae, and is primarily composed of gallotannin (50-70%) such as gallic acid, pentagalloyl glucose and the like.
  • gallotannin 50-70% such as gallic acid, pentagalloyl glucose and the like.
  • the present invention has found that an extract of Galla Rhois can inhibit the differentiation of adipocytes based on the mechanism of autophagocytosis and prevent triglycerides from accumulating in the differentiated adipocytes.
  • the present invention has confirmed in an obesity-induced animal model that the Galla Rhois extract exhibits such inhibitory activities of reducing the accumulation of abdominal fats, suppressing the accumulation of liver and epididymal fats, and inhibiting body weight gain. It has been proved that thus confirmed inhibitory activities of the Galla Rhois extract is identical to or higher than those of the prior art obesity inhibitor well-known in the art. Until now it has been reported that the Galla Rhois extract is effective for the prevention and treatment of diabetes through a protective action on beta-cell damage, but there is no report that it can be used as an anti-obesity drug by inhibiting the differentiation of adipocytes and accumulation of fats in the differentiated adipocytes via autophagocytosis.
  • the present inventors have therefore endeavored to develop a new herbal medicine component having higher anti-obesity inhibitory activities than the prior art anti-obesity drugs without causing any side effects, and found that the extract and fraction of Galla Rhois inhibit the differentiation of adipocytes based on the mechanism of autophagocytosis, prevent triglycerides from accumulating in the differentiated adipocytes, and therefore, exhibit remarkable anti-obesity activities in an obesity-induced animal model without showing cytotoxicity.
  • the present invention is directed to overcoming the above deficiencies in the art.
  • One of the objectives of the present invention is to provide a composition for the prevention and treatment of obesity comprising an extract of Galla Rhois or fractions thereof as an active ingredient.
  • Another objective of the present invention is to provide health foods comprising an extract of Galla Rhois or fractions thereof as an active ingredient.
  • composition for the prevention and treatment of obesity comprising an extract of Galla Rhois or fractions thereof as an active ingredient.
  • a method of isolating the active ingredient from Galla Rhois is provided.
  • health foods comprising an extract of Galla Rhois or fractions thereof as an active ingredient.
  • the present invention relates to a composition for the prevention and treatment of obesity comprising an extract of Galla Rhois or fractions thereof as an active ingredient wherein the Galla Rhois is a nest of parasitic bug, Mellaphis chinensis Bell, formed in the leaves of Rhus javanica L. or other Anacardiaceae. Since the composition of the present invention can prevent the differentiation of preadipocytes into adipocytes via autophagocytosis and inhibit the accumulation of fats in the differentiated adipocytes, it can be effectively used for the prevention and treatment of obesity in the form of a food or a pharmaceutical composition without causing side effects.
  • Fig. 1 is a schematic diagram illustrating a method of isolating an ethylacetate fraction from an extract of Galla Rhois according to the present invention.
  • Fig. 2 is a schematic diagram illustrating a method of preparing an extract of Galla Rhois comprising active ingredients according to the present invention.
  • Fig. 3 is a graph showing autophagocytotic activities of the Galla Rhois fraction comprising active ingredients according to the present invention at Day 4 during the induction of adipocyte differentiation.
  • Fig. 4 is a graph showing autophagocytotic activities of the Galla Rhois fraction comprising active ingredients according to the present invention at Day 4 after the adipocyte differentiation.
  • Fig. 5 is a photograph showing autophagocytotic activities of the Galla Rhois extract comprising active ingredients according to the present invention at Day 4 after the adipocyte differentiation.
  • Fig. 6 is a graph showing inhibitory activities of the Galla Rhois extract comprising active ingredients according to the present invention on body weight gain in an obesity-induced animal model.
  • the present invention has found that a Galla Rhois extract and organic solvent fractions thereof inhibit the differentiation of preadipocytes into adipocytes via autophagocytosis, prevent the accumulation of fats in differentiated adipocytes, suppress the accumulation of body fats and body weight gain, and reduce blood triglyceride and low density lipoprotein levels in an obesity-induced animal model. Therefore, the present invention relates to the Galla Rhois extract which can be used as a composition for the prevention and treatment of obesity.
  • Galla Rhois is extracted with alcohol, to thereby obtain an alcohol extract.
  • the alcohol suitable for the present invention may be preferably selected from the group consisting of methanol, ethanol, isopropyl alcohol, propyl alcohol, butyl alcohol and mixtures thereof.
  • the alcohol extract is suspended in water, subjected to fractionation with normal hexane, dichloromethane and ethylacetate in order, and then, concentrated under reduced pressure, to thereby obtain an ethylacetate fraction.
  • the ethylacetate fraction is then subjected to silica gel chromatography so as to obtain active fractions.
  • the silica gel chromatography is carried out according to a gradient elution system by using a mixed solvent of ethylacetate and methanol (98:5 ⁇ 80:20), to thereby obtain the active fractions (Rf: 4.5, in a mixed solvent (3:7) of hexane and ethylacetate).
  • purified active ingredient is a compound represented by the following Formula 1.
  • n is an integer of from 1 to 2. It has been confirmed that thus obtained Galla Rhois alcohol extract and fraction thereof inhibit the differentiation of preadipocytes (3T3-L1) into adipocytes via autophagocytosis, prevent the accumulation of fat in the differentiated adipocytes, and suppress body weight gain in an obesity-induced animal model. Therefore, the composition comprising the Galla Rhois extract and fraction thereof can be effectively used in the prevention and treatment of obesity by inhibiting the differentiation of adipocytes via autophagocytosis and suppressing the accumulation of fats in differentiated adipocytes.
  • composition comprising the Galla Rhois extract or fraction thereof as an active ingredient can be clinically administered as various oral formulations, for example, tablets, troches, lozenge, soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs.
  • the tablet and capsule formulations can be prepared by using binders such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch or sweet potato starch; and lubricating agents such as magnesium strearate, calcium stearate, sodium stearylfumaric acid or polyethylene glycol.
  • the capsule formulations can be prepared by using liquid carriers such as fatty oil in addition to the additives described above.
  • composition comprising the Galla Rhois extract or fraction thereof as an active ingredient can be clinically administered via various parental routes including subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection and the like.
  • the parental formulations can be prepared by mixing the composition with stabilizers or buffering agents and filling the resulting solution in an ampule or a vial as a unit dosage form.
  • the typical daily dose of the Galla Rhois extract or fraction thereof comprising active ingredients of the present invention may range from 10 to 50 mg/kg body weight per day and can be administrated in a single dose or in divided dose.
  • the amount of the active ingredient actually administrated ought to be determined in light of various relevant factors including the conditions to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom. Therefore, the above dose should not be construed as a limitation to the scope of the invention in any way.
  • the term "health foods” as used herein refers to foods that are prepared by adding the active ingredient to food stuffs including beverages, teas, flavorings, chewing gums, snacks and the like, and exert specific favorable effects on health when taken in. Since the health foods utilize foods as a raw material different from general medicines, they are free from side effects that may be occurred by administering a medicine for a long time, and thus, can be taken in as a food for preventing obesity or an adjunctive for treating obesity.
  • the health foods comprising the Galla Rhois extract or fraction thereof according to the present invention as an active ingredient may be used in combination with other foods or food additives.
  • the amount of the active ingredient and other additives mixed can be appropriately determined within a conventional scope according to the objective for using (prophylactic or therapeutic).
  • the Galla Rhois extract or fraction thereof can be contained in the amount of
  • the content of the Galla Rhois extract or fraction thereof can be lowered to the above range. Further, since the Galla Rhois extract or fraction thereof has no problem with safety and side effects, the content thereof can be also increased over the above range.
  • composition of the present invention can be added.
  • Representative examples of the foods may include meat, sausage, bread, chocolate, candies, snacks, biscuits, pizza, instant noodles, other noodles, chewing gums, dairy products including ice creams, soups, beverages, tea, drinks, alcoholic beverages, vitamin complex and the like.
  • the present invention includes all kinds of health foods being acceptable in common sense.
  • the health drinks of the present invention can further comprise various flavorings or natural carbohydrates as an additive like other ordinary drinks.
  • the natural carbohydrate may be exemplified by monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol.
  • a sweetening agent natural sweetening agents such as thaumatin and stevia extracts, or synthetic sweetening agents such as saccharin and aspartame can be used.
  • the natural carbohydrates can be used in the amount ranging from 0.01 to 0.04 g, preferably 0.02 to 0.03 g based on 100 mL of the composition according to the present invention.
  • the health foods of the present invention can further comprise various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid as a thickener, pH regulators, stabilizing agents, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks and the like.
  • the health foods of the present invention can further comprise fruit flesh for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages.
  • the ingredients can be used alone or in combination thereof.
  • the amount of the additives added is of little important, but it is preferable to add the additives in the amount of 0.01 to 0.1 part by weight based on 100 parts by weight of the composition according to the present invention.
  • Preparation Example 3 Isolation of active ingredients The ethlyacetate fraction 5 g obtained in Preparation Example 2 was subjected to silica gel chromatography (gradient elution from 95:5 to 80:20 of ethylacetate : methanol) using a glass column (5x50 cm) packed with silica beads, to separate 7 fractions.
  • MPLC medium- pressure liquid chromatography
  • Combiflash RF TELEDYNE ISCO
  • RediSep 130 gram vol.
  • n is an integer of from 1 to 2).
  • Preparation Example 4 Preparation of a Galla Rhois extract
  • 1 kg of Galla Rhois was grinded, mixed with 3 L of a 50% acetone solution, extracted at 60 " C for 3 hours with gentle stirring.
  • Example 1 Autophagocytotic activity of a Galla Rhois fraction on preadipocytes and adipocytes 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% serum and antibiotics, and cultured in a 5% CO2 incubator at 37 °C .
  • Mouse preadipocyte cell line 3T3-L1 was purchased from ATCC (American Type Culture Collection), DMEM, trypsin, FBS (fetal bovine serum) and BCS (bovine calf serum) were purchased from GibcoTM Invitrogen Corporation, and AdipoRedTM Assay Reagent was purchased from Cambrex.
  • 3T3-L1 cells were inoculated into a 96- well plate at a concentration of 1 x 10 4 cells/ ml and cultured until confluence reaches 100%.
  • the cells were treated with an MDI (0.5 mM 3-isobutyl-l-methyxanthine, 1 ⁇ M dexamethason, 10 ⁇ g/mL insulin, Sigma) cocktail. Two days later, the culture medium was replaced with a fresh, and the cells were treated with insulin at the same concentration. Two days later, the culture medium was replaced with a fresh DMEM supplemented with 10% serum, and the cells were further cultured for 4 days.
  • MDI 0.5 mM 3-isobutyl-l-methyxanthine, 1 ⁇ M dexamethason, 10 ⁇ g/mL insulin, Sigma
  • the cells were treated with an ethylacetate fraction of Galla Rhois at a concentration of 7.5 to 125 ⁇ g/mL. Further, the ethylacetate fraction of Galla Rhois was added to the cells with the same concentration simultaneously with the treatment of DMEM so as to examine autophagocytosis activity after the differentiation.
  • the cells treated for 4 days after the induction of differentiation were fixed with 4% paraformaldehyde for 15 minutes, washed with a phosphate buffered saline (PBS) twice, and treated with 0.05 mM monodansyl cadaverine (MDC) for 60 minutes at 37 ° C .
  • the cells were washed with PBS twice and treated with 1 uM ethidium bromide as an internal standard for 10 minutes.
  • DMSO dimethyl sulfoxide
  • Fluorometer analysis monodansyl cadaverine; excitation 356 ran/ emission 545 nm, ethidium bromide; excitation 530 nm/ emission 590 nm
  • Autophagocytosis activity of the Galla Rhois ethylacetate fraction was determined in these cells depending on the change in concentration [Figs. 3 to 5]. The results are shown in the following Table 1.
  • the Galla Rhois fraction exhibited significantly high autophagocytotic activity in a concentration dependent manner during the induction of differentiation and after the differentiation.
  • Example 2 Inhibitory effects on the differentiation of preadipocytes and accumulation of fats in adipocytes
  • 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% serum and antibiotics, and cultured in a 5% CO 2 incubator at 37 ° C .
  • Murine preadipocyte cell line 3T3-L1 was purchased from ATCC (American Type Culture Collection), DMEM, trypsin, FBS (fetal bovine serum) and BCS (bovine calf serum) were purchased from GibcoTM Invitrogen Corporation, and AdipoRedTM Assay Reagent was purchased from Cambrex.
  • 3T3-L1 cells were inoculated into a 96- well plate at a concentration of 1 ⁇ 10 4 cells/ ml and cultured until reaching 100% confluence.
  • the cells were treated with an MDI (0.5 mM 3-isobuty 1-1 -methy xanthine, 1 ⁇ M dexamethason, 10 ⁇ g/mL insulin, Sigma) cocktail. Two days later, the culture medium was replaced with a fresh one, and the cells were treated with insulin at the same concentration. Two days later, the culture medium was replaced with a fresh DMEM supplemented with 10% serum, and the cells were further cultured for 4 days.
  • MDI 0.5 mM 3-isobuty 1-1 -methy xanthine, 1 ⁇ M dexamethason, 10 ⁇ g/mL insulin, Sigma
  • the cells were treated with a methanol extract and an ethylacetate fraction of Galla Rhois, respectively, at a concentration of 6.25 to 100 ⁇ g/mL. Further, each of the methanol extract and ethylacetate fraction of Galla Rhois was added to the cells with the same concentration simultaneously with the treatment of DMEM so as to examine inhibitory effect on fat accumulation in adipocytes.
  • the adipocytes were fixed with 4% formaldehyde for 5 hours, washed with a phosphate buffered saline (PBS) twice, and treated with a reagent for quantification of intracellular lipid (AdipoRed). After 10 minutes, the resulting cells were subjected to fluorometer analysis (excitation 485 nm; emission 535 nm) and observed under a fluorescence microscope at 488 nm. An EC50 value for each fraction was calculated. In order to examine whether the Galla Rhois extract inhibits fat accumulation in adipocytes, triglyceride was detected by using a TRIGLYZME assay kit (Shinyang, Seoul, Korea) according to the manufacturer's instruction.
  • the Galla Rhois extract (Preparation Example 1) and ethylacetate fraction (Preparation Example 2) showed the highest inhibitory effects on the adipocyte differentiation and fat accumulation in differentiated adipocytes.
  • Example 3 Therapeutic effect of a Galla Rhois extract on obesity in an animal model After 6-week old C57/BL mice (Central Lab. Animal Inc., 18-20 g body weight) were divided into 10 groups, one group of mice were fed a normal diet, and mice in nine other groups were fed a high-fat diet containing 45% fat. After the induction of obesity for 6 weeks, the first and second groups were administered with distilled water (D. W), the third group was administered with 2 mg/kg of Xenical (Roche), and the forth group was administered with 2.4 mg/kg of Reductil (Abbott). The fifth to seventh groups were orally administered everyday with the Galla Rhois extract at a different concentration (10 mg/kg, 20 mg/kg, 40 mg/kg) for 6 weeks, respectively.
  • D. W distilled water
  • the third group was administered with 2 mg/kg of Xenical (Roche)
  • the forth group was administered with 2.4 mg/kg of Reductil (Abbott).
  • the fifth to seventh groups were orally administered everyday with
  • the eighth to tenth groups were orally administered gallotannin everyday at a different concentration (5 mg/kg, 10 mg/kg, 20 mg/kg) for 6 weeks, respectively.
  • the body weight and amount of dietary intake were measured once a week.
  • blood samples were taken from each mouse group, and the levels of triglycerides, neutral lipids and cholesterols in the blood were measured. Further, after the mouse was sacrificed, the liver and epididymis were extracted therefrom, and the level of triglycerides was measured. The results are shown in the following Tables 3 and 4.
  • the Galla Rhois extract exhibited inhibitory effects on the increase in triglycerides, cholesterols and LDL-cholesterols in the obesity- induced mouse in a concentration dependent manner, which confirms that the Galla Rhois extract has prophylactic and therapeutic activities on obesity.
  • the Galla Rhois extract exhibited inhibitory effects on the increase in abdominal and epididymal fats in the obesity- induced mouse in a concentration dependent manner, which confirms that the Galla Rhois extract has prophylactic and therapeutic activities on obesity.
  • the Galla Rhois extract of the present invention exhibits 3- to 4-fold higher inhibitory effect on obesity than the banaba extract.
  • Toxicity test for the Galla Rhois methanol extract and ethylacetate fraction thereof according to the present invention as an active ingredient was carried out as follows.
  • Each of the Galla Rhois methanol extract and ethylacetate fraction was dissolved in 0.5% sodium carboxymethly cellulose, diluted with water, orally administered to mice (10 mice per group) at a concentration of 0, 4, 20, 100, 500 mg/kg, respectively, and then, observed for 7 days. There was no mouse died with single administration and 7 days-repeated administration of the Galla Rhois methanol extract and ethylacetate fraction.
  • a tablet comprising 15 mg of a Galla Rhois extract, an ethlyacetate fraction or a mixture thereof as an active ingredient was prepared according to the following method.
  • the active ingredient 250 g was mixed with lactose 175.9 g, potato starch 180 g and colloidal silicic acid 32 g. After a 10% gelatin solution was added thereto, the resulting mixture was passed through a 14 mesh sieve. Thus sieved mixture was dried, mixed with potato starch 160 g, talc 50 g and magnesium stearate 5 g, and then, formulated into a tablet.
  • Preparation Example 2 Preparation of capsules
  • the active ingredient (Galla Rhois extract, ethlyacetate fraction or a mixture thereof) 500 mg was filled into a soft gelatin capsule, to thereby obtain a capsule.
  • Preparation Example 3 Preparation of beverages
  • the active ingredient (Galla Rhois extract, ethlyacetate fraction or a mixture thereof) 500 mg was dissolved in a proper amount of water, and mixed with vitamin C as a subsidiary ingredient; citric acid, sodium citrate and oligosaccharide as a sweetness additive; and a proper amount of sodium benzoate as a preservative. Then, a total amount of the mixture was adjusted to 100 mL by adding water, to thereby obtain a composition for beverages. At this time, taurine, myoinositol, folic acid, pantoteic acid and the like can be added thereto alone or in combination thereof.

Abstract

The present invention relates to a composition comprising a Galla Rhois extract for the prevention and treatment of obesity based on the mechanism of autophagocytosis. In particular, the present invention has found that the Galla Rhois extract or fraction thereof exhibits inhibitory effects on the proliferation of adipocytes by inducing autophagocytosis thereof, and therefore, provides a method of isolating active ingredients capable of being used as a composition for the prevention and treatment of obesity from Galla Rhois, and a composition for the prevention and treatment of obesity which comprises a Galla Rhois extract or a fraction thereof. That is, the Galla Rhois extract according to the present invention exerts beneficial activities of inducing autophagocytosis of adipocytes in vitro, inhibiting body weight gain in an obesity-induced animal model, reducing the body weight of a body weight gain-induced animal model, and lowering the levels of triglycerides and low density lipoproteins in the blood. Therefore, the Galla Rhois extract according to the present invention can be effectively used as a pharmaceutical composition and health foods for the prevention of obesity and complications thereof and treating the same.

Description

[DESCRIPTION] [Invention Title]
COMPOSITION COMPRISING GALLA RHOIS EXTRACT FOR PREVENTION AND TREATMENT OF OBESITY BY MEANS OF AUTOPHAGIC MECHANISM
[Technical Field]
The present invention relates to a composition comprising a Galla Rhois extract for the prevention and treatment of obesity based on the mechanism of autophage inducing system.
[Background Art]
Obesity is a body condition referring to a state when an excess body fat relative to body weight has accumulated in the body due to the imbalance between energy supply and consumption by inappropriate control by the adipocytes, which are present about 20 billion or more in the body. If the number of adipocytes is increased and energy and triglycerides are excessively accumulated therein, the size of adipocytes is increased, leading to obesity. Primary function of adipocytes is to store and release energy in the body. When energy supply exceeds energy consumption, most of the excess energy is converted into triglycerides and accumulated in the adipocytes. Meanwhile, when energy is exhausted, the accumulated triglycerides are degraded into free fatty acids and glycerol to be used as an energy source. The accumulation of triglycerides in the adipocytes due to the energy surplus is a major cause of obesity. There have been various efforts made to treat obesity as follows: a method of preventing the excessive supply of energy by inhibiting fat absorption into the body and giving a feeling of satiety; a method of degrading fats by using shogaol, caffenine, theophylline and the like that are isolated from ginger as an anti-obesity substance capable of accelerating fat degradation and inhibiting fat accumulation; and a method of degrading fats through the activation of adenylate cyclase and inhibition of phosphodiesterase.
With regard to the inhibition of adipocyte differentiation and fat accumulation, there has been reported a plant extract which is capable of preventing obesity and diabetes by inhibiting preadipocyte differentiation. However, there is no investigation on the functional mechanism to inhibit the accumulation of fats and triglycerides in differentiated adipocytes, which leads to a technical limitation. Meanwhile, Korean Patent No. 588469 discloses a method of inhibiting the differentiation of adipocytes and accumulation of fats in the differentiated adipocytes by using shogaol isolated from ginger. The patent merely discloses that shogaol inhibits the accumulation of fats in adipocytes, but there was no teaching regarding the effect of preventing obesity or reducing body weight in an obesity- induced animal model, thus limiting its applicability.
Therefore, there is a strong need for the development of a novel material from a natural substance which is capable of inhibiting the differentiation of adipocytes and accumulation of fats in the differentiated adipocytes while maintaining its unharmfulness to human body and without any side effect. Further, there remains a need to investigate the inhibitory effect of the new material in an obesity-induced animal model and verify that it shows the inhibitory effects identical to or higher than those of the prior art anti-obesity drug.
Different from natural death or necrosis which involves in the death of a whole cell, autophagocytosis, a new type of cell death, relates to the death and regeneration of intracellular organelles such as mitochondria, endoplasmic reticulum and the like. It was believed that these intracellular organelles are usually maintained under static conditions, but recent study has discovered that they undergo a continuous process of apoptosis and regeneration. The old intracellular organelle undergone a degenerative change becomes extinct through such a process and is provided as a nutrient source for the regeneration of a new intracellular organelle. Autophage inducing system (autophagocytosis) can prevent the death of a whole cell or induce the same via the apoptosis and regeneration process of intracellular organelles. In the meantime, physiological functions and clinical significance of the continuous death and regeneration of intracellular organelles via autophagocytosis were not clear, but recent studies have suggested several evidences on the relationship between autophagocytosis and various kinds of diseases or aging. It has been found that autophagocytosis plays an important role in the development of cancer, degenerative nervous diseases, and the like, but there is no report on the relationship between autophagocytosis and obesity.
Meanwhile, Galla Rhois is a nest of a parasitic bug, Mellaphis chinensis Bell, formed in the leaves of Rhus javanica L. or other Anacardiaceae, and is primarily composed of gallotannin (50-70%) such as gallic acid, pentagalloyl glucose and the like. The present invention has found that an extract of Galla Rhois can inhibit the differentiation of adipocytes based on the mechanism of autophagocytosis and prevent triglycerides from accumulating in the differentiated adipocytes. Further, the present invention has confirmed in an obesity-induced animal model that the Galla Rhois extract exhibits such inhibitory activities of reducing the accumulation of abdominal fats, suppressing the accumulation of liver and epididymal fats, and inhibiting body weight gain. It has been proved that thus confirmed inhibitory activities of the Galla Rhois extract is identical to or higher than those of the prior art obesity inhibitor well-known in the art. Until now it has been reported that the Galla Rhois extract is effective for the prevention and treatment of diabetes through a protective action on beta-cell damage, but there is no report that it can be used as an anti-obesity drug by inhibiting the differentiation of adipocytes and accumulation of fats in the differentiated adipocytes via autophagocytosis.
[Disclosure] [Technical Problem]
The present inventors have therefore endeavored to develop a new herbal medicine component having higher anti-obesity inhibitory activities than the prior art anti-obesity drugs without causing any side effects, and found that the extract and fraction of Galla Rhois inhibit the differentiation of adipocytes based on the mechanism of autophagocytosis, prevent triglycerides from accumulating in the differentiated adipocytes, and therefore, exhibit remarkable anti-obesity activities in an obesity-induced animal model without showing cytotoxicity.
Therefore, the present invention is directed to overcoming the above deficiencies in the art. One of the objectives of the present invention is to provide a composition for the prevention and treatment of obesity comprising an extract of Galla Rhois or fractions thereof as an active ingredient. Another objective of the present invention is to provide health foods comprising an extract of Galla Rhois or fractions thereof as an active ingredient.
[Technical Solution]
According to an embodiment of the present invention, there is provided a composition for the prevention and treatment of obesity comprising an extract of Galla Rhois or fractions thereof as an active ingredient. According to another embodiment of the present invention, there is provided a method of isolating the active ingredient from Galla Rhois.
According to still another embodiment of the present invention, there is provided health foods comprising an extract of Galla Rhois or fractions thereof as an active ingredient.
[Advantageous Effects]
The present invention relates to a composition for the prevention and treatment of obesity comprising an extract of Galla Rhois or fractions thereof as an active ingredient wherein the Galla Rhois is a nest of parasitic bug, Mellaphis chinensis Bell, formed in the leaves of Rhus javanica L. or other Anacardiaceae. Since the composition of the present invention can prevent the differentiation of preadipocytes into adipocytes via autophagocytosis and inhibit the accumulation of fats in the differentiated adipocytes, it can be effectively used for the prevention and treatment of obesity in the form of a food or a pharmaceutical composition without causing side effects.
[Description of Drawings]
The embodiments of the present invention will be described in detail with reference to the following drawings.
Fig. 1 is a schematic diagram illustrating a method of isolating an ethylacetate fraction from an extract of Galla Rhois according to the present invention.
Fig. 2 is a schematic diagram illustrating a method of preparing an extract of Galla Rhois comprising active ingredients according to the present invention. Fig. 3 is a graph showing autophagocytotic activities of the Galla Rhois fraction comprising active ingredients according to the present invention at Day 4 during the induction of adipocyte differentiation.
Fig. 4 is a graph showing autophagocytotic activities of the Galla Rhois fraction comprising active ingredients according to the present invention at Day 4 after the adipocyte differentiation.
Fig. 5 is a photograph showing autophagocytotic activities of the Galla Rhois extract comprising active ingredients according to the present invention at Day 4 after the adipocyte differentiation. Fig. 6 is a graph showing inhibitory activities of the Galla Rhois extract comprising active ingredients according to the present invention on body weight gain in an obesity-induced animal model.
[Best Mode] Hereinafter, the present invention will be explained in more detail.
The present invention has found that a Galla Rhois extract and organic solvent fractions thereof inhibit the differentiation of preadipocytes into adipocytes via autophagocytosis, prevent the accumulation of fats in differentiated adipocytes, suppress the accumulation of body fats and body weight gain, and reduce blood triglyceride and low density lipoprotein levels in an obesity-induced animal model. Therefore, the present invention relates to the Galla Rhois extract which can be used as a composition for the prevention and treatment of obesity.
First, Galla Rhois is extracted with alcohol, to thereby obtain an alcohol extract. The alcohol suitable for the present invention may be preferably selected from the group consisting of methanol, ethanol, isopropyl alcohol, propyl alcohol, butyl alcohol and mixtures thereof. The alcohol extract is suspended in water, subjected to fractionation with normal hexane, dichloromethane and ethylacetate in order, and then, concentrated under reduced pressure, to thereby obtain an ethylacetate fraction.
The ethylacetate fraction is then subjected to silica gel chromatography so as to obtain active fractions. Here, the silica gel chromatography is carried out according to a gradient elution system by using a mixed solvent of ethylacetate and methanol (98:5 → 80:20), to thereby obtain the active fractions (Rf: 4.5, in a mixed solvent (3:7) of hexane and ethylacetate). Among the active fractions, the fraction being detected as a spot identical to tannic acid on TLC is concentrated and purified with MPLC (hexane : ethylacetate = 9:1 to 2:8).
Thus purified active ingredient is a compound represented by the following Formula 1.
[Formula 1]
Figure imgf000008_0001
n wherein R1 is
Figure imgf000008_0002
(here, n is an integer of from 1 to 2). It has been confirmed that thus obtained Galla Rhois alcohol extract and fraction thereof inhibit the differentiation of preadipocytes (3T3-L1) into adipocytes via autophagocytosis, prevent the accumulation of fat in the differentiated adipocytes, and suppress body weight gain in an obesity-induced animal model. Therefore, the composition comprising the Galla Rhois extract and fraction thereof can be effectively used in the prevention and treatment of obesity by inhibiting the differentiation of adipocytes via autophagocytosis and suppressing the accumulation of fats in differentiated adipocytes.
Further, the composition comprising the Galla Rhois extract or fraction thereof as an active ingredient can be clinically administered as various oral formulations, for example, tablets, troches, lozenge, soluble or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs. The tablet and capsule formulations can be prepared by using binders such as lactose, sucrose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch or sweet potato starch; and lubricating agents such as magnesium strearate, calcium stearate, sodium stearylfumaric acid or polyethylene glycol. The capsule formulations can be prepared by using liquid carriers such as fatty oil in addition to the additives described above. Further, the composition comprising the Galla Rhois extract or fraction thereof as an active ingredient can be clinically administered via various parental routes including subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection and the like. The parental formulations can be prepared by mixing the composition with stabilizers or buffering agents and filling the resulting solution in an ampule or a vial as a unit dosage form. The typical daily dose of the Galla Rhois extract or fraction thereof comprising active ingredients of the present invention may range from 10 to 50 mg/kg body weight per day and can be administrated in a single dose or in divided dose. However, it should be understood that the amount of the active ingredient actually administrated ought to be determined in light of various relevant factors including the conditions to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom. Therefore, the above dose should not be construed as a limitation to the scope of the invention in any way. Further, the term "health foods" as used herein refers to foods that are prepared by adding the active ingredient to food stuffs including beverages, teas, flavorings, chewing gums, snacks and the like, and exert specific favorable effects on health when taken in. Since the health foods utilize foods as a raw material different from general medicines, they are free from side effects that may be occurred by administering a medicine for a long time, and thus, can be taken in as a food for preventing obesity or an adjunctive for treating obesity.
The health foods comprising the Galla Rhois extract or fraction thereof according to the present invention as an active ingredient may be used in combination with other foods or food additives. The amount of the active ingredient and other additives mixed can be appropriately determined within a conventional scope according to the objective for using (prophylactic or therapeutic).
The Galla Rhois extract or fraction thereof can be contained in the amount of
0.01 to 100 weight% based on the weight of a total food composition. However, when taken in for a long time for the purpose of managing body weight and preventing obesity, the content of the Galla Rhois extract or fraction thereof can be lowered to the above range. Further, since the Galla Rhois extract or fraction thereof has no problem with safety and side effects, the content thereof can be also increased over the above range.
There is no limitation to the kind of foods to which the composition of the present invention can be added. Representative examples of the foods may include meat, sausage, bread, chocolate, candies, snacks, biscuits, pizza, instant noodles, other noodles, chewing gums, dairy products including ice creams, soups, beverages, tea, drinks, alcoholic beverages, vitamin complex and the like. The present invention includes all kinds of health foods being acceptable in common sense.
The health drinks of the present invention can further comprise various flavorings or natural carbohydrates as an additive like other ordinary drinks. The natural carbohydrate may be exemplified by monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetening agent, natural sweetening agents such as thaumatin and stevia extracts, or synthetic sweetening agents such as saccharin and aspartame can be used. The natural carbohydrates can be used in the amount ranging from 0.01 to 0.04 g, preferably 0.02 to 0.03 g based on 100 mL of the composition according to the present invention.
Besides the above-mentioned additives, the health foods of the present invention can further comprise various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid as a thickener, pH regulators, stabilizing agents, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks and the like. Moreover, the health foods of the present invention can further comprise fruit flesh for the manufacture of natural fruit juice, fruit juice beverages and vegetable beverages. The ingredients can be used alone or in combination thereof. The amount of the additives added is of little important, but it is preferable to add the additives in the amount of 0.01 to 0.1 part by weight based on 100 parts by weight of the composition according to the present invention.
[Mode for Invention]
Preparation Example 1: Preparation of a Galla Rhois extract
After domestic Galla Rhois produced in Jeju island and its vicinity (Korea) was dried in the shade and minced, 1 kg of the washed Galla Rhois was extracted with 10 - 20 L of methanol twice to five times at room temperature, to thereby obtain 232.5 g of a methanol extract (yield: 23.3%) [Fig. I].
Preparation Example 2: Preparation of a Galla Rhois fraction
The methanol extract 232.5 g obtained in Preparation Example 1 was suspended in 2 L of distilled water, subjected to serial fractionation with the same amount of each normal hexane, dichloromethane and ethylacetate, and then, concentrated under reduced pressure, to thereby obtain 106.52 g of an ethylacetate fraction (yield: 10.7%) [Fig. I].
Preparation Example 3: Isolation of active ingredients The ethlyacetate fraction 5 g obtained in Preparation Example 2 was subjected to silica gel chromatography (gradient elution from 95:5 to 80:20 of ethylacetate : methanol) using a glass column (5x50 cm) packed with silica beads, to separate 7 fractions. Among the fractions, the fractions 2 and 3 (Rf= 4.5, in a mixed solvent (3:7) of hexane and ethylacetate) being detected as a spot identical to tannic acid on a TLC plate were concentrated and completely dried, followed by medium- pressure liquid chromatography (MPLC, Combiflash RF, TELEDYNE ISCO) using a RediSep (130 gram vol.) flash column (gradient elution from 9:1 to 2:8 of hexane : ethylacetate). Thus obtained subfraction 100 mg was purified with preparative
HPLC using a reverse phase column packed with octadecylated silica (gradient elution from 20:80 to 70:30 of methanol : water), to thereby separate active fractions (Rf= 4.5, in a mixed solvent (3:7) of hexane and ethylacetate) at 20-35 minutes in which tannin was detected, which were combined and dried under reduced pressure. When analyzed by using tannic acid as a standard, the active fraction separated above included 99.5 weight% of tannin based on the weight of the fraction.
Thus purified tannin was dissolved in a 1.5 N hydrochloric acid solution and subjected to hydrolysis in a 100 °C water bath for 30 minutes, to obtain gallic acid, which was analyzed by HPLC. Further, the purified tannin was dissolved in methanol, mixed with the same amount of 2 M sulfuric acid, subjected to methanolysis in a 100 °C water bath for 30 minutes, to obtain methylgallate, which was analyzed by HPLC. As a result, it has been found that 38.4% of gallic acid is generated from tannin through the hydrochloric acid hydrolysis, and 13.1% of gallic acid and 28.1% of methlygallate are generated from tannin through the methanolysis. Therefore, it has been discovered that tannin included in the ethylacetate fraction purified as an active fraction of Galla Rhois is a compound represented by the following Formula 1 to which gallic acid is coupled. [Formula 1]
Figure imgf000013_0001
wherein R1 is
Figure imgf000014_0001
(here, n is an integer of from 1 to 2).
Preparation Example 4: Preparation of a Galla Rhois extract In order to establish a standard process of preparing a Galla Rhois extract comprising active ingredients, 1 kg of Galla Rhois was grinded, mixed with 3 L of a 50% acetone solution, extracted at 60 "C for 3 hours with gentle stirring. Thus obtained filtrate 2.2 L was distilled under reduced pressure at 45 °C, mixed with 1 L of a mixed solvent (butanol : toluene = 8:2), and then, subjected to liquid-liquid extraction, to remove a water layer. The resulting solution was dried under reduced pressure, mixed with 2 L of a mixed solvent (water : hexane = 1:1), and then, subjected to liquid-liquid extraction, to remove a hexane layer. The resulting water layer was mixed with 1 L of a mixed solvent (butanol : toluene = 8:2) and subjected to liquid-liquid extraction. Thus separated butanol : toluene layers were combined and totally dried under reduced pressure. The resulting residue was completely dissolved in 200 mL of a 50% ethanol solution, dried under reduced pressure, completely dissolved in 200 mL of a 95% ethanol solution, and then, dried under reduced pressure, to thereby completely remove the residual solvent.
Example 1: Autophagocytotic activity of a Galla Rhois fraction on preadipocytes and adipocytes 3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% serum and antibiotics, and cultured in a 5% CO2 incubator at 37 °C . Mouse preadipocyte cell line 3T3-L1 was purchased from ATCC (American Type Culture Collection), DMEM, trypsin, FBS (fetal bovine serum) and BCS (bovine calf serum) were purchased from Gibco™ Invitrogen Corporation, and AdipoRed™ Assay Reagent was purchased from Cambrex.
3T3-L1 cells were inoculated into a 96- well plate at a concentration of 1 x 104 cells/ ml and cultured until confluence reaches 100%. In order to induce the differentiation of preadipocytes into adipocytes, the cells were treated with an MDI (0.5 mM 3-isobutyl-l-methyxanthine, 1 μM dexamethason, 10 μg/mL insulin, Sigma) cocktail. Two days later, the culture medium was replaced with a fresh, and the cells were treated with insulin at the same concentration. Two days later, the culture medium was replaced with a fresh DMEM supplemented with 10% serum, and the cells were further cultured for 4 days. In order to examine autophagocytosis activity during the induction of differentiation, when treated with the MDI cocktail, the cells were treated with an ethylacetate fraction of Galla Rhois at a concentration of 7.5 to 125 μg/mL. Further, the ethylacetate fraction of Galla Rhois was added to the cells with the same concentration simultaneously with the treatment of DMEM so as to examine autophagocytosis activity after the differentiation.
The cells treated for 4 days after the induction of differentiation were fixed with 4% paraformaldehyde for 15 minutes, washed with a phosphate buffered saline (PBS) twice, and treated with 0.05 mM monodansyl cadaverine (MDC) for 60 minutes at 37 °C . The cells were washed with PBS twice and treated with 1 uM ethidium bromide as an internal standard for 10 minutes. After that, the cells were lysed with dimethyl sulfoxide (DMSO), subjected to Fluorometer analysis (monodansyl cadaverine; excitation 356 ran/ emission 545 nm, ethidium bromide; excitation 530 nm/ emission 590 nm), and observed under a fluorescence microscope. Autophagocytosis activity of the Galla Rhois ethylacetate fraction was determined in these cells depending on the change in concentration [Figs. 3 to 5]. The results are shown in the following Table 1.
[Table 1]
Autophagocytotic activity of a Galla Rhois ethylacetate fraction on the preadipocyte differentiation and adipogenesis
Figure imgf000016_0001
As can be shown in Table 1, the Galla Rhois fraction exhibited significantly high autophagocytotic activity in a concentration dependent manner during the induction of differentiation and after the differentiation.
Example 2: Inhibitory effects on the differentiation of preadipocytes and accumulation of fats in adipocytes
3T3-L1 preadipocytes were maintained in DMEM supplemented with 10% serum and antibiotics, and cultured in a 5% CO2 incubator at 37 °C . Murine preadipocyte cell line 3T3-L1 was purchased from ATCC (American Type Culture Collection), DMEM, trypsin, FBS (fetal bovine serum) and BCS (bovine calf serum) were purchased from Gibco™ Invitrogen Corporation, and AdipoRed™ Assay Reagent was purchased from Cambrex. 3T3-L1 cells were inoculated into a 96- well plate at a concentration of 1 χ 104 cells/ ml and cultured until reaching 100% confluence. In order to induce the differentiation of preadipocytes into adipocytes, the cells were treated with an MDI (0.5 mM 3-isobuty 1-1 -methy xanthine, 1 μM dexamethason, 10 μg/mL insulin, Sigma) cocktail. Two days later, the culture medium was replaced with a fresh one, and the cells were treated with insulin at the same concentration. Two days later, the culture medium was replaced with a fresh DMEM supplemented with 10% serum, and the cells were further cultured for 4 days. In order to examine inhibitory effects on adipocyte differentiation, when treated with the MDI cocktail, the cells were treated with a methanol extract and an ethylacetate fraction of Galla Rhois, respectively, at a concentration of 6.25 to 100 μg/mL. Further, each of the methanol extract and ethylacetate fraction of Galla Rhois was added to the cells with the same concentration simultaneously with the treatment of DMEM so as to examine inhibitory effect on fat accumulation in adipocytes.
After the differentiation for 8 days, the adipocytes were fixed with 4% formaldehyde for 5 hours, washed with a phosphate buffered saline (PBS) twice, and treated with a reagent for quantification of intracellular lipid (AdipoRed). After 10 minutes, the resulting cells were subjected to fluorometer analysis (excitation 485 nm; emission 535 nm) and observed under a fluorescence microscope at 488 nm. An EC50 value for each fraction was calculated. In order to examine whether the Galla Rhois extract inhibits fat accumulation in adipocytes, triglyceride was detected by using a TRIGLYZME assay kit (Shinyang, Seoul, Korea) according to the manufacturer's instruction.
The results are shown in the following Table 2. [Table 2]
Inhibitory effects of a Galla Rhois extract and solvent fractions on adipocyte differentiation and fat accumulation
Figure imgf000018_0001
Referring to the results shown in Table 2, the Galla Rhois extract (Preparation Example 1) and ethylacetate fraction (Preparation Example 2) showed the highest inhibitory effects on the adipocyte differentiation and fat accumulation in differentiated adipocytes.
Example 3: Therapeutic effect of a Galla Rhois extract on obesity in an animal model After 6-week old C57/BL mice (Central Lab. Animal Inc., 18-20 g body weight) were divided into 10 groups, one group of mice were fed a normal diet, and mice in nine other groups were fed a high-fat diet containing 45% fat. After the induction of obesity for 6 weeks, the first and second groups were administered with distilled water (D. W), the third group was administered with 2 mg/kg of Xenical (Roche), and the forth group was administered with 2.4 mg/kg of Reductil (Abbott). The fifth to seventh groups were orally administered everyday with the Galla Rhois extract at a different concentration (10 mg/kg, 20 mg/kg, 40 mg/kg) for 6 weeks, respectively. Further, the eighth to tenth groups were orally administered gallotannin everyday at a different concentration (5 mg/kg, 10 mg/kg, 20 mg/kg) for 6 weeks, respectively. The body weight and amount of dietary intake were measured once a week. Six weeks after the administration, blood samples were taken from each mouse group, and the levels of triglycerides, neutral lipids and cholesterols in the blood were measured. Further, after the mouse was sacrificed, the liver and epididymis were extracted therefrom, and the level of triglycerides was measured. The results are shown in the following Tables 3 and 4.
[Table 3]
Effects of a Galla Rhois extraction on blood neutral lipid, triglyceride and cholesterol levels in an obesity-induced mouse
Figure imgf000019_0001
As illustrated in Table 3, the Galla Rhois extract exhibited inhibitory effects on the increase in triglycerides, cholesterols and LDL-cholesterols in the obesity- induced mouse in a concentration dependent manner, which confirms that the Galla Rhois extract has prophylactic and therapeutic activities on obesity.
[Table 4]
Inhibitory effects of a Galla Rhois extract on the accumulation of abdominal and epididymal fats in an obesity-induced mouse
Figure imgf000020_0001
Referring to the results shown in Table 4, the Galla Rhois extract exhibited inhibitory effects on the increase in abdominal and epididymal fats in the obesity- induced mouse in a concentration dependent manner, which confirms that the Galla Rhois extract has prophylactic and therapeutic activities on obesity.
Example 4: Comparison with other herb medicinal extracts
The contents of active ingredients in the Galla Rhois methanol extract according to the present invention and a Banaba ethanol extract (Wellness Banaba Inc., mail-order purchase] were analyzed as follows, and the results are shown in the following Table 5.
1. Direct analysis: each extract of 50 mg was accurately weighed, transferred to 10 mL of a mobile phase, treated by ultrasonication for 1 minute, and centrifuged to separate a supernatant. Thus separated supernatant was analyzed with highspeed liquid chromatography.
2. Hydrolysis: each extract of 50 mg was accurately weighed, dissolved in 10 mL of a 1.5 M HCl solution, and transferred to a sealable test tube. After the test tube was completely sealed, it was subjected to hydrolysis in a 100 °C water bath for 30 minutes and centrifuged to separate a supernatant. Thus separated supernatant was analyzed with high-speed liquid chromatography.
3. Methanolysis: each extract of 50 mg was accurately weighed, dissolved in 5 mL of methanol, mixed with the same amount of 2 M sulfuric acid, and transferred to a sealable test tube. After the test tube was completely sealed, it was subjected to methanolysis in a 100 °C water bath for 30 minutes and centrifuged to separate a supernatant. Thus separated supernatant was analyzed with high-speed liquid chromatography.
The operating conditions for the high-speed liquid chromatography were as follows.
Figure imgf000021_0001
[Table 5]
Figure imgf000022_0001
In order to demonstrate the superiority of the Galla Rhois extract according to the present invention by comparing its active ingredients with those of the banaba extract already known to have the inhibitory effect on adipocyte differentiation and anti-obesity effect, the inhibitory effects of the Galla Rhois methanol extract on 3T3- Ll differentiation and fat accumulation were compared those of the banaba extract according to the same method as described in Example 2. The results of showing the inhibitory effect of each extract on 3T3-L1 differentiation and fat accumulation are shown in Table 6. [Table 6]
Figure imgf000022_0002
As can be seen in Table 6, it has been confirmed that the Galla Rhois extract of the present invention exhibits 3- to 4-fold higher inhibitory effect on obesity than the banaba extract.
Example 5: Toxicity test
Toxicity test for the Galla Rhois methanol extract and ethylacetate fraction thereof according to the present invention as an active ingredient was carried out as follows.
Each of the Galla Rhois methanol extract and ethylacetate fraction was dissolved in 0.5% sodium carboxymethly cellulose, diluted with water, orally administered to mice (10 mice per group) at a concentration of 0, 4, 20, 100, 500 mg/kg, respectively, and then, observed for 7 days. There was no mouse died with single administration and 7 days-repeated administration of the Galla Rhois methanol extract and ethylacetate fraction.
Preparation Example 1: Preparation of tablets
A tablet comprising 15 mg of a Galla Rhois extract, an ethlyacetate fraction or a mixture thereof as an active ingredient was prepared according to the following method. The active ingredient 250 g was mixed with lactose 175.9 g, potato starch 180 g and colloidal silicic acid 32 g. After a 10% gelatin solution was added thereto, the resulting mixture was passed through a 14 mesh sieve. Thus sieved mixture was dried, mixed with potato starch 160 g, talc 50 g and magnesium stearate 5 g, and then, formulated into a tablet.
Preparation Example 2: Preparation of capsules The active ingredient (Galla Rhois extract, ethlyacetate fraction or a mixture thereof) 500 mg was filled into a soft gelatin capsule, to thereby obtain a capsule.
Preparation Example 3: Preparation of beverages The active ingredient (Galla Rhois extract, ethlyacetate fraction or a mixture thereof) 500 mg was dissolved in a proper amount of water, and mixed with vitamin C as a subsidiary ingredient; citric acid, sodium citrate and oligosaccharide as a sweetness additive; and a proper amount of sodium benzoate as a preservative. Then, a total amount of the mixture was adjusted to 100 mL by adding water, to thereby obtain a composition for beverages. At this time, taurine, myoinositol, folic acid, pantoteic acid and the like can be added thereto alone or in combination thereof.

Claims

[CLAIMS] [Claim 1]
A pharmaceutical composition for the prevention and treatment of obesity comprising a Galla Rhois extract or a fraction thereof as an active ingredient.
[Claim 2]
A method of isolating active ingredients from Galla Rhois comprising the following steps of:
1) preparing an alcohol extract by extracting Galla Rhois with alcohol; 2) suspending the alcohol extract in water, fractionating the resulting suspension with normal hexane, dichloromethane and ethylacetate in order, and concentrating under reduced pressure, to obtain an ethylacetate fraction;
3) purifying the ethylacetate fraction by silica gel column chromatography to isolate an active fraction (Rf: 4.5, in a mixed solvent (3:7) of hexane and ethylacetate) represented by the following Formula 1: [Formula 1]
Figure imgf000025_0001
wherein R1 is , and R2 is
(here, n is an integer of from 1 to 2).
[Claim 3] The method according to Claim 2, wherein the alcohol is selected from the group consisting of methanol, ethanol, isopropyl alcohol, propyl alcohol, butyl alcohol and mixtures thereof.
[Claim 4] A health food for preventing and improving obesity which is characterized as comprising a Galla Rhois extract or a fraction thereof as an active ingredient.
PCT/KR2008/006294 2007-11-13 2008-10-24 Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism WO2009064082A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2007-0115749 2007-11-13
KR20070115749 2007-11-13
KR10-2008-0104394 2008-10-23
KR1020080104394A KR101078002B1 (en) 2007-11-13 2008-10-23 Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism

Publications (1)

Publication Number Publication Date
WO2009064082A1 true WO2009064082A1 (en) 2009-05-22

Family

ID=40638896

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2008/006294 WO2009064082A1 (en) 2007-11-13 2008-10-24 Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism

Country Status (1)

Country Link
WO (1) WO2009064082A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112444590A (en) * 2019-09-02 2021-03-05 江阴天江药业有限公司 Method for constructing UPLC fingerprint of gallnut medicinal material, fingerprint constructed by method and application of fingerprint

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009094A1 (en) * 2002-07-24 2004-01-29 Ohio University Methods and compositions for treating diabetes mellitis
US20070254948A1 (en) * 2004-08-23 2007-11-01 Yuko Fukui Lipase Inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004009094A1 (en) * 2002-07-24 2004-01-29 Ohio University Methods and compositions for treating diabetes mellitis
US20070254948A1 (en) * 2004-08-23 2007-11-01 Yuko Fukui Lipase Inhibitors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHOI, M.-Y. ET AL.: "Effect of Rhus Chinensis Gall extract on liver function, plasma lipid composition and antioxidant system in rats with high fat diet.", J. KOREAN SOC. FOOD SCI. NUTR., vol. 28, no. 3, 1999, pages 632 - 637 *
FROHLICH, B. ET AL.: "Gallotannin biosynthesis: two new galloyltransferases from Rhus typhina leaves preferentally acylating hexa- and heptagalloylglucoses.", PLANTA., vol. 216, 2002, pages 168 - 172 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112444590A (en) * 2019-09-02 2021-03-05 江阴天江药业有限公司 Method for constructing UPLC fingerprint of gallnut medicinal material, fingerprint constructed by method and application of fingerprint
CN112444590B (en) * 2019-09-02 2022-12-02 江阴天江药业有限公司 Method for constructing UPLC fingerprint of gallnut medicinal material, fingerprint constructed by method and application of fingerprint

Similar Documents

Publication Publication Date Title
JP6222626B2 (en) Fructose absorption inhibitor
RU2576032C2 (en) Compounds, compositions and methods for cerebral health protection in neurodegenerative disorders
US20080044453A1 (en) Therapeutic Agent for Treatment of Autoimmune Diseases
US8029831B2 (en) Formulations containing thymoquinone for urinary health
US20230125075A1 (en) Composition Containing Paper Mulberry Extracts
KR20090084439A (en) A composition comprising extract of sophora flavescens or prenylated flavonoids compounds isolated therefrom preventing or treating diabetic complication
EP1618875B1 (en) Composition for inhibition or prevention of bone density lowering
JP2009298769A (en) Fat accumulation-suppressing composition
KR101785495B1 (en) Composition comprising Chrisanthemum indicum extract or fraction for treating, improving or preventing obesity or obesity-related disease
KR20160123130A (en) Composition comprising Chrisanthemum indicum extract or fraction for treating, improving or preventing obesity or obesity-related disease
KR102080410B1 (en) Composition for Preventing, Treating or Improving of Andropause Syndrome Comprising Elderberry Extracts
WO2008150074A2 (en) The composition comprising the extracts, fractions and the isolated compounds of rhus verniciflua for prevention and treatment of diabetic complications
KR101078002B1 (en) Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism
KR101303306B1 (en) Composition comprising an extract of Akebiae Caulis for preventing and treating obesity
KR101050003B1 (en) Composition for inhibiting obesity, containing the extract as an active ingredient
WO2009064082A1 (en) Composition comprising galla rhois extract for prevention and treatment of obesity by means of autophagic mechanism
KR100839185B1 (en) Composition comprising plantamajoside for treating or preventing diabete and diabetic complication
KR101332074B1 (en) Composition Comprising Esculetin for Inhibition of Bone Loss
KR101910099B1 (en) Compositions for improving lipid metabolism or anti-obesity as an active ingredient extracted from an immature persimmon by pressurized hydrothermal method
KR101436213B1 (en) Compositions for prevention and/or treatment of obesity comprising extracts of Boehmeria sieboldiana
KR101503583B1 (en) Compositions for anti-obesity comprising extract of Vitis amurensis ruprecht
KR20100021139A (en) Pharmaceutical and food composition for preventing or improving cerebrovascular diseases containing phytoceramide
KR102448274B1 (en) Composition for preventing, ameliorating or treating bone disease comprising crude polysaccharide fraction of Psoralea corylifolia extract as effective component
KR100992995B1 (en) Novel uses of piperonal
KR101543998B1 (en) Composition for treating and improving fatty liver and hyperlipidemia comprising extract of olive as an active ingredient

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08850211

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08850211

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)