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)
  • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to extracts obtained from the seeds of plants of the genus Phaseolus , and the process for the preparation thereof.
• this invention relates to extracts of Phaseolus vulgaris seeds, characterised by a content in ⁇ -amylase inhibitors and phytohaemagglutinins in established ratios which reduce the absorption of glucose originating from starches in the diet, and reduce the appetite after repeated administration.
• ⁇ -Amylase inhibitor is a glycoprotein contained in the seeds of kidney beans ( Phaseolus vulgaris ) which inhibits the enzymatic activity of amylase of animal origin, and especially human amylase, in a differentiated, species-dependent way.
• This inhibitor which was purified for the first time by Marshall and Lauda in 1974 (J. Biol. Chem., 250 (20), 8030-8037, 1975), has attracted interest because of the effects which its pancreatic amylase inhibiting activity can exert on the intestinal absorption of glucose (deriving from enzymatic hydrolysis of starch), and above all for its potential application in the diet industry.
• Carbohydrates are an important source of calories and contribute to the synthesis of fats in individuals that are predisposed to obesity or Type II diabetes.
• the availability of food for survival was intermittent, so the ability to accumulate energy in excess of the amount required for immediate use was essential.
• the adipose cells developed in different parts of the body, are among the sites where energy is accumulated, so that it is easily available when the body needs it. This physiological system, orchestrated by endocrine and neurone secretions, enables humans to survive for long periods, even in the absence of food.
• ⁇ -Amylase inhibitors have long been identified in different legumes and corn, and specific clinical trials have been conducted in last years, with mixed results. Depending on the preparation process used for the concentration and isolation of these inhibitors, the results have been contradictory, as many commercial preparations proved to lack effective activity in vivo. According to the first studies of Layer, Carlson and Di Magno (Gastroenterology, 88(6): 1895, 1902, 1985), this problem is apparently due to the high degree of dilution of the inhibitor in highly impure preparations; in fact, preparations of purified inhibitor are proved to be active on ⁇ -amylase when are directly introduced into the intestinal lumen.
• the products according to the invention are prepared by extraction with an aqueous or hydroethanolic medium and precipitation with suitable mixtures of ethanol and water.
• the process of the invention comprises extraction of the biomass with buffers having a pH ranging between 3 and 6.5, preferably pH 3.5-5.5, and even more preferably pH 4, at temperatures of between 2 and 25° C., and preferably between 4 and 18° C., and subsequent separation of the extract from the biomass by centrifugation.
• buffers having a pH ranging between 3 and 6.5, preferably pH 3.5-5.5, and even more preferably pH 4, at temperatures of between 2 and 25° C., and preferably between 4 and 18° C.
• Suitable buffers for the extraction are typically phosphate, citrate or acetate buffers or dicarboxylic aminoacid buffers, preferably phosphate or citrate buffer. Buffered water-alcohol mixtures can also be used as extraction solvent.
• the biomass can be further extracted three more times with a suitable quantity of buffer, and in any case until its ⁇ -amylase inhibitor and phytohaemagglutinin content is exhausted.
• the combined extracts are clarified by filtration or centrifugation and concentrated in vacuum at a temperature of between 25° and 35° C., preferably 30° C., or by ultrafiltration (10,000 Da cut-off) to a volume corresponding to approx. 10% of the weight of the extract after centrifugation.
• the concentrated aqueous extract is then precipitated with ethanol added to a final concentration of between 60 and 70% v/v, preferably 65% v/v, operating at a temperature of between 18° and 30° C., and preferably between 20° and 25° C.
• the obtained precipitate can be centrifuged and/or filtered, redissolved in demineralised water and re-precipitated in 60% ethanol to reduce the saline part. Alternatively, it can be diafiltered through a membrane with a 10,000 Da cut-off.
• the sediment of the precipitation, which constitutes the extract according to the invention, is dried.
• the efficacy of the extracts has been proved in rats treated with doses of 200 and 400 mg/Kg a day, with free access to the food consisting of a special starch-enriched diet.
• the extracts according to the invention reduce food consumption significantly, while water consumption remains unchanged.
• the product according to the invention is perfectly tolerated, and can be incorporated into pharmaceutical or diet formulations at doses ranging between 50 and 1.000 mg, to be taken at main meals.
• the extract can be incorporated in drinkable forms or the like, to be taken as appetite suppressants.
• the suspension was centrifuged, and the aqueous centrifugate was concentrated 7.5 times (dry residue: 15.0% w/w).
• the concentrate was diluted with 95% ethanol to a concentration of 65% ethanol to obtain a precipitate which was recovered by centrifugation at +22° C.
• the collected solid was dried under vacuum at a temperature not exceeding 50° C.
• the obtained product (yield 2.36%) has an ⁇ -amylase inhibiting activity of 1.050 U/mg, a haemagglutinating activity of 9,000 HAU/g, and an HPLC titre of 7.3% w/w.
• the suspension was centrifuged, and the aqueous centrifugate was concentrated 10.5 times (dry residue: 17.1% w/w).
• the concentrate was diluted with 95% ethanol to a concentration of 65% ethanol to obtain a precipitate which was recovered by centrifugation at +22° C.
• the collected solid was dried under vacuum at a temperature not exceeding 50° C.
• the obtained product (yield 3.5%) has an ⁇ -amylase inhibiting activity of 1,600 U/mg, a haemagglutinating activity of 18,600 HAU/g and an HPLC titre of 10.0% w/w.
• the suspension was centrifuged, the clear liquid phase discarded, and the sediment subjected to a new extraction cycle with 750 mL of water.
• the liquid phase of the second extraction was combined with the first, and concentrated 4.3 times (dry residue: 4.78% w/w).
• the concentrate was diluted with 95% ethanol to a concentration of 70% ethanol to obtain a precipitate which was recovered by centrifugation at +22° C.
• the collected solid was dried under vacuum at a temperature not exceeding 50° C.
• the obtained product (yield 0.88%) has an ⁇ -amylase inhibiting activity of 1,570 U/mg, a haemagglutinating activity of 27,000 HAU/g, and an HPLC titre of 13.6% w/w.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Natural Medicines & Medicinal Plants (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Botany (AREA)
• Mycology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Alternative & Traditional Medicine (AREA)
• Biotechnology (AREA)
• Medical Informatics (AREA)
• Microbiology (AREA)
• Epidemiology (AREA)
• Polymers & Plastics (AREA)
• Food Science & Technology (AREA)
• Nutrition Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• Obesity (AREA)
• Child & Adolescent Psychology (AREA)
• Medicines Containing Plant Substances (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Extraction Or Liquid Replacement (AREA)
• Peptides Or Proteins (AREA)

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