EP1931701A1 - Agent et préparations l'incluant pour l'inhibition de lipases et/ou de phospholipases dans les fluides corporels, les cellules et les tissus - Google Patents

Agent et préparations l'incluant pour l'inhibition de lipases et/ou de phospholipases dans les fluides corporels, les cellules et les tissus

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Publication number
EP1931701A1
EP1931701A1 EP05823512A EP05823512A EP1931701A1 EP 1931701 A1 EP1931701 A1 EP 1931701A1 EP 05823512 A EP05823512 A EP 05823512A EP 05823512 A EP05823512 A EP 05823512A EP 1931701 A1 EP1931701 A1 EP 1931701A1
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EP
European Patent Office
Prior art keywords
protein
composition
cells
phospholipases
tissues
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP05823512A
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German (de)
English (en)
Inventor
Upadhyay Shakti
Yadav Raman
Ansari Arif
Rao Harinarayana
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Reliance Life Sciences Pvt Ltd
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Reliance Life Sciences Pvt Ltd
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Publication date
Application filed by Reliance Life Sciences Pvt Ltd filed Critical Reliance Life Sciences Pvt Ltd
Publication of EP1931701A1 publication Critical patent/EP1931701A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a protein and its compositions for inhibiting or reducing lipase and /or phospholipase enzymes in body fluids, cells, and tissues.
  • the protein as described in the present invention and its composition are useful for the prevention or treatment of clinical manifestations and diseases caused as a consequence of. lipase and phospholipase enzyme activities in the body fluids, cells, and tissues.
  • the present invention relates to the isolation, purification, characterization of the protein, which is an inhibitor of lipase and/or phospholipase enzymes in body fluids, cells and tissues.
  • the present invention also relates to a stable composition of the purified protein against trypsin inactivation.
  • Lipases and phospholipases are key control elements in mammalian metabolism. They share many common features that set them apart from other metabolic enzyme classes, most importantly their association with "two-dimensional" substrates, i.e., lipid droplets, lipoproteins, phospholipid layers, biomembranes, and the resulting implications for their cleavage mechanism and regulation.
  • pancreatic lipase is believed to be effective in causing a partial hydrolysis of triglycerides to obtain fatty acids and monoglycerides that, together with the bile acids, form complexes, which are then absorbed through the intestinal mucosa.
  • Hepatic lipase (HL) and lipoprotein lipase (LPL) are the two major lipolytic enzymes responsible for the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Both lipases are attached to the vascular endothelium via cell surface proteoglycans.
  • HL is primarily involved in the metabolism of chylomicron remnants, intermediate density lipoproteins, and high-density lipoproteins
  • LPL catalyzes the hydrolysis of triglycerides from chylomicrons and very low-density lipoproteins.
  • HL and LPL appear to serve as ligands that mediate the interaction of lipoproteins to cell surface receptors and/or proteoglycans.
  • LPL Accumulation and distribution of triglyceride-rich lipoprotein-associated fatty acids at extra- hepatic sites is facilitated by LPL.
  • the enzyme is also involved in several non-lipolysis associated functions, including the cellular uptake of whole lipoprotein particles and lipophilic vitamins.
  • the tissue-specific variations of LPL expression have been implicated in the pathogenesis of various lipid disorders, obesity, and atherosclerosis.
  • LPL expressed by cells of the vascular wall, particularly macrophages have identified additional actions of the enzyme that contribute to the promotion of foam cell formation and atherosclerosis. Development of drugs specifically acting on the cholesteryl ester transfer protein and lipoprotein lipase systems, are being explored.
  • lipases as drug targets for the treatment of metabolic syndrome and cardiovascular disorders is increasingly recognized. It is now believed that the front line therapy for diseases related to lipid absorption and metabolism should be to inhibit or reduce lipase activity in the body fluids, cells, and tissues.
  • Lipase inhibitors have been reported from various natural products, especially from microbial sources.
  • the example of such inhibitors include lipstatin and Panclicins A-E from Streptomyces species or their synthetic derivatives that inhibit the hydrolysis of triglycerides and cholesterol esters (Hochuli et al., Lipstatin, and Inhibitor of Pancreatic Lipase, Produced by Streptomyces Toxytricini, IL Chemistry and Structure Elucidation, J. Antibiot.
  • LPL has been shown to be involved in the pathogenesis of atherosclerosis (Mead et al, Lipoprotein Lipase, a Key Role in Atherosclerosis? , FEBS Lett., 1999 Nov. 26, 462(1-2): 1-6). Inhibition of LPL is believed to prevent the atherosclerotic process (Zimmerman et al., Lipoprotein Lipase Mediates the Uptake of Glycated LDL in Fibroblasts, Endothelial Cells, and Macrophages, Diabetes, 50, 1643-1653, 2001).
  • Phospholipases specifically act on and hydrolyse membrane phospholipids and generate mediators implicated in signal transduction and inflammatory processes.
  • the role of phospholipase A2 (PLA2) is well known in the generation of arachidonic acid, which is responsible for leukotriene and prostaglandin synthesis; PLA2 inhibitors have been proposed as drugs for variety of inflammatory and degenerative diseases.
  • Lipoprotein-associated phospholipase A2 has been shown to be involved in atherosclerosis and its inhibition is being proposed for its treatment (Leach et al., Lipoprotein-Associated PLA2 Inhibition—A Novel, Non- Lipid Lowering Strategy for Atherosclerosis Therapy, Farmaco, 2001 Jan-Feb, 56(l-2):45-50).
  • the present invention relates to a protein and composition comprising the same for inhibiting or reducing lipases and/or phospholipase enzymes in body fluids, cells, and tissues.
  • the protein as described in the present invention and the composition comprising of the same are useful for the prevention or treatment of clinical manifestations and diseases caused as a consequence of lipase and/or phospholipase enzyme activities in the body fluids, cells, and tissues.
  • the protein having lipase and /or phospholipase inhibitory activity can be synthesized, produced by recombinant technology or isolated from natural sources.
  • the protein is isolated from the seeds of plant species belonging to Moringa genus. -
  • the protein is purified by a simple, efficient and cost-effective process.
  • the protein is protected from trypsin inactivation by combination with trypsin inhibitors.
  • compositions for inhibition of lipases and/or phospholipases comprises of protein as described in the present invention in a therapeutically effective amount and pharmaceutically inert adjuvants, diluents or carriers.
  • compositions for inhibition of lipases and/or phospholipases comprising of protein as described in the present invention may also be combined with other active ingredients.
  • the protein as described in the present invention or composition comprising the same is believed to have the ability to inhibit lipases and/or phospholipases under physiological conditions, and thereby would have corresponding effectiveness for prevention or treatment of metabolic syndrome, cardiovascular disorders, and inflammatory diseases.
  • the protein as described in the present invention or the compositions comprising proteins are useful as for inhibition of lipase and/or phospholipase in the body fluids, cells, and tissues for the prevention and treatment of metabolic syndrome, cardiovascular disorders, and inflammatory diseases.
  • the protein as described in the present invention or the composition comprising a protein can be used for prevention or treatment of metabolic disorders like obesity, diabetes, and atherosclerosis.
  • the protein as described in the present invention or the composition comprising a protein can be used in inhibiting or reducing accumulation of lipids in monocytic cells, vascular cells, hepatocytes, and adipose tissues.
  • the protein as described in the present invention or the composition comprising a protein can be used for prevention or treatment of inflammatory diseases, such as arthritis, atherosclerosis, and septic shock, that are caused by the activation and/or the action of phospholipases.
  • inflammatory diseases such as arthritis, atherosclerosis, and septic shock
  • the protein as described in the present invention or the composition comprising a protein can be used for skin and hair care and cosmetic preparations.
  • the protein as described in the present invention or the composition comprising a protein can be used to prevent or treat cellular and tissue damage caused by microbial pathogens secreting lipases and/or phospholipases.
  • composition comprising a protein can be used in veterinary medicine for the treatment and prophylaxis of diseases caused or aggravated by lipase and/or phospholipase activity in the body fluids, cells and tissues.
  • the present invention also provides the pharmaceutical formulations comprising protein either alone or a suitable pharmaceutically acceptable adjuvant useful in inhibition of lipases and/or phospholipases in the body fluids, cells, and tissues for the prevention and treatment of metabolic syndrome, cardiovascular disorders, and inflammatory
  • the present invention also relates to the delivery of the stable protein so as to protect from trypsin inhibition. In the preferred embodiments the present invention also provides for an oral delivery of the isolated protein.
  • the present invention also provides the manner of manufacture of medicaments comprising of protein as described in the present invention in a therapeutically effective amount either alone or in combination with pharmaceutically acceptable adjuvant.
  • the protein as described in the present invention may also be combined with other active ingredients.
  • the present invention relates to a protein containing 5 - 100 amino acid residues and having a molecular weight ranging from 0.5 - 10 kD, with or without glycosylation.
  • the protein has inhibitory or reducing effect on lipase and/or phospholipase enzyme activities.
  • the protein may be synthesized or produced through recombinant DNA technology or it may be isolated from plant material.
  • the protein as disclosed in the present invention is isolated from species belonging to genus Moringa, more preferably it is isolated from seeds of plant Moringa.
  • the protein can be isolated by the method as disclosed herein later under the examples.
  • the protein isolated is then purified by suitable techniques.
  • the purified protein is then characterized by SDS PAGE and LC -MS techniques for the following sequence.
  • the protein has partial sequence ID as following SEQ. ID. NO. 1 CGQQLRNISPPQRCPSLRQAVQLAHQQQGQGPQQVRQMYR Further this protein was studied for lipase inhibitory activity in presence of synthetic and natural substrate.
  • Moringa seed protein was further analyzed for its activity in presence of trypsin.
  • trypsin inhibitor such as Soya bean (Glycine max) , Lima Bean (Phaseolus limensis), Prosopis julifiora seed, sunflower seed (Helianthus annus) and combinations thereof were selected for combination with the lipase/phospholipase inhibitor of the present invention.
  • the protein was studied for lipase inhibition in presence of soya protein.
  • the present invention also relates to the compositions for inhibition of lipases and/or phospholipases comprising of protein as described in the present invention in a therapeutically effective amount and pharmaceutically inert adjuvants, diluents or carriers.
  • the compositions for inhibition of lipases or phospholipases comprising of protein as described in the present invention may also be combined with other active ingredient or ingredients.
  • the protein as described in the present invention or composition comprising the same is believed to have the ability to inhibit lipases and/or phospholipases under physiological conditions, and thereby would have corresponding effectiveness for prevention or treatment of metabolic syndrome, cardiovascular disorders, and inflammatory diseases.
  • the protein as described in the present invention or the compositions comprising protein are useful for inhibition of lipases and/or phospholipases in the body fluids, cells, and tissues for the prevention and treatment of metabolic syndrome, cardiovascular disorders, and inflammatory diseases.
  • the protein as described in the present invention or the composition comprising a protein can be used for prevention or treatment of metabolic disorders like obesity, diabetes, and atherosclerosis.
  • the protein as described in the present invention or the composition comprising a protein can be used in inhibiting or reducing accumulation of lipids in monocytic cells, vascular cells, hepatocytes, and adipose tissues.
  • the protein as described in the present invention or the composition comprising a protein can be used to prevent or treat cellular and tissue damage caused by microbial pathogens secreting lipases and /or phospholipases.
  • the protein as described in the present invention or the composition comprising a protein can be used for skin and hair care and cosmetic preparations.
  • the protein as described in the present invention or composition comprising the same can be administered in any conventional oral, buccal, nasal, by inhalation spray in unit dosage form, parenteral, (for example, intravenous, intramuscular, subcutaneous intrastemal or by infusion techniques), topical (for example, powder, ointment or drop), transdermal, intracisternal, intravaginal, intraperitoneal, intravesical, or rectal.
  • parenteral for example, intravenous, intramuscular, subcutaneous intrastemal or by infusion techniques
  • topical for example, powder, ointment or drop
  • transdermal intracisternal
  • intravaginal intraperitoneal
  • intravesical intravesical
  • rectal transdermal
  • the compound of the present invention and at least one other pharmaceutically active agent may be administered either separately or in the pharmaceutical composition comprising both. It is generally preferred that such administration be oral. However, if the subject being treated is unable to swallow, or oral administration is otherwise impaired or undesirable, parenteral or trans
  • formulations according to the present invention for reducing lipase and/or phospholipase activity in body fluids, cells, and tissues will comprise, as the essential active ingredient, the protein of the present invention.
  • the present invention provides formulations for reducing lipase and/or phospholipase activity in body fluids, cells, and tissues comprising the protein of the present invention, it ca be formulated either alone or in combination with a known pharmaceutically acceptable and inert adjuvant, diluent or carrier.
  • a formulation comprising the protein according to the present invention can be formulated together with one or more routine additives, carriers, assistants, and the like. It can be formulated for oral administration and can be used in the field of pharmaceuticals. Examples of suitable forms for oral administration include tablets, capsules, granules, fine granules, spherules, syrups, and drinks. In the preferred embodiments it is formulated in the form of spherules. In most preferred embodiments the spherules are enteric coated.
  • suitable carrier materials are water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkyleneglycols, petroleum jelly, etc.
  • the pharmaceutical preparations can be made up in a solid form (e.g., as tablets, dragees, suppositories or capsules) or in a liquid form (e.g., as solutions, suspensions or emulsions).
  • the pharmaceutical preparations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure, or buffers. They can also contain other therapeutically valuable substances.
  • the essential ingredients are mixed with one or more pharmaceutically-acceptable vehicles, carriers, excipients, binders, antiseptics, anti-oxidants, stabilizers, taste corrigents, buffers, and the like, followed by formation into a desired unit dosage form.
  • adjuvants that can be incorporated in tablets, capsules, or the like, upon formulation according to the present invention include: binders such as gum arabic, corn starch, and gelatin; lubricants such as magnesium stearate; excipients, such as crystalline cellulose; swelling agents, such as gelatinized starch and arginic acid; sweeteners, such as sucrose, lactose, and saccharin; and taste corrigents, such as peppermint and cherry.
  • a liquid carrier such as oil, can also be incorporated together with the above adjuvants.
  • tablets can be coated with shellac, sugar, or any acidic pH resistant polymer.
  • Syrups and elixirs can be added with sucrose as a sweetener, methylparaben or propylparaben as an antiseptic, and/or peppermint or orange flavor as a taste corrigent.
  • the formulation can be used as a medicament for lowering total serum lipid cholesterol, and for the treatment of obesity, ischemic heart diseases, arteriosclerosis, cerebrovascular dementia, diabetes, angiopathic Parkinson's diseases, inflammatory diseases, and the like.
  • the formulation described in the present invention may be administered once or few times a day in an amount of about 10 to 2000 mg/day in terms of dry weight.
  • the active ingredients of the formulation according to the present invention can be added to various foods for the reduction of the serum lipid level or the total blood cholesterol level or accumulation of fat in tissues.
  • foods to which the active ingredients according to the present invention can be added include tea beverages, juice, coffee, drinks, carbonated beverages, chewing gum, candies, caramels, chocolates and ice creams.
  • protein or the composition comprising of protein as described in the present invention may be useful as veterinary medicine for the treatment and prophylaxis of diseases caused or aggravated by lipase and/or phospholipase activity in the body fluids, cells, and tissues in animals.
  • This invention further relates to a method for inhibiting or reducing lipase and/or phospholipase activity in body fluids, cells, and tissues by administration of a formulations comprising of the protein.
  • the present invention is based on our discovery that the protein is a potent inhibitor of lipases and/or phospholipases using specific enzyme assays as disclosed herein later. Further, the invention is also based on our observation that the protein remains stable during formulation and thereby it would retain its activity. After confirmation of the invitro results, animal studies were conducted for its lipase activity.
  • Powdered sample of Moringa seed was soaked in water.
  • the extract was filtered and then the clear extract was mixed with 0.1 M phosphate buffer (1:1).
  • the solution was then centrifuged, supernatant collected and subjected to acetone precipitation (chilled acetone) for 30 min at room temperature.
  • the resulting solution was centrifuged at precipitate collected and dissolved in 0.1 M phosphate buffer, pH 8.0. This solution was mixed well and subjected to TCA precipitation.
  • the solution was again centrifuged.
  • the sticky precipitate was finally dissolved in 0.1 M Phosphate buffer, pH 8.0 by vortexing.
  • the above protein solution was then purified by gel filtration chromatography to obtain pure protein using Sephadex G-50 column equilibrated with 5OmM, pH-8.0 phosphate buffer.
  • the collected fractions were tested for protein activity by Bradford microplate-based assay and the purity of protein was confirmed by running on 17% SDS-PAGE (Laemmli, 1970) gels stained with Coomassie Brilliant blue.
  • SDS-PAGE (17%) was performed with the protein isolated from Moringa seeds and stained with Coomassi blue. 5 +/- 1 kD band was cut and transferred to a siliconized tube and washed and destained in 200 ⁇ L 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 ⁇ L of 10 mM dithiolthreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 ⁇ L 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h.
  • the reagent was removed and the gel pieces dehydrated in 100 ⁇ L acetonitrile.
  • the acetonitrile was removed and the gel pieces rehydrated in 100 ⁇ L 0.1 M ammonium bicarbonate.
  • the pieces were dehydrated in 100 ⁇ L acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation.
  • the gel pieces were rehydrated in 20 ng/ ⁇ L trypsin in 50 mM ammonium bicarbonate on ice for 10 min. Any excess trypsin solution was removed and 20 ⁇ L 50 mM ammonium bicarbonate added.
  • the sample was digested overnight at 37 oC and the peptides formed extracted from the polyacrylamide in two 30 . ⁇ L aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 25 ⁇ L for MS analysis.
  • the LC-MS system consisted of a Finnigan LCQ ion trap mass spectrometer system with a Protana nanospray ion source interfaced to a self-packed 8 cm x 75 um id Phenomenex Jupiter 10 um C18 reversed-phase capillary column. 0.5-5 ⁇ L volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.25 ⁇ L/min.
  • the nanospray ion source was operated at 2.8 kV.
  • the digest was analyzed using the double play capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in sequential scans.
  • the partial sequence ID of the protein was identified as shown below:
  • Enzyme assay was performed by method described by Winkler and Stuckmann, 1979, with modification where there was use of pancreatic lipase. Assay was designed, using a 96-well format. The substrate used in this assay was p-nitrophenol palmitate (Sigma, Cat No- N-2752). 4.5 mg of p-nitrophenol palmitate was dissolved in 200 ⁇ l of N, N-dimethylformamide (Sigma, Cat No, D-4551) and volume made up to 10 ml with 0.1 M .Ph 8.0-phosphate buffer.
  • Lipase (Sigma, Cat No, L-3126) sample was prepared by dissolving the enzyme in O.lM-phosphate buffer at a concentration of 5mg/ml.
  • the reaction mixture consisted of substrate solution-150 ⁇ l; phosphate buffer (pH 8.0, 0.1 M) - 40 ⁇ l and lipase solution- lO ⁇ l.
  • the reaction mixture was incubated at 37 0 C and optical density was measured at 405 nm after incubation.
  • Enzyme activity was presented in the form of international unit (IU).
  • One enzyme unit of lipase is defined as that quantity releasing lnm of free phenol from the substrate (p-nitro phenol palmitate)/ ml/min under the standard assay condition (Winkler K. W. and Stuckman M, 1979 Glycogen hyaluronate and some other polysaccharides greatly enhances the formation of exolipase by Serratia marcescens, J. of Bacteriology 138: 663-670). It is derived from standard graph of p-nitro phenol.
  • Inhibition assay was performed in a dose dependent manner .
  • concentration of the protein checked 40 ⁇ g-0.156 ⁇ g/ml reaction mixture.
  • the assay was similar to assay described above except 40 ⁇ l of inhibitor solution was used instead of phosphate buffer in control. Released p- nitro phenol was recorded at 405 nm. Enzyme inhibition was presented in the term of percentage inhibition simply on the basis of change in international unit (IU), which was calculated from standard graph. Result: Table- 1
  • Moringa protein was able to inhibit the pancreatic lipase even at very low concentration.
  • Lipase activity was also measured by titrating free fatty acids liberated in the reaction mixture by following a modified method of "Ishiia C et al, 1988, Inhibition of lipase by proteins and their inhibitory mechanism, Nippon Shokuhin Kogyo Gakkaishi, 35 (6), 430-439.
  • the reaction was performed in tubes by shaking at 37°C for 30 min.
  • the reaction mixture was prepared with 1.5 ml of Mcllvaine buffer, pH-7.0, 0.24 ml of olive oil, and 0.5 ml of inhibitor solution and water all in final volume of 5.5 ml . After 5 min preincubation, the reaction was then started by adding 0.5 ml of enzyme solution (5.0 mg) .
  • Moringa seed protein was able to inhibit the pancreatic lipase during the hydrolysis of natural substrate olive oil.
  • the lipase inhibitory activity was performed after tryptic cleavage of Moringa seed protein.
  • the isolated protein 500 ⁇ g/ml estimated by Bradford method
  • trypsin 0.5%) in 50 mM, Tris buffer, pH-9.0 & sample buffer for 24 hrs at 37 0 C in 1:1:2 ratio. After incubation all the samples were allowed for thermal inactivation at 7O 0 C for 10 min.
  • the lipase inhibitory assay was performed as described earlier except 40 ⁇ l of test solution (trypsin treated/ untreated) was used instead of phosphate buffer in control. Released p-nitro phenol was recorded at 405 nm.
  • Lipase activity was presented in the term of percentage inhibition simply on the basis of change in international unit (IU), which was calculated from standard graph. Result: It was observed that Moringa seed protein lost the lipase inhibitory activity after tryptic cleavge which suggests the lipase inhibitory activity present of protein. Table-3
  • Moringa seed protein was protected against trypsin performed using various concentration of soya protein.
  • the Moringa seed protein (49.1 ⁇ g/100ul estimated by Bradford method) was incubated with 100 ⁇ l trypsin (0.5%) solution and various concentration 100 ⁇ l of soya protein in 50 mM, buffer , pH-9.0 & sample buffer for 24 hrs at 37O 0 C in 1:1:1:1 ratio. After incubation all the samples were allowed for thermal inactivation at 70°C for 10 min.
  • the assay was performed as described earlier except 40 ⁇ l of test solution (treated/ untreated) was used instead of phosphate buffer in control. Released p-nitro phenol was recorded as OD at 405 nm. Lipase activity was presented in the term of percentage inhibition simply on the basis of change in international unit (IU), which was calculated from standard graph.
  • IU international unit
  • composition of the protein Moringa seed protein 50.0 gms
  • Microcrystalline Cellulose 336.0 gms
  • Lactose 84.0 gms
  • 10% starch paste was prepared using Maize starch ( 30.0 gms ) & required amount of water.
  • Starch Paste was added in the mixer & mixing was continued until dough was formed with a consistency suitable for extrusion.
  • the dough mixture was charged into a Fuji Paudal EXDS - 60 extruder fitted with a 0.8 mm radial screen. The extrude were dried using drying oven at 40°C for 8 hrs.
  • Coating was done in two steps viz 1) Seal coating 2) Enteric coating.
  • Seal coating was done by using HPMC as a film forming polymer & enteric coating was done with the help of Eudragit L 30 D 55 as an enteric polymer & Yellow iron oxide as coloring agent.
  • the spherules were filled in size 0 hard gelatin transparent capsules.
  • the in-vitro protein analysis was done on Coated Spherules as well as on Filled capsules by using Brad ford Method.
  • Sample A* Spherules sample
  • Sample B** Filled Capsules sample Graph was plotted Cone, v/s Absorbance

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une protéine et des préparations comprenant ladite protéine pour l'inhibition de lipases et/ou de phospholipases dans les fluides corporels, les cellules et les tissus, pour un traitement prophylactique et thérapeutique du syndrome métabolique, des troubles cardio-vasculaires et des maladies inflammatoires. La protéine est soit isolée à partir d'une espèce de plantes, soit synthétisée, soit produite par technologie d'ADN recombinant.
EP05823512A 2005-10-05 2005-10-05 Agent et préparations l'incluant pour l'inhibition de lipases et/ou de phospholipases dans les fluides corporels, les cellules et les tissus Withdrawn EP1931701A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IN2005/000330 WO2007039911A1 (fr) 2005-10-05 2005-10-05 Agent et préparations l’incluant pour l'inhibition de lipases et/ou de phospholipases dans les fluides corporels, les cellules et les tissus

Publications (1)

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EP1931701A1 true EP1931701A1 (fr) 2008-06-18

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EP05823512A Withdrawn EP1931701A1 (fr) 2005-10-05 2005-10-05 Agent et préparations l'incluant pour l'inhibition de lipases et/ou de phospholipases dans les fluides corporels, les cellules et les tissus

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EP (1) EP1931701A1 (fr)
JP (1) JP2009511466A (fr)
WO (1) WO2007039911A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2025242A1 (fr) * 2007-07-25 2009-02-18 Universität Hohenheim Aliment et additif d'alimentation pour les herbivores, et son procédé de fabrication
US20230070929A1 (en) * 2020-05-21 2023-03-09 Agence Francaise Pour Le Developpement D'al Ula Extract of moringa peregrina seed cake, method for obtaining same and use thereof in cosmetic or nutricosmetic compositions

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2776519B1 (fr) * 1998-03-24 2000-11-17 Serobiologiques Lab Sa Utilisation d'au moins un extrait proteique de graines de plantes du genre moringa et composition cosmetique et/ou pharmaceutique correspondante
US20040147723A1 (en) * 2001-07-19 2004-07-29 Nicolas Mermod Moringa seed proteins
FR2834642A1 (fr) * 2002-01-14 2003-07-18 Philippe Thurot Composition pour lutter contre le cholesterol et ameliorer la digestion et le transit intestinal

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007039911A1 *

Also Published As

Publication number Publication date
WO2007039911A1 (fr) 2007-04-12
JP2009511466A (ja) 2009-03-19

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